Histone H4


Accurate prediction of inducible transcription factor binding intensities in vivo

DNA sequence and local chromatin landscape act jointly to determine transcription factor (TF) binding intensity profiles. To disentangle these influences, an experimental approach, called protein/DNA binding followed by high-throughput sequencing (PB-seq), was developed that allows the binding energy landscape to be characterized genome-wide in the absence of chromatin. These methods were applied to the Drosophila Heat Shock Factor (HSF), which inducibly binds a target DNA sequence element (HSE) following heat shock stress. PB-seq involves incubating sheared naked genomic DNA with recombinant HSF, partitioning the HSF-bound and HSF-free DNA, and then detecting HSF-bound DNA by high-throughput sequencing. PB-seq binding profiles were compared with ones observed in vivo by ChIP-seq and statistical models were developed to predict the observed departures from idealized binding patterns based on covariates describing the local chromatin environment. It was found that DNase I hypersensitivity and tetra-acetylation of H4 were the most influential covariates in predicting changes in HSF binding affinity. The extent was investigated to which DNA accessibility, as measured by digital DNase I footprinting data, could be predicted from MNase-seq data and the ChIP-chip profiles for many histone modifications and TFs. GAGA element associated factor (GAF), tetra-acetylation of H4, and H4K16 acetylation were found to be the most predictive covariates. Lastly, an unbiased model was generated of HSF binding sequences, which revealed distinct biophysical properties of the HSF/HSE interaction and a previously unrecognized substructure within the HSE. These findings provide new insights into the interplay between the genomic sequence and the chromatin landscape in determining transcription factor binding intensity (Guertin, 2010).

The PB-seq technique combined with EMSA and competition assays provides a straightforward, yet versatile and powerful framework for characterizing all potential binding sites in a genome, regardless of tissue specificity, developmental stage, or environmental conditions. Comparing in vitro and in vivo binding profiles, in the context of pre-induction genomic chromatin landscape, revealed DNase I hypersensitivity, H4 tetra-acetylation, and GAF as critical features that modulate cognate element binding intensity in vivo. Furthermore, DNase I sensitivity was found to be strongly influenced by high GAF occupancy and histone acetylation, while repressive factors were minimally influential in the statistical models. Finally, the full set of potential genomic binding sites provided a rich data set that was used to build more detailed sequence models, which tease apart substructure and features that are lost with traditional PSSM models (Guertin, 2010).

One initially surprising observation from this study was that 40% of the in vivo HSF peaks were not found in vitro. It is believed that the limited dynamic range for quantifying in vitro binding affinity may be responsible for the lack of detectable in vitro peaks. Although in vitro binding was quantified over an order of magnitude (40-400 pM), the experimental concentrations of HSF and genomic DNA and the depth of sequencing do not permit the detection of lower affinity HSF binding sites. For instance, only eleven sequence tags would be predicted to underlie a hypothetical 5 nM HSF binding site, and these would not be distinguishable from background. Upon further examination, it was found that the composite HSE representing those in vivo binding sites that were not found in vitro is more degenerate than those found using both assays. Moreover, the in vivo sites that were not found using PB-seq were also more accessible in vivo, in support of the hypothesis. Performing PB-seq at a range of protein and DNA concentrations, or increasing sequence coverage would expand the dynamic range of quantification by PB-seq (Guertin, 2010).

The notion that motif accessibility is driving inducible TF binding in vivo is supported by independent studies of distinct TFs: STAT1, HSF, glucocoticoid receptor (GR), and GATA1. These studies show that the chromatin landscape prior to TF binding influences inducible TF binding. In the one study, it was found that a large fraction of STAT1 induced binding sites contained H3K4me1/me3 marks prior to interferon-gamma (IFN-γ) induced STAT1 binding. Previously studies found that inducible HSF binding sites are marked by active chromatin compared to sites that remain HSF-free. A more recent study has shown that inducibly bound GR sites are marked by DNase I hypersensitive chromatin prior to GR binding. Likewise, the permissive chromatin state at GATA1 binding sites is established even in GATA1 knock out cells. While these correlations are instructive, no previous attempt has been made to model inducible TF binding using biological measurements of chromatin landscape present prior to TF binding. Recent models have successfully inferred TF binding profiles using DNA sequence and chromatin landscape data, generated at the same time the TF is bound. However, these models do not distinguish between the influence TFs have upon local chromatin and the chromatin features that permit TF binding. In contrast, this study modeled the changes between HSF in vitro binding (PB-seq) and in vivo binding (ChIP-seq) landscapes as a function of the non-heat shock chromatin state. This produced a quantitative model describing the important features that modulate the in vivo HSF binding intensity. Moreover, the use of a rules ensemble model enabled the capture of potential interactions between these chromatin features (Guertin, 2010).

This study reveals that DNase I hypersensitivity and acetylation of H4 and H3K9 are strong predictors of inducible HSF binding intensities, however the molecular events and factors that precede TF occupancy to maintain accessible chromatin remain poorly characterized. For instance, the degree to which pioneering factors or flanking DNA sequence, individually or in combination, maintain or restrict accessibility remains unclear. A recent study highlights the biological consequences of maintaining the inaccessibility of TF binding sites, in order to repress expression of tissue-specific transcription factors in the wrong tissues. The authors found that ectopic expression of CHE-1, a zinc-finger TF that directs ASE neuron differentiation, in non-native C. elegans tissue is not sufficient to induce neuron formation (Tursun, 2011). However, combining ectopic CHE-1 expression with knockdown of lin-53 did modify the expression patterns of CHE-1 target genes in non-native tissue, effectively converting germ line cells to neuronal cells (Tursun, 2011). LIN-53 has been implicated in recruitment of deacetylases, and deacetylase inhibitor treatment mimics lin-53 depletion, suggesting that LIN-53 is actively maintaining CHE-1 target sites inaccessible in germ cells (Guertin, 2010).

Alternatively, functional TF binding sites could be actively maintained in the accessible state. HSF binding within ecdysone genes has a functional role in shutting down their transcription, and activating ecdysone-inducible genes containing inaccessible HSEs causes chromatin changes that are sufficient to allow HSF binding. In this special case of HSF-bound ecdysone genes, active transcription and the corresponding histone marks are mediating access to HSEs, in order for HSF to bind and repress transcription upon heat shock. A more recent study has shown that activator protein 1 (AP1) actively maintains chromatin in the accessible state, so that Glucocorticoid receptor can bind to cognate elements (Guertin, 2010).

Although TF accessibility to critical genomic sites appears to be actively maintained, many binding sites may be a non-functional result of fortuitous TFBS recognition. It has long been hypothesized that the binding affinities for TF/DNA interactions are sufficiently strong to allow promiscuous binding at the cellular concentrations of TFs and DNA. There are roughly 32,000 HSF molecules per tetraploid S2 cell and the dissociation constants for trimeric-HSF/HSE interactions are in the picomolar range; therefore much of the in vivo HSF binding may be non-functional promiscuous binding. Additional investigation will further illuminate the role of chromatin context in TF binding and the mechanisms by which programmed developmental or environmental chromatin changes permit or deny TF binding (Guertin, 2010).

Elucidating the rules that govern accessibility is essential for predicting in vivo occupancy of TFs. Diverse transcription factors, from a broad spectrum of organisms, bind their sequences based on site accessibility. This study found that chromatin accessibility as measured by DNase I hypersensitivity could be inferred using ChIP-chip data for various histone modifications and transcription factors. Although the model can infer accessibility based on chromatin composition, the mechanism by which accessibility originates is not addressed. Previous studies have shown that activators, such as HSF, glucocorticoid receptor, and androgen receptor bind to their cognate sites and direct a concomitant increase in local acetylation, DNase I hypersensitivity, and nucleosome depletion. Androgen receptor also acts to position flanking nucleosomes marked by H3K4me2. These post-TF binding chromatin changes that occur are the result of acetyltransferase and nucleosome remodeler recruitment, both of which functionally interact with activators. For instance, both GR and GATA1 interact with the nucleosome remodeling complex Swi/Snf. Concomitant increases in locus accessibility likely allow large molecular complexes such as RNA Pol II and coactivators to access the region that in turn can reinforce and maintain active and accessible chromatin (Guertin, 2010).

Thorough biophysical characterization of TF binding site properties is critical for accurate predictions of TF binding sites, underscoring the need for more complete models of TF binding. While the commonly used position-specific scoring matrix model makes the assumption of base independence, recent work has revealed that richer models providing for interactions between positions are necessary. The current model captures critical features of the HSF/HSE interaction that are lost with simpler computational models, namely the interdependencies between the sub-binding sites of each HSF monomer. Consistent with this model, a series of in vitro experiments with S. cerevisiae, D. melanogaster, A. thaliana, H. sapien and D. rerio HSFs indicate that HSF from each of these species can bind to discontinuous HSEs containing canonical pentamers that contain intervening five base pair gaps; interestingly, however, C. elegans HSF strictly binds to continuous HSEs that do not contain gaps. The complex interactions between positions within a binding site are a critical aspect of inferring whether a polymorphism or mutation affects TF binding. These features should prove useful in providing degenerate HSE sequences for optimal co-crystallization of trimeric HSF and DNA and inferring changes in DNA sequence that affect HSF binding within and between species (Guertin, 2010).

In conclusion, the data and models presented in this study reinforce both the importance of chromatin landscape in modulating in vivo TF binding intensity and how genome wide, chromatin free, binding assays contribute to the understanding of TF sequence binding specificity (Guertin, 2010).

Transcriptional Regulation

The abnormal oocyte (abo) gene of Drosophila is a peculiar maternal effect gene whose mutations cause a maternal-effect lethality that can be rescued by specific regions of heterochromatin during early embryogenesis. An increase in the dosage of specific regions of heterochromatin, denoted ABO, to either the mutant mother or the zygote, increases embryonic survival rates. abo encodes an evolutionary conserved chromosomal protein that localizes exclusively to the histone gene cluster and binds to the regulatory regions of such genes. A significant increase of histone transcripts occurs in eggs of abo mutant mothers and a partial rescue of the abo maternal-effect defect takes place with deficiencies of the histone gene cluster. On the basis of these results, it is suggested that the Abo protein functions specifically as a negative regulator of histone transcription and a molecular model is proposed to account for the ability of heterochromatin to partially rescue the abo maternal-effect defect. This model proposes that increased doses of specific heterochromatic regions titrate out abnormally high levels of histones present in embryos from mutant abo mothers and that a balanced pool of histones is critical for normal embryogenesis in Drosophila (Berloco, 2001).

The abo gene consists of a 1,974-bp sequence containing a putative TATA box, a CAAT box, and an ORF, interrupted by a small intron, and producing a single 1.8-kb transcript. This transcript encodes a putative 509-aa protein. The abo1 mutation is due to the insertion of an incomplete Doc transposable element into the coding region of the abo gene producing a larger transcript than the wild type, whereas that abo2 mutation is caused by a P[ry+] insertion into the 5' promoter region and does not produce a detectable transcript (Berloco, 2001).

A computer database search (the BLASTP program) found no known protein motifs in the conceptually translated Abo protein. However, 25.3% identity and 51.9% similarity was found to the DET1 protein, a nuclear located negative regulator of light-mediated gene expression in Arabidopsis, whose putative homologs are present also in Oryza sativa and Lycopersicon esculentum. Intriguingly, 24% identity and 44% similarity was found to the putative human hCP43420 protein from the Celera Human Report and to a putative mouse protein. Considering the evolutionary distance, the homology between these proteins appears significant. They share stretches of homology across their entire lengths and are very similar in charge, distribution of hydrophilic residues, and overall amino acid composition. In particular, the human and mouse proteins appear strikingly identical, with few differences in the nucleotide sequences of their encoding genes (Berloco, 2001).

The homology with DET1 suggests that the Abo protein might also be a transcriptional regulator and therefore might bind specific target sequences. To test this, bacterially produced Abo protein was used as an antigen to raise a polyclonal antibody in mice. Both the polytene chromosomes from salivary glands and the mitotic chromosomes of neuroblasts from wild-type larvae stain for Abo protein. A strong signal exclusively localized on the 39E region on polytene chromosomes was seen. In mitotic metaphase chromosomes, a unique strong signal is present on the constriction on the base of the left arm of the second chromosome. In both cases, the signal is localized at the position of the histone gene cluster, as confirmed by sequential immunostaining with the anti-Abo antibodies and in situ hybridization of the cDm500 probe, which contains the histone cluster. These results clearly demonstrate that the regions with exclusive binding affinity for Abo contain the histone clusters in both the polytenes and mitotic chromosomes (Berloco, 2001).

To identify Abo-binding sites in the histone repeat unit, the X-ChIP (formaldehyde-crosslinked-chromatin immunoprecipitation) method was applied by using polyclonal anti-Abo antibodies. 12 overlapping primer pairs were designed that amplify 400- to 500-bp fragments spanning the whole Drosophila histone repeat unit and they were used to amplify the DNA immunoprecipitated from chromatin of early embryos (0-4 h old) and SL-2 cultured cells. Binding was found of Abo protein in early embryos to the promoter regions of H2A-H2B and H3-H4. In SL-2 cells, Abo binds to an additional site in an H1 promoter fragment. These results show clearly that Abo protein binding is restricted to the three main regulatory regions of the repeat unit containing the histone gene promoters (Berloco, 2001).

The functional significance of the interaction of abo with the promoters of histone genes was addressed by a quantitation of histone transcripts in unfertilized eggs from heterozygote abo1/abo2 and abo1/abo+ mothers. The results show that abo mutations affect histone transcription. Much higher levels of H2A and H2B were found in eggs from mutant mothers than in eggs from their heterozygous sisters. The amount of H3 and H4 transcripts was significantly higher, whereas variations in the amount of H1 transcripts were not detectable. These results strongly suggest that abo is a negative regulator of histone genes. This possibility was further examined by testing the genetic effects of deficiencies of the entire histone gene cluster on the abo1 maternal effect. The results clearly show that the histone deficiencies [Df(2)DS5 and Df(2)DS6] induce a strong suppression of the abo1 maternal-effect defect, thus giving strong support to the suggestion that Abo negatively regulates histone gene expression (Berloco, 2001).

Taken together, these studies reveal that abo is a negative regulator of H2A, H2B, H3, and H4 expression during oogenesis. Hence, the deleterious maternal-effect defect induced by the abo mutations is probably due to an excess of these histones. The regulation of histone expression has been extensively studied in different species. The 5' flanking regions contain cis elements that interact with transacting factors. These transacting factors differ among species and, more surprisingly, also differ among the different classes of histone genes. It has been proposed that the coordinate expression of the histone genes probably depends on the interaction of a protein complex with the different transacting factors. In this context, the uniqueness of the Abo protein location on the histone genes in different Drosophila species and its strong evolutionarily conservation suggest that this protein probably plays a basic role in regulating histone gene expression. However, differential histone gene expression in early embryogenesis of several species has been seen. In Drosophila, specific histone classes are also known to be differentially expressed. For example, it has been shown that the maternal histone H1 transcript is not translated in early embryogenesis and is replaced by the HMG-D chromosomal protein. Intriguingly, the lack of any effect on H1 histone maternal transcription by the abo mutations and the lack of binding to its promoter by Abo in early embryos suggest that the regulation of histone H1 in both ovaries and embryos could not involve the abo gene. However, Abo does bind to the H1 promoter in SL-2 cells (representing late embryonic tissue), suggesting that Abo is probably involved in transcriptional regulation of histone H1 later in embryogenesis. Moreover, the differential enhancement of transcripts found in eggs from abo mutant mothers suggests that Abo could be more important for H2A and H2B repression than H3 and H4 repression during oogenesis (Berloco, 2001).

The data suggest a simple direct model for explaining an intriguing aspect of this gene, namely its interaction with the specific heterochromatic regions termed ABO elements. According to the model, homozygous abo mothers produce eggs with disproportionately high levels of H2A, H2B, H3, and H4 histones, which affect egg viability. Increasing doses of the ABO regions may titrate out these histones, reducing their negative effect. It is predicted that the abo and ABO-counteracting effects are produced by modulations in chromatin structure. Histones could be involved in such effects, as suggested by growing evidence showing that modified histones have differential chromosomal distributions, and hence they could play a role in the formation of heterochromatic domains. In fact H4 histone acetylated at lysine 4 and H3 histone methylated at lysine 9 are both present along the mitotic heterochromatin of Drosophila, with patterns of distribution indicating preferential binding for some regions (Berloco, 2001).

In conclusion, the characterization of abo opens the possibility of using this gene as an entry point to dissect the regulatory machinery of histone expression by looking at Abo-interacting molecules. Moreover, it could be a paradigm for experimental approaches to study the biological role of the heterochromatin. In D. melanogaster, other maternal-effect mutations closely linked to abo have been isolated. Preliminary experiments provide evidence that these abo-like mutations produce defects that can be compensated by discrete heterochromatic elements similar to ABO. It is possible that these other genes, like abo, may also encode transregulators of histone genes or other essential genes encoding chromosomal proteins (Berloco, 2001).

Epigenetic Silencing of Core Histone Genes by HERS in Drosophila

Cell cycle-dependent expression of canonical histone proteins enables newly synthesized DNA to be integrated into chromatin in replicating cells. However, the molecular basis of cell cycle-dependency in the switching of histone gene regulation remains to be uncovered. This study reports the identification and biochemical characterization of a molecular switcher, HERS (histone gene-specific epigenetic repressor in late S phase), for nucleosomal core histone gene inactivation in Drosophila. HERS protein is phosphorylated by a cyclin-dependent kinase (Cdk) at the end of S-phase. Phosphorylated HERS binds to histone gene regulatory regions and anchors HP1 and Su(var)3-9 to induce chromatin inactivation through histone H3 lysine 9 methylation. These findings illustrate a salient molecular switch linking epigenetic gene silencing to cell cycle-dependent histone production (Ito, 2012).

Histones are fundamental components of chromatin that maintain and regulate appropriate chromatin conformation to support all genomic DNA-dependent processes, including transcription, replication, repair, and mitosis). It is now accepted that histone proteins play a prime role in epigenetic regulation, serving as substrates for chromatin modifications (Ito, 2012).

The genes encoding canonical histones (H1, H2A, H2B, H3, and H4) are present in multiple copies and transcriptionally inactive in the quiescent cell state. Unlike the vast majority of the genes transcribed by RNA polymerase II, multiple copies of the histone genes are organized in gene clusters. In Drosophila, the histone gene cluster is composed of about 100 copies of tandemly arranged nucleosomal core (H2A, H2B, H3, and H4) and linker (H1) histone gene cassettes. The histone gene cluster is localized at a subnuclear compartment, histone locus body (HLB) that is presumed to contain factors essential for coordinated regulation of all histone gene copies. In early S phase of the cell cycle, histone genes are activated to supply histone proteins for the integration of newly synthesized DNA into nucleosomes. Coordinated protein production is required for all of the canonical core histones to produce optimal amounts of histone octamers in response to cellular requirements. Therefore, regulatory sequences appear to be common in the nucleosomal core histone gene loci to ensure their synchronized expression (Ito, 2012).

The cell cycle-dependent expression of canonical histones might be achieved through conserted action of cell-cycle, phase-specific nuclear factors at multiple levels of regulation. Recent studies have shown that the nuclear protein ataxia-telangiectasia locus (NPAT) plays a key role in the induction of histone genes. NPAT associates with histone loci and is functionally activated in early S phase by cyclin-dependent kinase 2 (Cdk2)/cyclin E (CycE), a kinase complex known to promote G1/S transition. Recently, fly homeotic gene mxc was identified as Drosophila NPAT ortholog. At the end of S phase, histone gene transcription is rapidly turned down and histone mRNAs are destabilized, ending the histone protein production. Although it has been assumed that dephosphorylation of NPAT is critical for histone gene silencing, the mechanisms of transcriptional suppression of histone genes remain to be identified (Ito, 2012).

During genetic screening of transcriptional coregulators in Drosophila, a critical factor has been identified that switched off nucleosomal core histone gene expression at the end of S phase through epigenetic inactivation of chromatin. Therefore, this factor was designated histone gene-specific epigenetic repressor in late S-phase (HERS) and presumed to function as a repressor of canonical core histone gene loci (Ito, 2012).

In contrast to NPAT, HERS protein abundance appears to be cell cycle-dependent because HERS was undetectable at the G1/S transition and only emerged in late S phase. Cdk-dependent phosphorylation of HERS is required for its association with core histone gene regulatory regions through sequence-specific DNA binding. This study has identified Cdk1/CycA as a prospective kinase complex that may phosphorylate HERS in vivo. However, because it has not been confirmed whether the Cdk1 inhibitor that was used here does not affect Drosophila Cdk2 or Cdk1/CycB activities, the possibility cannot be excluded that these Cdk complexes may also phosphorylate HERS. Owing to the cell cycle-specific expression and phosphorylation of HERS, its localization on the HIS-C appears to be limited to the completion of replication in the late S phase. This is further supported by HERS localization on the histone gene loci in the polytene chromosomes of salivary glands, in which DNA replication is completed and histone genes are inactive. Moreover, phosphorylation also significantly enhances HERS protein stability. Therefore, it is conceivable that dephosphorylation terminates HERS action and targets the protein for degradation (Ito, 2012).

HERS shuts down core histone gene expression through recruitment of the Su(var)3-9/HP1 repressor complex, a universal and normally abundant epigenetic silencer. Molecular interaction has been confirmed only between HERS and Su(var)3-9 but not with other tested H3K9 methyltransferases, G9a and dSETDB1, although genetic interaction of HERS was also observed with G9a and dSETDB1 in the Drosophila experimental system. It has been proposed that histone H3K9-specific methyltransferases function independently at their target loci, whereas they act in a sequential manner at the same gene locus. Moreover, it has been reported that G9a and dSETDB1 are active in early development, whereas Su(var)3-9 predominantly acts from early embryogenesis to later stages of Drosophila development. Thus, the possibility cannot be excluded that other histone H3K9 methyltransferases may also support HERS function (Ito, 2012).

The motif organization of HERS protein is atypical compared to other reported cell-cycle regulators. Even though phosphorylated HERS appears to act as a DNA binding factor, it lacks known motifs associated with DNA binding, protein-protein interaction, or other molecular interactions. Instead, this protein encompasses only one known motif, the BAH domain. The BAH domain is present in many chromatin-related regulators, including ORC1, polybromo, DNMT1, and yeast Sir3. The only reported role for the BAH domains in yeast Sir3 and Orc1 is that of nonselective association with nucleosomes. Although functional and physical interactions of the BAH domains were assessed for different histones modified at particular residues (for example, H3K9 methylation), it has not yet been possible to delineate any specific role of the HERS BAH domain at this stage. Nevertheless, it is conceivable that nonselective association with nucleosomes through the BAH domain may facilitate or stabilize HERS binding to specific regulatory DNA sequences in histone gene loci (Ito, 2012).

Although it remains unclear whether Drosophila HERS acts as a regulator of other cellular events, it was found that HERS knockdown in insect S2 cells resulted in cell-cycle arrest at the G2/M phase. Furthermore, when HERS was overexpressed in mammalian cell lines (HEK293 and Y1 cells), cell-cycle arrest was observed together with polyploidy. Besides other possible implications, this suggests an existence of mammalian HERS homolog(s). Interestingly, several uncharacterized mammalian proteins bearing the BAH domain have been documented. As mammals have three HP1 protein family members (α, β, and γ) with different functions, it is conceivable that mammalian HERS may consist of multiple members or isoforms with distinct cellular functions (Ito, 2012).

Gene-specific transcriptional mechanisms at the histone gene cluster revealed by single-cell imaging

To bridge the gap between in vivo and in vitro molecular mechanisms, this study dissected the transcriptional control of the endogenous histone gene cluster (His-C) by single-cell imaging. A combination of quantitative immunofluorescence, RNA FISH, and FRAP measurements revealed atypical promoter recognition complexes and differential transcription kinetics directing histone H1 versus core histone gene expression. While H1 is transcribed throughout S phase, core histones are only transcribed in a short pulse during early S phase. Surprisingly, no TFIIB or TFIID was detectable or functionally required at the initiation complexes of these promoters. Instead, a highly stable, preloaded TBP/TFIIA 'pioneer' complex primes the rapid initiation of His-C transcription during early S phase. These results provide mechanistic insights for the role of gene-specific core promoter factors and implications for cell cycle-regulated gene expression (Guglielmi, 2013).

This study found that whereas the four core histone genes are cotranscribed, the timing and pattern of H1 expression is quite distinct despite the tight promoter arrangement of all five His genes. The core histone genes are expressed through short but intense bursts in the very beginning of S phase, whereas histone H1 is expressed throughout S phase. It is speculated that this separation in the timing, distinct factor requirements, and pattern of H1 expression is in keeping with the differential functions assigned to these gene products. The core histone genes encode proteins destined to form a tight nucleosomal octamer, whereas histone H1 operates alone by binding to internucleosomal spacer regions to mediate higher-order condensed chromatin. The differential pattern of H1 transcription versus core histones could reflect a mechanism to achieve greater flexibility and access to chromatin during DNA replication to allow S phase checkpoint control. For example, a common event that can instigate S phase checkpoint arrest is DNA damage requiring repair. The continuous transcription of H1 throughout S phase, coupled with its much shorter mRNA half-life, makes the system more responsive to checkpoint arrest. One can imagine that a tight but highly responsive control of H1 could be a simple mechanism to avoid a buildup of excess H1 protein pools, thereby limiting the formation of higher-order, less accessible chromatin for repair enzymes to reach damaged regions of DNA. On the other hand, building an abundant and stable pool of H2A, H2B, H3, and H4 mRNA early in S phase could be a way to maintain core nucleosome-mediated chromatin integrity during S phase arrest. It is speculateb that perhaps such a constant flux of nucleosome production might be a mechanism to prevent the loss of valuable epigenetic information encoded in pools of modified nucleosomes that must be transmitted to newly formed chromatin (Guglielmi, 2013).

The transcription of both H1 and core histone genes becomes activated at early stages of S phase, suggesting that they are both likely responsive to S phase signaling initiated by the CyclinE-Cdk2 complex . However, it is unclear how core histone gene transcription is shut down early in S phase while the arrest of H1 transcription only occurs at the end of S phase. It has also been proposed that the DNA binding factor HERS silences histone gene transcription by formation of heterochromatin at the His-C locus toward the end of S phase. This mechanism, if involved, could not account for the early S phase silencing of the core histone genes, but could represent a more long-term silencing of the locus outside of S phase (Guglielmi, 2013).

Another intriguing feature of core histone gene expression revealed by these studies is the unusual pattern of transcription dynamics during S phase. A number of studies have concluded that transcription is largely a stochastic process. By contrast, the current studies of the His-C cluster point to a more concerted and deterministic type of mechanism that may be at play. Various studies measured the in vivo binding rates of transcription factors. Other studies of RNA Pol II transcription in living cells have found that initiation can in some cases be rather inefficient relative to elongation. However the binding of linchpin core promoter factors such as TBP to specific endogenous genes in living cells had not been measured. These studies of the endogenous histone genes in Drosophila cells revealed highly efficient initiation by RNA Pol II and very long dwell times (hours) for TBP. Indeed, it was found that TBP is involved in an unusually stable TBP-TFIIA complex that enhances the TBP dwell time at the His locus (Guglielmi, 2013).

It is imagined that such a stable marking of the His-C locus that occurs well before the onset of transcription during S phase can act to rapidly deploy the transcription machinery by serving as a landing pad for the other PIC components, including RNA Pol II. Such a preloaded highly stable pioneer TBP-TFIIA complex may provide an efficient mechanism to rapidly direct high levels of His gene transcription activation needed during the short window of early S phase expression. A stable bookmarked TBP/TFIIA complex could also serve as an insulator protecting the His gene promoter from heterochromatin formation, thereby facilitating transcription initiation at the start of S phase. Such a mechanism could likewise be useful at certain stages of development when spikes of gene activation must occur in narrow time intervals that are followed by efficient shut down. Curiously, there has been one other example of a TBP/TFIIA complex that can be purified from P19 embryonal carcinoma cells but not from more differentiated cells. Why this dimer complex is so abundant in these cells is not clear, but it may point to a more general role of TBP-TFIIA substituting for TFIID as a physiologically relevant alternative transcription initiation complex (Guglielmi, 2013).

The rapid and coordinated mechanism of on/off switches modulating transcription initiation reported in this study is quite distinct from the popular models invoking paused polymerases and reliance on post-initiation events to regulate transcription output described in a number of recent studies. It seems likely that diverse mechanisms may have evolved to deal with timing and dynamics of transcription dependent on cell type and developmental context (Guglielmi, 2013).

Finally, the use of alternative PIC subunits even within the His-C locus was most surprising. First, the dependence of H1 transcription initiation on the TBP-related factor TRF2 was confirmed, whereas H2A, H2B, H3, and H4 depend on the prototypic TBP for their expression. This differential use of core promoter recognition factors could play a key role in the transcriptional control of the His genes, particularly in combination with other gene-specific promoter binding factors responsible for directing S phase-triggered His gene transcription. Perhaps even more surprising was the observation that no TFIIB or TAFs could be detected binding to the His gene promoters. It cannot be entirely rule out that TFIIB has such a fast on/off rate that it was not possible to measure its presence at this locus. However, an equally plausible explanation would be that the PIC at these promoters is substantially different from canonical initiation complexes and neither TFIIB nor TAFs are required to form an active PIC at these promoters. It is also possible that there is another, as yet unidentified functional substitute for TFIIB operating at these promoters. At this stage it cannot be rationalized why the His genes would have evolved to require such a seemingly distinct PIC instead of merely adapting more classical mechanisms such as gene specific DNA binding activators to orchestrate the differential expression of H1 versus core histones during S phase. In any case, the pre-eminence of a highly stable preloaded TBP/TFIIA complex and potential changes in PIC subunit composition suggest that whatever form of PIC does assemble at the His-C locus represents a significant departure in composition and likely functional specificity from the prototypic housekeeping or canonical RNA Pol II promoter recognition apparatus. Perhaps the notion that a universal invariant 'basal or general' core promoter machinery is all that is required to mediate transcriptional regulation within a single cell type in eukaryotic organisms is a concept that has outlived its usefulness (Guglielmi, 2013).

In summary, single live-cell gene expression analysis unmasked a set of unexpected mechanisms regulating transcription of an endogenous gene cluster. Single-cell imaging of core promoter factors in unsynchronized populations of living cells also revealed a surprisingly efficient temporal control of transcription executed by a unique set of stably prebound promoter recognition complexes that significantly expands understanding of the diverse molecular mechanisms that have evolved to accommodate gene-specific transcription controlling physiologically critical processes in animal cells (Guglielmi, 2013).

Processing of the 3' end of Drosophila histone pre-mRNAs

Nuclear extracts from Drosophila Kc cells were used to characterize 3' end processing of Drosophila histone pre-mRNAs. Drosophila Stem-loop binding protein (SLBP) plays a critical role in recruiting the U7 snRNP (Dominski, 2003) to the pre-mRNA and is essential for processing all five Drosophila histone pre-mRNAs. The Drosophila processing machinery strongly prefers cleavage after a fourth nucleotide following the stem-loop and favors an adenosine over pyrimidines in this position. Increasing the distance between the stem-loop and the histone downstream element (HDE) does not result in a corresponding shift of the cleavage site, suggesting that in Drosophila processing the U7 snRNP does not function as a molecular ruler. Instead, SLBP directs the cleavage site close to the stem-loop. The upstream cleavage product generated in Drosophila nuclear extracts contains a 3' OH, and the downstream cleavage product is degraded by a nuclease dependent on the U7 snRNP, suggesting that the cleavage factor has been conserved between Drosophila and mammalian processing. A 2'O-methyl oligonucleotide complementary to the first 17 nt of the Drosophila U7 snRNA was not able to deplete the U7 snRNP from Drosophila nuclear extracts, suggesting that the 5' end of the Drosophila U7 snRNA is inaccessible. This oligonucleotide selectively inhibited processing of only two Drosophila pre-mRNAs and had no effect on processing of the other three pre-mRNAs. Together, these studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing share common features, there are also significant differences, likely reflecting divergence in the mechanism of 3' end processing between vertebrates and invertebrates (Dominski, 2005).

Metazoan replication-dependent histone pre-mRNAs do not contain introns, and the only processing reaction necessary to generate mature histone mRNAs is a single endonucleolytic cleavage of the mRNA precursors (pre-mRNAs) to form the 3' end. Studies on 3' end processing were initially carried out in Xenopus oocytes using synthetic pre-mRNAs and sea urchin histone genes and later were facilitated by the development of an in vitro system based on nuclear extracts from mammalian cells. Replication-dependent histone pre-mRNAs contain two cis elements required for 3' end processing: a highly conserved stem-loop structure consisting of a 6-bp stem and a 4-nt loop and a less conserved histone downstream element (HDE) located ~15 nt 3' of the stem-loop. Mammalian histone pre-mRNAs are cleaved between the two elements, 5 nucleotides downstream of the stem-loop. The stem-loop is recognized by the stem-loop binding protein (SLBP), also referred to as the hairpin binding protein (HBP). The HDE interacts with the U7 snRNP, which contains an ~60-nt U7 snRNA, and this interaction is primarily mediated by base-pairing between the HDE and the 5' end of U7 snRNA. In vitro studies in mammalian nuclear extracts suggest that SLBP stabilizes binding of the U7 snRNP to the pre-mRNA and is essential in processing of only those pre-mRNAs that do not form sufficiently stable duplexes with the U7 snRNA. This role of SLBP in mammalian processing is most likely mediated by ZFP100, a 100-kDa zinc finger protein associated with the U7 snRNP and interacting with the SLBP/stem-loop complex. In addition to bridging the two factors bound to their respective sequence elements, ZFP100 may also play other roles in 3' end processing, possibly including the recruitment of the cleavage factor (Dominski, 2005).

Purification of the U7 snRNP from mammalian cells resulted in identification of two novel Sm-like proteins: Lsm10 and Lsm11, which replace the D1 and D2 Sm proteins present in the spliceosomal snRNPs. Lsm11 interacts in vitro with ZFP100 and plays a key role in recognizing the unique sequence of the Sm binding site in U7 snRNA. Orthologs of Lsm10 and Lsm11 are also found in the Drosophila U7 snRNP, demonstrating that the unique structure of the U7 snRNP in vertebrates and invertebrates is conserved. A counterpart of ZFP100 has not been yet identified in the Drosophila genome, suggesting that ZFP100 is either weakly conserved between vertebrates and invertebrates or processing of histone pre-mRNAs in Drosophila does not require this protein (Dominski, 2005).

Nuclear extracts from Drosophila S-2 and Kc cultured cells and embryos are capable of 3' end processing of presynthesized Drosophila histone pre-mRNAs. Nuclear extracts from Kc cells are also capable of cotranscriptional processing of histone pre-mRNAs. Unlike the auxiliary role played by SLBP in mammalian in vitro processing, Drosophila SLBP is indispensable for processing of all Drosophila histone pre-mRNAs. This observation suggests that Drosophila SLBP plays a much more important role in recruiting the U7 snRNP to the pre-mRNA than it does in the mammalian processing. This study uses an in vitro system based on Drosophila nuclear extracts to characterize 3' end processing of Drosophila histone pre-mRNAs and to define differences and similarities in processing between this model invertebrate processing system and processing in mammalian nuclear extracts (Dominski, 2005).

These studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing occur with similar chemistry and both require SLBP and the U7 snRNP, the two mechanisms differ significantly in the relative importance of these trans-acting factors and in the specification of the cleavage site (Dominski, 2005).

Drosophila nuclear extracts cleave histone pre-mRNAs after the fourth nucleotide following the stem-loop and prefer an adenosine preceding the cleavage site. Consistent with this, all natural Drosophila histone pre-mRNAs contain an adenosine in this position. If the fourth nucleotide is changed to a pyrimidine, cleavage is also efficient after an adenosine at the third position but not after an adenosine located 5 nt downstream of the stem-loop, i.e., at the site exclusively utilized during mammalian processing. Sea urchin histone mRNAs, the only other invertebrate histone mRNAs with the characterized 3' ends, terminate with an ACCA consensus sequence. Thus, cleavage after the fourth nucleotide following the stem-loop may be a general feature of 3' end processing of invertebrate histone pre-mRNAs. Both Drosophila and mammalian processing machineries are similar in their extreme resistance to EDTA, generation of a 3' hydroxyl group at the end of the upstream cleavage product, and degradation of the downstream cleavage product by a U7 snRNP dependent activity. These results suggest that both processing machineries utilize the same or a highly related cleavage factor in 3' end processing of histone pre-mRNAs (Dominski, 2005).

In mammalian processing, the site of cleavage is determined by the position of the HDE, and moving the HDE, and, hence, the U7 snRNP, away from the stem-loop by as few as 4 nt results in a corresponding shift of the cleavage site. This observation led to the hypothesis that U7 snRNP recruits the cleavage factor to the pre-mRNA and acts as a molecular ruler to specify the cleavage site. SLBP bound to the stem-loop facilitates binding of the U7 snRNP to the HDE but does not play a direct role in recruitment of the cleavage factor. Consistent with this model, removal of SLBP, or using a substrate that cannot bind SLBP, reduces processing activity but does not abolish it (Dominski, 2005).

In contrast to mammalian processing, processing of Drosophila histone pre-mRNA is absolutely dependent on SLBP. In addition, increasing the distance between the stem-loop and the HDE by 4 or 8 nt in Drosophila histone pre-mRNA moved the cleavage site only 1 nt upstream from its normal position and did not abolish processing at the normal site. Larger insertions between the stem-loop and the HDE resulted in low efficiency cleavage further away from the stem-loop, but cleavage at these sites was still dependent on SLBP. This is in direct contrast to mammalian histone processing, where cleavage at the distant sites is independent of SLBP. Thus, in Drosophila processing the U7 snRNP does not function as a molecular ruler, but instead SLBP plays the critical role in specifying the cleavage site (Dominski, 2005).

To explain the observed differences between processing in Drosophila and mammalian nuclear extracts, it is proposed that within the Drosophila processing complex SLBP tightly interacts with the U7 snRNP, and this interaction is essential for bringing the U7 snRNP to the pre-mRNA. The two factors remain associated even if their respective binding sites are separated by a larger distance, likely by looping out the inserted nucleotides. The mutant pre-mRNAs are preferentially cleaved close to the stem-loop, reflecting the critical role of SLBP in forming the processing complex, although the precise position of the cleavage site and efficiency of processing depends on the size of the insert. In mammalian processing, the region between the stem-loop and the HDE is either rigidified, thus precluding looping out the inserted nucleotides, as previously suggested, or the interaction between SLBP and the U7 snRNP is relatively weak and disrupted by larger insertions, so binding of the U7 snRNP to the pre-mRNA depends solely on the base-pairing interaction. It is likely that in Drosophila processing the cleavage factor is recruited to histone pre-mRNA by interaction with both the U7 snRNP and SLBP, and neither factor is competent to carry out this function individually (Dominski, 2005).

In mammalian nuclear extracts, processing of histone pre-mRNAs is efficiently inhibited by relatively short 2'O-methyl oligonucleotides complementary to the 5' end of the mammalian U7 snRNA. These oligonucleotides, including a 10-mer, are also very efficient in depleting the U7 snRNP from nuclear extracts and were successfully used to affinity purify U7 snRNP from mammalian cells, demonstrating that the 5' end of the mammalian U7 snRNA is readily accessible. In contrast, two relatively long oligonucleotides, alphaDa, complementary to the first 17 nt of the Drosophila U7 snRNA, and alphaDb, complementary to nt 4-23, were not effective in depleting the U7 snRNP from Drosophila nuclear extracts. These results suggest that the 5' end of U7 snRNA is not accessible in the Drosophila U7 snRNP (Dominski, 2005).

Surprisingly, the alphaDa 2'O-methyl oligonucleotide abolished processing of the dH3* and dH1* pre-mRNAs (hybrid pre-mRNAs consisting of the stem-loop and cleavage site from the mouse H2a-614 pre-mRNA) but did not significantly affect processing of the other three Drosophila histone pre-mRNAs. Three additional oligonucleotides complementary to the regions of the U7 snRNP located closer to the Sm binding site effectively blocked processing of all five histone pre-mRNAs. It is not understood why processing of only two Drosophila pre-mRNAs is affected by the alphaDa oligonucleotide and which features of the HDEs make processing of the Drosophila pre-mRNAs either sensitive or resistant to this oligonucleotide. Selective inhibition of processing by the alphaDa oligonucleotide depending on the type of pre-mRNA used in the reaction suggests that blocking of the U7 snRNA must occur during processing. One possibility is that the U7 snRNP is initially recruited to the pre-mRNA solely by SLBP bound to the pre-mRNA, and later this interaction is followed by formation of a duplex between the HDE and the U7 snRNA, as a result of unmasking of the 5' end of U7 snRNA. The alphaDa oligonucleotide might block binding of the U7 snRNA to the HDE in the hybrid dH1* and dH3* pre-mRNAs, but not in the other pre-mRNAs, during this later step, while the other oligonucleotides block binding to all the HDEs (Dominski, 2005).

Overall, thes studies indicate that the structure of the 5' end of the Drosophila U7 snRNA and the mechanism of its initial interactions with the HDE differ significantly from the recognition of the HDE in processing of mammalian histone pre-mRNAs (Dominski, 2005).

In vitro processing of all five Drosophila histone pre-mRNAs is absolutely dependent on SLBP. This study has demonstrated that SLBP is essential for recruitment of the U7 snRNP to the pre-mRNA. The necessity of SLBP for recruitment of the U7snRNP to the Drosophila pre-mRNAs suggests that either Drosophila HDEs are unable to form a strong duplex with the U7 snRNA or that the interaction of the U7 snRNP with the SLBP/pre-mRNA complex is necessary to promote base-pairing by making the 5' end of U7 snRNA accessible (Dominski, 2005).

Both the 5' end of the Drosophila U7 snRNA and Drosophila HDEs are AU rich, allowing a number of possible base-pair schemes for making a duplex between the two RNAs. It is hypothesized that the most likely alignment used during processing is the one that allows formation of the largest number of base pairs between the purine core of the HDE and the CUCUUU sequence in the U7 snRNA and not necessarily the alignment that allows formation of the overall most stable duplex. The CUCUUU sequence is highly conserved among all known U7 snRNAs and is involved in recognition of the purine core in sea urchin and mammalian histone pre-mRNAs. A 3-nt mutation within the purine core of the hybrid dH3* pre-mRNA abolishes processing, whereas a 6-nt mutation within the AU-rich region immediately downstream of the purine core only partially inhibits processing. These results support the interpretation that base-pairing between the U7 snRNA and the purine core is critical, whereas formation of additional base in other regions increases the efficiency of Drosophila processing. It is also possible that the base-pairing interaction is limited to the purine core and the CUCUUU sequence in the U7 snRNA, whereas the AU-rich sequences in the U7 snRNA and the HDE are brought together by protein-protein interactions (Dominski, 2005).

This study demonstrated that the HDE of the hybrid dH3* pre-mRNA can abolish processing of the full-length substrate, presumably by sequestering the U7 snRNP, only when present at very high concentrations. Interestingly, this weak interaction of Drosophila HDEs with the U7 snRNP is sufficient to recruit a 5'-3' exonuclease that specifically degrades the downstream cleavage product in a U7 dependent manner. Thus, the endonucleolytic cleavage must require much stronger binding of the U7 snRNP to the pre-mRNA, while degradation of the DCP by an exonuclease may require only loose association of the HDE with the U7 snRNP (Dominski, 2005).

The most notable difference between histone pre-mRNA processing in Drosophila and mammalian nuclear extracts is the absolute dependence of Drosophila processing on SLBP and the role of SLBP in specifying the cleavage site close to the stem-loop. The Drosophila U7 snRNP does not function as a molecular ruler in processing and this feature most likely reflects a critical role of SLBP in recruiting the cleavage factor as well as the U7 snRNP, to histone pre-mRNA. These data suggest that SLBP and the U7 snRNP may form a tight complex on the histone pre-mRNA, and this complex remains stable even in the presence of large insertions between the stem-loop and the HDE (Dominski, 2005).

The similarities in the chemistry of the cleavage reaction, including preference for an adenosine preceding the cleavage site and generation of the 3'OH group in the presence of EDTA, as well as degradation of the downstream cleavage product by a U7-dependent 5'-3' exonuclease suggest that the cleavage factor has been conserved between Drosophila and mammalian processing. It will be of interest to determine whether there are factors unique to only one of these two types of organisms emphasizing long evolutionary distance and the divergence between vertebrates and invertebrates (Dominski, 2005).

Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation

Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop-binding protein (SLBP), an RNA-binding protein involved in the histone pre-mRNA processing. The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. This study shows that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine were used to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA (Zhang, 2014).

U7 snRNA mutations in Drosophila block histone pre-mRNA processing and disrupt oogenesis

Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem–loop structure generated by an endonucleolytic cleavage involving the U7 snRNP, which interacts with histone pre-mRNAs through base-pairing between U7 snRNA and a purine-rich sequence in the pre-mRNA located downstream of the cleavage site. Null mutations of the single Drosophila U7 gene were generated and U7 snRNA was demonstrated to be required in vivo for processing all replication-associated histone pre-mRNAs. Mutation of U7 results in the production of poly A+ histone mRNA in both proliferating and endocycling cells because of read-through to cryptic polyadenylation sites found downstream of each Drosophila histone gene. A similar molecular phenotype also results from mutation of Slbp, which encodes the protein that binds the histone mRNA 3' stem–loop. U7 null mutants develop into sterile males and females, and these females display defects during oogenesis similar to germ line clones of Slbp null cells. In contrast to U7 mutants, Slbp null mutations cause lethality. This may reflect a later onset of the histone pre-mRNA processing defect in U7 mutants compared to Slbp mutants, due to maternal stores of U7 snRNA. A double mutant combination of a viable, hypomorphic Slbp allele and a viable U7 null allele is lethal, and these double mutants express polyadenylated histone mRNAs earlier in development than either single mutant. These data suggest that SLBP and U7 snRNP cooperate in the production of histone mRNA in vivo, and that disruption of histone pre-mRNA processing is detrimental to development (Godfrey, 2006).

Chromosome duplication during the cell cycle requires the production of histones during S phase to package newly replicated DNA into chromatin. Bulk histone production during S phase is achieved through the biosynthesis of replication-dependent histone mRNAs, which are cell-cycle regulated and accumulate only in S phase. In animal cells these histone mRNAs are unique: The 3' end terminates in a conserved 26-nt sequence that forms a stem–loop rather than in a poly A+ tail. Since histone genes lack introns, the only processing step required for mature histone mRNA production is endonucleolytic cleavage of the pre-mRNA to form the 3' end of the mRNA. Much of the cell-cycle regulation of histone mRNAs is post-transcriptional and is mediated by the 3' end of the mRNA. Thus, a complete understanding of cell-cycle-regulated histone mRNA production requires a full understanding of the factors required for histone pre-mRNA processing (Godfrey, 2006).

The processing of histone pre-mRNAs requires two cis elements and a number of trans-acting factors. The cis elements are the stem–loop at the 3' end of histone mRNA and a purine-rich region downstream of the cleavage site, termed the histone downstream element (HDE). A protein called stem–loop binding protein (SLBP) or hairpin binding protein (HBP) specifically binds the 3' end of histone mRNA. SLBP is required for histone pre-mRNA processing in vivo and accompanies the mRNA to the cytoplasm, where it promotes the translation of the histone mRNA. The HDE binds U7 snRNP by base-pairing with the 5' end of U7 snRNA. In mammals, SLBP, the U7 snRNP, and a U7 snRNP-associated zinc finger protein called ZFP100 cooperate to recruit an endonuclease complex that cleaves the pre-mRNA. Recent evidence indicates that CPSF73, a component of the complex that mediates AAUAAA-directed cleavage prior to polyadenylation, is the likely endonuclease. This revealed some unexpected overlap in the machinery carrying out histone pre-mRNA processing and canonical polyadenylation (Godfrey, 2006).

The U7 snRNA is a small RNA (55–70 nt) that, like the spliceosomal snRNAs, contains both a trimethyl guanosine cap and an Sm binding site, which is essential for its function. The Sm site in these snRNAs stably binds a complex of seven related proteins of the LSm/Sm family to form the core snRNP particle. Proteins of the LSm/Sm family share a common tertiary structure called the Sm fold that assembles into hexameric or heptameric rings capable of binding single-stranded RNA. The U snRNPs contain a heptameric Sm ring, with each of the seven individual subunits making a specific contact with a residue in the Sm binding site of the snRNA. The heptameric Sm ring of spliceosomal snRNPs contains the proteins SmB/B', SmD1, SmD2, SmD3, SmE, SmF, and SmG. In contrast, the U7 snRNP contains five of these Sm proteins (B/B1, D3, E, F, G) and two novel Sm proteins called LSm10 and LSm11 that replace SmD1 and SmD2 of the spliceosomal snRNPs. The Sm site found in U7 snRNAs is distinct from the Sm site in spliceosomal snRNAs and is responsible for incorporation of LSm10 and LSm11 into the U7 snRNP. In addition to the Sm fold that participates in ring formation, LSm11 contains an NH2 terminal extension that makes contacts with ZFP100 and possibly other components of the histone pre-mRNA processing machinery (Godfrey, 2006).

The role of U7 snRNP in histone pre-mRNA processing has been examined primarily in nuclear extract systems that support the processing of synthetic histone pre-mRNAs, and by monitoring the processing of histone pre-mRNAs injected into Xenopus ooctyes. Complementary mutations in U7 snRNA and the HDE provided early evidence that base-pairing between the 5' end of U7 and the HDE was an important part of U7 snRNP function. Furthermore, blocking the 5' end of the U7 snRNA with a complementary oligonucleotide specifically inhibits processing of synthetic histone pre-mRNAs in nuclear extracts. However, the contribution of U7 snRNA to endogenous histone mRNA biosynthesis and whether this contribution is important for animal development have not been examined. To explore these issues, U7 snRNA mutations in Drosophila were generated and characterized (Godfrey, 2006).

Drosophila SLBP, U7 snRNA, and U7 snRNP specific proteins Lsm10 and Lsm11, have all been identified, and steps have been taken to characterize them genetically. Mutations in the Drosophila Slbp gene block normal histone pre-mRNA processing during embryonic development and result in production of polyadenylated histone mRNAs as a consequence of read-through past the normal processing site. This occurs because each of the five Drosophila histone genes contains cryptic polyadenylation sites downstream of the HDE that are utilized in the absence of SLBP. Null mutations of Slbp cause lethality during larval and pupal stages, presumably because of the histone processing defects, although the precise cause of lethality is not known. Slbp mutant cells are capable of replicating chromatin, likely because the inappropriate polyadenylated mRNAs are translated. A hypomorphic Slbp mutant allele that produces reduced amounts of SLBP protein results in the production of both normal and poly A+ histone mRNAs during embryogenesis, but does not cause lethality. However, these viable mutant females lay eggs that contain reduced amounts of histone mRNA and protein and do not develop. Thus, SLBP is required during both zygotic development and oogenesis (Godfrey, 2006).

This study compared mutations in the U7 snRNA gene, and the resulting phenotypes were compared with those caused by mutation of Slbp. The results indicate that U7 snRNA is required for normal histone mRNA biosynthesis during Drosophila development and that, like Slbp mutations, loss of U7 snRNA results in the production of polyadenylated histone mRNAs. However, unlike Slbp null mutants, U7 null mutants are viable, but both males and females are sterile. This difference in terminal phenotype is most likely because the maternal supply of U7 snRNA delays the onset of the histone processing defect in U7 mutants relative to Slbp mutants, which do not have a significant maternal supply of SLBP protein. Both U7 and SLBP are required for normal histone mRNA biosynthesis in the female germ line, and mutation of either gene disrupts oogenesis. These data indicate that loss of SLBP and U7 cause similar molecular phenotypes in Drosophila and suggest that early expression of this molecular phenotype prevents normal development (Godfrey, 2006).

Drosophila octamer elements and Pdm-1 dictate the coordinated transcription of core histone genes

This study reveals a set of divergent octamer elements in Drosophila core histone gene promoters. These elements recruit transcription factor POU-domain protein in Pdm-1, which along with co-activator dmOct-1 coactivator in S-phase (dmOCA-S), activates transcription from at least the Drosophila histone 2B (dmH2B) and 4 (dmH4) promoters in a fashion similar to the transcription of mammalian histone 2B (H2B) gene activated by octamer binding transcription factor 1 (Oct-1) and Oct-1 coactivator in S-phase (OCA-S). The expression of core histone genes in both kingdoms is coordinated; however, although the expression of mammalian histone genes involves subtype-specific transcription factors and/or co-activator(s), the expression of Drosophila core histone genes is regulated by a common module (Pdm-1/dmOCA-S) in a directly coordinated manner. Finally, dmOCA-S is recruited to the Drosophila histone locus bodies in the S-phase, marking S-phase-specific transcription activation of core histone genes (Lee, 2010).

Given that Oct-1 and Pdm-1 share similarities in their evolutionarily conserved POU-domains, as do the human OCA-S and putative dmOCA-S components, a similar trans-regulatory network is proposed in the Drosophila H2B gene activation pathway in which dmOCA-S abets the role of Pdm-1; thus, silencing individual dmOCA-S subunits may bring about phenotypes similar to those observed in Pdm-1-silenced cells. In particular, glyceraldehyde-3-phosphate dehydrogenase (p38/GAPDH) and lactate dehydrogenase (p36/LDH) were shown to be OCA-S components absolutely required for hH2B transcription (Zheng, 2003; Dai, 2008). DmGapdh1/2, dmLdh, and abnormal wing discs [Awd, the non-metastatic protein 23 in human (nm23) homolog] were subject to RNAi. The expression of these proteins was silenced to relative completion with 37 nm dsRNA doses, which was accompanied by coordinately repressed expression of dmH2B and dmH4 genes. The repressed dmH2B expression was attributed to reduced promoter activity of the dmH2B gene, for the activities of the ectopic dmH2B promoter-luciferase reporter were severely reduced upon silencing the protein expression of each of the tested dmOCA-S components (dmGapdh1/2, dmLdh, and Awd). Collectively, these results suggest an existence of a dmOCA-S co-activator in Drosophila that comprises of the above-tested proteins and functions in the dmH2B gene regulation pathway by abetting the role of Pdm-1 (Lee, 2010).

Although more than 800 million years apart in evolution, Drosophila and human share similarities in histone expression pathways, albeit with species-specific features. In Drosophila, core histone gene promoters contain multiple evolutionarily diversified Pdm-1 binding sites, contributing to the optimal histone expression​; on the other hand, a single prototype octamer ATTTGCAT mediates the hH2B transcription, and Oct-1 association with this element, which is conserved among vertebrates, is kept under strict sequence requirement. This strategy may be also employed by other Oct-1-dependent genes and their cognate coactivators. These species-specific features could imply significance for metazoan evolution (Lee, 2010).

Histone biosynthesis and DNA replication are coupled and essential for cell viability. Thus, impeding the function of Pdm-1/dmOCA-S would lead to S-phase defects and likely be detrimental to cell viability. In the case of dmGapdh and dmLdh, their roles in glycolysis and their moonlighting nuclear functions abetting the role of Pdm-1 also dictate negative consequences on the cellular ability to progress through the S-phase in a loss-of-function situation (Lee, 2010).

Efficient RNAi-mediated protein knockdown requires mRNA destruction and decay of the preexisting protein, which was realized at 72 h for Pdm-1 and 88A) with coordinately repressed expression of core histone genes; however, the cell cycle profiles were not affected or only marginally affected at this point, which was statistically insignificant. Thus, the histone expression defects at 72 h were primary defects. It was reasoned that a most likely scenario is that despite depriving cells of histone transcription at 72 h by a Pdm-1 deficiency, pre-existing histone protein levels are above a threshold that supports DNA replication. Indeed, H2B protein levels at 72 h were largely normal or slightly reduced. Beyond 72 h, however, Pdm-1-silenced cells also exhibited a prominent S-phase defect at 96 h, and cell viability defects manifested at 84 h with a more drastic viability decrease at 96 h (Lee, 2010).

The cell viability defects might be related to a mechanism coupling histone expression to S-phase progression. The more drastic histone expression defects at 84 h as compared with that at 72 h could be due to additive effects of a primary Pdm-1 loss-of-function and secondary cell viability and S-phase defects, which fed back. Histone transcription defects as a result of the Pdm-1/dmOCA-S deficiency would deprive cells of histone proteins in a long run, thus providing insufficient histone protein levels for chromatin assembly, which ultimately leads to DNA replication, S-phase, and cell viability defects. It is proposed that although histone transcription defects at 72 h were primary defects, the later-manifested cell viability and S-phase defects were secondary defects due to reduced histone protein levels as exemplified by that of H2B beyond 72 h (Lee, 2010).

Coordinated histone expression in diverse species is needed to maintain balanced expression of core histone genes, known to sustain genome integrity. In mammalian cells, eliminating OCA-S function by silencing p38/GAPDH expression led to H2B expression defect, and a lagged histone H4 expression defect manifested after a severe cell cycle arrest; eliminating H2B expression by gene deletion in yeast also led to cell cycle arrest and subsequent expression defects of other core histone genes. These observations led to a thought that coordinated histone expression was regulated through an S-phase feedback mechanism, which was later revised (Yu, 2009; Lee, 2010).

In Drosophila cells, the RNAi-mediated silencing of Pdm-1 or dmOCA-S components led to concerted and directly coordinated expression defects of all core histone genes and as primary transcription effects due to a missing Pdm-1/dmOCA-S function. In mammalian cells, the core histone genes employ distinct promoter elements and associated (co)factors, but their expression remains highly coordinated through an uncharacterized mechanism that is indirect but still does not involve S-phase feedback. Distinct histone expression coordination mechanisms in fly and mammalian cells might be of crucial relevance in metazoan evolution (Lee, 2010 and references therein).

The expression of metazoan-specific linker histone H1 gene is largely S-phase-specific, but the H1 expression output is not tightly coupled with that of core histone genes. The TATA-less Drosophila H1 gene utilizes a TRF-containing complex but not the prototype TBP-containing TFIID complex for its expression. This study found that Drosophila core histone genes, at least that of dmH2B and dmH4, contain TFIID in line with recruitment of TBP to dmH3 and dmH4 promoters and the idea that the basal transcription machineries of Drosophila core histone genes use the prototype TFIID. TRF-containing complexes have not been known to function in a cell cycle-dependent manner; it is of interest to investigate if the S-phase-specific Drosophila H1 expression is conferred upon by S-phase-specific factors that ought to be distinct from Pdm-1/dmOCA-S because the Drosophila H1 expression was not coordinately repressed with that of core histone genes in Pdm-1-deficient cells (Lee, 2010).

Mammalian histone genes are organized into Cajal bodies in a process facilitated by nuclear protein, ataxia-telangiectasia locus (NPAT), a cyclinE/cdk2 substrate that conveys the cyclinE/cdk2 signaling to histone transcription machineries. In contrast, Drosophila cells contain distinct nuclear domains dubbed HLB, which host all the histone genes and contain a cyclinE/cdk2-dependent phospho-epitope recognized by the MPM-2 monoclonal antibody in S-phase. Drosophila HLB are often in close proximity to, but never overlapped with Drosophila Cajal bodies (Lee, 2010 and references therein).

That cyclinE/cdk2 signaling is conserved from Drosophila to human, and that the HLB foci are associated with nascent histone transcripts prompted an investigation of S-phase-specific recruitment of dmOCA-S (represented by nuclear dmGapdh) to HLB. A higher percentage of HLB (~90%) foci as compared with dmOCA-S (~70%) foci in the early S-phase, i.e.,~80% of HLB foci are nuclear dmGapdh-positive, suggests that the dmOCA-S function (dmGapdh-HLB nuclear co-localization) is likely downstream of cyclinE/cdk2 signaling. The increase in the foci size and maximal degree of HLB-dmOCA-S co-localization coincided with the peak of dmH2B and dmH4 mRNA levels in the mid-S-phase, in line with the notion that the HLB foci size is proportional to histone expression levels (Lee, 2010 and references therein).

None of dmOCA-S components possesses a consensus sequence(s) for cdk; the fly cyclinE/cdk2 signaling might be conveyed to histone genes via an unidentified molecule(s) (dmNPAT), for which the phospho-epitope-containing protein recognized by MPM-2 monoclonal antibody is a potential candidate (Lee, 2010 and references therein).

Efforts to find dmNPAT have been fruitless; a dmNPAT gene might likely reside in so-far-unsequenced heterochromatic domains or possess sequences drastically divergent from vertebrate NPATs. Alternatively, an NPAT function may not have been acquired during insect evolution, necessitating histone genes to be organized into HLB and compelling cognate promoters to be regulated by diversified octamer elements and a Pdm-1/dmOCA-S module to ensure the directly coordinated expression of histone genes (Lee, 2010 and references therein).

The mechanistic aspects of co-activation by dmOCA-S seem to be similar to that of human OCA-S, in line with nuclear moonlighting transcription functions of metabolic enzyme conservation in metazoans. Conversely, some non-transcriptional functions of transcription factors or co-factors have been documented: e.g. in the cytoplasm, an isoform of co-activator OCA-B plays an essential non-transcriptional role for B-cell signaling (Lee, 2010).

Pdm-1 is a ubiquitous transcription factor for ubiquitous histone expression; the identified cell-specific roles of Pdm-1 in neuronal cell fate specification and Notch signaling might be due to cell-specific non-transcriptional functions of Pdm-1. Alternatively, cell-specific Pdm-1 phenotypes might be attributed to missing links between mutant pdm-1 alleles and alleles encoding cell-specific co-activators, especially in view of the fact that a given POU-domain is rich in separable surfaces that provide contacts for distinct ubiquitous or tissue-specific co-activators. Non-transcriptional development regulators may also interact with partners through different surfaces. Thus, it is not surprising that mutant alleles of even a ubiquitously expressed gene, be it specifying a non-transcription or transcription function, may lead to tissue-specific phenotypes (Lee, 2010).

The work toward dissecting cis- and trans-regulatory networks of the Drosophila histone transcription regulation pathway(s) will provide a new paradigm for studying transcriptional regulator changes implied in evolution and development as well as permit a broader range of questions to be asked about a cohort of genes involved in histone expression and related DNA replication-dependent transcription and its tight coupling with S-phase progression and about roles of the individual dmOCA-S components as novel S-phase-specific players in above processes in a genetically tractable organism (Lee, 2010).

Histone H4 and acetylation

During periods of active DNA replication and chromatin assembly, newly synthesized histone H4 is deposited in a diacetylated form. In Tetrahymena, a specific pair of residues, lysines 4 and 11, has been shown to undergo this modification in vivo. Presumably, this reaction is catalyzed, at least in part, by histone acetyltransferases (HAT) of the B type, cytoplasmic enzymes displaying strong preference for free, non-chromatin-bound H4. To investigate which lysine acetylation sites are preferred in the H4 of other organisms, a cytoplasmic HAT B activity was prepared from Drosophila embryos and used to acetylate H4 from several species. When either H4 or synthetic NH2-terminal peptides from Tetrahymena was used, acetate was preferentially incorporated at lysine 11 with little, if any, incorporation at other conserved, acetylatable lysines. Drosophila H4 acetate incorporation occurred preferentially on lysine 12, the residue analogous to lysine 11 in Tetrahymena. These data show remarkable preference for lysine 11/12 by the Drosophila HAT B activity in vitro and provide support for the assertion that this activity functions to acetylate new H4, at least in part, for deposition and chromatin assembly in vivo (Sobel, 1994).

Newly synthesized histone H4 is deposited in a diacetylated isoform in a wide variety of organisms. In Tetrahymena a specific pair of residues, lysines 4 and 11, have been shown to undergo this modification in vivo. The analogous residues, lysines 5 and 12 are acetylated in Drosophila and HeLa H4. These data strongly suggest that deposition-related acetylation sites in H4 have been highly, perhaps absolutely, conserved. In Tetrahymena and Drosophila newly synthesized histone H3 is also deposited in several modified forms. For H3, like H4, a specific, but distinct, subset of lysines is acetylated in these organisms. In Tetrahymena, lysines 9 and 14 are highly preferred sites of acetylation in new H3 while in Drosophila, lysines 14 and 23 are strongly preferred. No evidence has been obtained for acetylation of newly synthesized H3 in HeLa cells. Thus, although the pattern and sites of deposition-related acetylation appear to be highly conserved in H4, the same does not appear to be the case for histone H3 (Sobel, 1995).

Carnitine is a well-known naturally occurring compound, very similar to butyrate, with an essential role in intermediary metabolism mainly at the mitochondrial level. Since butyrate inhibits the enzyme histone deacetylase and is capable of suppressing position-effect variegation in Drosophila, carnitine was tested for a similar function in the nucleus. Carnitine derivatives are capable of suppressing the position-effect variegation, albeit with different efficiencies. The carnitine derivatives interact lethally with Su-var(2-)1(01), a mutation that induces hyperacetylation of histones, while hyperacetylated histones accumulate in both the nuclei of HeLa cells and Drosophila polytene chromosomes treated with the same compounds. These results strongly suggest that the carnitine derivatives suppress position-effect variegation by a mechanism similar to that of butyrate. It is suggested that carnitines may have a functional role in the nucleus, probably at the chromatin level (Fanti, 1994).

The Drosophila nucleosome remodeling factor NURF utilizes the energy of ATP hydrolysis to perturb the structure of nucleosomes and facilitate binding of transcription factors. The ATPase activity of purified NURF is stimulated significantly more by nucleosomes than by naked DNA or histones alone, suggesting that NURF is able to recognize specific features of the nucleosome. The interaction between NURF and nucleosomes is impaired by proteolytic removal of the N-terminal histone tails and by chemical cross-linking of nucleosomal histones. The ATPase activity of NURF is also competitively inhibited by each of the four Drosophila histone tails expressed as GST fusion proteins. A similar inhibition is observed for a histone H4 tail substituted with glutamine at four conserved, acetylatable lysines. These findings indicate a novel role for the flexible histone tails in chromatin remodeling by NURF, and this role may, in part, be independent of histone acetylation (Georgel, 1997).

What are the structural determinants of nucleosomes that are important for the activity of NURF? Based on the loss of the nucleosome-stimulated ATPase activity of NURF and the diminution of the DNase I footprint when the histone tails are removed by limited proteolysis, it is suggested that the flexible tails of the Drosophila core histones are critical elements for interaction with NURF. This conclusion is strengthened by the inhibition of NURF ATPase activity by GST-histone fusions. The effects of cross-linking the core histones in nucleosomes are also consistent with a contribution from the histone tails, although contributions from the globular domains of the nucleosome core histones cannot be excluded by this technique. Finally, a minor role for nucleosomal DNA is indicated by the modest inhibitory effects of DNA on the nucleosome-stimulated ATPase activity of NURF. These several determinants, individually insufficient for stimulating the ATPase activity of NURF, may be required in a combinatorial manner for achieving ATP-dependent perturbation of nucleosome structure. It will be of interest to relate the recognition of these determinants to one or more subunits of the NURF complex, and to analyze how this recognition is transduced to nucleosomal reorganization coupled with the utilization of chemical energy. Although a discrete supercomplex of NURF and a nucleosome has not been detected by native gel electrophoresis, it will also be important, when sufficient amounts of NURF become available for systematic studies, to define the interactions between NURF and nucleosomes quantitatively by biophysical methods, and to determine the histone composition of the remodeled nucleosome (Georgel, 1997).

The requirement for the Drosophila histone tails in nucleosomal interactions with NURF and the lack of strong binding specificity for structured DNA, a property of the SWI/SNF complex, provides further evidence for separate modes of action for the NURF and SWI/SNF chromatin remodeling complexes, which share related ATPase subunits ISWI and SWI2/SNF2, and the ability to alter chromatin structure in vitro in an ATP-dependent manner. Genetic studies have shown that the histone H2A/H2B tails and the histone H3/H4 tails are essential for viability in yeast. For histones H3 and H4, the tails are also important for repression of basal transcription, for telomeric and silent mating locus repression and for activation and repression of some genes. The H3 and H4 tails have been shown to bind in vitro with the yeast silencing information regulators SIR3 and SIR4, providing direct evidence that these extended regions may form specific binding sites for protein regulators of nucleosome structure and function. Tup1, a repressor of transcription of yeast a-cell specific genes, has also been demonstrated to interact directly with the tails of histones H3 and H4. Together with the present findings, these results suggest that the flexible tails of the histone octamer serve as common sites of interaction with several distinct nuclear protein complexes that affect nucleosome stability in a positive or negative manner (Georgel, 1997).

Other biochemical studies have demonstrated that the basic histone tails partially restrict binding of transcription factors to nucleosomal DNA. This restricted accessibility of nucleosomal DNA imposed by the histone tails can be alleviated upon neutralization of charged lysines by acetylation. However, as indicated by the ability of the GST-yH4 (Q5,8,12,16) mutant protein to retain competitive inhibition of the nucleosome-stimulated ATPase activity of NURF, the four acetylatable lysines of histone H4 in yeast do not seem to be of crucial importance for interaction with NURF, as measured by the ATPase assay. These lysine positions are strictly conserved in the Drosophila histone H4 tail and also undergo acetylation. Hence, the remaining conserved amino acid residues of the Drosophila histone H4 tail are likely to be involved in the interaction with NURF, and this interaction, at least for histone H4, could be independent of the state of lysine acetylation. It should be noted that these results do not exclude an interaction between NURF and other lysine residues of the histone tails that are not subject to acetylation. Nonetheless, the observed ability of hyperacetylated nucleosomes to stimulate the ATPase activity of NURF as well as normal nucleosomes, in the case of both HeLa cell and Drosophila histones, is consistent with the possibility that NURF may act independently of the histone acetylation pathway of nucleosome destabilization. It will be of interest to elucidate, by site-directed mutagenesis, the precise nature of the interaction between NURF and the histone tails, to understand the mechanism by which this interaction leads to nucleosomal reorganization and to define the parallel or sequential nature of the pathways of nucleosome reorganization by chromatin remodeling and histone modifying activities (Georgel, 1997).

A number of activators are known to increase transcription by RNA polymerase (pol) II through protein acetylation. While the physiological substrates for these acetylases are poorly defined, possible targets include general transcription factors, activator proteins and histones. Using a cell-free system to reconstitute chromatin with increased histone acetylation levels, a direct test was performed for a causal role of histone acetylation in transcription by RNA pol II. Chromatin, containing either control or acetylated histones, was reconstituted to comparable nucleosome densities and characterized by electron microscopy after psoralen cross-linking, as well as by in vitro transcription. Chromatin was reconstituted using histones from either TSA-treated CV-1 cells, which accumulates hyperacetylated histone isoforms, or from untreated cells, in which the histones are primarily non-acetylated onto a 7.75 kb plasmid containing an hsp26 minigene. This process involves the prior depletion of the endogenous histones present in the chromatin assembly extract such that chromatin is assembled quantitatively from the input, exogenous histones. The chromatin assembly reaction generates complex chromatin containing many non-histone proteins and enzymatic activities. While H1-containing control chromatin severely represses transcription of a model hsp26 gene, highly acetylated chromatin is significantly less repressive. Acetylation of histones, and particularly of histone H4, affects transcription at the level of initiation (Nightingale, 1998).

The Drosophila Polycomb and trithorax group proteins act through chromosomal elements such as Fab-7 to maintain repressed or active gene expression, respectively. A Fab-7 element is switched from a silenced to a mitotically heritable active state by an embryonic pulse of transcription. Here, histone H4 hyperacetylation has been found to be associated with Fab-7 after activation, suggesting that H4 hyperacetylation may be a heritable epigenetic tag of the activated element. Activated Fab-7 enables transcription of a gene even after withdrawal of the primary transcription factor. This feature may allow epigenetic maintenance of active states of developmental genes after decay of their early embryonic regulators (Cavalli, 1999).

Fab-7-dependent chromosomal memory of silent or open chromatin states occurs in transgenic Drosophila lines such as FLW-1 and FLFW-1. These lines carry a heat shock-inducible GAL4 driver (hsp70-GAL4) regulating a GAL4-dependent lacZ reporter (UAS-lacZ) flanked by Fab-7 and the mini-white gene. Silencing imposed by Fab-7 on the flanking reporter genes is dependent on the components of the PcG, since heterozygous mutant PcG genes show a relief of white gene repression. Conversely, white gene activity requires the trxG because heterozygous mutations in the different members tested result in a down-regulation of expression. A GAL4 pulse during embryogenesis can impose a mitotically stable reprogramming of the Fab-7 cellular memory module (CMM) from a silenced to an open chromatin state. The maintenance of the activated Fab-7 state is dependent on trithorax (trx) but not on Polycomb (Pc). In a heterozygous Pc- background, Fab-7 can be switched by a GAL4 pulse and be stably maintained, resulting in strong white expression. In contrast, a trx- mutation completely abolishes the mitotic transmission (Cavalli, 1999).

To assess whether the epigenetically activated Fab-7 state correlates with a permanent loss of PcG proteins from the chromatin template, a strong GAL4 induction pulse was administered during embryogenesis in the FLFW-1 line. Polytene chromosomes of third instar larvae were immunostained with antibodies directed against PcG proteins. Surprisingly, all of the PcG proteins tested, Polycomb (Pc) and Posterior sex combs (Psc), Polyhomeotic (Ph), and Polycomb-like (Pcl), are still strongly bound to the Fab-7 transgene irrespective of the epigenetic state. Thus, an epigenetically activated state can be stably propagated in the presence of the protein components of the PcG. These data support previous observations that have demonstrated binding of Pc at cytological sites containing potentially active genes in polytene chromosomes and binding of Ph and Psc proteins at an actively transcribed gene in Drosophila Schneider cells. It has been reported that certain PcG genes may function as activators in specific tissues and at specific developmental times by genetic analyses. Although a role for Pc protein in the maintenance of the activated state of Fab-7 is not observed, it may be possible that other PcG proteins are involved in this process (Cavalli, 1999).

If it is not the removal of PcG repressors on the template, what is the epigenetic tag that marks the activated Fab-7 state? A single embryonic GAL4 pulse was administered to FLFW-1 embryos, and histone acetylation of the Fab-7 transgene as a possible mark was analyzed by immunostaining polytene chromosomes of third instar larvae with specific antibodies against the tetra-acetylated form of H4 and H3 histones. Hyperacetylation of histone H4 is detected at the Fab-7 transgene location in larvae derived from activated embryos, but not from control embryos raised at 18°C. In contrast to H4, no hyperacetylation of histone H3 could be detected in activated FLFW-1 individuals (Cavalli, 1999).

Patterning transcription factors, like the products of many segmentation genes, act only shortly on their downstream genes during early Drosophila development, whereas the PcG/trxG memory system subsequently maintains the embryonically programmed patterns. For this reason, a test was performed to see whether embryonically activated Fab-7 can maintain expression of the reporter gene lacZ in the absence of the primary transcription factor GAL4. The leakiness of the hsp70 promoter prevents the complete disappearance of GAL4 protein during single fly development. To overcome this problem, use was made of the fact that activated Fab-7 can be efficiently propagated through meiosis in the line FLW-1. This allows the crossing out of the GAL4 driver to test lacZ expression in the complete absence of GAL4 in subsequent generations. Upon crossing out GAL4 in activated flies, 20% to 25% of the GAL4-less embryos show substantial levels of homogeneous beta-galactosidase expression in all embryonic cells in two consecutive generations. This percentage correlates well with the fraction of adults showing meiotically stable white derepression. Unfortunately, it is not possible to also test for the meiotic inheritance of H4 hyperacetylated states because of a staining pattern with endogenous bands at the insertion site of the transgene in the FLW-1 line. However, the functional analysis demonstrates that epigenetic inheritance of an active Fab-7 chromatin state results in transcriptional activity of the UAS-lacZ reporter even in the absence of the GAL4 transactivator (Cavalli, 1999).

A weak expression of lacZ in the absence of GAL4 may arise from a heritable loss of PcG-mediated repression, thereby neutralizing the silencing ability of Fab-7 and consequently reflecting the ground state of a nonrepressed chromatin template. If this were the case, it might be expected that in flies carrying an UAS-lacZ construct without Fab-7 (pU/l5), a similar weak homogeneous beta-Gal staining pattern would be observed in all embryos in the absence of GAL4. To test this point, beta-Gal staining was examined in two independent lines carrying the pU/l5 construct but no GAL4 driver. In both cases, most of the embryos were not stained or were stained in a weakly variegated fashion in random cells. This strongly suggests that meiotic inheritance of Fab-7 CMM-activated states does not simply reflect lifting of PcG-mediated silencing but rather the inheritance of an active chromatin state, which is competent for transcriptional activation (Cavalli, 1999).

These findings show in a functional manner that trxG protein complexes recruited at a CMM relieve the requirement for the activating factor for transcriptional maintenance. Hyperacetylation of histone H4 has been identified as an epigenetic mark for the activated Fab-7 state. Unlike the short-lived H4 hyperacetylation induced by transient gene activation at late developmental stages, the mark set at embryonic stages is mitotically stable and inheritable. An important maintenance function of the PcG and trxG protein complexes at CMMs might be to protect epigenetic marks from erasure (Cavalli, 1999).

Histone H4, acetylation and dosage compensation

Dosage compensation ensures that males with a single X chromosome have the same amount of most X-linked gene products as females with two X chromosomes. In Drosophila, this equalization is achieved by a twofold enhancement of the level of X chromosome transcription in males, relative to each X chromosome in females. The products of at least five genes, maleless (mle), male-specific lethal 1, 2, and 3 (msl-1, msl-2, msl-3) and males absent on the first (mof), are necessary for dosage compensation. MOF transcript is found in larvae and adults of both sexes. The proteins produced by these genes form a complex that is preferentially associated with numerous sites on the X chromosome in the somatic cells of males, but not females. Binding of the dosage compensation complex to the X chromosome correlates with a significant increase in the presence of a specific histone isoform (histone 4 acetylated at lysine 16), on the chromosome. Experimental results and sequence analysis suggest that the mof gene encodes an acetyl transferase that plays a direct role in the specific histone acetylation associated with dosage compensation. The predicted amino acid sequence of MOF exhibits a significant level of similarity to several other proteins, including the human HIV-1 Tat interactive protein Tip60, the human monocytic leukemia zinc finger protein MOZ and the yeast silencing proteins SAS3 and SAS2. Also studied has been the role played by the various components of the complex in the targeting of MOF to the X chromosome. To this end, indirect cytoimmunofluorescence was used to monitor the binding of these components in males carrying either complete or partial loss-of-function mutations as well as in XX individuals in which formation of the dosage compensation complex has been induced by genetic means (Gu, 1998 and Hilfiker, 1997).

While the link between acetylation of histone H4 at lysine 16 and the dosage compensated male X chromosome points to an involvement of the modification early on, a causal relationship between the two phenomena has been difficult to established. It has been considered possible that the same principle that promotes an increase in accessibility of genes on the male X chromosome to the transcription machinery may also increase the availability of the nucleosome substrate to a ubiquitous acetyltransferase. In this scenario, H4 acetylation would not be causal to the increased expression of X-linked genes in males, but both phenomena would profit from a common, yet unidentified cause. It has now been shown that acetylation of nucleosomes at H4 lysine 16 can lead to a remarkable relief of nucleosomal repression (Akhtar, 2000). Mof has been demonstrate to act is a histone acetyltransferase that acetylates chromatin specifically at histone H4 lysine 16. This acetylation relieves chromatin-mediated repression of transcription in vitro and in vivo if Mof is targeted to a promoter by fusion to a DNA-binding domain. Acetylation of chromatin by MOF, therefore, appears to be causally involved in transcriptional activation during dosage compensation. Dosage compensation in Drosophila therefore presents a strong case for a direct role of H4 acetylation on gene transcription in vivo (Akhtar, 2000).

Hyperacetylation of histone H4 can lead to an increased access of transcription factors to nucleosomal DNA, to an unfolding of the nucleosomal fiber, and to activation of transcription on chromatin templates. However, hyperacetylation is an experimentally induced condition in cells that is not observed under physiological conditons. Histones are mainly monoacetylated in vivo, and the determinant of functional states appears to reside in the site specificity of acetylation rather than the extent of the modification. This has been nicely illustrated by the visualization of histone H4 on Drosophila polytene chromosomes: acetylation of H4 at lysine 16 paints the hyperactive X chromosome, while acetylation at lysine 12 is a hallmark of inactive heterochromatin. Site-specific acetylation may conceivably affect interactions of the histone H4 N terminus with either vicinal nucleosomes or nonhistone proteins. It is likely that these interactions may lead to alterations of the folding of the nucleosomal fiber and degree of chromatin compaction. Interactions of the H4 N-terminal amino acids K16-N25 with the H2A/H2B heterodimer occur in the nucleosome crystal, but it is unclear yet whether these would contribute to the folding of a physiological chromatin fiber. The quest for interacting factors with potential to discriminate between particular histone isoforms is ongoing, with no results reported to date (Akhtar, 2000 and references therein).

A cDNA fragment containing the putative Mof catalytic domain (aa 518 to 827) was expressed and it was determined that the recombinant peptide can acetylate Drosophila histones with a preference for histone H4. This pattern is similar to that for a related yeast protein, Esa1p. Active full-length Mof could not be expressed, Mof was isolated as a component of a partially purified MSL complex. Tissue culture cells were used for the initial characterization of the MSL complex. S2 cells are male, based on the following criteria: they do not express the Sxl (Sex-lethal) gene product, which is necessary for female differentiation, and they express Msl2, a limiting component of the dosage compensation machinery whose synthesis is prevented by Sxl. S2 cells can be stably transfected, allowing the use of commercially available antibodies recognizing epitope tags. Transient transfection of S2 cells with Msl2 tagged at its carboxy terminus with the HA epitope reveals that the localization of the HA epitope is coincident with the location of endogenous Mof. After selection with hygromycin, most cells exhibit HA staining on the male X chromosome, the location of which is revealed by antibodies to H4Ac16 (Smith, 2000).

Immunoprecipitation of nuclear extracts from Msl2-HA cells with the 12CA5 (anti-HA) antiserum results in the same proteins as those obtained from S2 cells with an Msl1 antiserum. In salivary gland nuclei, Mle is released from the male X chromosome with RNase treatment. Furthermore, the roX1 and roX2 RNAs are found along the X chromosome with a distribution that mimics that of the MSL complex. Therefore, attempts were made to obtain a partially purified complex containing Mle and a roX RNA and to see whether the presence of either of these components depended on the other. 'RNA-friendly' conditions were developed to increase the chances of purifying Mle and roX RNA-containing complex. The method involved a cell line expressing Flag-tagged MSL3 and sonication under low-salt conditions, immunoprecipitation with Flag antibodies followed by peptide elution, and a second immunoprecipitation with either an MSL antibody or with the corresponding preimmune serum. By using this two-step procedure, a faint band was detected by silver staining that corresponds to Mle protein. Clear enrichment of Mle was seen in the Msl1 immunoprecipitate relative to the preimmune serum. However, following a brief treatment with 0.4 M NaCl, the Mle levels were significantly reduced (Smith, 2000).

To determine if roX RNAs are expressed in S2 cells, Northern blot analysis was performed and it was observed that roX2, but not roX1, is expressed in these cells, consistent with the observation that roX1 is dispensable in flies. The size of the major roX2 transcript observed by Northern analysis was ~ 600 nucleotides. To test if roX2 RNA is present in the Mle-containing immunoprecipitates, RNA was extracted from the immunoprecipitation pellets and a RT-PCR was performed with roX2-specific primers in the linear range. The results show a clear enrichment of roX2 RNA in the immune over the preimmune serum precipitates (Smith, 2000).

The MSL complex specifically acetylates lysine 16 of histone H4. When MSL-containing immunoprecipitates were incubated with nucleosomal substrates, significant acetyltransferase activity toward histone H4 was detected. Msl1 immunoprecipitates from S2 nuclear extracts and 12CA5 immunoprecipitates from Msl2-HA nuclear extracts contain H4-specific acetyltransferase activity, while control immunoglobulin G or 12CA5 immunoprecipitates from S2 cells do not. To demonstrate that the acetyltransferase activity of the MSL complex is ascribable to Mof, complexes were purified containing either wild-type Mof or a protein produced by the mutant allele mof1. This allele is a point mutation resulting in a glycine-to-glutamic acid replacement at the most highly conserved residue of the acetyl-CoA binding domain (G691E). Wild-type Mof-HA or G691E Mof-HA were overexpressed in S2 cells and immunoprecipitated with anti-HA antibodies to obtain complexes with only transfected Mof fusion proteins. Immunoprecipitates from G691E cells have markedly reduced acetylation, consistent with the conclusion that Mof is the sole acetyltransferase in the MSL complex (Smith, 2000).

Given the specificity of the MSL complex toward H4, it was of intereset to determine which particular lysines were acetylated. When acetylated histones were separated by acid-urea gel electrophoresis, predominantly monoacetylated H4 was detected. A similar acid-urea gel was blotted to PVDF, and the mono-acetylated band was subjected to microsequencing. Counts were found at lysine 16, while other potential acetylation sites (at position 5, 8, or 12) were unlabeled. This result provides a causative link between the presence of histone H4 acetylated at lysine 16 and the MSL complex on the X chromosome in Drosophila males (Smith, 2000).

Dosage compensation works to heighten the activity of the single X chromosome in males. This heightened expression of the X chromosome in males is accomplished through the action of male-specific lethal (MSL) proteins. Immunostaining of chromosomes shows that the MSL proteins are associated with all female chromosomes at a low level but are sequestered to the X chromosome in males. Histone-4 Lys-16 acetylation follows a similar pattern in normal males and females, being higher on the X and lower on the autosomes in males than in females. However, the staining pattern of acetylation and the mof gene product, a putative histone acetylase, returns to a uniform genome-wide distribution as found in females and in males that are mutant for the msl gene. Gene expression on the autosomes correlates with the level of histone-4 acetylation. With minor exceptions, the expression levels of X-linked genes are maintained with either an increase or decrease of acetylation, suggesting that the MSL complex renders gene activity unresponsive to H4Lys16 acetylation. Evidence has also been found for the presence of nucleation sites for the association of the MSL proteins with the X chromosome rather than individual gene binding sequences (Bhadra, 1999).

Mutations in Drosophila ISWI, a member of the SWI2/SNF2 family of chromatin remodeling ATPases, alter the global architecture of the male X chromosome. The transcription of genes on this chromosome is increased 2-fold relative to females due to dosage compensation, a process involving the acetylation of histone H4 at lysine 16 (H4K16). Blocking H4K16 acetylation suppresses the X chromosome defects resulting from loss of ISWI function in males. In contrast, the forced acetylation of H4K16 in ISWI mutant females causes X chromosome defects indistinguishable from those seen in ISWI mutant males. Increased expression of MOF, the histone acetyltransferase that acetylates H4K16, strongly enhances phenotypes resulting from the partial loss of ISWI function. Peptide competition assays have revealed that H4K16 acetylation reduces the ability of ISWI to interact productively with its substrate. These findings suggest that H4K16 acetylation directly counteracts chromatin compaction mediated by the ISWI ATPase (Corona, 2002).

SU(VAR)3-7 links heterochromatin and dosage compensation in Drosophila

In Drosophila, dosage compensation augments X chromosome-linked transcription in males relative to females. This process is achieved by the Dosage Compensation Complex (DCC), which associates specifically with the male X chromosome. It has been found that the morphology of this chromosome is sensitive to the amounts of the heterochromatin-associated protein SU(VAR)3-7. This study examined the impact of change in levels of SU(VAR)3-7 on dosage compensation. It was first demonstrated that the DCC makes the X chromosome a preferential target for heterochromatic markers. In addition, reduced or increased amounts of SU(VAR)3-7 result in redistribution of the DCC proteins MSL1 and MSL2, and of Histone 4 acetylation of lysine 16, indicating that a wild-type dose of SU(VAR)3-7 is required for X-restricted DCC targeting. SU(VAR)3-7 is also involved in the dosage compensated expression of the X-linked white gene. Finally, it was shown that absence of maternally provided SU(VAR)3-7 renders dosage compensation toxic in males, and that global amounts of heterochromatin affect viability of ectopic MSL2-expressing females. Taken together, these results bring to light a link between heterochromatin and dosage compensation (Spierer, 2008).

Drosophila uses two systems of whole chromosome regulation: dosage compensation mediating the two fold up-regulation of male X-linked genes and the Painting of fourth, POF, regulating the mainly heterochromatic fourth chromosome. Binding of POF to the fourth chromosome is dependent on the heterochromatic protein HP1). POF and HP1 colocalize on fourth chromosome-linked genes and both are involved in the global regulation of the fourth chromosome. It has been proposed that POF stimulates and HP1 represses genes expression and that the interdependent binding of these two proteins precisely tunes output from the fourth chromosome (Spierer, 2008).

Dosage compensation targets the male X chromosome to correct the unbalance between the unique X chromosome of males and the two X chromosomes of females. To compensate for the resulting disparity in X chromosome-linked gene expression, most X-linked genes in males are hyperactivated. The Dosage Compensation Complex (DCC) consists of five proteins called the MSLs for Male Specific Lethal (MSL1, MSL2, MSL3, MLE and MOF) as well as two non-coding RNAs, roX1 and roX2. In males, the expression of MSL2 mediates the stabilization of the other proteins and the assembly of the DCC specifically on the X chromosome. This results in an enrichment of acetylation of histone H4 at lysine 16 (H4K16ac) on the male X chromosome, due to the MOF protein of the complex. The histone mark could in part explain the subsequent hypertranscription of X-linked genes in males. In females, the Sex-lethal gene turns off the dosage compensation system by repressing the Msl2 translation (Spierer, 2008).

One of the most intriguing issues of dosage compensation is the specific recognition of the male X chromosome by the DCC. Searches for X chromosomal DNA sequences determining DCC binding failed to identify a consensus sequence. Global mapping of MSL proteins on the X chromosome has demonstrated that the DCC associates primarily with genes rather than intergenic regions, displays a 3'- bias and correlates with transcription. Moreover, the MSL complex is attracted to genes marked by H3K36 trimethylation, a mark of active transcription. Furthermore, the levels of DCC proteins MSL1 and MSL2 are critical for correct targeting to the X chromosome. Over-expression of both msl1 and msl2 results in inappropriate MSLs binding to the chromocenter and chromosome 4. MSL2, deleted of its C-terminal part, binds as a complex with MSL1 to the heterochromatic chromocenter. roX RNAs are also key components for X chromosome targeting since roX1roX2 mutants cause relocation of MSLs complex to autosomal regions and the chromocenter. These data reveal an unpredicted physical association between the MSL complex and heterochromatic regions (Spierer, 2008 and references therein).

H4K16 acetylation is not the only chromatin mark distinguishing the Drosophila male X chromosome from the autosomes. Phosphorylation of H3 at serine 10, catalyzed by JIL-1, is a histone modification highly enriched on the male X chromosome. The JIL-1 kinase interacts with the DCC and is involved in dosage compensation of X-linked genes. Interestingly, Jil-1 mutant alleles affect both condensation of the male X chromosome and expansion of heterochromatic domains, providing evidence for a dynamic balance between heterochromatin and euchromatin. Other general modulators of chromatin state, as ISWI or NURF, are also required for normal X chromosome morphology in males. The NURF complex and MSL proteins have opposite effects on X chromosome morphology and on roX transcription (Spierer, 2008 and references therein).

An intriguing genetic interaction has been discovered between the heterochromatic proteins SU(VAR)3-7 and HP1, and dosage compensation (Spierer, 2005). Su(var)3-7 encodes a protein mainly associated with pericentromeric heterochromatin and telomeres, but also with a few euchromatic sites. Specific binding to pericentric heterochromatin requires the heterochromatic protein HP1 (Spierer, 2005). HP1 localizes to heterochromatin through an interaction with methylated K9 of histone H3 (H3K9me2), a heterochromatic mark mainly generated by the histone methyltransferase SU(VAR)3-9. SU(VAR)3-7 interacts genetically and physically with HP1 and with SU(VAR)3-9. Su(var)3-7, Su(var)205, encoding HP1, and Su(var)3-9 are modifiers of position effect variegation (PEV), the phenomenon of gene silencing induced by heterochromatin. These three genes enhance or suppress the PEV depending on their doses and thus are considered as encoding structural components of heterochromatin. Strikingly, the amounts of SU(VAR)3-7 and HP1 affect male X chromosome morphology more dramatically than other chromosomes. Reduced doses of SU(VAR)3-7 or HP1 result in bloating of the X chromosome specifically in males (Spierer, 2005). Increased doses of SU(VAR)3-7 cause the opposite phenotype, a spectacular condensation of the X chromosome associated with recruitment of other heterochromatin markers. Some unique feature of the male X chromosome makes it particularly sensitive to change in SU(VAR)3-7 amounts. In addition, knock-down of Su(var)3-7 results in reduced male viability leading to a 0.7 male/female ratio in the progeny of Su(var)3-7 homozygous mutant mothers (Delattre, 2004). The possibility of interaction between activating and repressive chromatin factors on the male X chromosome led to an analysis of the impact of SU(VAR)3-7 on dosage compensation (Spierer, 2008).

This study shows that wild-type levels of SU(VAR)3-7 are required for male X chromosome morphology, X chromosome-restricted DCC targeting, expression of P(white) transgenes in males and for coping with increased MSL1 and MSL2 levels. Evidence is provided for interplay between heterochromatin and dosage compensation in Drosophila (Spierer, 2008).

This work reveals a connection between heterochromatin and dosage compensation in Drosophila. SU(VAR)3-7 is implicated in male X chromosome morphology, in correct distribution of the DCC, in the expression of the dosage compensated white gene and in male viability. This study describes some of the complex interactions between SU(VAR)3-7 and the DCC and illustrates the ability of heterochromatin/DCC balance to affecting chromatin conformation and protein distribution. The results support a model whereby the activating dosage compensation system in Drosophila is influenced by chromatin silencing factors (Spierer, 2008).

Reduced levels of SU(VAR)3-7 induce bloating of the male X chromosome, whereas increased levels cause condensation of the male X chromosome. Moreover, at high dose, SU(VAR)3-7, normally restricted to heterochromatin, invades preferentially the male X chromosome and, to a lesser extent, the autosomes. These observations led to an investigation of the features rendering the male X chromosome particularly sensitive to SU(VAR)3-7. This paper examined the genetic interaction between a gene essential for dosage compensation, mle, and Su(var)3-7 on the morphology of the male X chromosome. Bloating and shrinking of the X chromosome both require the presence of the DCC, and assembly of the DCC in females is sufficient to make their X chromosomes preferential targets for SU(VAR)3-7, when in excess. The dosage compensation system is thus responsible for the sensitivity of the male X chromosome to changes in SU(VAR)3-7 amounts. One explanation for the X chromosome sensitivity is that increased levels of H4K16 acetylation induced by the DCC render chromatin of the male X chromosome more accessible to chromatin factors and more sensitive to disturbances than other chromosomes. The possibility cannot be excluded that SU(VAR)3-7-induced X chromosome defects are indicators of a more general effect of the protein on all chromosomes as described for ISWI: Null mutations in the gene encoding ISWI cause aberrant morphology of the male X chromosome but not of autosomes and females X chromosomes, but expression of a very strong dominant negative form of ISWI in vivo leads indeed to decondensation of all chromosomes in both sexes. Nevertheless other data in this study, to be described below, favour the hypothesis whereby X chromosome defects result from a specific interaction between SU(VAR)3-7 and dosage compensation (Spierer, 2008).

Male X chromosome sensitivity to SU(VAR)3-7 was rather unexpected, as in a wild-type context, in contrast to over-expression conditions, no preferential binding of SU(VAR)3-7 to the male X chromosome was detected. The absence of detectable SU(VAR)3-7 enrichment on the male X polytene chromosome from third instar larvae may be due either to lack of sensitivity of the immunostaining procedure or to observations made in inappropriate tissues or development stages. Similar puzzling observations have been made for HP1, which is not preferentially seen on the male X polytene chromosomes, although reduced HP1 induces bloating of the male X chromosome. In cultured cells however, a moderate HP1 enrichment was detected with the DamID technique on the male X chromosome and not on the female X chromosomes, suggesting that HP1 participates in the structure of the male X chromosome (Spierer, 2008).

A striking and novel result of this study is that precise wild-type amounts of the heterochromatic protein SU(VAR)3-7 are required to restrict MSLs binding to the X chromosome. In Su(var)3-7 mutants, it was observed that the MSL proteins are recruited to the chromocenter. Furthermore, when SU(VAR)3-7 is present in excess, MSLs are massively delocalized from the X chromosome to many sites on autosomes (Spierer, 2008).

Two hypotheses are proposed. First, the effect of SU(VAR)3-7 on the MSLs distribution is indirect and due to the regulation of the expression of a component of the DCC. Indeed, increased amounts of MSL1 and MSL2 lead to MSLs binding on autosomes and at chromocenter, and MSLs delocalization from the X chromosome to autosomes and chromocenter is detectable in roX1roX double mutants. A careful regulation of MSLs and roX RNAs concentration is therefore important to restrict DCC activity to appropriate targets. In addition, increased levels of MSL2, or of both MSL2 and MSL1, result in a diffuse morphology of the X chromosome. This phenotype resembles the bloated X chromosome of Su(var)3-7 and Su(var)2-5 mutants, suggesting that the amounts of MSL2 and MSL1 are downregulated by the heterochromatic proteins. Expression of many euchromatic genes are under the control of the HP1 protein, leading the idea of testing whether changes in SU(VAR)3-7 amounts modify the expression of roXs, msl1 and msl2 or the stability of MSL1 and MSL2. Quantitative RTPCR and Western blots did not detect significant changes in the amounts of DCC components. In HP1 mutant msl1 transcription is also not affected. These results speak against the hypothesis of regulation of expression of a DCC component by a SU(VAR)3-7/HP1 complex (Spierer, 2008).

The second hypothesis is that SU(VAR)3-7 modifies the MSLs distribution by changing the chromatin state of the X chromosome and of the pericentric heterochromatin. Changes in chromatin conformation or epigenetic marks could modify affinity of the DCC for entry sites. Numerous entry sites on the X chromosome have been described, and a hierarchy of entry sites has been suggested with different affinities for the DCC. Even cryptic binding sites on autosomes and at the chromocenter are recognized by the DCC in certain conditions. It is proposed that increasing SU(VAR)3-7 amounts on the X chromosome results in an enrichment of HP1 and H3K9 dimethylation, and leads to a more compact heterochromatic-like structure of the X chromosome which then blocks access to the high-affinity entry sites. The free DCC, chased from the X chromosome sites turns toward low-affinity sites present on autosomes, but not toward those embedded into the chromocenter. Indeed, cryptic chromocenter sites become more inaccessible by heterochromatin compaction, a phenomenon also responsible for the enhancement of variegation by increased SU(VAR)3-7 levels. Inversely, the absence of SU(VAR)3-7 induces a more relaxed chromatin state at the chromocenter (Spierer, 2005), thus increasing affinity of the entry sites embedded into heterochromatin, and allowing MSLs binding at the chromocenter. Similar recruitments of MSLs at heterochromatin have been described in the literature in three situations: (1) in roX1roX2 mutants, (2) in presence of excess of MSL2 and in (3) C-terminal truncated MSL2 mutants. This means that cryptic entry sites present in heterochromatin become more accessible to the MSLs either in a Su(var)3-7 mutants or if DCC composition is modified. The explanation of heterochromatin affinity for the MSLs remains obscure. On the X chromosome, the Su(var)3-7 mutation induces the bloated morphology resembling that described as a result of decreased levels of silencing factors as HP1, ISWI and NURF, or of increased MSLs levels. The current study and others suggest that male X chromosome morphology depends on the balance between silencing and activating complexes. The simultaneous existence of the repressive SU(VAR)3-7/HP1 proteins and the MSLs complex may provide a set of potential interactions that cumulatively regulate dosage compensation (Spierer, 2008).

Several arguments support a role for SU(VAR)3-7 in dosage compensation. Reduced male viability in the progeny of Su(var)3-7 homozygous females is a first argument for a function played by the protein specifically in males. The results also show that wild-type amounts of SU(VAR)3-7 are required to cope with increased MSL1 and MSL2 levels. In absence of maternal SU(VAR)3-7 product, the transgenes expressing MSL1 and MSL2 become toxic to males, whereas no lethality is observed with wild-type or half amounts of SU(VAR)3-7. This suggests that SU(VAR)3-7 is required very early in development to counteract an excess of MSL1 or MSL2 activity. Corroborating this effect, it was determined that the global amount of heterochromatin affects the viability of females engineered to expressing msl2. The presence of the highly heterochromatic Y chromosome kills half of the females expressing msl2. It has been proposed that the Y chromosome functions as a sink for heterochromatic factors as SU(VAR)3-7 and HP1). A Y chromosome added to XX females could sequester heterochromatic proteins, and induce lethality in a context of female dosage compensation. All these data lead to the conclusion that SU(VAR)3-7 is required for the viability of dosage-compensated flies. Two explanations are proposed: 1) Either SU(VAR)3-7 is required to restrict DCC on the X chromosome and the lethality induced by the lack of SU(VAR)3-7 is due to the MSLs ectopic activity outside of the X chromosome (at heterochromatin), or 2) SU(VAR)3-7 is required on the dosage compensated X chromosome and, in this case, the Su(var)3-7 mutant lethality results from malfunctioning of the DCC on the X (Spierer, 2008).

To discriminate between these two hypothesis, expression of X-linked genes was examined in Su(var)3-7 mutants. Although small changes are visible, the RT-PCR analysis did not sufficient to allow the concluion that the lack of SU(VAR)3-7 affects significantly the levels of transcripts of seven X-linked genes. If they exist, changes were indeed expected to be very small. For MSLs mutations, the magnitude of the decrease is very modest considering the severe failure of dosage compensation (around 1.5). Taking into account that the Su(var)3-7 mutation induces only 30% lethality among males, expected changes in transcript accumulation are predicted to be even smaller. Moreover, transcripts analysis was done in male larvae and some slight biological variations between the three samples cannot be avoided though great care was taken on samples homogeneity. Finally, normalizing to internal autosomal genes RNA could also introduce a bias. It is believed that in the case, quantitative RT-PCR experiment was not the appropriate method to detect very small changes of expression (Spierer, 2008).

In consequence, an alternative system was used to test the implication of SU(VAR)3-7 on dosage compensation. The effect of increased or decreased Su(var)3-7 expression on the dosage compensated expression of the white gene carried by P transgenes was determined. Strikingly, it was observed that lack and excess of SU(VAR)3-7 decreases the white expression specifically in males, and never in females. This is a strong indication that the wild type dose of SU(VAR)3-7 is required for correct dosage compensated expression of the white gene. Interestingly, Su(var)3-7 over-expression affects white expression when the gene is localized on the X chromosome and not on autosomes, although white is still partially dosage compensated on autosomes. This may result from the combination of two phenomena: On the X chromosome, excess of SU(VAR)3-7 induces preferential enrichment of heterochromatic silencing proteins and partial loss of MSLs. On autosomes, heterochromatic proteins recruitment is less visible and, in addition, the MSLs are massively present. Consequently the dosage compensation of a P(white) transgene linked to the X chromosome is more likely to be perturbed by excess of SU(VAR)3-7 than an autosomal insertion (Spierer, 2008).

In sum, in this study has revealed a role for SU(VAR)3-7 on global X chromosome morphology with an impact on the distribution of MSLs proteins, thus highlighting the contribution of SU(VAR)3-7 to the intriguing issue of X specific DCC targeting. It appears also that SU(VAR)3-7 is required for the viability of dosage compensated flies and the expression of a dosage compensated X-linked gene, suggesting a puzzling interplay between heterochromatin and the DCC. SU(VAR)3-7 plays a subtle role on dosage compensation: Flies need SU(VAR)3-7, especially the maternal protein, for correct dosage compensation but, at the same time, excess of SU(VAR)3-7 has a negative effect on dosage compensation. Future interest will focus on the fascinating issue of the molecular nature of heterochromatin/DCC intersection (Spierer, 2008).

Genome-wide analysis reveals MOF as a key regulator of dosage compensation and gene expression in Drosophila

Dosage compensation, mediated by the MSL complex, regulates X-chromosomal gene expression in Drosophila. This study reports that the histone H4 lysine 16 (H4K16) specific histone acetyltransferase MOF displays differential binding behavior depending on whether the target gene is located on the X chromosome versus the autosomes. More specifically, on the male X chromosome, where MSL1 and MSL3 are preferentially associated with the 3' end of dosage compensated genes, MOF displays a bimodal distribution binding to promoters and the 3' ends of genes. In contrast, on MSL1/MSL3 independent X-linked genes and autosomal genes in males and females, MOF binds primarily to promoters. Binding of MOF to autosomes is functional; H4K16 acetylation and the transcription levels of a number of genes are affected upon MOF depletion. Therefore, MOF is not only involved in the onset of dosage compensation, but also acts as a regulator of gene expression in the Drosophila genome (Kind, 2008).

Consistent with previous MSL1 and MSL3 profiling studies, MSL1, MSL3, MOF, and H4K16Ac display enrichment to 3' end of genes in SL-2 cells. Surprisingly, MOF displays a bimodal binding pattern on genes residing on the X chromosome, associating with both the 3' ends of dosage-compensated genes as well as with promoter regions (Kind, 2008).

Recent observations on individual X-chromosomal target genes using transgene analysis in vivo have revealed that there are at least two classes of sites; transcription-independent 'high-affinity sites' such as roX2 and transcription-dependent 'low-affinity sites' such as mof or CG3016. Integrating the observations obtained from the genome-wide binding and RNAi-mediated knockdown analysis shown in this study, it appears that MOF plays a central role in targeting the MSL complex to 'low-affinity sites' where recruitment of MSL1 and MSL3 is found to be dependent on the presence of MOF. This is in contrast to the 'high-affinity sites' where partial complexes of MSL1/MSL2 can be recruited independently of MOF, MSL3, and MLE (Kind, 2008).

Interestingly, MOF was found to bind not only to the male X chromosome, but also to autosomes and female chromosomes. Different from the bimodal binding pattern of MOF on the male X chromosome, in Kc cells, MOF is enriched to promoters of all chromosomes similarly to the situation on the male autosomes in SL-2 cells. However, although the binding pattern between the X and the autosomes in Kc cells looks practically identical, the amplitude of promoter binding is significantly higher on the X chromosome than on the autosomes in Kc cells, as is the case in SL-2 cells. It is possible that X-chromosomal genes have as-yet-unidentified sequence elements that contribute toward MOF binding to promoters of X-chromosomal genes in males and females. Alternatively, since reduced amount of MSL1 is expressed in females and MSL1 displays low-level promoter binding on the X chromosome in SL-2 cells, it may contribute to higher amplitude of MOF binding on X chromosomal genes in both SL-2 and Kc cells compared to autosomes. Since the gene density on the X chromosome is similar to that of other chromosomes (except for the fourth chromosome), this does not explain the higher amplitude of MOF binding on the X chromosome. It is therefore possible that MOF, in addition to its role in facilitating transcriptional elongation by acetylating gene loci in an MSL context, is also involved in transcriptional initiation in an MSL-independent manner, perhaps by interaction with additional factors. Another interesting possibility is that the enrichment of MOF to promoters may provide a reservoir of enzyme, held in check by other factors, to be readily used by the MSL proteins or other promoter-bound complexes when needed for modulating transcription levels (Kind, 2008).

Intriguingly, the MSL3 profile across gene loci appeared very similar to that of H4K16Ac, suggesting a role for MSL3 in activation and/or stabilization of H4K16Ac on X-linked genes. In support of this hypothesis, MSL3 has been shown to stimulate MOF's HAT activity in vitro. MSL3 has been shown to bind H3K36 trimethylated (H3K36me3) nucleosomes, and H3K36me3 (which also peaks at 3' end of genes, similar to MSLs) was shown to influence MSL binding. In S. cerevisiae, Eaf3 recognition of H3K36me3 has been shown to direct Rpd3(S) to actively transcribed genes to deacetylate histones in the wake of polymerase II, preventing spurious transcription within genes from cryptic promoters. It has been proposed that the MSL complex on the X chromosome may compete for the Rpd3(S) complex, thereby increasing the overall H4K16Ac levels by reducing the turnover rates of this modification (Kind, 2008).

Since the 3' ends of genes are indispensable for MSL target recognition on the X chromosome (Kind, 2007), it is proposed that MSL1 and MSL2 initially target 3' regions by occasional recognition of degenerative DNA target elements, possibly made accessible by low levels of H4K16Ac brought about by MOF occupancy of the promoter. MSL3 may serve to stabilize the association of MSL1/MSL2 with dosage-compensated genes by binding to H3K36me3, which in turn may lead to the recruitment and stimulation of MOF to the body of the gene. It has also been proposed that local recycling of RNA polymerase II could result in enhanced mRNA production. MOF, with its enrichment to promoter-proximal and 3' regions, is a likely candidate to bridge such a loop formation. Gene structural studies should reveal whether such a gene-loop formation is involved in the process of dosage compensation (Kind, 2008).

This study presents four independent lines of evidence that show that MOF is involved in H4K16Ac of a large number of genes in the male and female genome. 1) MOF binding significantly correlates with H4K16Ac of all chromosomes in both SL-2 and Kc cells. 2) The H4K16Ac profile across genes correlates strongly with the diversified binding of MOF between the X chromosome (peaking toward the 3' end of genes), and autosomes (peaking toward the 5' end of genes. 3) Depletion of MOF results in a marked decrease in H4K16Ac of a number of genes on both the X chromosome and the autosomes. 4) In MOF-depleted SL-2 and Kc cells a more than 50% reduction in total H4K16Ac levels is found by mass specterometery analysis (Kind, 2008).

Several studies have implied a structural role for histone acetylation and H4K16Ac acetylation in particular, in the packaging of DNA into chromatin. Interestingly, H4K16Ac has been shown to cause an increase in the α-helical content of histone H4, and to prevent 30 nm chromatin-fiber formation and crossfiber interactions. H4K16Ac might therefore serve a structural role, imparting a relaxed chromatin state that, in turn, reduces the energy required for RNA polymerase II to affect transcription through a nucleosomal template and thereby enhancing elongation efficiency (Kind, 2008).

Regulation of ubiquitously expressed (housekeeping) genes on the X chromosome by the MSL complex probably necessitates a state of continual association with its target binding sites. Elevated levels of H4K16Ac are reached on the X chromosome presumably by constant activation of MOF by MSL1 and MSL3. On the autosomes, since MOF appears to be present independently of other MSL proteins, it does not associate to the interior of gene loci but is instead promoter bound, similar to its behavior on the X chromosome in the MSL1-depleted condition (Kind, 2008).

Assuming that MOF is involved in general transcription regulation, apart from dosage compensation, it is not surprising that MOF is required for most H4K16 acetylation. Similarly, MOF in mammals has been found to be responsible for most, if not all, H4K16Ac. Interestingly, in line with a possible role for MOF in the G2/M cell-cycle checkpoint in mammals it was found that in both SL-2 and Kc cells, MOF-bound targets are significantly enriched for certain cell-cycle functional categories. It would therefore be very interesting to study gene regulation by MOF in a cell-cycle context in synchronized cells (Kind, 2008).

The role of MOF mediated H4K16Ac on the autosomes remains speculative. One possibility: H4K16Ac modification on autosomes by MOF may create an opportunity for transcription initiation/reinitiation, rather than being an essential mark for transcriptional activity itself. This could also explain why it was observed that, although MOF is generally bound to active genes, approximately 30% of the autosomal bound genes are affected by MOF depletion. MOF's presence on autosomal genes may therefore provide a minimal landscape of H4K16Ac, maintaining a local environment with relatively open chromatin structure, presumably similar to the condition of mating type loci in. Upon transcriptional cues, those genes would be able to rapidly and efficiently respond to meet the cell's requirements, as would be the case for cell cycle-related genes (as discussed above) (Kind, 2008).

Another possibility: MOF may work together with as-yet-uncharacterized proteins, which may allow RNA polymerase II to move efficiently through the chromatin template similar to the situation on the X chromosome. In fully elucidating the molecular mechanisms behind this process, a vital step will be the characterization of additional protein complexes associated with MOF, apart from the MSL complex. It is proposed that such complexes, comprising different trans-activating or repressive factors, may modulate MOF's HAT activity resulting in differential transcriptional outputs. Furthermore, MOF binding to promoters may allow efficient and rapid response to cellular events by recruitment/exclusion of H4K16 binding proteins or, more generally, by unique H4K16Ac-induced conformational changes to the chromatin fiber. Interestingly, one of the evolutionary conserved interacting partners of MOF is WDS, a protein in mammals shown to associate with histone H3 lysine 4 methylation, a histone mark enriched at promoters. It would be interesting to study the potential involvement of WDS or other promoter-bound factors in recruiting MOF to promoters (Kind, 2008).

In summary, it has been shown that the MSL complex members do not conform to a uniform binding behavior on their target genes on the X chromosome: MSL1 and MSL3 are enriched at the 3' end of genes, while MOF shows a bimodal distribution with enrichment at promoter-proximal regions as well as 3' ends. The data reveal that MOF plays a central role in the targeting process on low-affinity sites where recruitment of MSL1 and MSL3 appear to be dependent on the presence of MOF, in contrast to high-affinity sites such as roX2 where targeting of MSL1 appears to be MOF independent. Furthermore, the previously unappreciated binding of MOF to promoter-proximal regions on X-chromosomal as well as autosomal sites provides an opportunity to investigate additional roles of this enzyme in other cellular processes (Kind, 2008).

X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila

The evolution of sex chromosomes has resulted in numerous species in which females inherit two X chromosomes but males have a single X, thus requiring dosage compensation. MSL (Male-specific lethal) complex increases transcription on the single X chromosome of Drosophila males to equalize expression of X-linked genes between the sexes. The biochemical mechanisms used for dosage compensation must function over a wide dynamic range of transcription levels and differential expression patterns. It has been proposed that the MSL complex regulates transcriptional elongation to control dosage compensation, a model subsequently supported by mapping of the MSL complex and MSL-dependent histone 4 lysine 16 acetylation to the bodies of X-linked genes in males, with a bias towards 3' ends. However, experimental analysis of MSL function at the mechanistic level has been challenging owing to the small magnitude of the chromosome-wide effect and the lack of an in vitro system for biochemical analysis. This study used global run-on sequencing (GRO-seq) to examine the specific effect of the MSL complex on RNA Polymerase II (RNAP II) on a genome-wide level. Results indicate that the MSL complex enhances transcription by facilitating the progression of RNAP II across the bodies of active X-linked genes. Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome (Larschan, 2011).

To investigate how the MSL complex specifically increases transcription of X-linked genes, GRO-seq was performed in SL2 cells, a male Drosophila cell line that has been extensively characterized for MSL function. To show the average enrichment across genes, a 3-kb 'metagene' profile was plotted in which the internal regions were rescaled so that all genes appear to have the same length. Analysis was restricted to expressed genes that were sufficiently large (>2.5 kb) so that gene-body effects could be clearly assessed (822 X-linked genes, 3,420 autosomal genes), and all gene profiles were normalized by their copy number as determined by analysis of SL2 DNA content. High correlation coefficients were observed between replicate libraries. The metagene profiles revealed a prominent 5' peak of paused RNAP II consistent with previous chromatin immunoprecipitation (ChIP) and analysis of short 5' RNAs. In addition, a peak of RNAP II density downstream of the metagene 3' processing site is evident, possibly due to slow release in regions of transcription termination. The 3' peak is present even when the influence of neighbouring gene transcription is eliminated (Larschan, 2011).

The central question with regard to dosage compensation is how genes on the X chromosome differ on average from genes on autosomes. Overall, it was found that RNAP II density on active X-linked genes was higher than on autosomal genes, specifically over gene bodies. The increase in tag density over the bodies of X-linked genes compared to autosomal genes was approximately 1.4-fold, consistent with previous estimates of MSL-dependent dosage compensation. RNAP II ChIP was performed in SL2 cells, confirming higher occupancy on X-linked genes compared to autosomes but with lower resolution and reduced sensitivity. Therefore, GRO-seq was performed to analyse X and autosomal differences (Larschan, 2011).

To measure how X and autosomes differed on average in the distribution of elongating RNAP II, genes were segmented into their 5' 500 bp and the remainder of the coding region. The remainder of the coding region was subdivided further into 5' and 3' segments (25% and 75%, respectively). Using this segmentation, RNAP II pausing and elongation were quantitated separately on the basis of the unscaled GRO-seq signal. The pausing index (PI) was previously defined as the ratio of the GRO-seq signal at the 5' peak to the average signal over gene bodies. Herewe calculated the PI was calculated for X and autosomal genes as the ratio of the 5' peak to the first 25% of the remaining gene body, and no statistically significant difference was found when the two groups were compared (Larschan, 2011).

To examine separately transcription elongation across gene bodies, the elongation density index (EdI) was defined as the ratio of tag density in the 3' region of each gene compared to its 5' region after the first 500 bp. In contrast to the analysis of 5' pausing, statistically significant differences was found in EdI between X and autosomes. This conclusion was robust to how the 5' and 3' regions of genes were divided. As defined, the average PI (log scale) is a positive number because RNAP II is generally enriched at 5' ends compared to gene bodies; the average EdI (log scale) is a negative number, as the relative density of RNAP II typically decreases from the beginning to the end of gene bodies. It is concluded that X-linked genes, on average, show a significantly smaller decrease in RNAP II density along their gene bodies when compared to autosomal genes (Larschan, 2011).

To measure the specific contribution of the MSL complex to the increase in RNAP II within X-linked gene bodies, MSL2 RNA interference (RNAi) was used to reduce complex levels in male SL2 cells. Excellent correlations between replicate data sets were observed. To confirm the X-specific effect of MSL2 RNAi, the distributions of the GRO-seq signal (averaged over the bodies of genes excluding the 5' peak) were computed for all genes before and after RNAi. When comparing X versus autosomes, a preferential decrease was found on the X chromosome, with an average control:MSL RNAi ratio of 1.4. MSL-dependent changes in average GRO-seq density showed a weak but statistically significant correlation with changes in steady-state messenger RNA levels assayed by expression array or mRNA-Seq10. These results confirm that MSL-dependent changes in steady-state RNA levels reflect differences in active transcription on the X chromosome (Larschan, 2011).

In addition to assessing the average decrease of X-linked RNAP II density after MSL2 RNAi, it was asked whether any genes showed strong MSL-dependence, a hallmark of the roX genes that encode RNA components of the complex. It was found that roX2 showed a strong loss in GRO-seq density (ninefold) after MSL2 RNAi, as predicted. Interestingly, in the untreated or control RNAi samples, there is a prominent GRO-seq peak downstream of the major roX2 3' end, coincident with an MSL recruitment site. roX1 expression is low in this isolate of SL2 cells, and no other expressed genes on X or autosomes showed strong MSL dependence in these assays (Larschan, 2011).

Next the average RNAP II density was compared along X and autosomal metagene profiles after control and MSL2 RNAi. Unlike the initial analysis of X and autosomes, where different gene populations were compared, here it was possible to examine the same genes in the presence and absence of the MSL complex. It was found that after MSL2 RNAi, the density of elongating RNAP II over the bodies of X-linked genes decreased, approaching the level on autosomes. The presence of the MSL complex affected RNAP II density starting just downstream of the 5' peak and continuing through the bodies of X-linked genes. Thus, GRO-seq functional data correlate with physical association of the MSL complex, which is biased towards the 3' ends of active genes on the male X chromosome (Larschan, 2011).

To quantify the differences in density of engaged RNAP II in the presence and absence of the MSL complex, the PI and EdI were calculated for each gene, followed by the PI and EdI ratios comparing MSL2 and control RNAi treatment. It was found that both X and autosomes increased PI and decreased EdI after MSL2 RNAi treatment. However, in each case the change was larger on X than on autosomes, and the most profound difference was an MSL-dependent change in EdI on X compared with autosomes. EdI was computed, as before, by defining the 5' and 3' regions as 25% and 75%, respectively, of the gene body after removing the 5' peak, but the difference was statistically significant for all other values until the 3' end was reached. When these analyses were performed separately for two independently prepared sets of GRO-seq libraries, the results were also statistically significant. It is concluded that the MSL complex causes the transcriptional elongation profiles of X-linked genes to differ from those of autosomal genes (Larschan, 2011).

To visualize the location along gene bodies at which the MSL complex functions, control:MSL2 RNAi GRO-seq ratios were calculated and a metagene profile was generated. Here, values above zero represent higher relative amounts of engaged RNAP II in the presence of the MSL complex compared to after RNAi treatment. In contrast, values below zero represent a relative increase in engaged RNAP II after MSL2 RNAi. In the absence of the MSL complex, there is a relative increase in the amount of RNAP II localized to the 5' ends of both autosomal and X-linked genes, perhaps due to relocalization of RNAP II from the bodies of X-linked genes. A limitation of the GRO-seq assay is that it is not possible to distinguish between initiating and 5' paused polymerase, so a definitive role cannot be assigned for this 5' increase in RNAP II after MSL2 RNAi treatment. However, relative RNAP II levels over autosomal gene bodies do not increase, indicating that any relocalized enzyme in this experiment is likely to remain paused rather than progressing across transcription units. This is consistent with a model in which the functional outcome of MSL2 RNAi is to shift RNAP II density away from productive transcription through X-linked gene bodies (Larschan, 2011).

The local effect of the MSL complex was plotted to compare it to the status of histone 4 lysine 16 (H4K16) acetylation catalysed by the MOF component of the MSL complex. H4K16 acetylation typically is enriched at the 5' ends of most active genes in mammals and flies; in contrast, a 3' bias of this mark is a distinctive characteristic of the dosage compensated male X chromosome in Drosophila. Interestingly, there is an overall coincidence across gene bodies between the MSL-complex-dependent GRO-seq signal and the presence of H4K16 acetylation. How might H4K16 acetylation biased towards the 3' end of genes generate the improved transcriptional elongation indicated by the GRO-seq results? During transcription elongation, nucleosomes are thought to comprise a barrier to the progress of RNAP II and several well-studied elongation factors, including Spt6 and the FACT complex, are proposed to function by removing nucleosomes that block RNAP II progression and replacing them in the wake of transcription. Interestingly, H4K16 acetylation of nucleosomes has been observed to act in opposition to the formation of higher-order chromatin structure in vitro. Thus, H4K16 acetylation is likely to reduce further the steric hindrance to RNAP II progression through chromatin. Improving the entry of RNAP II into the bodies of genes may allow 5', gene-specific events to proceed at an increased but still regulated rate. Furthermore, reduction in the repressive effect of nucleosomes could increase mRNA output by improving the processivity of RNAP II on each template. Available methodologies cannot distinguish between these mechanisms in vivo, and therefore future approaches will be required to assess their relative contributions to dosage compensation (Larschan, 2011).

In addition to increasing the transcription of X-linked genes for dosage compensation, the MSL complex also positively regulates the roX noncoding RNA components of the complex, to promote their male specificity. roX1 expression is low in the SL2 cell line, but GRO-seq data indicate that active transcription of roX2 is highly dependent on MSL2 as predicted. Interestingly, there is a strong GRO-seq peak at the 3' roX2 DHS (DNaseI hypersensitive site), which contains sequences important for targeting the MSL complex to the X chromosome. Sites of roX gene transcription are thought to be critical for MSL complex assembly. Therefore, it is possible that paused RNAP II at the roX2 DHS could promote an open chromatin structure that facilitates MSL complex targeting or incorporation of noncoding roX2 RNA into the complex (Larschan, 2011).

In summary, it is proposed that the MSL complex functions on the male X chromosome to promote progression and processivity of RNAP II through the nucleosomal template. Improving transcriptional output downstream of typical gene-specific regulation makes biological sense when compensating the diverse set of genes found along an entire chromosome (Larschan, 2011).

Depletion of histone deacetylase 3 antagonizes PI3K-mediated overgrowth through the acetylation of histone H4 at lysine 16

Histone acetylation is one of the best-studied gene modifications and has been shown to be involved in numerous important biological processes. This study has demonstrated that the depletion of histone deacetylase 3 (Hdac3) in Drosophila melanogaster results in a reduction in body size. Further genetic studies showed that Hdac3 counteracts the overgrowth induced by InR, PI3K or S6K over-expression, and the growth regulation by Hdac3 is mediated through the deacetylation of histone H4 at lysine 16 (H4K16). Consistently, the alterations of H4K16 acetylation (H4K16ac) induced by the over-expression or depletion of males-absent-on-the-first (MOF), a histone acetyltransferase that specifically targets H4K16, results in changes in body size. Furthermore, H4K16ac was found to be modulated by PI3K signaling cascades. The activation of the PI3K pathway caused a reduction in H4K16ac, whereas the inactivation of the PI3K pathway results in an increase in H4K16ac. The increase in H4K16ac by the depletion of Hdac3 counteracts the PI3K-induced tissue overgrowth and PI3K-mediated alterations in the transcription profile. Overall, these studies indicated that Hdac3 serves as an important regulator of the PI3K pathway and reveals a novel link between histone acetylation and growth control (Lv, 2012).

Core histone modifications are known to play an essential role in the regulation of chromatin organization and transcription. These modifications include acetylation, methylation, phosphorylation, ubiquitination, sumoylation and poly(ADP-ribosyl)ation. Histone acetylation is one of the best-studied modifications and is thought to be involved in both the initiation and elongation steps of transcription. The acetylation of the core histone tails alters the folding dynamics of nucleosomal arrays and 30-nm chromatin fibers and recruits specific chromatin remodeling complexes that exert the specific function(s) of chromatin (Lv, 2012).

The acetylation of histones is regulated by two highly conserved classes of histone enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which catalyze the addition and removal, respectively, of acetyl groups on histone lysine residues. Reversible histone acetylation and deacetylation are highly regulated processes that are crucial for chromatin reorganization and the regulation of gene transcription in response to extracellular conditions. The balance between the acetylation and deacetylation of histones serves as a key regulatory mechanism for gene expression and governs numerous developmental processes and disease states (Lv, 2012).

HDACs have been classified into four subfamilies based on their homologs and functional similarities. Hdac3 is a class HDAC that shares homology with yeast Rpd3. This protein is reportedly present in the nuclear, cytoplasmic and membrane fractions. The knockout of Hdac3 in mice leads to embryonic lethality before day 9.5. The inactivation of Hdac3 has been shown to delay cell cycle progression and result in cell cycle-dependent DNA damage, inefficient repair and increased apoptosis in mouse embryonic fibroblasts. Hdac3 has also been shown to be up-regulated in various tumor types. However, the precise function and underlying molecular mechanism of Hdac3 in these processes remain largely unknown (Lv, 2012).

The Drosophila ortholog to human Hdac3 is known to be Hdac3 or dHDAC3 (Johnson, 1998). This study used Drosophila to investigate the function of Hdac3 during development. Depletion of Hdac3 in Drosophila results in a reduction in both organ and body sizes. Hdac3 controls growth through the regulation of H4K16 deacetylation. Alterations in H4K16ac through the ectopic expression of MOF, a histone acetyltransferase that specifically targets H4K16, result in changes of cell/body size. It was also found that H4K16ac is modulated by PI3K signaling. Increasing the level of H4K16ac by depleting Hdac3 effectively reverses the PI3K-induced tissue overgrowth and alterations in the transcription profile (Lv, 2012).

Hdac3 is a component of the nuclear receptor co-repressor complex containing N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors), both of which are recruited by nuclear hormone receptors to regulate gene transcription). Several substrates were found to be targets of Hdac3, including histones and non-histone proteins. Among the targets affected by Hdac3, this study found that H4K16ac is a critical epigenetic modification associated with animal growth, as demonstrated not only by the finding that alterations in H4K16ac were closely associated with Hdac3-induced organ/body growth but also by the finding that mutating H4K16 directly affected Hdac3-induced growth. Furthermore, transgenic lines in which MOF, the specific histone H4K16 HAT, was over-expressed or depleted exhibited similar changes in cell/body size, thus confirming that H4K16ac plays an essential role in animal growth. Histone H4K16 acetylation is known to function as a dual switch for higher-order chromatin and protein-histone interactions and has been shown to regulate embryonic stem cell self-renewal and cellular life span. Recent work in has suggested that H4K16ac in Drosophila not only is critical for the acetylation of H4K5, H4K8 and H3K9, which are hallmarks of active chromatin, but also exerts an effect on H3K9 methylation and the association of HP1 with chromatin, which are hallmarks of heterochromatin. It is therefore presumed that the changes in H4K16ac affect higher-order chromatin and alter the transcription of genes related to growth. However, the exact mechanism by which H4K16ac regulates the transcription of genes related to growth needs to be further investigated (Lv, 2012).

One of the main findings in this work is the genetic interaction between Hdac3/H4K16ac and the PI3K pathway. The PI3K pathway is a highly conserved signal transduction cascade from flies to humans. Previous studies have identified a number of the components of this signaling pathway. However, the mechanisms by which this pathway regulates nuclear events, such as gene transcription, remain largely unknown. This work shows that PI3K signaling modulates the acetylation of H4K16. This finding was supported by results showing that the activation of PI3K caused a corresponding reduction in H4K16ac, whereas the inactivation of the PI3K pathway resulted in an increase in H4K16ac. Furthermore, the introduction of the H4K16A mutant, in which H4K16 cannot be acetylated, further enlarged the PI3K-induced increase in ommatidial size, confirming the function of histone H4K16ac in PI3K signaling (Lv, 2012).

Although the exact mechanism by which PI3K regulates H4K16ac is still unknown, this study demonstrates that the loss of Hdac3 inhibited PI3K-mediated overgrowth, thus suggesting that PI3K targets the activity of Hdac3 and subsequently affects H4K16ac. This hypothesis is supported by the observations that Drosophila Hdac3 can form a complex with Akt and that the complex of human Hdac3 with the deacetylase activation domain (DAD), the human SMRT co-repressor and inositol tetraphosphate is required for the activation of Hdac3 enzymatic functionality. The observation that the depletion of Hdac3 decreased the level of phospho-Akt and affected the subcellular localization of GFP-PH also supported this possibility. However, the observation that Hdac3 depletion failed to counteract the PI3K-induced hyperphosphorylation of Akt while completely rescuing the decrease in H4K16ac and the tissue overgrowth induced by the PI3K over-expression indicated that Hdac3 likely counteracts the PI3K-induced tissue overgrowth by modulating the level of H4K16ac (Lv, 2012).

The hyperactivation of the PI3K pathway is known to be associated with many types of human cancer. A number of HDAC inhibitors have been developed and applied in clinical trials to inhibit tumor growth. However, the molecular mechanisms of these HDAC inhibitors in cancer prevention remain to be elucidated. The present study found that the over-expression of PI3K decreases H4K16ac in vivo. Further studies have shown that increasing the level of H4K16ac by depleting Hdac3 can antagonize the PI3K-induced tissue overgrowth. This finding, therefore, may provide further insight into the mechanisms by which the HDAC inhibitors inhibit tumor growth (Lv, 2012).

Chromatin state marks cell-type- and gender-specific replication of the Drosophila genome

Duplication of eukaryotic genomes during S phase is coordinated in space and time. In order to identify zones of initiation and cell-type- as well as gender-specific plasticity of DNA replication, replication timing, histone acetylation, and transcription was profiled throughout the Drosophila genome. Two waves of replication initiation were identified with many distinct zones firing in early-S phase, along with multiple, less defined peaks at the end of S phase, suggesting that initiation becomes more promiscuous in late-S phase. A comparison of different cell types revealed widespread plasticity of replication timing on autosomes. Most occur in large regions, but only half coincide with local differences in transcription. In contrast to confined autosomal differences, a global shift in replication timing occurs throughout the single male X chromosome. Unlike in females, the dosage-compensated X chromosome replicates almost exclusively early. This difference occurs at sites that are not transcriptionally hyperactivated, but show increased acetylation of Lys 16 of histone H4 (H4K16ac). This suggests a transcription-independent, yet chromosome-wide process related to chromatin. Importantly, H4K16ac is also enriched at initiation zones as well as early replicating regions on autosomes during S phase. Together, this study reveals novel organizational principles of DNA replication of the Drosophila genome and suggests that H4K16ac is more closely correlated with replication timing than is transcription (Schwaiger, 2009).

The high resolution of these replication timing profiles allowed zones of replication initiation to be identified throughout S phase, that were confirmed in combination with measuring small nascent strand abundance. This revealed that sites of early initiation are rather distinct, which manifests in the timing profile as single peaks or a few peaks clustered together in early-S phase, followed by long stretches of the replication timing profile without changes in slope. Late initiation zones often reside in close proximity to other late initiation zones. The feature of distinct peaks of early initiation in Drosophila is very distinct from mammalian genomes (Hiratani, 2008), where many more sites of initiation of similar timing are clustered together, resulting in large regions up to several megabase pairs of early replication timing (Schwaiger, 2009).

Interestingly, the frequency of initiation appears discontinuous with high rates in early-S, a reduced frequency in mid-S, and again increased appearance of initiation sites in late-S phase. The high frequency and proximity of late-firing initiation zones suggest that late regions are replicated by many proximal late-firing origins of replication. This finding is particularly interesting in light of a recent report that suggested the absence of a checkpoint to control for the completion of DNA replication before mitosis (Torres-Rosell, 2007). This would in turn require a mechanism that mediates rapid replication of unreplicated regions in late-S phase, which could be achieved by a promiscuous activation of many proximal origins. Interestingly, replicative stress that reduces replication fork progression leads to a decrease in inter-origin distance through activation of normally dormant origins. It is conceivable that a similar situation is encountered in late replicating regions (Schwaiger, 2009).

Since the previously reported correlation between replication timing and transcription in Drosophila was not absolute, the percentage of the genome that replicates in a tissue-specific fashion remained to be tested quantitatively. For example, the general correlation could be driven by housekeeping genes that are active in most cells, resulting in a uniform replication timing program. This study showed that dynamic replication timing differs significantly between two Drosophila cell types, affecting at least 20% of autosomal DNA. It was also shown by two different methodologies that this plasticity of DNA replication coincides with transcription differences in only half of all cases (Schwaiger, 2009).

Early replication was shown previously to correlate with transcription levels over 180 kb, leading to the suggestion that replication timing integrates transcription over large regions. Consistent with this model, it was found that dynamic replication timing often occurs in large (~100 kb) regions encompassing many genes. Interestingly, genes with related function often cluster together in the Drosophila genome, and such clusters tend to be similarly 100 kb in size. In mammalian genomes, this clustering appears functionally related to chromatin structure, suggesting that widespread open chromatin at developmentally regulated multigene loci could lead to early replication or vice versa. This, in turn, might increase the potential of gene expression over large regions as in the case of genes important for wing disc development in Cl8 cells, where early replication could render the locus poised for activation (Schwaiger, 2009).

Localized differences in gene expression of a fraction of genes in a large region might also account for replication timing differences. Indeed, some, but not all, genes in differentially replicating regions are strongly differentially expressed between the two cell types. Thus, while gene expression could account for much of the observed changes on autosomes, a considerable fraction does not display transcriptional changes. It seems unlikely that the analysis missed such changes since noncoding transcription was measured as well as RNA polymerase abundance (Schwaiger, 2009).

The relation between replication timing and chromatin structure has been controversial. Transcription itself involves an opening of chromatin structure, and thus early replication could in many situations be downstream from transcriptional activation. However, previous work using injected plasmids suggested a role for early replication in mediating increased levels of histone acetylation. This led to a model in which replication timing mediates an open chromatin structure required for transcription. This suggestion is compatible with the genome-wide analysis, where a preferential location of H4K16ac was observed not only to active genes, but also to early replicating regions that are not transcribed. It is possible that early replication and elevated H4K16ac at inactive genes will result in a more open chromatin confirmation compared with late replicating inactive genes. This might render them more responsive to downstream activating cues, and thus replication timing could modulate the sensitivity to activators. This process could also function in maintenance of an active state through cell division. Importantly, however, this mechanism does not override the parallel process of transcription-coupled acetylation, as late replicating genes that are actively transcribed are still hyperacetylated (Schwaiger, 2009).

Interestingly, a strong abundance of H4K16ac was observed at sites of initiation during S phase. Several single-gene studies have suggested a positive function of histone acteylation for origin activity. Other reports, however, did not support this model. Recent maps of human replication initiation suggest that early origins are marked by H3K9/K14 acetylation (Lucas, 2007). However, no genome-wide correlation between active chromatin marks and early origin firing was observed in S. cerevisiae, where specific sequences function as origins of replication. This study identified a preferential localization of H4K16ac to initiation zones throughout the Drosophila genome compatible with a function of acetylation. In this study, focus was placed on acetylation of H4K16 because this residue has been functionally linked to higher-order chromatin compaction and chromatin opening on the dosage-compensated X in Drosophila (Schwaiger, 2009).

It has been proposed that origins of replication lie frequently between promoters of active genes, which would make transcription and replication fork progression co-oriented. Furthermore, transcription and replication are thought to be coordinated in the nucleus to be spatially and temporally separated. It thus seems plausible that the enrichment of H4K16ac in initiation zones reflects location between highly acetylated, active promoters. According to this model, proximity to active promoters would result in an open chromatin confirmation through increased H4K16ac, which in turn enhances origin firing (Schwaiger, 2009).

Importantly, however, enrichment for H4K16ac was observed at initiation zones that are not proximal to active genes, arguing against a simple process that is solely transcription-coupled. Open chromatin structure, reflected and potentially even mediated by H4K16ac, could make DNA more accessible for efficient initiation of DNA replication and thus provide a sequence-independent component that could contribute to origin localization and activity. While these are testable models, they do require a fine-mapping of actual origins at a resolution higher than the current detection of zones of initiation at the level of several kilobases (Schwaiger, 2009).

This analysis reveals the almost complete absence of late replication on the single X chromosome in male Drosophila cells. About 90% of female late replicating regions on the X replicate early in males, while autosomes show no advanced replication. Such chromosome-wide advance in replication timing has not been observed previously. In mammals, transcriptional inactivation of one of the female X chromosomes correlates with its late replication, reflecting the efficient silencing of this chromosome and increased chromatin compaction. In contrast, dosage compensation in flies involves the twofold up-regulation of genes already active in females and an open chromatin state mediated by H4K16ac. Interestingly, this study showed that advanced replication of the dosage-compensated X occurs mostly outside of transcriptionally activated regions and thus is unlikely to be accounted for by transcriptional changes. Importantly, the local increases in H4K16ac, which are detected throughout the male X chromosome, can be directly related to this loss of late replication. Reduction of the responsible Histone-Acetyltransferase Mof leads to a block in cell division, making it difficult to test this model. Notably, slightly delayed replication of the X chromosome was detected in the few cells that were in S phase in the knockdown population. While this is compatible with a model that Mof-mediated H4K16 acetylation advances replication of intergenic regions on the male X chromosome, the predominant effect on the cell cycle precluded further analysis (Schwaiger, 2009).

This suggests a transcription-independent, chromatin-dependent process, which leads to early replication chromosome-wide. While this likely reflects a different chromatin compaction, it is tempting to speculate that it also reflects a particular nuclear organization as the dosage-compensated X chromosome has been shown to associate directly with nuclear pores (Schwaiger, 2009).

Together these findings provide new principles of the replication timing program of the Drosophila genome and its dynamics relative to histone acetylation and transcription. The data further support a model in which open chromatin structure is a general feature of early replication timing and could potentially even advance replication of entire chromosomes (Schwaiger, 2009).

Acetylation and phosphorylation at heat shock gene promoters

The regulatory elements of the hsp26 promoter are well-known from in vivo and in vitro studies. A proximal regulatory element includes the TATA box, proximal heat shock element (HSE) and an adjacent GAGA element, while a distal regulatory site corresponds to the distal HSE and GAGA binding sites. Monitoring the ability of the transcription machinery to associate with the promoter in chromatin, it was found that Heat shock factor, a crucial regulator of heat shock gene transcription, profits most from histone acetylation. Templates with mutated hsp26 promoters were assembled into control and acetylated chromatin and analysed for their transcription potential. A template bearing only the TATA box supports a very low level of transcription, even in the absence of chromatin; there is no discernible transcription from chromatinized templates even after prolonged exposure. Addition of the proximal HSEs however, results in significantly increased transcription from both mock assembled and chromatinized templates, confirming the important role of the activator HSF. This minimal promoter, containing only proximal HSEs and the TATA box, clearly shows increased transcription from the acetylated template. The addition of GAGA elements to the promoter enhances transcription significantly, but to a similar degree on control and acetylated chromatin templates. Interestingly, the GAGA elements do not increase transcription in the mock assembled control, confirming that GAGA factor is involved in overcoming chromatin-mediated transcriptional repression, although the mechanism employed does not profit from histone acetylation. These results suggest that the HSE and TATA box are the significant sequence elements for the increased transcription observed in acetylated chromatin. Thus histone acetylation can modulate activator access to their target sites in chromatin, and provide a causal link between histone acetylation and enhanced transcription initiation of RNA pol II in chromatin (Nightingale, 1998).

Phosphorylation of histone H3 correlates with transcriptionally active loci

Posttranslational modifications of the N-terminal tails of the core histones within the nucleosome particle are thought to act as signals from the chromatin to the cell for various processes. The experiments presented here show that the acetylation of histones H3 and H4 in polytene chromosomes does not change during heat shock. In contrast, the global level of phosphorylated H3 decreases dramatically during a heat shock, with an observed increase in H3 phosphorylation at the heat shock loci. Additional experiments confirm that this change in phosphorylated H3 distribution is dependent on functional heat shock transcription factor activity. These experiments suggest that H3 phosphorylation has an important role in the induction of transcription during the heat shock response (Nowak, 2000).

The acetylation of the N-terminal tails is the best-studied modification of the core histones. Several transcription factors, such as GCN5, and the TAFII250 subunit of TFIID, as well as subunits of the RNA polymerase complex show intrinsic histone acetyltransferase (HAT) activity, which suggests a potential role for histone acetylation in either the activation or maintenance of transcription. The acetylation of the N-terminal tail domains of core histones H3 and H4 at various lysine residues is essential for the normal implementation of various cellular processes, such as promoter-transcription factor association, gene transcription, and dosage compensation (Nowak, 2000 and references therein).

Phosphorylation of serine 10 of the N-terminal arm of histone H3 has been shown to be essential for proper mitotic chromosomal condensation and segregation. In addition, recent studies have outlined the possibility that histone H3 phosphorylation may have a role in the regulation of transcription. Ser 10 H3 phosphorylation is found to rapidly increase in quiescent cells during mitogenic stimulation, as well as during immediate-early gene induction via the epidermal growth factor (EGF)-signaling pathway. In addition, recent experiments performed in vitro have suggested that EGF-stimulated H3 phosphorylation may act as a signal for histone acetyltransferase binding and subsequent acetylation of a particular locus during transcription initiation (Nowak, 2000 and references therein).

Acetylation of core histones H3 and H4 at lysines 14 and 8, respectively, has been linked to gene transcription. In addition, deacetylation of core histones is thought to have a role in silencing specific loci. Because of the near-total repression of cellular gene products during a heat shock, it might be expected that the distribution of acetylated H3 and H4 would radically change during thermal stress in a manner reflective of the transcriptional profile of the cell. Because acetylation of H3 at Lys 14 of the N-terminal arm has been described as essential for transcription, the distribution of acetylated H3 was examined by staining polytene chromosomes with an antibody specific for Lys 14 acetylated histone H3. Lys 14 acetylated H3 staining is observed at the puffs, which are active sites of transcription in polytene chromosomes, and distributed throughout the chromosomes in discrete bands before heat shock. One locus, subdivision 62A, which becomes puffed during larval development in response to ecdysone, is intensely labeled with the Lys 14 acetylated H3 antibody. In addition, other chromosomal subdivisions such as 89B display Lys 14 acetylated H3 staining but are not puffed before heat shock. The chromosomal subdivision 93D, which is known to become puffed during heat shock, is Lys 14 acetylated but not puffed before heat shock. Examination of polytene chromosomes from larvae that were subjected to a 20-min heat shock shows that the 87A and 87C heat shock puffs, which contain the hsp70gene cluster, are stained by the anti-Lys 14 acetylated antibody, although the staining at these puffs appears to be less intense and rather diffuse. This might not represent a reduction in the level of acetylation, but rather a decrease in signal intensity due to the large puffing at the heat shock loci. After heat shock, the overall number of discrete stained bands does not appear to change significantly and regions that were stained before heat shock, such as 89B, remain acetylated. Loci with acetylated H3 staining that were puffed before heat shock, such as 62A, are no longer puffed after heat shock but remain acetylated. The observation that the heat shock genes are acetylated before heat shock, at a time when they are not transcribed, and non-heat shock genes, which are not transcribed during heat shock, are acetylated during heat shock, suggests that the presence of Lys 14-acetylated H3 does not necessarily denote an actively transcribed locus (Nowak, 2000).

Examination of H3 acetylation during EGF stimulation raises the issue that antibodies against Lys 14 acetylated H3 may show decreased recognition of their epitope when other modifications, such as phosphorylation, coexist on the same histone tail. This problem can be overcome by using antibodies against histone H3 acetylated at lysines 9 and 14. To ensure that these results were not caused by this potential artifact, the distribution of hyperacetylated H3 was examined using antibodies against H3 acetylated at lysines 9 and 14 on the N-terminal tail before and after heat shock. The results suggest that the distribution of diacetylated H3 is similar to the distribution of Lys 14 acetylated H3 before and after heat shock. Diacetylated H3 staining appears to be more widespread than monoacetylated staining, which is probably caused by the antibody's recognition of acetylation of H3 at lysine 9. The intensity of staining of the Lys 9,14-acetylated H3 antibody at several of the heat shock puffs examined appears to be similar to that observed with the Lys 14-acetylated H3 antibody. These results suggest that the diffuse staining at the heat shock puffs is not an artifact attributed to the masking of the acetylated Lys 14 epitope by Ser 10 phosphorylation (Nowak, 2000).

H4 acetylation was also examined using antibodies specific for Lys 8-acetylated histone H4 to stain polytene chromosomes isolated from third instar larvae. The distribution of Lys 8 acetylated histone H4 is similar to that of acetylated H3, with H4 acetylation observed in discrete bands in nonpuffed regions, such as subdivision 89B, and at ecdysone-induced puffed regions, such as 62A, before heat shock. Chromosomal subdivisions 87A and 87C, which contain the hsp70 heat shock genes, are acetylated before and after heat shock. Similar to acetylated H3, heat shock does not significantly affect the observed distribution of Lys 8 acetylated H4 in polytene chromosomes. Taken together, the above results suggest that the acetylation state of H3 and H4 does not change substantially during heat shock and that a gene locus can be acetylated when it is not actively transcribed (Nowak, 2000).

The absence of a drastic change in H3 acetylation during heat shock is rather surprising, given current models that indicate that H3 acetylation is a crucial step in transcription initiation. This would lead to the expectation that the heat shock loci would not be acetylated before heat shock and should become intensely acetylated during thermal stress. To determine if other histone modifications occur during the heat shock response, whether changes in histone H3 phosphorylation occur after temperature elevation was examined. Stimulation of quiescent cells with EGF leads to rapid and transient phosphorylation of histone H3 at Ser 10 of the N-terminal arm in vivo. This EGF-mediated phosphorylation of H3 is targeted to a small subpopulation of total histone H3 that is acetylated at the Lys 14 position. In addition, in vitro studies have shown that phosphorylated H3 may serve as an affinity-increasing substrate for HAT activity in H3 acetylation, which raises the possibility that phosphorylation may be tied to transcription. If histone phosphorylation were implicated in transcription, then the distribution of phosphorylated H3 might change in response to heat shock and would most likely be localized primarily to the heat shock puffs while disappearing from other loci after heat shock. Because histone H3 phosphorylation is a robust marker for mitotic cells, analysis of the distribution of phosphorylated H3 in polytene chromosomes, rather than isolation of phosphorylated H3 from whole cell extracts, allows for the examining of phosphorylation of H3 in a nonmitotic environment. To examine whether the heat shock-induced puffs contain N-terminal phosphorylated H3 molecules, polytene chromosomes were stained with antibodies specific for Ser 10 phosphorylated histone H3. Before heat shock, phosphorylated H3 staining is found in discrete bands throughout the chromosomes, with the most intense staining observed in the naturally occurring ecdysone-induced developmental puffs. After a 20-min heat shock at 37°C, phosphorylated H3 staining is not distributed throughout the chromosomes but is instead concentrated at a few specific sites. The most prominent of these regions corresponds to chromosomal divisions 63BC, 67B, and 87AC. These regions contain the hsp83 gene, the hsp22, hsp23, hsp26, and hsp27 gene cluster, and hsp70 gene clusters, respectively. These regions become reproducibly puffed during the heat shock response. Although in some chromosomes examined there are several non-heat shock loci that remain slightly phosphorylated during heat shock, the intensity of staining at these regions is much lower than the staining observed at the heat shock loci (Nowak, 2000).

The regions of the chromosome where the heat shock genes are located do not contain histone H3 phosphorylated at Ser 10 before heat shock. After temperature elevation, the only puffs that possess phosphorylated histone H3 are the heat shock puffs. The appearance of phosphorylated histone H3 in the heat shock puffs, accompanied by the nearly complete reduction of staining at all other loci during heat shock, leads to the conclusion that the presence of the Ser 10 phosphorylated isoform of histone H3 might be required for the transcriptional activation of the heat shock genes (Nowak, 2000).

Induction of the heat shock genes and cessation of normal gene expression is rapid and reproducible in response to heat shock. Transcription run-on assays reveal that after only 1 min at 37°C, the levels of many normal cellular gene transcripts have greatly diminished, with the heat shock gene transcripts dominating the population of total mRNA in the cell. Following a heat shock, the normal pattern of gene expression within the cell is restored gradually over time. Therefore an examination was made of the change in phosphorylated histone H3 staining over time during and after heat shock, to determine whether or not the appearance of phosphorylated H3 closely parallels the induction of transcription of the heat shock genes and whether or not the non-heat shocked H3 distribution might be restored following recovery from heat shock. After only 1 min at 37°C, there is a noticeable change in the distribution of Ser 10 phosphorylated H3. The level of global H3 phosphorylation decreases, with several regions remaining intensely phosphorylated. Within 5 min of incubation at 37°C, many of the less intense regions of staining have disappeared. After 10 min at 37°C, the only remaining intense regions of staining are those at the heat shock puffs. When larvae were allowed to recover at room temperature from a 20-min heat shock at 37°C, H3 phosphorylation reappears in several non-heat shock loci after 10 min of recovery. After 30 min of recovery from heat shock, the number and distribution of loci that contained phosphorylated H3 appears to be virtually indistinguishable from normal (i.e., non-heat shocked) chromosomes. This restoration of the normal (non-heat shocked) H3 phosphorylation pattern closely mimics previously described restoration of normal gene expression in cells experiencing thermal stress (Nowak, 2000).

During heat shock, the heat shock transcription factor (HSF) rapidly trimerizes in solution, localizes to the heat shock loci, binds to heat shock response promoter elements (HSEs), and induces the expression of the heat shock gene products. The appearance of phosphorylated H3 at the heat shock loci could therefore be due to HSF recruitment of a specific histone kinase on binding to the HSEs of the heat shock genes. To test this hypothesis, the staining pattern of phosphorylated H3 was examined in polytene chromosomes isolated from hsf4-mutant larvae, which lack functional HSF at restrictive temperatures and do not respond to thermal stress. Before heat shock, the distribution of phosphorylated H3 in hsf4-mutant chromosomes is similar to wild-type chromosomes, with staining observed in discrete bands and at the developmental puffs. In contrast to wild-type chromosomes, histone H3 at the heat shock loci does not become phosphorylated in hsf4-mutant chromosomes during heat shock, which suggests that phosphorylation of histone H3 at the heat shock loci depends on functional HSF activity. In addition, no H3 phosphorylation was detected in the rest of the genome during heat shock in hsf4 mutants, suggesting that repression of normal transcription and loss of H3 phosphorylation at non-heat shock loci does not require the presence of an active HSF protein (Nowak, 2000).

To determine if the loss of the HSF transcription factor could also alter the distribution of acetylated H3 and H4 during heat shock, acetylation of each of these histones was examined in hsf4-mutant polytene chromosomes. The distribution of Lys 14 acetylated histone H3 before and after heat shock in hsf4 mutants was indistinguishable from the wild-type distribution, with staining observed at both the developmental puffs and nonpuffed regions. H3 acetylation was observed at the 87A and 87C chromosomal subdivisions, which normally are puffed during heat shock but these regions do not become puffed in hsf4-mutant chromosomes. Examination of acetylated H3 using antibodies for Lys 9- and Lys 14-acetylated H3 shows a pattern similar to that observed for the Lys 14 acetylated H3 antibody. In addition, H4 acetylation does not change after heat shock in hsf4 mutants. Because the heat shock genes are not induced in hsf4 mutants during thermal stress and because hsf4-mutant chromosomes are acetylated, but not phosphorylated after heat shock, it is concluded that H3 phosphorylation, and not acetylation, depends on the presence of a functional heat shock transcription factor (Nowak, 2000).

How might acetylation and phosphorylation of histones H3 and H4 work together to promote transcription of a particular gene? The data suggest that acetylated histones might define a particular locus that is primed for possible phosphorylation and subsequent transcription. This acetylated locus would attract transcription factors that interact with the acetylated residues on histones H3 and H4, known to be essential for proper association of several transcription factors with their promoters. Once bound to this locus, the transcription factor would then recruit a particular histone, which phosphorylates Ser 10 of the N-terminal arm of histone H3. The most logical site of phosphorylation would be an H3 molecule with a Lys 14 acetylated N-terminal arm, a species that has been shown to exist in vivo. The presence of this dimodified H3 would define that locus as 'active' for transcription (Nowak, 2000).

There are several kinases known to localize to specific loci on polytene chromosomes that phosphorylate H3 in vitro, such as JIL-1 on the X chromosome and P-TEFb kinase at the heat shock loci (Lis, 2000). This raises the possibility that the specificity of a kinase for activation of a particular gene through H3 phosphorylation might be regulated by the specific transcription factors that control expression of this gene. It has yet to be determined whether phosphorylation of H3 is required for assembly of the RNA polymerase II complex or if phosphorylation is a by-product of complex formation and polymerase procession during transcription. If phosphorylation of H3 were indeed the critical step in activating gene transcription, then a reasonable hypothesis is that deactivation of a particular gene would be dependent on either regulated or unregulated phosphatase activity to remove the activating phosphate group from the N-terminal tails of H3. The disappearance of phosphorylated H3 at nontranscribing loci and appearance of phosphorylated H3 at actively transcribing loci during heat shock suggests that a functional transcription complex might actively maintain the phosphorylated state of histone H3, which would be subject to ready dephosphorylation by either passive or regulated phosphatase activity in a nontranscribing state (Nowak, 2000).

The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin; H2Av variant replacement is followed by H4 Lys 12 acetylation as necessary steps before H3 Lys 9 methylation and HP1 recruitment

Activation and repression of transcription in eukaryotes involve changes in the chromatin fiber that can be accomplished by covalent modification of the histone tails or the replacement of the canonical histones with other variants. The histone H2A variant of Drosophila melanogaster, Histone H2A variant (H2Av), localizes to the centromeric heterochromatin, and it is recruited to an ectopic heterochromatin site formed by a transgene array. His2Av behaves genetically as a PcG gene and mutations in His2Av suppress position effect variegation (PEV), suggesting that this histone variant is required for euchromatic silencing and heterochromatin formation. His2Av mutants show reduced acetylation of histone H4 at Lys 12, decreased methylation of histone H3 at Lys 9, and a reduction in HP1 recruitment to the centromeric region. Neither H2Av accumulation nor histone H4 Lys 12 acetylation is affected by mutations in either Su(var)3-9 or Su(var)2-5. The results suggest an ordered cascade of events leading to the establishment of heterochromatin, requiring the recruitment of the histone H2Av variant followed by H4 Lys 12 acetylation as necessary steps before H3 Lys 9 methylation and HP1 recruitment can take place (Swaminathan, 2005).

Recent results suggest that H3 trimethylated at Lys 27 facilitates Pc binding to silenced regions and this modification is carried out by the Enhacer of zeste [E(z)] protein present in the ESC-E(z) complex. Since a reduction in Pc on polytene chromosomes was observed in His2Av mutants, whether recruitment of the ESC-E(z) complex is also impaired in these mutants was examined. In wild type, E(z) can be observed at multiple sites throughout the genome. The levels and localization of E(z) do not appear to be altered in the His2Av810 mutant compared to wild type. Whether H3 Lys 27 methylation is affected by mutations in His2Av was examined. The levels and distribution of this modification appear to be the same in polytene chromosomes from wild-type and His2Av810 mutant larvae. This result was confirmed by Western analysis, which shows equal levels of H3 trimethylated at Lys 27 in wild-type and His2Av810 mutant larvae. These results suggest that H2Av is required upstream of Pc recruitment in the process of Pc-mediated silencing. Since neither recruitment of the E(z) complex nor H3 Lys 27 methylation seem to be affected in His2Av mutants, H2Av replacement might take place after H3 Lys 27 methylation and before Pc recruitment. Alternatively, Pc repression might require at least two parallel and independent pathways, one involving H2Av recruitment and a second one leading to H3 Lys 27 methylation, both of which might be required for proper Pc recruitment (Swaminathan, 2005).

Formation of heterochromatin requires deacetylation of H3 Lys 9 followed by methylation of the same residue and recruitment of HP1. The heterochromatin of Drosophila chromosomes is enriched in dimethylated and trimethylated histone H3 in the Lys 9 residue. To analyze the possible role of H2Av in heterochromatin assembly, the localization was examined of H3 dimethylated at Lys 9 in polytene chromosomes from larvae carrying a mutation in the His2Av gene. Antibodies against histone H3 dimethylated in Lys 9 stain the pericentric heterochromatin in wild-type larvae. Interestingly, polytene chromosomes from His2Av810 mutants show a decrease in the amount of methylated H3 Lys 9, whereas the presence of Su(Hw), used as a control, is the same in chromosomes from wild-type and His2Av810 mutant larvae. Since modification of this residue is important for HP1 recruitment, whether localization of HP1 in heterochromatin is also affected by mutations in His2Av was examined. In wild-type larvae, HP1 localizes preferentially to the pericentric heterochromatin of the chromocenter, but accumulation of HP1 is dramatically reduced in the His2Av810 mutant (Swaminathan, 2005).

To confirm these results, Western analyses of protein extracts obtained from wild-type and His2Av mutant larvae was carried out using antibodies against HP1 and histone H3 dimethylated in Lys 9. The results show little or no accumulation of histone H3 methylated in Lys 9, and lower levels of HP1 in the His2Av810 mutant. Methylation of histone H3 at the Lys 9 residue is carried out by the Su(var)3-9 histone methyltransferase, and HP1 is encoded by the Su(var)2-5 gene. In order to ensure that the observed effects on the levels of HP1 or the methylation of H3 Lys 9 were not caused by alterations in transcription of Su(var)3-9 or Su(var)2-5 due to the His2Av mutation, quantitative RT-PCR analyses of RNA obtained from wild-type and His2Av810 mutant third instar larvae were carried out . The results show that there are no significant changes in the levels of Su(var)3-9 or HP1 RNAs in His2Av810 mutant larvae when compared to wild type. These results and those from immunocytochemistry analyses confirm a role for H2Av in the methylation of H3 Lys 9 and subsequent recruitment of HP1 (Swaminathan, 2005).

Based on the observed effects of His2Av mutations on H3 Lys 9 methylation and HP1 recruitment, it appears that the presence of H2Av in heterochromatin might be required prior to these two events. To confirm this hypothesis, the pattern of H2Av distribution on polytene chromosomes from larvae carrying mutations was examined in the Su(var)2-5 and Su(var)3-9 genes. In both cases, H2Av localization appears normal, suggesting that the presence of H2Av is required prior to H3 Lys 9 methylation and HP1 recruitment during the establishment of heterochromatin (Swaminathan, 2005).

Phosphorylation of histone H4 Ser1 regulates sporulation in yeast and is conserved in fly and mouse spermatogenesis

Sporulation in Saccharomyces cerevisiae is a highly regulated process wherein a diploid cell gives rise to four haploid gametes. This shows that histone H4 Ser1 is phosphorylated (H4 S1ph) during sporulation, starting from mid-sporulation and persisting to germination, and is temporally distinct from earlier meiosis-linked H3 S10ph involved in chromosome condensation. A histone H4 S1A substitution mutant forms aberrant spores and has reduced sporulation efficiency. Deletion of sporulation-specific yeast Sps1, a member of the Ste20 family of kinases, nearly abolishes the sporulation-associated H4 S1ph modification. H4 S1ph may promote chromatin compaction, since deletion of SPS1 increases accessibility to antibody immunoprecipitation; furthermore, either deletion of Sps1 or an H4 S1A substitution results in increased DNA volume in nuclei within spores. H4 S1ph is present during Drosophila melanogaster and mouse spermatogenesis, and similar to yeast, this modification extends late into sperm differentiation relative to H3 S10ph. Thus, H4 S1ph may be an evolutionarily ancient histone modification to mark the genome for gamete-associated packaging (Krishnamoorthy, 2006).

Whether H4 S1ph and H3 S10ph also occur during male meiosis and spermatogenesis in was investigated Drosophila. In spermatocytes undergoing the first meiotic division, metaphase I chromosomes stained strongly against both H4 S1ph and H3 S10ph antibodies. The H4 S1ph signal was detected on meiotic chromosomes from prophase through telophase for both meiosis I and meiosis II cells. In contrast, H3 S10ph staining was only prominent in metaphase and decreased substantially in anaphase and telophase. The H3 S10ph signal was also not detected in prophase spermatocytes. Immunofluorescence staining of Drosophila male germ cells undergoing spermatid differentiation revealed certain parallels between differentiation of yeast spores and male gametes in the behavior of H4 S1ph and H3 S10ph. During Drosophila spermatogenesis, as in yeast sporulation, H4 S1ph persisted until late in the terminal differentiation stages, while H3 S10ph levels were strongly reduced by the time meiosis was completed. Thus, in round and early elongating haploid spermatids, nuclei stained brightly for H4 S1ph, while staining for H3 S10ph was nearly undetectable in the same cells. The H4 S1ph epitopes persisted in spermatid nuclei as cells grew flagella and elongated and were still detected in nuclei undergoing chromatin compaction and nuclear shaping. Staining with H4 S1ph antibody gradually diminished at the later stages of nuclear elongation and shaping and was not detected in mature spermatids awaiting individualization. Staining for H3 S10ph was not detected in elongating spermatid nuclei at any stage (Krishnamoorthy, 2006).

Thus, similarities in the processes of sporulation and spermatogenesis, particularly in the drastic reduction in nuclear volume in both processes, led to an examination of whether H4 S1ph might correlate with the timing of chromatin compaction during spermatogenesis. H4 S1ph extending well beyond the time that meiosis-associated H3 S10ph is reduced during Drosophila and mouse spermatogenesis. In these metazoans, H3 S10ph and H4 S1ph both occur during meiotic divisions and thus may play a role in chromosome condensation. However, following meiotic divisions, the H3 S10ph is dramatically lowered, while H4 S1ph persists during the early stages of the developing spermatids when the genome begins to be compacted. This is true in mouse, as H4 S1ph continues to be present beyond the meiotic divisions and begins to be reduced contemporaneously with replacement of histones by the highly basic transition proteins. Although it is not yet yet known whether H4 S1ph has a role in genome compaction in metazoans, the data indicate that H4 S1ph has an additional role beyond meiotic divisions, as is the case in yeast. One clear difference between H4 S1ph in lower eukaryotes compared with metazoans is the persistence of the mark in mature spores and elimination only after germination. Thus, while H4 S1ph may directly promote stable chromatin compaction in mature spores, its role in metazoans may help to compact the genome connected to histone replacement by basic transition proteins. While many histone modifications have been correlated with broad genomic mechanisms such as transcription and DNA repair, the role of only a few modifications has been elucidated in higher-level biological processes. In this case, a central biological process, gametogenesis, is critically controlled in yeast by a single histone modification. The similarities observed between yeast and metazoans in the persistence of H4 S1ph after the decline of H3 S10ph emphasizes its importance (Krishnamoorthy, 2006).

Methylation of Histone H4

Drosophila Pr-Set7 function was characterized based on the the availability of a P-element disruption of the 5' UTR region of the corresponding gene. A mutation in Drosophila pr-set7 is lethal: second instar larval death coincides with the loss of Histone H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincides with condensed chromosomal regions on polytene chromosomes, including chromocenter and euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail (Nishioka, 2002).

A mammalian histone methyltransferase (HMT) has been identified that is specific for lysine 20 of histone H4. This enzyme, PR-Set7, resides as a single polypeptide and is highly specific for nucleosomal histones. It was also shown that methylation of H4-K20 is associated with silent, transcriptionally inactive regions within euchromatin. Methylation of histone H4-K20 may maintain this higher order chromatin structure by inhibiting the acetylation of histone H4-K16. Taken together, these studies help to shed light on mechanisms that regulate chromatin structure through a series of concerted enzymatic reactions that ultimately 'mark' functionally distinct chromatin domains (Nishioka, 2002).

To identify and analyze HMTs present in human cells that specifically methylate histone H4, nuclear extracts from HeLa cells were fractionated on several chromatographic resins. Fractions from the columns were assayed for HMT activity using as substrates either core histone polypeptide or mono- and oligo-nucleosomes, in the presence and absence of histone H1. The separation of proteins in the DEAE-cellulose flowthrough (unbound) fraction on a negatively charged column (phosphocellulose) resulted in the resolution of two HMT activities, each with a different substrate and histone specificity. The histone H3-specific activity was eluted from the column at a lower salt concentration and was able to methylate core histone polypeptides as well as oligonucleosomes. This activity was specific for the K9 residue of H3 and was identified as Suv39h1. The other major HMT activity was eluted from the phosphocellulose column at a higher salt concentration and exclusively methylated nucleosomal histone H4. Further separation of the H4-specific HMT on a gel filtration column demonstrated that the activity had an apparent native mass of approximately 70 kDa. The final step of the purification scheme, fractionation on a Heparin agarose column, showed that the H4 HMT activity correlated with the appearance of a single polypeptide of approximately 40 kDa. It was later found by gel-filtration analysis that the enzymatically active 40 kDa protein resides as a homodimeric complex (Nishioka, 2002).

The purified native enzyme was subjected to further analysis, in order to more clearly define its substrate specificity. Assays were conducted with known substrates for several previously characterized protein methyltransferases, and it was found that the newly purified enzyme was highly specific for nucleosomal histone H4. A reaction mixture that contained nucleosomal histone H4, 3H-labeled S-adenosyl methionine (SAM), and the purified enzyme was then subjected to Edman degradation, and this analysis demonstrated that the target site for methylation is lysine 20. Moreover, when an HMT assay was carried out using nucleosomes reconstituted with an H4 species that contained an alanine in place of a lysine at position 20 (K20A), the newly purified HMT was unable to methylate the substrate, demonstrating further that this enzyme is specific for H4-K20 (Nishioka, 2002).

Mass spectrometric analysis of peptides derived from the protein that coeluted with the nucleosomal H4-specific HMT activity allowed probes to be generated with which to isolate a full-length cDNA clone. cDNA sequence analysis revealed that the activity was encoded by a gene that is absent in lower eukaryotes but is present in worms, flies, and vertebrates. The cDNA sequence matched perfectly with a sequence deposited in GenBank referred to as PR/SET domain containing protein 07 (accession number AAL40879). For simplicity, the enzyme was termed PR-Set7 (Nishioka, 2002).

Because a PR-Set7 homolog is present in Drosophila as a gene product of CG3307 (see Figure S1A), and because methylation of H4-K20 can be detected in the fruit fly, Drosophila was chosen as a model system to analyze the biological significance of this modification. The catalytic SET domain of Drosophila pr-set7 is about 40% identical in amino acid composition to that of human PR-Set7 (Nishioka, 2002).

Drosophila and mammalian PR-Set7 specifically methylate lysine 20 of histone H4 exclusively within a nucleosomal context. Although histone proteins have long been recognized to be methylated at specific residues in vivo, the enzymes that catalyze the modification reaction and the functions of these modifications have only recently begun to be revealed. Prior to this study, the function(s) of lysine methylated histone H4 was obscure, but was largely believed to be associated with transcriptionally active rather than repressed genes. However, this study has established that methylated H4-K20 is associated with silent chromatin. In support of the 'histone code hypothesis' methylation at H4-K20 inhibits acetylation of H4-K16 and vice versa. Consistent with the notion that an enzyme that alters the establishment of silent chromatin should have a tremendous impact on gene expression, these studies establish that the absence of methyl H4-K20 in vivo impairs the development and viability of a multicellular organism. Based upon the available evidence, the view is favored that a lack of, or diminishment of, H4-K20 methylation may alter patterns of gene expression, by perturbing a generally repressive, higher order chromatin structure that critically depends upon H4-K20 methylation (Nishioka, 2002).

The enzymatic activity of PR-Set7 is contained within a single polypeptide of ~40 kDa that appears to exist as a homodimer, because the native protein elutes from a gel filtration column with an apparent mass of ~70 kDa. The results demonstrate that PR-Set7 is an H4-K20-specific HMT, since the enzyme does not methylate any other residue on histone H4 or on any other histone. In agreement with previous studies demonstrating that the SET domain can be a signature for lysine-HMTs, PR-Set7 contains a SET domain, and a single substitution of a conserved arginine to glycine within the SET domain abolishes its enzymatic activity. Interestingly, PR-Set7 is devoid of the Pre- and Post-SET domains, demonstrating that these domains, although important for the functions of other HMTs, are not absolutely required for HMT activity. PR-Set7 is highly specific for nucleosomes, since no activity could be demonstrated when histones were used as a substrate. This lack of activity on nonnucleosomal histones is not likely to be due to the absence of the Pre- and Post-SET domains, because an HMT has been isolated with specificity for H3-K4 that exclusively methylates free histones and lacks both of these domains (Nishioka, 2002).

The histone H4 lysine 20 monomethyl mark, set by PR-Set7 and stabilized by L(3)mbt, is necessary for proper interphase chromatin organization

Drosophila PR-Set7 or SET8 is a histone methyltransferase that specifically monomethylates histone H4 lysine 20 (H4K20). L(3)MBT has been identified as a reader of methylated H4K20. It contains several conserved domains including three MBT repeats binding mono- and dimethylated H4K20 peptides. Depletion of PR-Set7 was found to block de novo H4K20me1 resulting in the immediate activation of the DNA damage checkpoint, an increase in the size of interphase nuclei, and drastic reduction of cell viability. L(3)mbt, in contrast, stabilizes the monomethyl mark, as L(3)mbt-depleted S2 cells show a reduction of more than 60% of bulk monomethylated H4K20 (H4K20me1) while viability is barely affected. Ploidy and basic chromatin structure show only small changes in PR-Set7-depleted cells, but higher order interphase chromatin organization is significantly affected presumably resulting in the activation of the DNA damage checkpoint. In the absence of any other known functions of PR-Set7, the setting of the de novo monomethyl mark appears essential for cell viability in the presence or absence of the DNA damage checkpoint, but once newly assembled chromatin is established the monomethyl mark, protected by L(3)mbt, is dispensable (Sakaguchi, 2012).

In these studies it was established that PR-Set7 sets the H4K20 monomethyl mark in vivo and that at least in S2 cells K20 is the only amino acid that is methylated. It was further found that depleting PR-Set7 in Drosophila S2 cells leads to the activation of the DNA damage checkpoint and within about 10 days to cell death. When the DNA damage checkpoint is abrogated by double knock-down of PR-Set7 and the checkpoint genes mei-41 or grp, the half/life of the cells is increased by 1 to two days, but ultimately the cells still die, suggesting that whatever is perturbed in the absence of PR-Set7 cannot be repaired. No double strand breaks were observed when staining for anti-phosphorylated histone H2A. This does not agree with results observed in vertebrate cells and may be because, in Drosophila, H2Av is not phosphorylated in the absence of H4K20me1 or the specific epitope is obscured. Alternatively, double strand breaks may not exist and the checkpoint is activated because of abnormal chromatin organization or because protein complexes are not removed in a timely manner as observed in Saccharomyces cerevisiae (Sakaguchi, 2012).

In this context it is interesting to note that in vertebrates H4K20 me2 is implicated in double strand break repair. Because H4K20me1 is the likely substrate for Suv4-20H1 and H2, the di- and trimethyltransferases, an additional link between H4K20 methylation and double strand breaks seems to exist. However, besides potentially setting the monomethyl mark at double strand breaks, PR-Set7 would have to have additional functions, because in both flies and vertebrates PR-Set7 mutants have a substantially stronger phenotype than the loss of the Suv4-20 enzymes (Sakaguchi, 2012).

The increase in nuclear volume, together with the changes in the number of FISH signals per nucleus observed in interphase cells following PR-Set7 RNAi would be consistent with a role for PR-Set7 in chromosome compaction and higher-order chromatin organization. Interestingly, mass spectrometry experiments show that the H4K20 monomethyl mark is set at the G2/M transition well after newly synthesized histone H4 is incorporated into chromatin in S phase. These findings suggest that the abnormalities in chromosome compaction and organization evident in interphase nuclei might be due to defects arising during the G2/M transition. Consistent with this possibility, cells depleted for only Pr-Set7 appear to arrest mostly in early mitosis. But unlike what is observed in larval brains, there is also a subset of cells that arrest in S phase; these may represent cells that despite the abnormalities in higher order chromatin organization are able to continue through the cell cycle until a checkpoint is activated during S. The discrepancy between the brain and tissue culture cells may be a reflection of differences in their cell cycle and developmental potential (Sakaguchi, 2012).

Results from several laboratories suggest that PR-Set7 function is coupled to DNA replication based on its targeting to the dividing fork via its interaction with PCNA. The current findings indicate that while abnormalities in chromatin organization and compaction appear to accumulate after growth without Pr-Set7 activity, these defects are inconsistent with massive disruptions in de novo nucleosome assembly during replication. Instead, the DNA damage checkpoint activation must arise from more subtle abnormalities in chromatin or DNA structure (Sakaguchi, 2012).

As for the l(3)mbt, its functional requirement does not appear to overlap with that of PR-Set7, neither in tissue culture as shown in this study, nor in flies. In larvae the loss of l(2)mbt results in an expansion of the neuroblast pool and subsequent tumorous overgrowth of the optic lobe while in PR-Set7 mutants the cell cycle of neuroblasts arrests in early mitosis resulting in fewer cells. PR-Set7 is essential for de novo methylation of H4K20. While the loss of H4K20me1 could occur either because in the absence of L(3)mbt protection the H4K20me1 is lost, or it could be a secondary effect. Consistent with the latter explanation, recent results show that L(3)mbt binds to DNA boundary elements and affects the level of transcription of Salvador-Wart-Hippo pathway genes both positively and negatively. That L(3)mbt possibly controls expression of many genes is also supported by the observation that the transcription level of all genes tested was reduced compared to wild type (Sakaguchi, 2012).

Histone methylation by the Drosophila epigenetic transcriptional regulator Ash1

The establishment and maintenance of mitotic and meiotic stable (epigenetic) transcription patterns is fundamental for cell determination and function. Epigenetic regulation of transcription is mediated by epigenetic activators and repressors, and may require the establishment, 'spreading' and maintenance of epigenetic signals. Although these signals remain unclear, it has been proposed that chromatin structure and consequently post-translational modification of histones may have an important role in epigenetic gene expression. The epigenetic activator Ash1 has been shown to be a multi-catalytic histone methyl-transferase (HMTase) that methylates lysine residues 4 and 9 in H3 and 20 in H4. Transcriptional activation by Ash1 coincides with methylation of these three lysine residues at the promoter of Ash1 target genes. The methylation pattern placed by Ash1 may serve as a binding surface for a chromatin remodelling complex containing the epigenetic activator Brahma (Brm), an ATPase, and inhibits the interaction of epigenetic repressors with chromatin. Chromatin immunoprecipitation indicates that epigenetic activation of Ultrabithorax transcription in Drosophila coincides with trivalent methylation by Ash1 and recruitment of Brm. Thus, histone methylation by Ash1 may provide a specific signal for the establishment of epigenetic, active transcription patterns (Beisel, 2002).

Acetylation/de-acetylation, ubiquitination and methylation of histones (H1, H2A, H2B, H3, H4) have been correlated with the activation and silencing of transcription. Histone methylation occurs predominantly at arginine and lysine residues in the amino-terminal tails of H3 and H4. Arginine methylation mediates transcriptional activation by hormone receptors and probably other chromatin-dependent processes. By contrast, methylation of K9 and K4 in H3 and K20 in H4 has been linked to transcriptionally inactive chromatin, and corresponding HMTases have been identified. Methylation of H3 K4 has also been detected in transcription-competent chromatin, but the functional link between histone methylation and activation has not been dissected (Beisel, 2002).

To identify HMTases that establish activation-specific methylation patterns, a biochemical screen was used that identified Ash1, a member of the trithorax group of epigenetic activators as an HMTase. Ash1 contains a SET domain -- the 'signature motif' of lysine-specific HMTases -- flanked by cysteine-rich regions (pre-SET and post-SET domains). To confirm that Ash1 has HMTase activity, the ability to methylate histones was assessed in recombinant Ash1 derivatives Ash1DeltaN (deleted N terminus) and Ash1(SET) (containing the pre-SET, post-SET and SET domains only). The Ash1 derivatives methylate H3 and, to a lesser extent, H4 in polynucleosomes and histone core octamers. By contrast, 'free' H3 and H4 were methylated to a lesser extent compared with nucleosomes, even though free histones were present at a fivefold excess over polynucleosomal histones or when supplemented with DNA. These results suggest that Ash1 methylates H3 and H4. Since Ash1 used in the described HMTase assays was purified from eukaryotic cells, the HMTase activity of Ash1 could result from an associated rather than intrinsic activity. To test this, Ash1DeltaN was subjected to protein transfer membrane assays that detect intrinsic enzymatic activities in proteins. Ash1(SET) was separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred electrophoretically onto polyvinylidene fluoride (PVDF) membrane, and denatured/re-natured. Reconstituted Ash1(SET) methylates H3 and H4, suggesting that Ash1 has intrinsic HMTase activity (Beisel, 2002).

To identify the target amino acid residue(s) of Ash1, radiolabelled H3 was subjected to Edman-degradation. Scintillation counting of the released amino acid fractions detected radiolabelling of H3 K4 and K9. To support this, the ability of Ash1DeltaN to methylate peptides consisting of amino acids 1-20 of H3 [H3(1-20)] was tested. Ash1DeltaN methylates the peptides H3(1-20), H3(1-20)K4 (which contains H3 K4 but leucine residues instead of lysine residues at positions 9, 14 and 18) and H3(1-20)K9 (which contains H3 K9 but leucine residues instead of lysine residues at positions 4, 14 and 18). By contrast, H3(1-20)L4/L9 peptides, which contain leucine residues at position 4 and 9 of H3, are not significantly methylated, indicating further that Ash1 methylates H3 K4 and K9. Owing to the weak radiolabelling, the target(s) of Ash1 in H4 could not be identified by Edman-degradation. Since H4 K20 is the only H4 residue being methylated in vivo, a monoclonal antibody was generated against dimethylated H4 K20 [anti-dim(H4-K20)] to investigate whether Ash1 methylates H4 K20. H4 that was free, in histone core octamers or polynucleosomes, was methylated by Ash1 and analysed by Western blot analysis. Anti-dim(H4-K20) antibody recognizes Ash1-methylated H4, but not un-methylated H4, indicating that Ash1 methylates H4 K20 (Beisel, 2002).

Single amino acid point mutations ash110 and ash121 abolish Ash1 activator function in Drosophila. The mutation in ash110 (N1458I) resides within the SET domain, and in ash121 (E1357K) in the pre-SET domain. To assess whether these mutations affect HMTase activity, recombinant proteins were expressed and purified containing one of these mutations (Ash1DeltaN10, Ash1DeltaN21) and a third mutant (Ash1DeltaN1142) whose mutation (H1459K) resides in the SET domain and abolishes HMTase activity of SUV39H1. HMTase assays revealed that the mutants do not significantly methylate H3 and H4, indicating that the mutations abolish HMTase activity and that both the pre-SET and SET domains of Ash1 contribute to HMTase activity and transcriptional activation by Ash1 (Beisel, 2002).

To assess whether the mutations in Ash1 specifically inactivate HMTase activity or cause a general functional inactivation, the ability of mutant Ash1DeltaN to bind the known interaction partner Trx was investigated. Ash1DeltaN and the three mutants can interacte with Trx in vitro, suggesting that the inability of mutant Ash1 proteins to methylate histones is based on a specific inactivation of HMTase activity (Beisel, 2002).

To investigate the effect of ash110 and ash121 on transcriptional activation by Ash1 in Drosophila, transgenic flies were used carrying the Ash1-dependent reporter gene N18/15, which contains a 4-kilobase (kb) regulatory element of the bxd region from the Ash1 target gene Ubx fused to the mini-white gene. Ash1 supports activation of N18/15 transcription in the Drosophila eye. By contrast, N18/15 expression is significantly reduced in ash110/ + or ash121/+ heterozygous flies. Since Ash121 and Ash110 lack HMTase activity in vitro, these results imply that HMTase activity contributes to transcriptional activation by Ash1 in vivo (Beisel, 2002).

To dissect the functional relationship between transcriptional activation and histone methylation by Ash1, transcriptional activation by Ash1 was reconstituted in Drosophila S2 cells. To monitor transcription in chromatin, S2 (BCAT5) cells were generated that carry the stable integrated reporter gene BCAT5, which contains five DNA-binding sites for the yeast activator Gal4, a core promoter and the bacterial cat gene. To recruit Ash1 to chromatin, Ash1 derivatives were fused to the Gal4 DNA-binding domain (amino acids 1-147) [Gal4(DBD)]. BCAT5 cells were transfected with plasmids expressing fusion proteins comprising Gal4(DBD) and either wild type or mutant Ash1DeltaN. Gal4(DBD)-Ash1DeltaN activates BCAT5 expression 20-fold, whereas HMTase-inactive Ash1DeltaN derivatives did not. These results support the hypothesis that HMTase activity of Ash1 mediates activation of transcription (Beisel, 2002).

To link transcriptional activation by Ash1 to histone methylation, crosslinked chromatin immunoprecipitation (XChIP), which detects protein-DNA interactions in vivo, was used. Crosslinked chromatin was isolated from BCAT5 cells expressing Gal4(DBD)-Ash1DeltaN, Gal4(DBD)-Ash1DeltaN10 or Gal4(DBD)-Ash1DeltaN21, and immunoprecipitated by antibodies recognizing dimethylated H3 K4, H3 K9 or H4 K20. Precipitated DNA was purified and the enhancer/promoter of target genes was detected by polymerase chain reaction. All three antibodies precipitate chromatin containing the BCAT5 enhancer/promoter from cells in which Gal4(DBD)-Ash1DeltaN activates transcription. Methylation of these lysine residues was detectable 500 bp upstream of the enhancer/promoter and at the 3'-end of the cat gene. In cells expressing Gal4(DBD)-Ash1DeltaN10, methylation of H3 K4 was undetectable, but weak methylation of H3 K9 and H4 K20 could be observed. This finding supports current models proposing that transcriptional repression correlates with methylation of H3 K9 and H4 K20. As, however, H3 K9 and H4 K20 methylation is enhanced at the transcriptionally active (active) reporter, transcriptional activation by Ash1 correlates with de novo methylation of not only H3 K4 but also H3 K9 and H4 K20 (Beisel, 2002).

Methylation of H3 K9 at the transcriptionally silent (silent) enhancer/promoter implies that BCAT5 might be associated with HP1, which binds methylated H3 K9. This was tested by XChIP using anti-HP1 polyclonal antibody. The antibody precipitated the BCAT5 enhancer/promoter from cells expressing HMTase-inactive Gal4(DBD)-Ash1DeltaN10. By contrast, the enhancer/promoter was only weakly precipitated from cells in which Gal4(DBD)-Ash1DeltaN activates reporter expression. These results suggest that HP1 binds the silent enhancer/promoter and is removed/relocated from the reporter by Ash1-mediated histone methylation (Beisel, 2002).

To investigate whether Ash1 methylates histones to activate transcription of a natural target gene, methylation of Ubx was monitored in BCAT5 cells. Ubx is not expressed in S2-cells but PCR with reverse transcription (RT-PCR) indicates that transiently expressed Gal4(DBD)-Ash1DeltaN activates expression of this gene in BCAT5 cells. XChIP experiments indicate that H3 K4 is not methylated and that H3 K9 and H4 K20 are only weakly methylated at the silent Ubx promoter. By contrast, methylation of all three lysine residues is significantly enhanced when Ash1 activates BCAT5 expression, indicating that transcriptional activation of Ubx by Ash1 coincides with methylation of H3 K4, K9 and H4 K20 (Beisel, 2002).

Genetic data indicate that Ash1 activates Ubx expression in imaginal discs of the third leg. Therefore, to investigate histone methylation by Ash1 in the natural context of the activator, the methylation pattern of Ubx was examined in third leg discs by XChIP. Crosslinked chromatin was prepared from third leg discs dissected from third instar larvae. Chromatin immunoprecipitations indicated methylation of H3 K4, K9 and H4 K20 at the Ubx promoter, suggesting that Ash1-mediated methylation of all three lysine residues coincides with epigenetic activation of Ubx transcription in Drosophila (Beisel, 2002).

On the basis of the result that methylated lysine residues facilitate or inhibit the binding of proteins, an investigation was carried out to determine whether the trivalent (H3 K4, K9 and H4 K20) methylation pattern placed by Ash1 attracts or repels proteins to establish epigenetic activation. The XChIP experiments in indicate that the trivalent methylation pattern removes/relocates HP1 from chromatin. To support this finding, the interaction was investigated of HP1 with methylated H3 peptides and histone core octamers that had been methylated by Ash1 or Drosophila SU(VAR)3-9, which methylates H3 K9. HP1 binds H3 K9-methylated peptides and histone core octamers, as well as H3 K4/K9-methylated peptides. By contrast, HP1 does not bind Ash1-methylated core octamers, suggesting that the trivalent methylation pattern inhibits the interaction of HP1 with chromatin (Beisel, 2002).

Protein-protein interaction assays using H3(1-20) peptides methylated at H3 K4, K9 or H3 K4 and K9 (H3 K4/K9), and Drosophila embryonic nuclear extract or recombinant proteins, resulted in the identification of three proteins that exhibit differential binding to methylated peptides. Two of these proteins -- the epigenetic repressor Polycomb (Pc) and Caf-1 p55, a subunit of different protein complexes involved in, for example, epigenetic repression -- bind H3 K9-methylated peptides and histone core octamers, but show significantly reduced binding to H3 K4- or H3 K4/K9-methylated peptides and trivalently methylated histone core octamers. Furthermore, protein-binding assays indicate that Brm and Moira (Mor) interact with H3 K4/K9-methylated peptides. In contrast, both proteins were not recruited to peptides methylated at H3 K4 or H3 K9. Brm and Mor are subunits of a SWI/SNF-like chromatin remodelling complex, suggesting that this complex, rather than individual proteins, is recruited to K4/K9-methylated H3. These results imply that the trivalent methylation pattern established by Ash1 facilitates or prevents the interaction of proteins with methylated H3 during epigenetic activation. To support this finding, XChIP was used to investigate the interaction of Brm and repressors with Ash1 target genes. These analyses indicate that Brm and Mor are present at the active but not at silent promoters of Ash1 target genes in cells or third leg imaginal discs. By contrast, the repressors were only detected at silent promoters. Thus, transcriptional activation by Ash1 may coincide with the recruitment of Brm and Mor and the extinction of repressor binding at the promoter of Ash1 target genes (Beisel, 2002).

Collectively, these data indicate that the epigenetic activator Ash1 activates transcription by methylation of H3 K4, K9 and H4 K20 at the promoter of target genes. This suggests that epigenetic activation and silencing, which has been linked to methylated H3 K9, may correlate with different histone methylation patterns. Each of the three lysine residues targeted by Ash1 can be individually methylated by specific HMTases, resulting in transcriptional repression and probably activation (H3 K4). Combining these three modifications results in a novel biological readout: epigenetic activation. Why does the trivalent modification pattern generated by Ash1 mediate epigenetic activation? The results indicate that each modification of the pattern fulfils a specific function. Methylation of H3 K4 prevents the interaction of repressors (Pc, p55) with Ash1 target genes. Methylation of H4 K20 in addition to H3 K4 and H3 K9 prevents the interaction of HP1 with chromatin. Inhibition of repressor binding is an important mechanism, as epigenetic activators and repressors are expressed together during Drosophila development. Finally, methylation of H3 K4 and H3 K9 generates an interaction surface for a chromatin-remodelling complex. These results imply that a specific functional interplay between the epigenetic activators Ash1 and Brm mediates epigenetic activation of transcription. Ash1 initially binds target genes and generates the trivalent histone methylation pattern, which subsequently recruits a Brm-containing chromatin-remodelling complex. The activity of this complex may contribute to the establishment of epigenetic active chromatin structures (Beisel, 2002).

A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin

Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, Suv4-20h1 and Suv4-20h2 were identified as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, Suv4-20 is a novel position-effect variegation modifier. Together, these data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin (Schotta, 2004).

These data suggest H4-K20 trimethylation is a mark of silenced chromatin domains. Therefore whether this modification would indeed be important for gene silencing in well-described PEV models in Drosophila was investigated. A single, homozygous-viable P-element insertion (P{GT1}BG00814) into the third exon of Suv4-20 has been identified in the course of the Drosophila gene disruption project. H4-K20 trimethylation at polytene chromatin is nearly lost in homozygous mutant larvae, demonstrating that the P-element insertion (Suv4-20BG00814) represents a strong hypomorphic allele of Suv4-20. Because the Suv4-20 locus maps on the X chromosome, the classical PEV rearrangement In(1)wm4 cannot be used to analyze a potential modifier effect of Suv4-20. Therefore, another PEV rearrangement was analyzed that translocates a different marker, Stubble (Sb), close to pericentric heterochromatin (T(2;3)SbV). The dominant mutation Stubble induces short bristles, but heterochromatin-induced silencing of SbV results in wild-type (long) bristles. Homozygous Suv4-20BG00814 as well as control wild-type females were crossed to T(2;3)SbV males. In the progeny, the extent of SbV reactivation was determined as the ratio of short bristles (active SbV) to long bristles (inactive SbV). In males and females of the wild-type crosses, only 1%-2% of bristles show a Sb phenotype, indicating that SbV is largely inactivated. In contrast, SbV becomes derepressed in the progeny of Suv4-20BG00814 flies, because now ~25% of the bristles are short. This result classifies Suv4-20 as a dominant PEV modifier and further indicates a functional role for Suv4-20-dependent H4-K20 trimethylation in gene silencing (Schotta, 2004).

A role for the histone H4 in nucleosome remodeling by ISWI

The ATPase ISWI can be considered the catalytic core of several multiprotein nucleosome remodeling machines. Alone or in the context of nucleosome remodeling factor [the chromatin accessibility complex (CHRAC), or ACF] ISWI catalyzes a number of ATP-dependent transitions of chromatin structure that are currently best explained by its ability to induce nucleosome sliding. In addition, ISWI can function as a nucleosome spacing factor during chromatin assembly, where it will trigger the ordering of newly assembled nucleosomes into regular arrays. Both nucleosome remodeling and nucleosome spacing reactions are mechanistically unexplained. As a step toward defining the interaction of ISWI with its substrate during nucleosome remodeling and chromatin assembly a set of nucleosomes lacking individual histone N termini were generated from recombinant histones. The conserved N termini (the N-terminal tails) of histone H4 were found to be essential to stimulate ISWI ATPase activity, in contrast to other histone tails. Remarkably, the H4 N terminus, but none of the other tails, is critical for CHRAC-induced nucleosome sliding and for the generation of regularity in nucleosomal arrays by ISWI. Direct nucleosome binding studies did not reflect a dependence on the H4 tail for ISWI-nucleosome interactions. It is concluded that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate (Clapier, 2001).

Acf1, the largest subunit of CHRAC, regulates ISWI-induced nucleosome remodelling

The chromatin accessibility complex (CHRAC) was originally defined biochemically as an ATP-dependent 'nucleosome remodelling' activity. Central to its activity is the ATPase ISWI, which catalyses the transfer of histone octamers between DNA segments in cis. In addition to ISWI, four other potential subunits were observed consistently in active CHRAC fractions. The p175 subunit of CHRAC has been identified as Acf1, a protein known to associate with ISWI in the ACF complex. Interaction of Acf1 with ISWI enhances the efficiency of nucleosome sliding by an order of magnitude. Remarkably, it also modulates the nucleosome remodelling activity of ISWI qualitatively by altering the directionality of nucleosome movements and the histone 'tail' requirements of the reaction. The Acf1-ISWI heteromer tightly interacts with the two recently identified small histone fold proteins CHRAC-14 and CHRAC-16. Whether topoisomerase II is an integral subunit has been controversial. Refined analyses now suggest that topoisomerase II should not be considered a stable subunit of CHRAC. Accordingly, CHRAC can be molecularly defined as a complex consisting of ISWI, Acf1, CHRAC-14 and CHRAC-16 (Eberharter, 2001).

A heterodimeric complex of Acf1 and ISWI previously had been termed 'ACF'. In this context, Acf1 significantly increases the activity of ISWI in chromatin assembly. Since Acf1 has been identified as a component of CHRAC, the impact of Acf1 on ISWI-induced nucleosome sliding was examined. The directionality of nucleosome sliding differs depending on whether the reaction is catalysed by ISWI alone or by CHRAC. Flag-tagged ISWI and Acf1 were expressed from baculovirus vectors in insect cells, affinity purified and assayed for nucleosome sliding. In agreement with previous results, catalytic amounts (2-3 fmol) of ISWI move a mononucleosome from the center of a 248 bp rDNA fragment to the fragment end. No mobility was observed when the end-positioned nucleosome is exposed to ISWI. In contrast to the movement generated by ISWI, CHRAC catalyses nucleosome sliding from the end to the center of the DNA fragment. Strikingly, CHRAC-type directionality of nucleosome sliding is also obtained if Acf1 is added to ISWI, either after separate expression or by co-expression of both proteins in Sf9 cells. While Acf1 alone is inactive for nucleosome sliding, it boosts ISWI activity by at least an order of magnitude such that 10-fold lower enzyme concentrations (0.3-0.5 fmol) are required for nucleosome mobilization. Most importantly, Acf1 changes the directionality of sliding such that end-positioned nucleosomes move to central positions (Eberharter, 2001).

In order to determine whether Acf1 has an additional effect on the kinetics of nucleosome mobility under these conditions, a time course of nucleosome mobility was performed. The amounts of enzymes were chosen such that complete mobilization of the nucleosome was expected after 90 min (10-fold less ACF than ISWI). At any given time point throughout the reaction, the ratio of nucleosomes that had been mobilized to those that had not moved was determined. Nucleosome movement in the two reactions proceeds with similar speed, indicating that ACF is about an order of magnitude more efficient in nucleosome mobilization than ISWI alone. This could be explained most readily if Acf1 stimulates the ATPase activity of ISWI. To determine whether this was the case, the enzymes were compared in standard ATPase assays. ISWI alone shows a robust (7-fold) nucleosome stimulation of ATPase. This response to a nucleosomal structure remains unaltered if Acf1 is added, either after separate expression or through co-expression. While Acf1 alone does not show any sign of ATPase activity, it also does not stimulate the ATPase of ISWI significantly (7-fold stimulation over the free DNA level in all cases) (Eberharter, 2001).

Deletion of the H4 N-termini completely abolishes the ability of CHRAC to slide nucleosomes, whereas removal of any other histone tail has only minor effects. In contrast, ISWI-induced sliding not only requires the histone H4 N-termini (like CHRAC), but is also impaired if any of the other tails are deleted. Since Acf1 modulates the directionality of nucleosome sliding to resemble that of CHRAC, the histone tail dependence of ACF-induced nucleosomal sliding was tested. As expected, deletion of the H4 tail completely abolishes the remodelling activity of ACF. Removal of any other histone tail, however, has only little influence on the sliding activity of ACF. This result reinforces the notion of a qualitative alteration of the nucleosome remodelling activity of ISWI by Acf1, which points to altered interaction with the nucleosomal substrate (Eberharter, 2001).

Acf1 was first identified as a protein associated with ISWI to form the nucleosome assembly and spacing factor ACF. While either recombinant ISWI or Acf1 alone is only poorly active in an assembly system, the reconstitution of ACF from the two subunits increases the in vitro nucleosome assembly activity by some 30-fold. ISWI, expressed in a bacterial system, can, in principle, function autonomously in various cell-free remodelling assays. The direct comparison of the activity of factors expressed under similar conditions from baculovirus vectors shows that Acf1 enhances ISWI-induced nucleosome mobility by about an order of magnitude. In addition, the association of Acf1 has a striking qualitative effect as it alters the directionality of nucleosome sliding triggered by ISWI and affects the sensitivity of the ATPase towards deletion of the histone N-termini on the nucleosomal substrate. ISWI and Acf1 approach the nucleosome in a co-ordinated manner, leading to a new quality of interaction, such that Acf1 does not simply enhance the action of ISWI. Whether Acf1 interacts with DNA directly, effectively hindering the sliding of the nucleosome to the fragment end, remains to be explored. Upon association of ISWI with Acf1, 10-fold lower enzyme concentrations and correspondingly fewer ATP hydrolysis events are required to move a nucleosome as compared with free ISWI alone. It is possible that ACF has a higher affinity for the nucleosomal substrate, due to interaction domains contributed by Acf1. A decreased off-rate may lead to a higher processivity of the enzyme, converting the energy of ATP hydrolysis more effectively into directional nucleosome sliding. Testing this and alternative hypotheses will require more quantitative measurements of the parameters of the nucleosome sliding reaction (Eberharter, 2001).

The PHD finger and bromodomain are likely to be involved in Acf1 activity. PHD fingers are protein interaction surfaces found in many chromatin-bound regulators. Bromodomains are equally abundant among nuclear regulators. They are a hallmark of the remodelling ATPases of the SWI2/SNF2 type, but are absenty in ISWI. Bromodomains are known interactors of acetylated histone H4 N-termini. Nucleosome remodelling by ISWI critically depends on the integrity of the H4 tail on the nucleosomal substrate. It is possible that tandem PHD fingers and a bromodomain form a cooperative interaction unit, as has been suggested recently for the KRAB proteins. It will be interesting to see whether Acf1 interacts directly with the H4 tail during nucleosome mobilization and whether histone acetylation modulates this process. The function of the N-terminal WAC domain of Acf1, which has been implicated in targeting proteins to heterochromatin, is unknown (Eberharter, 2001).

Although CHRAC was first perceived due to its activity to render nucleosomal DNA accessible, it was soon discovered that CHRAC and the related ACF may have an important role in the assembly of regular nucleosomal arrays in vitro. Nucleosome mobility is not restricted to the assembly phase, but can also be observed within an ordered nucleosomal array. CHRAC may be involved mainly in the assembly and maintenance of nucleosomal arrays with dynamic properties. Several observations are in line with such a function. (1) The in vitro phenomenology shows that CHRAC and ACF can catalyse the assembly of dynamic nucleosomal arrays. (2) The restricted expression of ISWI, Acf1 and the CHRAC-14/16 pair during Drosophila embryonic development correlates with the time of most intense nuclear division. (3) Proteins with similarity to CHRAC-14 and CHRAC-16 have been found to associate with human DNA polymerase epsilon. (4) Mutation of ISWI in male flies leads to a striking abnormality of the structure of the male X chromosome, which is marked and perhaps sensitized by specific acetylation of the histone H4 N-terminus, although the additional presence of ISWI in NURF complicates the interpretation of the mutant phenotype. The outcome of rendering nucleosomes mobile may depend on the circumstances: in vitro, CHRAC can facilitate SV40 replication by promoting the access of T antigen to a nucleosomal origin. In contrast, an ACF-like complex contributes to the targeted repression in yeast, presumably by modulating nucleosome positions in the promoters of meiosis-specific genes (Eberharter, 2001 and references therein).

Histone tails modulate nucleosome mobility and regulate ATP-dependent nucleosome sliding by NURF

Nucleosome Remodeling Factor (NURF) is an ATP-dependent nucleosome remodeling complex that alters chromatin structure by catalyzing nucleosome sliding, thereby exposing DNA sequences previously associated with nucleosomes. How the unstructured N-terminal residues of core histones (the N-terminal histone tails) influence nucleosome sliding has been systematically studied. Bacterially expressed Drosophila histones were used to reconstitute hybrid nucleosomes lacking one or more histone N-terminal tails. Unexpectedly, it was found that removal of the N-terminal tail of histone H2B promote uncatalyzed nucleosome sliding during native gel electrophoresis. Uncatalyzed nucleosome mobility is enhanced by additional removal of other histone tails but is not affected by hyperacetylation of core histones by p300. In addition, the N-terminal tail of the histone H4 is specifically required for ATP-dependent catalysis of nucleosome sliding by NURF. Alanine scanning mutagenesis demonstrated that H4 residues 16-KRHR-19 are critical for the induction of nucleosome mobility, revealing a histone tail motif that regulates NURF activity. An exchange of histone tails between H4 and H3 impairs NURF-induced sliding of the mutant nucleosome, indicating that the location of the KRHR motif in relation to global nucleosome structure is functionally important. These results provide functions for the N-terminal histone tails in regulating the mobility of nucleosomes (Hamiche, 2001).

To examine how the histone N-terminal tails influence NURF-mediated nucleosome sliding, the ability of tailless nucleosomes to stimulate the ATPase activity of NURF was examined. Incubation of NURF with nucleosomes lacking histone H2A, H2B, or H3 tails individually or lacking both H2A and H2B tails shows simulation of ATPase activity like that generated by WT nucleosomes. By contrast, nucleosomes lacking the histone H4 tail alone or combined with removal of other histone tails show no stimulation of the ATPase activity of NURF. The effects of tailless nucleosomes on nucleosome sliding by NURF was investigated. For WT nucleosomes and nucleosomes lacking the histone H2A, H2B, or H3 tail, NURF-mediated nucleosome mobility similar to that reported for nucleosomes containing native full length histones was observed. Mononucleosomes reconstituted from full length recombinant Drosophila histones adopt four major positions (N1-N4) on the 359-bp hsp70 promoter when analyzed by native gel electrophoresis at low ionic strength. Nucleosomes N1 and N2 were depleted, whereas N3 was enriched, along with slower migration of the N4 'fragment end nucleosomes.' In contrast, nucleosomes lacking the H4 tail show little mobilization on reaction with NURF. N1 and N2 positions were retained, whereas N3 shows little enrichment (but the N4 nucleosomes show clear changes in migration). Taken together, the results demonstrate that the N-terminal tail of histone H4 regulates the activity of NURF (Hamiche, 2001).

An examination was carried out to see whether a glutathione S-transferase (GST)-histone H4 tail fusion could block nucleosome stimulation of the ATPase activity of NURF in trans. GST-H4 tail reduces stimulation of the ATPase activity of NURF by only 2-fold when introduced at 100-fold molar excess to nucleosomes. The ability of NURF to catalyze nucleosome sliding was not detectably affected by a 100-fold molar excess of GST-H4 tail, and higher concentrations of GST-H4 tail failed to show inhibition. It appears that the H4 tail is unable to influence NURF-induced nucleosome sliding when removed from the context of the nucleosome (Hamiche, 2001).

The x-ray crystal structure of the nucleosome core particle shows that the N-terminal tails of histones H3 and H2B exit through the aligned minor grooves of adjacent gyres of the DNA superhelix, whereas H4 and H2A tails exit in minor grooves from the top or bottom edges of the disc-like particle. Beyond the nucleosome core particle, the histone tails are disordered, having no visible interactions with the 147-bp DNA superhelix. Biophysical studies of nucleosome arrays in which the histone tails are removed by trypsinization or modified by acetylation indicate their involvement in the higher-order folding of chromatin. Moreover, contacts can be observed between the tails and the histone octamer of neighboring core particles in the x-ray crystal structure. However, there is evidence that the N-terminal histone tails also interact with core particle DNA in solution. The absence of histone N-terminal tails decreases the thermostability of the nucleosome and alters the equilibrium constants for dynamic DNA site accessibility in nucleosomes. Binding of sequence-specific transcription factors to the nucleosome is modulated by the presence of the histone tails, and photochemical and UV-laser crosslinking experiments reveal physical interactions between core histone tails and nucleosomal DNA. The histone tails not only make contact with DNA in the nucleosome core particle but also can preferentially interact with linker DNA. Stabilization of nucleosomes by histone tails is apparently effective only on intrinsically straight or bent, rather than flexible, DNA fragments (Hamiche, 2001 and references therein).

This study provides an additional perspective for the histone N-terminal tails. Deletion of the N-terminal tail of histone H2B promotes uncatalyzed nucleosome mobility when perturbed by native gel electrophoresis in Tris·glycine·EDTA (or Tris·borate·EDTA), and this effect is increased by deletion of the other histone tails. The observed changes in electrophoretic migration are likely to be caused by increased translational mobility of the histone octamer on DNA, although the additional possibility of increased conformational flexibility of the linker DNA is not excluded. Thus, uncatalyzed nucleosome positioning and mobility not only may depend on structured histone-DNA interactions in the nucleosome core particle but also could be modulated by interactions of the histone H2B tail and other histone tails with nucleosomal DNA. The proximity of the basic histone H2B tails to two adjacent DNA gyres of the nucleosome core particle may provide especially suitable interactions that restrict nucleosome mobility. In this respect, it is intriguing to recall genetic studies in which deletion of the first 20 amino acids of the H2B N-terminal tail bypassed the requirement for Swi-Snf in yeast (Hamiche, 2001).

It is of interest that quantitative hyperacetylation of core histones by p300 has no detectable effect on nucleosome positioning or nucleosome dynamics. These results, which suggest that histone acetylation by p300 has a significantly greater impact on higher-order folding of nucleosome arrays than on the positioning and mobility (or stability) of individual nucleosomes, concur with other findings of a similar nature. It is also noted that deletion of the histone H3 tail produces a slight retardation in electrophoretic migration irrespective of nucleosome positioning, raising the possibility that this histone tail affects the entry-exit angle of the linker DNA (Hamiche, 2001.

This study demonstrates the importance of the histone H4 tail in ATP-dependent nucleosome sliding catalyzed by NURF. This finding concurs with results showing that the histone H4 tail is required for induction of nucleosome sliding by the CHRAC chromatin remodeling complex and for stimulation of the ATPase activity of recombinant ISWI. Given that ISWI is a common component of NURF and CHRAC, it is likely that interactions between the H4 tail and ISWI are important for activating NURF. Full efficiency and positional specificity of nucleosome sliding require the participation of the largest NURF subunit, NURF301 (Hamiche, 2001).

The definition by alanine-scanning mutagenesis of histone H4 tail residues responsible for regulating NURF activity reveals that H4 tail residues 16-KRHR-19 are critical for the induction of ATP-dependent nucleosome sliding. The proper spatial location of this regulatory motif relative to the global structure of the nucleosome is also important, because interchanging the tails of H3 and H4 impairs nucleosome sliding by NURF. These findings, taken with the failure of a GST-H4 tail fusion protein to significantly inhibit NURF function, suggest that NURF probably interacts with H4 tail residues 16-KRHR-19 in complex with nucleosomal DNA. There is evidence that part of the N-terminal tails of histone H4 (and H3) can be organized in the nucleosome as DNA-bound polypeptide segments with alpha-helical character. It will be interesting to investigate the nature of the regulatory interaction between H4 16-KRHR-19 and NURF (ISWI). Given that H4 K16 and K20 are known sites of histone acetylation and methylation, respectively, it is possible that these modifications could influence the activities of NURF and other ISWI complexes (Hamiche, 2001).

Aside from the evident importance of histone H4 16-KRHR-19 in providing a key to the ATP-dependent catalytic activity of NURF, the involvement of the other core histone tails in catalyzed nucleosome sliding is unclear. Deletion of the histone H2B tail does not bypass the need for NURF to induce ATP-dependent nucleosome sliding under in vitro assay conditions, indicating that DNA-protein interactions within the nucleosome core particle are dominant. An attractive model for nucleosome sliding invokes the ATP-dependent induction and propagation of a DNA twist or bulge over the histone octamer, a process that necessitates the transient disruption of contacts between structured histone elements and core particle DNA. However, it is possible that the dissociation of the N-terminal tail of histone H2B and the other histone tails from nucleosomal DNA may facilitate the overall nucleosome sliding mechanism (Hamiche, 2001).

The histone modification pattern of active genes revealed through genome-wide chromatin analysis of Drosophila

The covalent modification of nucleosomal histones has emerged as a major determinant of chromatin structure and gene activity. To understand the interplay between various histone modifications, including acetylation and methylation, a genome-wide chromatin structure analysis was performed in Drosophila. A binary pattern of histone modifications was found among euchromatic genes, with active genes being hyperacetylated for H3 and H4 and hypermethylated at Lys 4 and Lys 79 of H3, and inactive genes being hypomethylated and deacetylated at the same residues. Furthermore, the degree of modification correlates with the level of transcription, and modifications are largely restricted to transcribed regions, suggesting that their regulation is tightly linked to polymerase activity (Schübeler, 2004).

ChIP analysis followed by hybridization to DNA microarrays was used to map the pattern of six different histone modifications in the Drosophila genome. The karyotypically stable Drosophila Kc cell line was used. Chromatin was purified after formaldehyde cross-linking (= input) and immunoprecipitated either with antibodies that recognize a specific histone modification or without the addition of antisera as a control. DNA enriched for a specific modification (= bound) and DNA from the input material was isolated, labeled with different fluorescent dyes, and hybridized to a DNA microarray. Enrichment for a histone modification via immunoprecipitation results in a stronger fluorescence signal from the bound fraction, whereas absence of the modification results in a stronger signal from the input fraction. Because the observed enrichments are antibody specific, the ratio of the two dyes represents a quantitative measure of the studied modification (Schübeler, 2004).

The principal findings include the following: (1) there is a binary pattern of histone modifications for euchromatic genes, with active genes consistently marked by all of the euchromatic histone modifications analyzed and the absence of any of these modifications on nontranscribed genes; (2) the level of transcript abundance is positively correlated with the degree of euchromatic histone modifications, and (3) the chromosomal extent of the modification coincides with, and is limited to, the transcribed region. The surprising observation of an 'all-or-none' pattern of histone modification for euchromatic genes suggests a concerted mechanism for the placing of these marks. For example, the euchromatic modifications could be restricted to nucleosomes containing a certain histone H3 variant. The replication-independent deposition of the H3 variant 3.3 raises the possibility that in Metazoa the majority of euchromatic histone H3 modifications may occur on H3.3. Indeed, histone H3.3 has recently been reported to be enriched in acetylated lysines and in methylated Lys 4 and Lys 79 (Schübeler, 2004).

Although it is currently unclear whether these euchromatic modifications can be set prior to nucleosome assembly and deposition, there is ample evidence for post-deposition modification of histones. For example, a link between the elongating polymerase complex and several histone-modifying enzymes, including Set1 (an H3-K4 methylase), Set2 (an H3-K36 methylase; see Drosophila Set2), and Sas3 (a HAT), has been demonstrated in S. cerevisiae. Furthermore, genetic evidence from S. cerevisiae suggests that Dot1, the H3-K79 methylase, may also be recruited to chromatin by the elongating polymerase complex. These findings in budding yeast indicate a coupling of histone modifications and transcription. This genome-wide analysis in Drosophila cells strongly supports these findings and further argues that such interactions may be an integral component of transcriptional elongation in metazoans (Schübeler, 2004).

More than 25 years ago, it was observed that chromatin of active genes is more sensitive to DNaseI digestion than that of inactive genes. Although, to date, the nature of this sensitivity has been elusive, it is proposed that this sensitivity reflects the presence of euchromatic tail modifications. Why does such a 'switch' between two chromatin configurations involve a large set of histone modifications? Each modification may participate in creating a chromatin structure that facilitates transcription, either by changing nucleosomal interactions or by serving as a binding substrate for other proteins. The use of multiple modifications would make such system more robust. Regardless, these results reveal a tight coupling between transcription and euchromatic histone modifications. On recruitment, these modifications may serve to facilitate polymerase elongation and reinitiation and to propagate the transcriptional state through cell division (Schübeler, 2004).

Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins

Polycomb group (PcG) and trithorax group (trxG) proteins act as antagonistic regulators to maintain transcriptional OFF and ON states of HOX and other target genes. To study the molecular basis of PcG/trxG control, the chromatin of the HOX gene Ultrabithorax (Ubx) was analyzed in UbxOFF and UbxONcells purified from developing Drosophila. PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all constitutively bound to Polycomb response elements (PREs) in the OFF and ON state. In contrast, the trxG protein Ash1 is only bound in the ON state; not at PREs but downstream of the transcription start site. In the OFF state, extensive trimethylation was found at H3-K27, H3-K9, and H4-K20 across the entire Ubx gene; i.e., throughout the upstream control, promoter, and coding region. In the ON state, the upstream control region is also trimethylated at H3-K27, H3-K9, and H4-K20, but all three modifications are absent in the promoter and 5' coding region. These analyses of mutants that lack the PcG histone methyltransferase (HMTase) E(z) or the trxG HMTase Ash1 provide strong evidence that differential histone lysine trimethylation at the promoter and in the coding region confers transcriptional ON and OFF states of Ubx. In particular, these results suggest that PRE-tethered PcG protein complexes act over long distances to generate Pc-repressed chromatin that is trimethylated at H3-K27, H3-K9, and H4-K20, but that the trxG HMTase Ash1 selectively prevents this trimethylation in the promoter and coding region in the ON state (Papp, 2006; Full text of article).

Previous studies have shown that PhoRC contains the DNA-binding PcG protein Pho that targets the complex to PREs, and dSfmbt, a novel PcG protein that selectively binds to histone H3 and H4 tail peptides that are mono- or dimethylated at H3-K9 or H4-K20 (H3-K9me1/2 and H4-K20me1/2, respectively) (Klymenko, 2006). PRC1 contains the PcG proteins Ph, Psc, Sce/Ring, and Pc. PRC1 inhibits nucleosome remodeling and transcription in in-vitro assays and its subunit Pc specifically binds to trimethylated K27 in histone H3 (H3-K27me3). PRC2 contains the PcG proteins E(z), Su(z)12, and Esc as well as Nurf55, and this complex functions as a histone methyltransferase (HMTase) that specifically methylates K27 in histone H3 (H3-K27) in nucleosomes (Papp, 2006).

This study used quantitative X-ChIP analysis to examine the chromatin of the HOX gene Ubx in its ON and OFF state in developing Drosophila larvae. Previous genetic studies had established that all of the PcG and trxG proteins analyzed in this study are critically needed to maintain Ubx OFF and ON states in the very same imaginal disc cells in which their binding to Ubx was analyzed in this study. The following conclusions can be drawn from the analyses reported in this study. (1) The PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all highly localized at PREs, but they are all constitutively bound at comparable levels in the OFF and ON state. (2) The trxG protein Ash1 is bound only in the ON state, where it is specifically localized ~1 kb downstream of the transcription start site. (3) In the OFF state, PRC2 and other unknown HMTases trimethylate H3-K27, H3-K9, and H4-K20 over an extended 100-kb domain that spans the whole Ubx gene. (4) In the ON state, comparable H3-K27, H3-K9, and H4-K20 trimethylation is restricted to the upstream control regions and Ash1 selectively prevents this trimethylation in the promoter and coding region. (5) Repressed Ubx chromatin is extensively tri- but not di- or monomethylated at H3-K27, H3-K9, and H4-K20. (6) Trimethylation of H3-K27, H3-K9, and H4-K20 at imaginal disc enhancers in the upstream control region does not impair the function of these enhancers in the ON state. (7) TBP and Spt5 are bound at the Ubx transcription start site in the ON and OFF state, but Kis is only bound in the ON state. This suggests that in the OFF state, transcription is blocked at a late step of transcriptional initiation, prior to the transition to elongation. A schematic representation of PcG and trxG protein complex binding and histone methylation at the Ubx gene in the OFF and ON state is presented (Papp, 2006).

Unexpectedly, ChIP analysis by qPCR used in this study and in a similar study by the laboratory of Vincent Pirrotta (V. Pirrotta, pers. comm. to Papp, 2006) reveals that the relationship between PcG and trxG proteins and histone methylation is quite different from the currently held views. Specifically, X-ChIP studies have reported that H3-K27 trimethylation is localized at PREs and this led to the model that recruitment of PRC1 to PREs occurs through H3-K27me3 (i.e., via the Pc chromodomain). In contrast, the current study and that by Vincent Pirrotta found H3-K27 trimethylation to be present across the whole inactive Ubx gene, both in wing discs and in S2 cells (V. Pirrotta, pers. comm. to Papp, 2006). No specific enrichment of H3-K27 trimethylation at PREs has been detected; rather, a reduction of H3-K27me3 signals is observed at PREs, consistent with the reduced signals of H3 that are detected at these sites. Consistent with these results, genome-wide analyses of PcG protein binding and H3-K27me3 profiles in S2 cells revealed that, at most PcG-binding sites in the genome, PcG proteins are tightly localized, whereas H3-K27 trimethylation is typically present across an extended domain that often spans the whole coding region. How could the differences between this study and the earlier studies be explained? It should be noted that in contrast to the qPCR analysis used in the current study, previous studies all relied on nonquantitative end-point PCR after 36 or more cycles to assess the X-ChIP results. It is possible that these experimental differences account for the discrepancies (Papp, 2006).

PhoRC, PRC1, and PRC2 are all tightly localized at PREs but they are all constitutively bound at the inactive and active Ubx gene. This suggests that recruitment of PcG complexes to PREs occurs by default. Although all three complexes are bound at comparable levels to the bxd PRE in the inactive and active state and PhoRC is also bound at comparable levels at the bx PRE, it should be pointed out that the levels of PRC1 and PRC2 binding at the bx PRE are about twofold reduced in the active Ubx gene compared with the inactive Ubx gene. Even though there is still high-level binding of PRC1 and PRC2 at the bx PRE, it cannot be excluded that the observed reduction in binding helps to prevent default PcG repression of the active Ubx gene. It is possible that transcription through the bx PRE reduces PRC1 and PRC2 binding at this PRE. Transcription through PREs has been proposed to serve as an 'anti-silencing' mechanism that prevents default silencing of active genes by PREs (Papp, 2006),

The highly localized binding of all three PcG protein complexes at PREs, together with earlier studies on PRE targeting of PcG protein complexes supports the idea that not only PhoRC but also PRC1 and PRC2 are targeted to PRE DNA through interactions with Pho and/or other sequence-specific DNA-binding proteins. In the case of trxG proteins, the binding modes are more diverse. In particular, recruitment of Trx protein to PREs and to the promoter is also constitutive in both states but recruitment of Ash1 to the coding region is clearly observed only at the active Ubx gene. At present, it is not known how Trx or Ash1 are targeted to these sites. It is possible that a transcription-coupled process recruits Ash1 to the position 1 kb downstream of the transcription start site (Papp, 2006).

In contrast to the localized and constitutive binding of PcG protein complexes and the Trx protein, it was found that the patterns of histone trimethylation are very distinct in the active and inactive Ubx gene. The results also suggest that the locally bound PcG and trxG HMTases act across different distances to methylate chromatin. For example, H3-K4 trimethylation is confined to the first kilobase of the Ubx coding region where Ash1 and Trx are bound, whereas H3-K27 trimethylation is present across an extended 100-kb domain of chromatin that spans the whole Ubx gene. This suggests that PRE-tethered PRC2 is able to trimethylate H3-K27 in nucleosomes that are as far as 30 kb away from the bxd or bx PREs. Unexpectedly, it was found that the H3-K9me3 and H4-K20me3 profiles closely match the H3-K27me3 profile. At present it is not known which HMTases are responsible for H3-K9 and H4-K20 trimethylation, but analysis of E(z) mutants indicate that these modifications may be generated in a sequential manner, following H3-K27 trimethylation by PRC2. The molecular mechanisms that permit locally tethered HMTases such as PRE-bound PRC2 to maintain such extended chromatin stretches in a trimethylated state are only poorly understood. However, a recent study showed that the PhoRC subunit dSfmbt selectively binds to mono- and dimethylated H3-K9 and H4-K20 in peptide-binding assays (Klymenko. 2006). One possibility would be that dSfmbt participates in the process that ensures that repressed Ubx chromatin is trimethylated at H3-K27, H3-K9, and H4-K20. For example, dSfmbt, tethered to PREs by Pho, may interact with nucleosomes of lower methylated states (i.e., H3-K9me1/2 or H4-K20me1/2) in the flanking chromatin and thereby bring them into the vicinity of PRE-anchored HMTases that will hypermethylate them to the trimethylated state (Papp, 2006).

These analyses suggest that H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region is critical for Polycomb repression. (1) Although H3-K27, H3-K9, and H4-K20 trimethylation is present at the inactive and active Ubx gene, it is specifically depleted in the promoter and coding region in the active Ubx gene. (2) Misexpression of Ubx in wing discs with impaired E(z) activity correlates well with loss of H3-K27 and H3-K9 trimethylation at the promoter and 5' coding region. It is possible that the persisting H3-K27 and H3-K9 trimethylation in the 3' coding region is responsible for maintenance of repression in those E(z) mutant wing discs cells that do not show misexpression of Ubx. (3) In haltere and third-leg discs of ash1 mutants, the promoter and coding region become extensively trimethylated at H3-K27 and H3-K9, and this correlates with loss of Ubx expression. Previous studies showed that Ubx expression is restored in ash1 mutants cells that also lack E(z) function. Together, these findings therefore provide strong evidence that Ash1 is required to prevent PRC2 and other HMTases from trimethylating the promoter and coding region at H3-K27 and H3-K9. The loss of H3-K4 trimethylation in ash1 mutants is formally consistent with the idea that Ash1 exerts its antirepressor function by trimethylating H3-K4 in nucleosomes in the promoter and 5' coding region, but other explanations are possible (Papp, 2006).

But how might H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region repress transcription? The observation that TBP and Spt5 are also bound to the promoter in the OFF state suggests that these methylation marks do not prevent assembly of the basic transcription apparatus at the promoter. However, the nucleosome remodeling factor Kis is not recruited in the OFF state, and transcription thus appears to be blocked at a late step of transcriptional intiation prior to elongation. It was found that the low-level binding of Pc in the coding region correlates with the presence of H3-K27 trimethylation; i.e., Pc and H3-K27me3 are both present in the OFF state, but are absent in the ON state. One possible scenario would thus be that H3-K27 trimethylation in the promoter and coding region permits direct recruitment of PRC1. According to this view, locally recruited PRC1 would then repress transcription; e.g., by inhibiting nucleosome remodeling in the promoter region. However, several observations are not easily reconciled with such a simple 'recruitment-by-methylation' model. First, peak levels of all PRC1 components are present at PREs and, apart from Pc, very little binding is observed outside of PREs. Second, excision of PRE sequences from a PRE reporter gene during development leads to a rapid loss of silencing, suggesting that transcriptional repression requires the continuous presence of PREs and the proteins that are bound to them. A second, more plausible scenario would therefore be that DNA-binding factors first target PcG protein complexes to PREs, and that these PRE-tethered complexes then interact with trimethylated nucleosomes in the flanking chromatin in order to repress transcription. For example, it is possible that bridging interactions between the Pc chromodomain in PRE-tethered PRC1 and H3-K27me3-marked chromatin in the promoter or coding region permit other PRE-tethered PcG proteins to recognize the chromatin interval across which they should act, e.g., to inhibit nucleosome remodeling in the case of PRC1 or to trimethylate H3-K27 at hypomethylated nucleosomes in the case of PRC2 (Papp, 2006).

The analysis of a HOX gene in developing Drosophila suggests that histone trimethylation at H3-K27, H3-K9, and H4-K20 in the promoter and coding region plays a central role in generating and maintaining of a PcG-repressed state. Contrary to previous reports, the current findings provide no evidence that H3-K27 trimethylation is specifically localized at PREs and could thus recruit PRC1 to PREs; widespread H3-K27 trimethylation is found across the whole transcription unit. The data presented in this study provide evidence that PREs serve as assembly platforms for PcG protein complexes such as PRC2 that act over considerable distances to trimethylate H3-K27 across long stretches of chromatin. The presence of this trimethylation mark in the chromatin that flanks PREs may in turn serve as a signal to define the chromatin interval that is targeted by other PRE-tethered PcG protein complexes such as PRC1. The results reported here also provide a molecular explanation for the previously reported antirepressor function of trxG HMTases; selective binding of Ash1 to the active HOX gene blocks PcG repression by preventing PRC2 from trimethylating the promoter and coding region. It is possible that the extended domain of combined H3-K27, H3-K9, and H4-K20 trimethylation creates not only the necessary stability for transcriptional repression, but that it also provides the molecular marks that permits PcG repression to be heritably maintained through cell division (Papp, 2006).

Chaperone-mediated assembly of centromeric chromatin

Every eukaryotic chromosome requires a centromere for attachment to spindle microtubules for chromosome segregation. Although centromeric DNA sequences vary greatly among species, centromeres are universally marked by the presence of a centromeric histone variant, centromeric histone 3 (CenH3), which replaces canonical histone H3 in centromeric nucleosomes. Conventional chromatin is maintained in part by histone chaperone complexes, which deposit the S phase-limited (H3) and constitutive (H3.3) forms of histone 3. However, the mechanism that deposits CenH3 specifically at centromeres and faithfully maintains its chromosome location through mitosis and meiosis is unknown. To address this problem, a soluble assembly complex has been biochemically purified that targets tagged CenH3 to centromeres in Drosophila cells. Two different affinity procedures led to purification of the same complex, which consists of CenH3, histone H4, and a single protein chaperone, RbAp48, a highly abundant component of various chromatin assembly, remodeling, and modification complexes. The corresponding CenH3 assembly complex reconstituted in vitro is sufficient for chromatin assembly activity, without requiring additional components. The simple CenH3 assembly complex is in contrast to the multisubunit complexes previously described for H3 and H3.3, suggesting that centromeres are maintained by a passive mechanism that involves exclusion of the complexes that deposit canonical H3s during replication and transcription (Furuyama, 2006a; full text of article).

RbAp48 is sufficient for centromeric chromatin assembly in vitro, but is it necessary for this process in vivo? RbAp48 is found in various chromatin-associated protein complexes, where it is thought to play a common role in mediating their interactions with histones. Although no mutations have been reported to eliminate Drosophila RbAp48 (NURFp55), mutations in other components of RbAp48-associated complexes are lethal [Nurf-38, E(z), sin3, and many others]; therefore, it would be expected that removal of RbAp48 would have pleiotropic effects. Indeed, knock-down of RbAp48 by RNAi in Drosophila S2 cells results in S phase arrest and derepression of various Rb/E2F target genes. These pleiotropic effects caused by reduction in RbAp48 levels would mask any centromere defect, and, in any case, such a defect would not be expected to occur immediately, because disruption of fission yeast RbAp48 did not affect chromosome segregation until the second round of mitosis (Furuyama, 2006a).

The single chaperone purified by using tagged CID contrasts with the multiple subunits found in purified chaperone complexes using tagged H3.1 and H3.3. The H3.1-specific replication-coupled assembly complex contains more than seven nonhistone subunits, and the H3.3-specific replication-independent complex contains at least five. Furthermore, H3.1- and H3.3-specific assembly reactions were performed in the presence of crude lysates, suggesting requirements for additional components that might restrict deposition to polymerase-driven processes. In contrast, both purified and reconstituted CID/H4-RbAp48 are sufficient for chromatin assembly in the absence of any other processes (Furuyama, 2006a).

The formation of chromatin from histones and DNA is a thermodynamically favorable reaction, and it is thought that histone chaperones are needed to prevent nonproductive aggregation between highly positively charged histones and highly negatively charged DNA in a dense protein environment. Both replication-coupled assembly of H3.1/H4 and transcription-coupled assembly of H3.3/H4 take place in the highly dynamic context of multisubunit polymerase transit, and assembly in both cases might require a large number of subunits to facilitate tethering of assembly complexes for rapid histone deposition. However, the basic assembly reaction appears to have minimal requirements, and conventional nucleosomes can be assembled in the presence of the NAP1 protein chaperone, polyglutamate, or high concentration of salt. It is suggested that the simplicity of CID/H4-RbAp48 reflects a simple in vivo situation in which assembly occurs in the absence of rapidly transiting polymerases and associated factors. Although both H3.1- and H3.3-specific complexes also contain RbAp48 and RbAp48 alone can assemble H3 nucleosomes, other components in these complexes might prevent spontaneous deposition at gaps in chromatin due to steric hindrance, whereas the much simpler CID/H4-RbAp48 would gain access to these chromatin gaps without impediment. In other words, H3- and H3.3-specific chromatin assembly complexes may have evolved to strictly couple their activities to replication and transcription, respectively, to increase the efficiency of these cellular processes, and to delineate assembly pathways of different histone 3 variants. There is precedence for such a variant-dependent exclusion mechanism: H3 appears to be prevented from assembling by replication-independent deposition anywhere in the genome, whereas H3.3 appears to deposit anywhere except at centromeres. When overproduced, CID deposits in a euchromatic pattern that is similar to that seen for H3.3, suggesting that CenH3s have fewer constraints than either H3 or H3.3 and that other chaperones in these complexes are the best candidates for mediating differential exclusion. Any CenH3 that incorporates in euchromatin at transient gaps created by transcription would be continuously replaced by transcription-coupled assembly of H3.3; in this way, CenH3 would be passively retained at centromeres but actively removed from transcriptionally active regions (Furuyama, 2006a).

Exclusion of H3 and H3.3 but not CenH3 from centromeric chromatin, such as by steric hindrance or RNA-mediated targeting, might help account for the deposition of CenH3s at a wide variety of sequences within a genome, including human neocentromeres, nematode holocentromeres, and gene-rich rice centromeres. Furthermore, budding yeast CenH3 (Cse4p) can localize properly to human centromeres and rescue a CENP-A depletion phenotype. Because of the high degree of divergence between Cse4p and CENP-A relative to the near invariance of H3, it is unlikely that a protein complex that normally recognizes CENP-A can associate with Cse4p and deposit it only at the centromeres. Rather, assembly of CenH3-H4 into centromeric chromatin in other organisms might be achieved by a simple H4-binding chaperone, such as RbAp48. Perhaps what distinguishes a CenH3 from a canonical H3 is that it is not accepted by H3- or H3.3-specific chaperone complexes (Furuyama, 2006a).

The efficient propagation of centromeric chromatin domains during every cell cycle requires the correct localization of CenH3s. The robustness and precision of this process is extraordinary; for example, the location of centromeres have not changed in this lineage for 30 million years. It has been proposed that the compact structure of the CENP-A/H4 protein tetramer leads to the perpetuation of correct CENP-A localization, but it is not clear how compactness by itself can facilitate the faithful recruitment of additional CENP-A/H4 protein tetramers during every cell division. The apparent simplicity of CenH3 assembly can provide a mechanism to delineate this assembly pathway from that of H3 and H3.3. Torsional stress induced at centromeres at anaphase may be an efficient mechanism to clear H3 or H3.3 from centromeres and to create gaps for CenH3 deposition. Thus, the assembly of centromeric nucleosomes at gaps, which are created by the very process that requires CenH3, would provide a robust self-enforcing mechanism to maintain centromeres indefinitely (Furuyama, 2006a).

Centromeres are chromosomal sites of microtubule binding that ensure correct mitotic segregation of chromosomes to daughter cells. This process is mediated by a special centromere-specific histone H3 variant (CenH3), which packages centromeric chromatin and epigenetically maintains the centromere at a distinct chromosomal location. However, CenH3 is present at low abundance relative to canonical histones, presenting a challenge for the isolation and characterization of the chaperone machinery that assembles CenH3 into nucleosomes at centromeres. To address this challenge, controlled overexpression of Drosophila CenH3 (CID) and an efficient biochemical purification strategy offered by in vivo biotinylation of CID was used to successfully purify and characterize the soluble CID nucleosome assembly complex. It consists of a single chaperone protein, RbAp48, complexed with CID and histone H4. RbAp48 is also found in protein complexes that assemble canonical histone H3 and replacement histone H3.3. This study highlights the benefits of the improved biotin-mediated purification method, and addresses the question of how the simple CID/H4-RbAp48 chaperone complex can mediate nucleosome assembly specifically at centromeres (Furuyama, 2006b).

A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities

Polycomb response elements (PREs) are specific cis-regulatory sequences needed for transcriptional repression of HOX and other target genes by Polycomb group (PcG) proteins. Among the many PcG proteins known in Drosophila, Pleiohomeotic (Pho) is the only sequence-specific DNA-binding protein. To gain insight into the function of Pho, Pho protein complexes were purified from Drosophila embryos and it was found that Pho exists in two distinct protein assemblies: a Pho-dINO80 complex containing the Drosophila INO80 nucleosome-remodeling complex, and a Pho-repressive complex (PhoRC) containing the uncharacterized gene product Scm-related gene containing four mbt domains (dSfmbt). Analysis of PhoRC reveals that dSfmbt is a novel PcG protein that is essential for HOX gene repression in Drosophila. PhoRC is bound at HOX gene PREs in vivo, and this targeting strictly depends on Pho-binding sites. Characterization of dSfmbt protein shows that its MBT repeats have unique discriminatory binding activity for methylated lysine residues in histones H3 and H4; the MBT repeats bind mono- and di-methylated H3-K9 and H4-K20 but fail to interact with these residues if they are unmodified or tri-methylated. These results establish PhoRC as a novel Drosophila PcG protein complex that combines DNA-targeting activity (Pho) with a unique modified histone-binding activity (dSfmbt). It is proposed that PRE-tethered PhoRC selectively interacts with methylated histones in the chromatin flanking PREs to maintain a Polycomb-repressed chromatin state (Klymenko, 2006).

The regulation of gene expression by Polycomb group (PcG) and trithorax group (trxG) proteins represents a paradigm for understanding the establishment and maintenance of heritable transcriptional states during development. PcG and trxG genes were first genetically identified as regulators that are required for the long-term maintenance of HOX gene expression patterns in Drosophila. PcG proteins keep HOX genes silenced in cells in which they must stay inactive, whereas trxG proteins maintain the active state of these genes in appropriate cells. This regulatory relationship is conserved in vertebrates, where PcG and trxG proteins also regulate HOX gene expression. In addition, mammalian PcG and trxG proteins have also been implicated in X-chromosome inactivation, hematopoietic development, control of cell proliferation, and oncogenic processes (Klymenko, 2006).

Drosophila HOX genes are among the best-studied target genes of the PcG/trxG system. Different studies have led to the identification of specific cis-regulatory sequences in HOX genes that are called Polycomb response elements (PREs) and are required for silencing by PcG proteins. PREs are typically several hundred base pairs in length, and they function as potent transcriptional silencer elements in the context of HOX reporter genes as well as in a variety of other reporter gene assays. This operational definition of PREs is complemented by their classification as DNA sequences to which PcG proteins bind, directly or indirectly. Among the 14 cloned Drosophila PcG genes, only Pleiohomeotic (Pho) and Pho-like (Phol) encode sequence-specific DNA-binding proteins. Pho and Phol bind the same DNA sequence, and while the two proteins act to a large extent redundantly, double mutants show severe loss of HOX gene silencing. DNA-binding sites for Pho and Phol are present in all PREs that have been characterized to date, and mutational analyses of these binding sites have shown that they are essential for silencing by PREs. In contrast, none of the other 12 characterized PcG proteins bind DNA in a sequence-specific manner. However, formaldehyde cross-linking studies showed that several of these proteins specifically associate with the chromatin of PREs in tissue culture cells and in developing embryos and larvae. Biochemical studies revealed that most of these non-DNA-binding PcG proteins are components of either PRC1 or PRC2, two distinct PcG protein complexes that have recently been purified and characterized. Specifically, PRC1 contains the PcG proteins Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), Sex combs extra/Ring (Sce/Ring), and Sex combs on midleg (Scm), whereas PRC2 contains the three PcG proteins Extra sex combs (Esc), Enhancer of zeste [E(z)], and Suppressor of zeste 12 [Su(z)12] (Klymenko, 2006).

What is the role of Pho and Phol at PREs? Biochemically purified PRC1 and PRC2 do not contain Pho or Phol. Several recent studies investigated possible physical interactions between Pho and PRC1 or PRC2 complex components. Based on coimmunoprecipitation and GST pull-down assays, it was proposed that Pho directly interacts with several different PRC1, PRC2, and SWI/SNF complex components. However, on polytene chromosomes of phol; pho double mutants, the binding of PRC1 and PRC2 to HOX genes and at most other loci is largely unperturbed (Brown, 2003), suggesting that, at least in this tissue, Pho and Phol are not strictly required for keeping PRC1 and PRC2 anchored to HOX genes (Klymenko, 2006).

To gain insight into the biological function of Pho, Pho-containing protein complexes were biochemically purified from Drosophila. The data show that Pho exists in two distinct multiprotein complexes that, contrary to expectation, do not contain any of the previously characterized PcG proteins. The functional analysis of one of these Pho complexes that was named PhoRC provides evidence that its binding to PREs is required for maintaining repressive HOX gene chromatin (Klymenko, 2006).

A tandem affinity purification (TAP) strategy was used to purify Pho protein complexes from Drosophila embryonic nuclear extracts. A transgene that expresses a TAP-tagged Pho fusion protein (Pho-TAP) was expressed under the control of the Drosophila alpha-tubulin promoter, and transgenic flies were generated. To test whether the Pho-TAP protein is functional, the transgene was introduced into the genetic background of animals homozygous for pho1, a protein-negative allele of pho. pho1 homozygotes die as pharate adults, but they are rescued into viable and fertile adults that can be maintained as a healthy strain if they carry one copy of the transgene expressing Pho-TAP. The Pho-TAP protein can thus substitute for the endogenous Pho protein, and this shows that the fusion protein is functional (Klymenko, 2006).

Proteins that are associated with the Pho-TAP protein were purified from embryonic nuclear extracts, following the TAP procedure. Seven different polypeptides that consistently copurified with the Pho-TAP bait protein in several independent purifications were identified through sequencing of peptides from individual protein bands by nanoelectrospray tandem mass spectrometry. In addition to Pho, the isolated protein assembly contains the product of CG31212, a protein that is most closely related to yeast INO80, the SWI/SNF2-like nucleosome-remodeling subunit in the yeast INO80 complex. The CG31212 locus as will therefore be referred to as dINO80. Five other subunits of the Pho complex were identified as Reptin (Rept), Pontin (Pon), Actin (Act), and the two actin-related proteins dArp5 and dArp8, which are encoded by CG7940 and CG7846, respectively. These five proteins represent the Drosophila homologs of five core subunits that assemble together with INO80 to form the yeast INO80 complex. Specifically, Rept and Pont are homologs of the yeast Rvb1 and Rvb2 AAA-ATPases that constitute a DNA helicase in the INO80 complex. Act, dArp5, and dArp8 are homologs of the Actin, Arp5, and Arp8 proteins, respectively, that are present in the yeast INO80 complex. Thus, it appears that a Drosophila dINO80 complex copurifies with Pho. In addition, the purified material also contained the product of CG16975, a protein that is not conserved in yeast but is closely related to the product of the murine Scm-related gene containing four mbt domains (Sfmbt); the CG16975 gene is referred to as dSfmbt. The characteristic features of mammalian Sfmbt and the Drosophila dSfmbt protein are four malignant brain tumor (MBT) repeats and a sterile alpha motif (SAM) domain. The Drosophila genome encodes two other proteins that contain MBT repeats and show a similar domain architecture, l(3)mbt and the PcG repressor Scm. Taken together, these findings suggest that Pho exists in multiprotein assemblies that contain a dINO80 complex and dSfmbt but, unexpectedly, none of the previously characterized PcG proteins (Klymenko, 2006).

Since the yeast genome does not contain any dSfmbt-related protein, it was asked whether dSfmbt and dINO80 are part of distinct Pho protein complexes. To this end, crude embryonic nuclear extracts were fractionated by glycerol gradient sedimentation and individual fractions were probed by Western blotting with antibodies against Pho, Pho-like, dINO80, and dSfmbt. The results show that dINO80 and dSfmbt are present in separate fractions of the gradient but that Pho and Pho-like are present in both dINO80- and dSfmbt-containing fractions. dSfmbt and dINO80 thus exist in distinct protein complexes in embryonic nuclear extracts. It should be noted that Pho and Pho-like are also present in fractions that do not contain dINO80 or dSfmbt. This suggests that Pho and Pho-like also exists in soluble protein assemblies that are distinct from the complexes identified in this study, but that these assemblies are not stable enough to be isolated as complexes in the purification scheme (Klymenko, 2006).

It was asked whether components of the purified Pho complexes are associated with PREs in vivo. To this end, chromatin immunoprecipitation (X-ChIP) assays were performed. Drosophila embryos were treated with formaldehyde and DNA that was cross-linked to Pho, dSfmbt, dINO80, Reptin, Pontin, or Ph was immunoprecipitated with antibodies against these proteins. Real-time quantitative PCR was used to measure the abundance of the following endogenous and transgene PREs in the immunoprecipitates. The bxd and iab-7 PREs in the HOX genes Ultrabithorax (Ubx) and Abdominal-B (Abd-B), respectively, are well-characterized, and Pho binds to these PREs in vitro and in vivo. It has been reported that PRED, a 572-bp core fragment of the bxd PRE, silences a Ubx-LacZ reporter gene in imaginal discs and in embryos but that point mutations in all six Pho protein-binding sites in this fragment (PRED pho mut) completely abolish its silencing capacity (Fritsch, 1999). Therefore X-ChIP assays were performed in transformed embryos that carried either the wild-type PRED or the mutated PRED pho mut reporter gene; this allowed direct comparison of protein binding at the transgenic PRE with protein binding at the endogenous bxd and iab-7 PREs in the same preparation of chromatin. Specific PCR primer sets allowed X-ChIP signals at the reporter gene PRE to be distinguished from signals at the endogenous bxd PRE. It was found that Pho, Ph, and, importantly, also dSfmbt are specifically bound at the endogenous bxd and iab-7 PREs but not at sequences flanking those PREs. In contrast, binding of dINO80, Reptin, or Pontin at any of the sequences analyzed (data not shown). Pho, dSfmbt, and Ph are also bound at the PRED fragment in the transgene was not detected, but, strikingly, binding signals of Pho, dSfmbt, and Ph are severely reduced at the mutated PRED pho mut fragment. Taken together, these data show that Pho-dSfmbt complexes are bound at PREs in vivo and that binding of these complexes to PREs requires DNA-binding sites for Pho. Since association of dINO80 complex components with PREs was not detected in this assay, further analysis focused on the characterization of Pho-dSfmbt complexes (Klymenko, 2006).

Therefore, this study shows that the PcG protein Pho exists in two stable protein complexes, a Pho-dINO80 complex and PhoRC. Biochemical and genetic analyses identify PhoRC as a novel PcG protein complex that has a different subunit composition and molecular function than the previously described PcG complexes PRC1 and PRC2. The following conclusions can be drawn from studies studies of PhoRC: (1) PhoRC contains Pho and dSfmbt, and these two proteins form a very stable complex that can be purified from embryos and reconstituted from recombinant proteins. (2) PhoRC is bound to PREs in vivo, and PRE-targeting of PhoRC requires intact Pho/Pho-like DNA-binding sites. (3) A dSfmbt knockout reveals that dSfmbt is a novel PcG protein that is critically needed for HOX gene silencing. (4) The MBT repeats of dSfmbt are a novel methyl-lysine-recognizing module that selectively binds to the N-terminal tails of histones H3 and H4 if they are mono- or di-methylated at H3-K9 or H4-K20, respectively. PhoRC thus contains sequence-specific DNA-binding activity via the Pho protein and methylated histone-binding activity via dSfmbt (Klymenko, 2006).

Pho and Pho-like are the only PcG proteins with sequence-specific DNA-binding activity. Therefore, it is likely that these factors might tether PRC1 or PRC2 to PREs. Unexpectedly, biochemical purification of Pho complexes revealed that Pho exists in stable assemblies with either the PcG protein dSfmbt or components of the Drosophila INO80 complex. However, native or recombinant Pho complexes that contain PRC1 or PRC2 components were not purified. Similarly, biochemically purified PRC1 and PRC2 also do not contain Pho. PhoRC, PRC1, and PRC2 thus seem to be separate biochemical entities (Klymenko, 2006).

Reconstitution of recombinant PhoRC shows that dSfmbt binds directly to Pho or to Pho-like to form stable dimeric complexes. Coimmunoprecipitation assays indicate that such interactions also take place in Drosophila, and it was found that dSfmbt is associated with Pho or Pho-like in vivo. Moreover, dSfmbt mutants and pho-like; pho double mutants show a comparable loss of HOX gene silencing with similar kinetics. These observations are consistent with dSfmbt being needed for repression by both Pho and Pho-like. Furthermore, the X-ChIP experiments show that Pho/Pho-like DNA-binding sites in PREs are critical for binding of both Pho and dSfmbt at PREs. These data thus suggest that PhoRC is tethered to PREs by Pho or Pho-like (Klymenko, 2006).

Binding of the PRC1 subunit Ph at the bxd PRE also depends on intact Pho protein-binding sites. Could dSfmbt in PRE-bound PhoRC interact with Scm or Ph, for example, through the C-terminal SAM domain and thereby tether PRC1 to PREs? In coimmunoprecipitation experiments, no association of dSfmbt with Ph or Scm was detected. These interactions, if they exist, might be either very weak or exist only transiently. Previous studies reported direct physical interactions between Pho and PRC1 or PRC2 subunits, respectively. A possible scenario could therefore be that multiple weak interactions between Pho and dSfmbt with PRC1 and/or with PRC2 subunits might help to stabilize the binding of these complexes to PREs. It is also possible that the lack of Ph binding to the PRE transgene with mutated Pho sites reflects an indirect role of PhoRC that does not involve direct physical interactions between PhoRC and PRC1. In this context, it is worth noting that, on polytene chromosomes, binding of Ph and other PRC1 components is largely unperturbed in animals that lack both Pho and Pho-like proteins (Klymenko, 2006).

Four consecutive MBT repeats are a key feature of the dSfmbt protein. Fluorescence polarization binding assays suggest that these MBT repeats selectively bind to the N-terminal tail of histones H3 and H4 if these are mono- or di-methylated, but not if the same sites are unmethylated or tri-methylated. This novel discriminatory methyl-lysine-binding activity of MBTs is in stark contrast to the well-documented preference of chromodomains for higher, i.e., tri-methylated, binding sites in histones and could constitute an important general function of chromatin-associated MBT-containing proteins. The dSfmbt methyl-lysine interaction seems to be specific for the H3K9 and H4K20 methylation sites since matched H3 peptides that are methylated at different lysine residues (i.e., H3-K4me instead of H3-K9me) or histone tail peptides in which the methylated lysine residue is embedded in the same amino acid sequence context (i.e., ARKmeS in H3-K27me instead of ARKmeS in H3-K9me) are bound with at least 20-fold lower affinity (Klymenko, 2006).

Since these results suggest that dSfmbt is targeted to HOX gene PREs primarily through interaction with Pho, it was reasoned that binding to methyl-lysine residues in histone tails is not a primary mechanism for targeting dSfmbt to HOX genes. Moreover, recent studies provide evidence that, in the PcG-repressed state, the silenced HOX gene Ubx is tri-methylated at H3-K9, H4-K20, and H3-K27 throughout the gene, whereas lower methylated states of these sites are largely absent. What, then, is the role of Sfmbt in binding histones that are mono- or di-methylated at H3-K9 and H4-K20 in silenced HOX genes? Mono- and di-methylation of H4-K20 are very abundant modifications in Drosophila chromatin, and mass spectroscopic analyses of histones in embryos imply that lower methylated forms of histone H4 (i.e., H4-K20me2) already exist prior to becoming incorporated into chromatin during S phase. It is therefore tempting to speculate that dSfmbt, tethered to PREs by Pho, scans the flanking HOX gene chromatin for nucleosomes that are only mono- or di-methylated at H3-K9 or H4-K20 and docks onto such nucleosomes through its MBT repeats. It is hypothesized that through this bridging interaction, nucleosomes of lower methylated states might be brought into proximity to PRE-bound PRC2 and other currently unknown HMTases that are responsible for local tri-methylation of H3-K9 and H4-K20 in silenced HOX genes. According to this model, PRE-bound PhoRC would act as a 'grappling hook' that tethers mono- and di-methylated histones in silenced HOX gene chromatin to PREs to ensure that they become hypermethylated to the tri-methylated state. Such a chromatin-scanning function might be particularly important during S phase, when newly incorporated histone octamers need to become fully tri-methylated in order to maintain silencing of HOX genes (Klymenko, 2006).

Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells

Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3 -- Centromere identifier). However, the mechanistic basis for centromere maintenance is unknown. Biochemical properties were investigated of CenH3 nucleosomes from Drosophila cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical 'beads-on-a-string' appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity (Dalal, 2007; full text of article).

Nucleosomal core histones mediate dynamic regulation of Poly(ADP-ribose) polymerase 1 protein binding to chromatin and induction of its enzymatic activity

Poly(ADP-ribose) polymerase 1 protein [PARP1; see Poly-(ADP-ribose) polymerase] mediates chromatin loosening and activates the transcription of inducible genes, but the mechanism of PARP1 regulation in chromatin is poorly understood. This study found that Drosophila PARP1 interaction with chromatin is dynamic and that PARP1 is exchanged continuously between chromatin and nucleoplasm, as well as between chromatin domains. Specifically, the PARP1 protein preferentially interacts with nucleosomal particles, and although the nucleosomal linker DNA is not necessary for this interaction, the core histones, H3 and H4, are critical for PARP1 binding. Histones H3 and H4 interact preferentially with the C-terminal portion of PARP1 protein, and the N-terminal domain of PARP1 negatively regulates these interactions. Finally, it was found that interaction with the N-terminal tail of the H4 histone triggers PARP1 enzymatic activity. Therefore, the data collectively suggests a model in which both the regulation of PARP1 protein binding to chromatin and the enzymatic activation of PARP1 protein depend on the dynamics of nucleosomal core histone mediation (Pinnola, 2007).

This paper provides the first insight into the nature of the association of the PARP1 protein with chromatin in vivo and in vitro. The dynamics between free and chromatin-bound PARP1 protein were characterized, and an additional mechanism for these interactions is suggested. It was also demonstrated that PARP1 associates with chromatin on a monucleosomal level in vivo. More specifically, H3 and H4 are preferential binding sites for the C-terminal domain of PARP1 and that DNA is not required for this association in vitro. Histone H4 works as a strong DNA-independent activator of pADPr enzymatic reaction, whereas other histones (especially H2A) inhibit H4-dependent PARP1 activation (Pinnola, 2007).

PARP1 protein is exchanged rapidly between chromatin regions in the nucleus. No difference was detected between the recovery rate of enzymatically inactive PARPe-EGFP protein and active PARP1-DsRed protein isoforms. Therefore, it is proposed that PARP1 enzymatic activity is not required for steady-state dynamics. However, PARP1 inactivation followed by due automodification of PARP1 molecules has been shown to be critical for PARP1 protein removal from chromatin. The existence of two distinct mechanisms controlling PARP1 interaction with chromatin were detected as a result of sucrose gradient purification experiments. That is, unmodified PARP1 molecules co-purifies with nucleosomes, as well as other fractions (Complex I), whereas PARP1 molecules modified with pADPr were segregated to a separate fraction (Complex II). Based on this finding, it is concluded that, indeed, two distinct mechanisms conjoin to control PARP1 molecule interaction with chromatin. One involves a protein-equilibrated binding via association-dissociation, and the other involves irreversible removal of PARP1 from chromatin after automodification. Based on an accepted model, the existence of Complexes I and II was expectedPARP1 protein is associated with chromatin in its inactive state (Complex I), and upon activation it becomes automodified, loses contact with chromatin, and establishes interactions with pADPr-binding proteins (Complex II). Interestingly, the fraction with Complex II also contains a significant amount of unmodified PARP1. This may suggest that there is a nucleoplasmic pool of unmodified PARP1 that can reversibly bind to pADPr (Pinnola, 2007).

A three step model is presented for the regulation of PARP1 protein enzymatic activity in chromatin. Step 1: PARP1 protein is broadly distributed in chromatin because of interaction with core histones in the context of nucleosome. PARP1 is inactive in this state because of inhibitory effect of histone H2A. Step 2: genotoxic stress-dependent PARP1 activation. The N-terminal domain of PARP1 protein serves as a sensor of the double-stranded breaks or nicks in genomic DNA. Upon binding of damaged DNA, it mediates conformational changes, which leads to disruption of interaction with histones and consequently to the activation of PARP1 enzymatic reaction. Step 3: DNA-independent PARP1 activation. Developmental or environmental signals induces local changes in the "histone modification core" and subsequently expose the N-terminal tail of histone H4 and/or hide histone H2A followed by H4-dependent PARP1 activation (Pinnola, 2007).

Similar to H1, PARP1 controls the establishment of silenced chromatin (Tulin, 2002). Recently, it has been shown that PARP1 and H1 work independently. Moreover, they antagonize each other in chromatin. This antagonistic interaction strongly suggests competition for the same binding sites. The site of linker histone binding is known to be the linker DNA in the context of nucleosomal array. Unlike H1, linker DNA is not crucial for PARP protein binding. This, in turn, suggests that if H1 and PARP compete for binding sites, they recognize different but overlapping, epitopes (Pinnola, 2007).

The ability of PARP1 to bind chromatin via nicks in double-stranded DNA, as well as noncanonical DNA structures, has been demonstrated in vitro. Still, the broad PARP1 localization in chromatin in vivo suggests an alternative mechanism for PARP1 protein binding. Histones H2A and H2B have been reported as preferential targets for PARP1 binding in vitro (Buki, 1995) and for enzymatic modification by PARP1. In the current experiments, unmodified PARP1 protein always co-purified with core histones, even after DNA digestion to mononucleosomes. It was also found that the C terminus of PARP1 preferentially binds histones H3 and H4 of histone octamers lacking DNA. The PARP1 C terminus contains the catalytic domain and the sequence required for homodimerization and thus activation. PARP1 C terminus binding to H3/H4 may serve to sequester the domains in PARP1 that are required for activation, and this could account for the broad localization of PARP1 in chromatin. Histone H4 activates, whereas histone H2A completely inhibits, PARP1 protein. These findings support the conclusion that the PARP1 protein is generally silent (enzymatically inactive) in chromatin, although a number of developmental and environmental stimuli could still activate it at specific loci. This activation is required for chromatin decondensation and transcriptional activation in these loci. PARP1 activation always correlates with changes of local histone modification {e.g. phosphorylation of histone H3 co-localized with pADPr in Drosophila puffs}. Therefore, it is hypothesized that changes in histone modification code promote conformational alteration of nucleosomes and therefore expose (or hide) specific domains of histones, which activate (or inhibit) PARP1 (Pinnola, 2007).

Structural basis of histone H4 recognition by p55

p55 is a common component of many chromatin-modifying complexes and has been shown to bind to histones. This study presents a crystal structure of Drosophila p55 bound to a histone H4 peptide. p55, a predicted WD40 repeat protein, recognizes the first helix of histone H4 via a binding pocket located on the side of a β-propeller structure. The pocket cannot accommodate the histone fold of H4, which must be altered to allow p55 binding. Reconstitution experiments show that the binding pocket is important to the function of p55-containing complexes. These data demonstrate that WD40 repeat proteins use various surfaces to direct the modification of histones (Song, 2008).

Since p55 is a common subunit of many complexes, whether disrupting the binding pocket in p55 affects the activity of two of these complexes was examined. The human Hat1 complex is composed of the Hat1p catalytic subunit and RbAp48 (the human ortholog of p55), and acetylates free but not nucleosomal histone H4 at Lys5 and Lys12. Histone acetyltransferase (HAT) activity was compared with Hat1 complexes containing wildtype or H4-binding pocket mutants of RbAp48. Two mutants of RbAp48, equivalent to the p55 mutants, were constructed based on sequence alignment between RbAp48 and p55. Leu31 in the N-terminal helix and Asp358 and Asp361 in the binding loop of RbAp48 were mutated to alanines. Hat1 complexes were expressed and copurified with either wild-type or one of the mutant RbAp48 subunits. Wild-type and mutant RbAp48 form equivalently stable complexes with the Hat1 subunit, and all complexes behaved similarly during gel filtration chromatography, indicating that these mutations do not disrupt folding or complex formation. The Hat1 complexes containing mutant RbAp48 show significantly less HAT activity than the wild-type Hat1 complex. This supports a role for the H4-binding pocket of p55 in the acetyltransferase activity of the Hat1 complex (Song, 2008).

p55 binding to a buried region of histone H4 is consistent with the function of some, but not all, p55-containing complexes. The Hat1 and CAF-1 complexes are involved in histone assembly; the p55 subunit in these complexes most likely binds to free histone H4. p55 is also found in the ATP-dependent chromatin remodeling complexes NURF and NuRD. It is tantalizing to propose that the first helix of histone H4 becomes accessible to p55 during the ATP-dependent chromatin remodeling process where substantial structural changes in the nucleosome might occur. Another p55-containing complex, PRC2, methylates histone H3 at Lys27 in the intact nucleosome where the first helix of histone H4 is buried. It is therefore of interest to examine the function of p55 in this complex (Song, 2008).

To examine this, attempts were made to form PRC2 complexes that contained wild-type and mutant forms of p55. However, mutant forms of p55 would not stably associate with the remaining subunits of PRC2. This suggests that the binding pocket of p55 might be interacting with other subunits of PRC2 rather than with histone H4 within this complex. This does not appear to be caused by a general defect in the ability of these mutants to form interactions, as these same mutants are able to form a stable complex with hHAT complex and bind to histone H3. This observation implies an interesting function of the histone H4-binding pocket of p55; it might be utilized in different ways depending upon the role for p55 in the complex. Elucidating the functional role for p55 in PRC2 will require further biochemical and structural studies (Song, 2008).

The results presented in this study reveal the molecular basis for histone H4 recognition by p55. The structure of p55 bound to the first helix of histone H4 suggests that the canonical histone fold has to be altered upon p55 binding. Moreover, the histone H4-binding pocket of p55 plays a critical role in the function of p55-containing complexes. Together these data suggest that p55 might serve as a multifunctional protein interaction platform within the many p55-containing complexes (Song, 2008).

DNA polymerase alpha interacts with PrSet7 and mediates H4K20 monomethylation in Drosophila

In human cells appropriate mono-methylation of histone H4 lysine20 by PrSet7/SET8 is important for the correct transcription of specific genes, and timely progression through the cell cycle. Over-methylation appears to be prevented through the interaction of PrSet7 with PCNA, which targets PrSet7 destruction via the CRL4cdt2 pathway, however the factors involved in positive regulation of its histone methylation remain undefined. This study presents biochemical and genetic evidence for a previously undocumented interaction between dPrSet7 and DNA polymerase-α in Drosophila. Depletion of the polymerase reduces H4K20 mono-methylation suggesting that it is required for the expression of dPrSet7 histone methylation activity. It was also shown that the interaction between PCNA and PrSet7 is conserved in Drosophila, but is only detectable in chromatin fractions. Consistent with this, S2 cells show a significant loss of chromatin bound dPrSet7 protein as S phase progresses. Based on these data it is suggested that interaction with the DNA polymerase represents an important route for the expression of PrSet7 histone methylase activity, by allowing loading of dPrSet7 onto chromatin or its subsequent activation (Sahashi, 2014).

Histone H4: Biological Overview | Evolutionary Homologs | Developmental Biology | References

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