Histone H1


Transcriptional Regulation

The function of the neuronal differentiation gene daughterless is required for the proper initiation of neuronal lineage development in all peripheral nervous system (PNS) lineages following the selection of neuronal precursor cells. Previous studies have shown that the ubiquitously expressed Da protein is required for the proper expression of neuronal precursor genes and lineage identity genes in the PNS of Drosophila embryos. These genes are required for differentiation and cell fate determination in the developing PNS. These findings, however, do not explain the failure of the nascent PNS precursors to undergo a normal cell cycle and divide in da mutants. Four genes whose products are required for various stages of the cell cycle are misexpressed in the PNS of da mutant embryos. Cyclin A, barren, disc proliferation abnormal and Histone H1 transcripts are significantly reduced or undetectable in the precursors of the PNS at stages 11 and 12. Precursors are still present at these stages in da mutants. This suggests that all aspects of PNS precursor differentiation examined so far are under the transcriptional control of da. Sensory organ precursors lacking Da may fail to express and/or accumulate other factors, such as critical differentiation genes, required for SOP entry into the cell cycle. It should be pointed out that these factors are unlikely to be the thus-far described neuronal precursor genes, as mutations in these genes do not result in any obvious cell cycle defects (Hassan, 1997).

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).

ISWI regulates higher-order chromatin structure and histone H1 assembly in vivo

Imitation SWI (ISWI) and other ATP-dependent chromatin-remodeling factors play key roles in transcription and other processes by altering the structure and positioning of nucleosomes. Recent studies have also implicated ISWI in the regulation of higher-order chromatin structure, but its role in this process remains poorly understood. To clarify the role of ISWI in vivo, defects in chromosome structure and gene expression were examined resulting from the loss of Iswi function in Drosophila. Consistent with a broad role in transcriptional regulation, the expression of a large number of genes is altered in Iswi mutant larvae. The expression of a dominant-negative form of ISWI leads to dramatic alterations in higher-order chromatin structure, including the apparent decondensation of both mitotic and polytene chromosomes. The loss of ISWI function does not cause obvious defects in nucleosome assembly, but results in a significant reduction in the level of histone H1 associated with chromatin in vivo. These findings suggest that ISWI plays a global role in chromatin compaction in vivo by promoting the association of the linker histone H1 with chromatin (Corona, 2007; full text of article).

Most studies of ISWI complexes in Drosophila and other organisms have focused on their ability to alter the structure or spacing of nucleosomes, the fundamental unit of chromatin structure. These findings reveal that ISWI also plays a global role in the regulation of higher-order chromatin structure. The Iswi mutations used in this study eliminate the function of multiple chromatin-remodeling complexes, including ACF, NURF, and CHRAC. Which of these complexes are required for the formation of higher-order chromatin structure? Loss of function mutations in Acf1 -- which encodes a subunit protein shared by ACF and CHRAC -- do not cause obvious defects in higher-order chromatin structure. By contrast, loss of function mutations in E(bx) -- which encodes a subunit specific to NURF -- cause male X chromosome defects similar to those observed in Iswi mutants. These findings suggest that ISWI modulates higher-order chromatin structure within the context of NURF, as opposed to ACF or CHRAC (Corona, 2007).

A striking correlation was observed between the severity of the chromosome defects resulting from the loss of ISWI function and the loss of the linker histone H1. This correlation suggests that ISWI regulates higher-order chromatin structure by promoting the association of histone H1 with chromatin. Histone H1 and other linker histones influence higher-order chromatin structure in vitro by stabilizing interactions between nucleosomes and chromatin fibers. Although the ability of histone H1 to promote chromatin compaction in vitro is well established, its function in vivo has been a topic of considerable debate. A protein with biochemical properties reminiscent of linker histones -- HHO1 -- is present in budding yeast; surprisingly, HHO1 is not essential for viability in yeast, and hho1 mutations have little effect on either gene expression or chromatin structure. Genetic studies in Tetrahymena have suggested roles for linker histones in chromatin condensation and gene expression, but the relevance of these studies to histone H1 function in higher eukaryotes remains unclear. Studies of histone H1 function in higher eukaryotes have been complicated by the presence of redundant genes encoding histone H1 or histone H1 subtypes. In spite of these difficulties, recent studies have revealed important roles for histone H1 in chromosome compaction in Xenopus and mice. Thus, the chromosome defects observed in Iswi mutants could easily result from inefficient incorporation of histone H1 into chromatin (Corona, 2007).

How might ISWI promote the association of histone H1 with chromatin? Since ISWI is not required for histone H1 synthesis, ISWI may directly promote the assembly of chromatin containing histone H1 following DNA replication. Recent biochemical studies provide support for this possibility: ACF promotes the ATP-dependent assembly of H1-containing chromatin in vitro. Loss of ACF1 function does not cause obvious changes in chromosome structure, however, suggesting that ACF either does not regulate higher-order chromatin structure in vivo or plays a redundant role in this process. It remains possible that ISWI promotes the assembly of histone-H1-containing chromatin within the context of NURF or another chromatin-remodeling complex (Corona, 2007).

The ability to promote histone H1 assembly is not a common property of all chromatin-remodeling factors, as illustrated by recent biochemical studies of CHD1. Like ACF and other ISWI complexes, the CHD1 ATPase promotes the assembly of regularly spaced nucleosomes in vitro. By contrast, CHD1 does not promote the incorporation of histone H1 during chromatin assembly in vitro. These biochemical studies provide a plausible explanation for why the loss of ISWI function leads to the loss of histone H1 without causing dramatic changes in nucleosome assembly in vivo (Corona, 2007).

In other organisms, depletion of histone H1 leads to a significant decrease in the nucleosome repeat length, presumably because of the failure to efficiently incorporate histone H1 during replication-coupled chromatin assembly. By contrast, the loss of ISWI function in salivary gland nuclei leads to a decrease in the amount of histone H1 associated with chromatin without causing dramatic changes in nucleosome repeat length. It is therefore tempting to speculate that ISWI promotes histone H1 incorporation via a replication-independent process. The association of histone H1 with chromatin is far less stable than that of core histones; histone H1 undergoes dynamic, global exchange throughout the cell cycle. Photobleaching experiments in Tetrahymena and vertebrates have suggested that the majority of histone H1 molecules associated with chromatin are exchanged every few minutes, but little is known about the factors that regulate this process. Based on the current findings, ISWI is an excellent candidate for a factor that regulates the dynamic exchange of histone H1 in vivo. Further work will be necessary to determine whether ISWI promotes histone H1 incorporation via replication-dependent or -independent mechanisms (Corona, 2007).

These findings suggest that acetylation of H4K16 may regulate the association of linker histones with chromatin in vivo. The histone H4 tail is required for the nucleosome-stimulated ATPase activity of ISWI, and for its ability to slide nucleosomes and alter their spacing in vitro. The region of the H4 tail that is critical for ISWI function in vitro is a DNA-bound basic patch (R17H18R19) adjacent to H4K16, the residue that is acetylated by the MOF histone acetyltransferase. The acetylation of H4K16 interferes with the ability of ISWI to interact with the histone H4 tail and alter the spacing of nucleosome arrays in vitro. Consistent with these findings, dosage compensation is necessary and sufficient for the decondensation of the X chromosome in Iswi mutant larvae, and genetic studies have revealed a strong functional antagonism between ISWI and MOF. Thus, H4K16 acetylation may function as a switch that regulates the histone H1 assembly mediated by ISWI (Corona, 2007).

Microarray studies revealed that ISWI is required for the proper expression of a large number of genes. These findings are consistent with numerous studies implicating ISWI in transcriptional regulation in vitro and in vivo. Does ISWI modulate transcription by altering higher-order chromatin structure? It is suspected that ISWI regulates transcription and higher-order chromatin structure via distinct mechanisms, since no obvious correlation is observed between the magnitude of the changes in gene expression and chromosome structure observed in Iswi mutant larvae. This is consistent with genetic studies in other organisms that have revealed that the loss of histone H1 does not cause dramatic changes in gene expression. No correlation was observed between the magnitude of transcriptional derepression and gene size in Iswi mutant larvae, as would be expected if ISWI relieved a general block to transcriptional elongation by Pol II. It should be noted, however, that relatively subtle, but biologically important, changes in gene expression may have escaped detection in the microarray studies. Further work will be necessary to clarify this issue and to determine whether ISWI regulates transcription and higher-order chromatin structure via distinct or related mechanisms (Corona, 2007).

Drosophila ISWI regulates the association of histone H1 with interphase chromosomes in vivo

Although tremendous progress has been made toward identifying factors that regulate nucleosome structure and positioning, the mechanisms that regulate higher-order chromatin structure remain poorly understood. Recent studies suggest that the ISWI chromatin-remodeling factor plays a key role in this process by promoting the assembly of chromatin containing histone H1. To test this hypothesis, the function of H1 was investigated in Drosophila. The association of H1 with salivary gland polytene chromosomes is regulated by a dynamic, ATP-dependent process. Reducing cellular ATP levels triggers the dissociation of H1 from polytene chromosomes and causes chromosome defects similar to those resulting from the loss of ISWI function. H1 knockdown causes even more severe defects in chromosome structure and a reduction in nucleosome repeat length, presumably due to the failure to incorporate H1 during replication-dependent chromatin assembly. These findings suggest that ISWI regulates higher-order chromatin structure by modulating the interaction of H1 with interphase chromosomes (Siriaco, 2009).

These findings provide direct evidence that H1 is a major determinant of interphase chromosome structure and support the proposal that ISWI regulates higher-order chromatin structure by promoting the association of H1 with chromatin. The incorporation of H1 during replication-coupled chromatin assembly has a particularly dramatic effect on chromatin compaction. After chromatin has been assembled, the continued association of H1 with chromosomes, while important, appears to have more subtle effects on chromosome structure (Siriaco, 2009).

An independent analysis of phenotypes resulting from the knockdown of Drosophila His1 by RNAi was recently reported (Lu, 2009). Consistent with the current data, the authors of this study found that histone H1 is essential for Drosophila development. However, they observed relatively mild defects in salivary gland polytene chromosome structure following H1 knockdown. These defects appear similar to the weakest phenotypes observed following H1 knockdown, that may reflect differences in the extent of H1 knockdown achieved in the current studies. On the basis of the analysis of fixed polytene chromosome squashes following H1 depletion, Lu (2009) concluded that H1 is required for the alignment of sister chromatids in polytene chromosomes. Although an even stronger disruption of the banding pattern of polytene chromosome squashes was observed following H1 knockdown, such defects were rarely observed via live analysis. The data therefore argue against a major role for H1 in sister chromatid alignment and illustrate the importance of using live analysis to study factors involved in the regulation of higher-order chromatin structure (Siriaco, 2009).

The incorporation of H1 during replication-coupled chromatin assembly increases the average distance between nucleosomes, thus leading to a decrease in genomewide nucleosome density. Accordingly, a significant decrease was observed in nucleosome repeat length (NRL) following H1 knockdown. By contrast, the loss of ISWI function leads to a dramatic reduction in the level of H1 associated with chromosomes without causing obvious changes in NRL. These data strongly suggest that ISWI promotes the association of H1 with salivary gland polytene chromosomes via a replication-independent mechanism. It remains possible that an additional role for ISWI in replication-coupled H1 assembly escaped detection in the genetic studies due to the failure to completely eliminate ISWI function during the stages of salivary gland development when the bulk of DNA replication occurs. Further experiments, including the analysis of fast-acting conditional ISWI alleles, will be required to address this issue (Siriaco, 2009).

How does ISWI promote the association of H1 with chromatin? By altering the structure, accessibility or fluidity of chromatin, ISWI may facilitate the binding of H1 to chromatin during dynamic exchange. Consistent with this possibility, it was found that inhibitors of oxidative phosphorylation lead to the dissociation of H1 from polytene chromosomes accompanied by its accumulation in the nucleoplasm. Alternatively, ISWI may stabilize the binding of H1 to chromatin by influencing its phosphorylation. H1 is phosphorylated in most organisms, including Drosophila. In both Tetrahymena and mammals, the phosphorylation of H1 weakens its association with chromatin, leading to an increased frequency of H1 exchange. Thus, ISWI may indirectly promote the association of H1 with chromatin by altering the level or activity of an H1 kinase or phosphatase (Siriaco, 2009).

The chromatin of stem cells is hyperdynamic, with both histone H1 and other chromatin-associated proteins undergoing highly elevated rates of exchange. This property of pluripotent cell types appears to be functionally important, since a mutant form of H1 that tightly binds chromatin blocks stem cell differentiation. These findings suggest that ISWI and other factors that regulate the association of H1 with chromatin may play important roles in the regulation of cellular pluripotency and differentiation. This possibility is intriguing in light of recent studies implicating ISWI in both nuclear reprogramming and stem cell self-renewal (Siriaco, 2009).

Previous studies have shown that the dosage compensation machinery antagonizes ISWI function via the acetylation of its nucleosome substrate on H4K16. Furthermore, increased linker histone exchange has been observed in active chromatin enriched in core histone acetylation. It is therefore tempting to speculate that the dynamic association of H1 with chromatin is modulated by the interplay of chromatin-remodeling and -modifying enzymes, thus providing a straightforward mechanism for creating rapid, readily reversible changes in higher-order chromatin structure and gene expression. Further work will be required to test this hypothesis and clarify the molecular mechanisms that regulate the association of H1 with chromatin in vivo (Siriaco, 2009).

ACF catalyses chromatosome movements in chromatin fibres

Nucleosome-remodelling factors containing the ATPase ISWI, such as ACF, render DNA in chromatin accessible by promoting the sliding of histone octamers. Although the ATP-dependent repositioning of mononucleosomes is readily observable in vitro, it is unclear to which extent nucleosomes can be moved in physiological chromatin, where neighbouring nucleosomes, linker histones and the folding of the nucleosomal array restrict mobility. In this study arrays were assembled consisting of 12 nucleosomes or 12 chromatosomes (nucleosomes plus linker histone) from defined components and subjected to remodelling by Drosophila ACF or the ATPase CHD1. Both factors increased the access to DNA in nucleosome arrays. ACF, but not CHD1, catalysed profound movements of nucleosomes throughout the array, suggesting different remodelling mechanisms. Linker histones inhibited remodelling by CHD1. Surprisingly, ACF catalysed significant repositioning of entire chromatosomes in chromatin containing saturating levels of linker histone H1. H1 inhibited the ATP-dependent generation of DNA accessibility by only about 50%. This first demonstration of catalysed chromatosome movements suggests that the bulk of interphase euchromatin may be rendered dynamic by dedicated nucleosome-remodelling factors (Maier, 2008).

Due to the abundance of linker histones in interphase chromatin, H1-containing nucleosome arrays are probably the most common and physiological substrate for ATP-dependent chromatin remodelling factors. It is therefore important to understand whether and how these complexes can deal with the linker histone. So far, the literature mostly suggested that linker histones hinder chromatin remodelling. Residual remodelling activity has largely been attributed to incomplete loading of the substrate with linker histones. Attempts were made to rule out this experimental shortcoming by tightly controlling the stoichiometric incorporation of linker histones into chromatin arrays. Yet, ACF was able to induce the movement of entire chromatosome units throughout extended arrays. Importantly, the inability of CHD1 to remodel H1-containing chromatin confirms the inhibitory nature of the chromatosome array. These data are in accordance with previous findings in a crude, undefined system that nucleosome movements can occur within H1-containing chromatin, but they present the first direct demonstration of ATP-dependent chromatosome mobility in a defined chromatin array (Maier, 2008).

The results are surprising in light of the documented impediments of linker histones on nucleosome remodelling. First, H1 binding limits the amount of free linker DNA, which is known to determine the efficiency of ACF-dependent remodelling (Yang, 2006; Gangaraju, 2007). Second, H1 is likely to compete with ISWI-type remodellers for nucleosomal binding sites. In addition, H1 is believed to constrain the path of DNA entering and exiting the nucleosome and may therefore hinder DNA translocation. Finally, the increased compaction promoted by linker histones might restrict the access of remodelling factors towards the chromatin fibre. According to both currently favoured models for the structure of the 30-nm fibre, the linker DNA and hence all points of access for remodelling enzymes are located inside the chromatin fibre. The cation concentrations in these experiments promote the compaction of the nucleosomal array (Maier, 2008).

In spite of these possible constraints, a considerable ACF- and ATP-dependent repositioning of chromatosomes was observed. It is considered that H1 purified from Drosophila embryos might carry modifications, decreasing its affinity for chromatin. For example, the extensive phosphorylation of linker histone C-termini interferes with DNA binding and relieves its inhibitory impact on SWI/SNF-dependent chromatin remodelling. However, mass spectrometrical analysis of histone H1 purified from Drosophila embryos did not reveal extensive phosphorylation. It is therefore considered unlikely that phosphorylation impacted the outcome of these experiments (Maier, 2008).

The inhibitory effect of histone H1 on nucleosome remodelling was apparent when CHD1 was used as a remodelling enzyme. Notably, CHD1's activity on nucleosome arrays was equal to that of ACF, ruling out a defective activity of CHD1. Rather, ACF appears particularly suited for coping with linker histones. This is supported by the observation that ACF can assist the assembly of H1-containing chromatin arrays, whereas CHD1 can only promote assembly of H1-free chromatin. Recently, the ISWI-containing remodelling factor NURF has been suggested to be involved in modulating the association of H1 with chromosomes in vivo (Corona, 2007). The ability to slide chromatosomes may thus be a more widespread property of remodelling enzymes (Maier, 2008).

How might ACF achieve chromatosome repositioning? ACF may directly catalyse the eviction of H1 before nucleosome sliding, and a number of reports indicate that nucleosome-remodelling factors can, in principle, disrupt the DNA interactions of other proteins than core histones. Although it was not possible to detect free linker histones during remodelling, the analysis does not exclude that a fraction of H1 is transiently dislocated to secondary sites on the nucleosome array or an acceptor site on ACF. In vivo, linker histone displacement may be facilitated by cooperating histone chaperones. ACF and the histone chaperone NAP1 can act in concert towards the assembly of H1-containing chromatin, and it is thus conceivable that in cells ACF may cooperate with chaperones to catalyse the reverse reaction, which is the eviction of linker histones. However, since no chaperone was included in this experiment, alternative mechanisms have to be considered (Maier, 2008).

Chromatosome movements might already be facilitated if only the linker histone's globular domain was transiently detached from the nucleosome, while the C-terminal tail remained associated with the linker DNA. Such a scenario is reminiscent of documented changes on H1 interaction due to transcription, where selective crosslinking in Drosophila showed that the globular domain but not the C-terminal tail of linker histones was reversibly displaced from chromatin. In line with these considerations, the C-terminal tail contributes to H1 binding to DNA and determines its residence time on chromatin in living cells (Maier, 2008).

The analysis of chromatosome positions by primer extension revealed that in the arrays H1 protects DNA from nuclease digestion only on one side of the nucleosome, suggesting an asymmetrical binding of H1. This asymmetrical interaction, combined with the repetitive nature of the 601 array, endows the entire array with directionality. Although the precise topography of the ACF-nucleosome complex is not known at present, it has suggested on the basis of site-directed DNA affinity labelling that the related ISW2 complex interacts with linker DNA only on one side of the nucleosome (Kagalwala, 2004; Dang, 2006). It is thus speculated that ACF may interact with nucleosomal linker on the side that is not contacted by the globular domain of H1, in order to initiate the remodelling reaction. Propagation of a 'looped segment' of DNA around the histone octamer would then lead to movement of the histone octamer and concomitant displacement of the globular domain. The domain would then have to relocate and bind to the new nucleosome dyad and DNA entry point. A testable prediction of this hypothesis is that nucleosome sliding in presence of H1 would be unidirectional (Maier, 2008).

It is not knowm at this point whether ACF distributively targets individual nucleosomes within a nucleosome array or rather remodels neighbouring nucleosomes processively. In the latter case the fibre ends may provide points of entry. However, restriction enzyme accessibility assays did not reveal a gradient of increased accessibility towards the ends of the array, as might be expected from such a scenario. In contrast ACF is known to remain bound to its initial substrate during chromatin assembly, and it was observed earlier that nucleosomes within extended arrays were repositioned by Drosophila embryonic extract in apparent synchrony. Further experiments are required to clarify this issue (Maier, 2008).

This study provides the first evidence that ATP-dependent nucleosome-remodelling factors can mobilize entire chromatosomes, even if they reside in extensive arrays. Hence, the majority of euchromatin might be characterized by mobile nucleosomes and chromatosomes (Maier, 2008).

Systematic protein location mapping reveals five principal chromatin types in Drosophila cells

Chromatin is important for the regulation of transcription and other functions, yet the diversity of chromatin composition and the distribution along chromosomes are still poorly characterized. By integrative analysis of genome-wide binding maps of 53 broadly selected chromatin components in Drosophila cells, this study shows that the genome is segmented into five principal chromatin types (see Chromatin types are characterized by distinctive protein combinations and histone modifications) that are defined by unique yet overlapping combinations of proteins and form domains that can extend over > 100 kb. A repressive chromatin type was identified that covers about half of the genome and lacks classic heterochromatin markers. Furthermore, transcriptionally active euchromatin consists of two types that differ in molecular organization and H3K36 methylation and regulate distinct classes of genes. Finally, evidence is provided that the different chromatin types help to target DNA-binding factors to specific genomic regions. These results provide a global view of chromatin diversity and domain organization in a metazoan cell (Filion, 2010).

By systematic integration of 53 protein location maps this study found that the Drosophila genome is packaged into a mosaic of five principal chromatin types, each defined by a unique combination of proteins. Extensive evidence demonstrates that the five types differ in a wide range of characteristics besides protein composition, such as biochemical properties, transcriptional activity, histone modifications, replication timing, DNA binding factor (DBF) targeting, as well as sequence properties and functions of the embedded genes. This validates the classification by independent means and provides important insights into the functional properties of the five chromatin types (Filion, 2010).

Identifying five chromatin states out of the binding profiles of 53 proteins comes out as a surprisingly low number (one can form approximately 1016 subsets of 53 elements). It is emphasized that the five chromatin types should be regarded as the major types. Some may be further divided into sub-types, depending on how fine-grained one wishes the classification to be. For example, within each of the transcriptionally active chromatin types, promoters and 3' ends of genes exhibit (mostly quantitative) differences in their protein composition and thus could be regarded as distinct sub-types. However, these local differences are minor relative to the differences between the five principal types that are described in this study. It cannot be excluded that the accumulation of binding profiles of additional proteins would reveal other novel chromatin types. It is also anticipated that the pattern of chromatin types along the genome will vary between cell types. For example, many genes that are embedded in 'BLACK' chromatin (defined in Kc167 cells) are activated in some other cell types. Thus, the chromatin of these genes is likely to switch to an active type (Filion, 2010).

While the integration of data for 53 proteins provides substantial robustness to the classification of chromatin along the genome, a subset of only five marker proteins (histone H1, PC, HP1, MRG15 and BRM), which together occupy 97.6% of the genome, can recapitulate this classification with 85.5% agreement. Assuming that no unknown additional principal chromatin types exist in some cell types, DamID or ChIP of this small set of markers may thus provide an efficient means to examine the distribution of the five chromatin types in various cells and tissues, with acceptable accuracy (Filion, 2010).

Previous work on the expression of integrated reporter genes had suggested that most of the fly genome is transcriptionally repressed, contrasting with the low coverage of PcG and HP1-marked chromatin. BLACK chromatin, which consists of a previously unknown combination of proteins and covers about half of the genome, may account for these observations. Essentially all genes in BLACK chromatin exhibit extremely low expression levels, and transgenes inserted in BLACK chromatin are frequently silenced, indicating that BLACK chromatin constitutes a strongly repressive environment. Importantly, BLACK chromatin is depleted of PcG proteins, HP1, SU(VAR)3-9 and associated proteins, and is also the latest to replicate, underscoring that it is different from previously characterized types of heterochromatin (identified as BLUE and GREEN chromatin in this study) (Filion, 2010).

The proteins that mark BLACK domains provide important clues to the molecular biology of this type of chromatin. Loss of Lamin (LAM), Effete (EFF) or histone H1 causes lethality during Drosophila development. Extensive in vitro and in vivo evidence has suggested a role for H1 in gene repression, most likely through stabilization of nucleosome positions. The enrichment of LAM points to a role of the nuclear lamina in gene regulation in BLACK chromatin, consistent with the long-standing notion that peripheral chromatin is silent. Depletion of LAM causes derepression of several LAM-associated genes (Shevelyov, 2009), while artificial targeting of genes to the nuclear lamina can reduce their expression, suggesting a direct repressive contribution of the nuclear lamina in BLACK chromatin. D1 is a little-studied protein with 11 AT-hook domains. Overexpression of D1 causes ectopic pairing of intercalary heterochromatin (Smith, 2010), suggesting a role in the regulation of higher-order chromatin structure. SUUR specifically regulates late replication on polytene chromosomes (Zhimulev, 2003), which is of interest because BLACK chromatin is particularly late-replicating. EFF is highly similar to the yeast and mammalian ubiquitin ligase Ubc4 that mediates ubiquitination of histone H3, raising the possibility that nucleosomes in BLACK chromatin may carry specific ubiquitin marks. These insights suggest that BLACK chromatin is important for chromosome architecture as well as gene repression and provide important leads for further study of this previously unknown yet prevalent type of chromatin (Filion, 2010).

In RED and YELLOW chromatin most genes are active, and the overall expression levels are similar between these two chromatin types. However, RED and YELLOW chromatin differ in many respects. One of the conspicuous distinctions is the disparate levels of H3K36me3 at active transcription units. This histone mark is thought to be laid down in the course of transcription elongation and may block the activity of cryptic promoters inside the transcription unit. Why active genes in RED chromatin lack H3K36me3 remains to be elucidated (Filion, 2010).

The remarkably high protein occupancy in RED chromatin suggests that RED domains are 'hubs' of regulatory activity. This may be related to the predominantly tissue-specific expression of genes in RED chromatin, which presumably requires many regulatory proteins. It is noted that the DamID assay integrates protein binding events over nearly 24 hours, so it is likely that not all proteins bind simultaneously; some proteins may bind only during a specific stage of the cell cycle. It is highly unlikely that the high protein occupancy in RED chromatin originates from an artifact of DamID, e.g. caused by a high accessibility of RED chromatin. First, all DamID data are corrected for accessibility using parallel Dam-only measurements. Second, several proteins, such as EFF, SU(VAR)3-9 and histone H1 exhibit lower occupancies in RED than in any other chromatin type. Third, ORC also shows a specific enrichment in RED chromatin, even though it was mapped by ChIP, by another laboratory and on another detection platform. Fourth, DamID of Gal4-DBD does not show any enrichment in RED chromatin (Filion, 2010).

RED chromatin resembles DBF binding hotspots that were previously discovered in a smaller-scale study in Drosophila cells. Discrete genomic regions targeted by many DBFs have recently also been found in mouse ES cells , hence it is tempting to speculate that an equivalent of RED chromatin may also exist in mammalian cells. Housekeeping and dynamically regulated genes in budding yeast also exhibit a dichotomy in chromatin organization which may be related to the distinction between YELLOW and RED chromatin. The observations that RED chromatin is generally the earliest to replicate and strongly enriched in ORC binding, suggest that this chromatin type may be not only involved in transcriptional regulation but also in the control of DNA replication (Filion, 2010).

This analysis of DBF binding indicates that the five chromatin types together act as a guidance system to target DBFs to specific genomic regions. This system directs DBFs to certain genomic domains even though the DBF recognition motifs are more widely distributed. It is proposed that targeting specificity is at least in part achieved through interactions of DBFs with particular partner proteins that are present in some of the five chromatin types but not in others. The observation that yeast Gal4-DBD binds its motifs with nearly equal efficiency in all five chromatin types suggests that differences in compaction among the chromatin types represent overall a minor factor in the targeting of DBFs. Although additional studies will be needed to further investigate the molecular mechanisms of DBF guidance, the identification of five principal types of chromatin provides a firm basis for future dissection of the roles of chromatin organization in global gene regulation (Filion, 2010).

Protein Interactions

A cell free system of Drosophila preblastoderm embryos was devised for the efficient assembly of cloned DNA into chromatin. The chromatin assembly system utilizes endogenous core histones and assembly factors and yields long arrays of regularly spaced nucleosomes with repeat length of 180 bp. Chromatin assembled with the preblastoderm embryo extract is deficient in histone H1, because of the absence of H1 in early embryos. Exogenous H1 can be incorporated during nucleosome assembly in vitro. When chromatin is reconstituted in the presence of H1, an increased nucleosome repeat length is observed, from 180 bp to about 197 bp, identical to the in vivo repeat length for postblastoderm chromatin. Regular spacing of nucleosomes with or without H1 is sufficient to maximally repress transcription from hsp70 and fushi tarazu gene promoters. There is a modest increase in the level of repression that is dependent on exogenous histone H1. These results show that optimal assembly or regularly spaced nucleosome cores is sufficient to maximally repress transcription in vitro, even in the absence of histone H1 (Becker, 1992).

Chromatin structure must be flexible to allow the binding of regulatory proteins and to accommodate different levels of gene activity. Chromatin assembled in a cell-free system derived from Drosophila embryos contains an activity that hydrolyses ATP to render entire nucleosome arrays mobile (See ISWI). Nucleosome movements (most likely their sliding) occurs even in the presence of the linker histone H1. Binding of more than one linker histone per nucleosome leads to a further compaction of chromatin or aggregation. This could explain the loss of accessability to endonuclease cleavage. The dynamic state of chromatin in the presence of the ATP hydrolysing factor and ATP globally increases the accessibility of nucleosomal DNA to incoming proteins. This increase can even take place in the presence of H1, but only when H1 is limited to one histone unit per nucleosome. Such an unprecedented demonstration of energy-dependent nucleosome mobility identifies a new principle that is likely to be fundamental to the mechanism of chromatin remodeling and the binding of regulatory proteins (Varga-Weisz, 1995).

Chromatin reconstituted in an extract taken from preblastoderm Drosophila embryos represses transcription by RNA polymerase II. Transcriptional repression of immobilized chromatin is largely due to nucleosome cores. When purified H1 is incorporated into chromatin (resulting in increased repeat lengths to 200-220 bp) the contribution of H1 to transcriptional repression is negligible. If more H1 is added no regularly spaced chromatin is obtained; only under these conditions is transcriptional inhibition by H1 apparent. It has been concluded that efficient repression of transcription by polymerase II in this system does not require the presence of histone H1 (Sandaltzopoulos, 1994).

The 5'-untranslated region of the Drosophila gypsy retrotransposon contains an "insulator," which disrupts the interactions between distally located enhancers and proximal promoter elements. The insulator effect is dependent on the suppressor of Hairy-wing (su[Hw]) protein, which binds to reiterated sites within the 350 base pairs of the gypsy insulator, and additionally acts as a transcriptional activator of gypsy. This study shows that the 350-base pair su(Hw) binding site-containing gypsy insulator behaves as a matrix/scaffold attachment region (MAR/SAR), involved in interactions with the nuclear matrix. In vitro experiments using nuclear matrices from Drosophila, murine, and human cells demonstrate specific binding of the gypsy insulator, not observed with any other sequence within the retrotransposon. Moreover, it is shown that the gypsy insulator, like previously characterized MAR/SARs, specifically interacts with topoisomerase II and histone H1, i.e. with two essential components of the nuclear matrix. Experiments within cells in culture demonstrate differential effects of the gypsy MAR sequence on reporter genes, namely no effect under conditions of transient transfection and a repressing effect in stable transformants, as expected for a sequence involved in chromatin structure and organization (Nabirochkin, 1998).

The presence of a MAR/SAR within gypsy is not totally unexpected, since "boundary" elements are in general regions which contain not only enhancer and insulating elements, but also matrix attachment domains. The rather original feature of the gypsy sequence is that all three domains, which in general are sufficiently "dispersed" so as to allow isolation of "pure" enhancers, MAR/SAR, or insulators, are in the present case "gathered" within a single and relatively short (350 bp) sequence. This rather uncommon situation might in fact be relevant to the pressure for compactness within retroviral sequences, as it is known that retroviruses can only package a limited amount of genetic information. A consequence of compaction is that the gypsy insulator and its associated components are most probably interacting, in vivo, with elements of the nuclear matrix. Accordingly, proteins of the nuclear matrix might play a role in the insulation process, and conversely the su(Hw) protein (which is essential for insulation) might interact with proteins of the matrix. Such interactions could actually account for the data on gypsy insulation and fit with previously proposed models for the gypsy effects (Nabirochkin, 1998).

A first series of data strongly suggested that the gypsy insulator, like all previously characterized insulators, essentially prevents interactions between distal enhancer and promoter, without any direct repressing effect on the enhancer itself. This directional effect can most easily be accounted for by the "looping model" involving generation of structural domains isolated one from the other by attachment of boundary sequences (MAR/SAR) to the nuclear matrix. Alternatively, a series of data on gypsy insulation (essentially in mod(mdg4) mutants) discloses bidirectional repressing effects, which can be accounted for by a model involving heterochromatinization. The present data (showing that the gypsy insulator behaves as a MAR/SAR) are clearly in agreement with the structural looping model, but also support the heterochromatinization model. Indeed, the gypsy MAR/SAR DNA per se, in the absence of su(Hw) protein, is involved in histone H1 nucleation (as shown in this paper), and it has been demonstrated that histone H1 nucleation is associated with both DNA compaction and transcriptional silencing. Additionally, Laemmli and co-workers have found that histone H1 can be removed from MAR/SAR domains by distamycin and distamycin-like proteins (D-like proteins, such as the high mobility group proteins); this has led to the proposal that MAR/SARs can activate or repress transcription of adjacent genes depending on the nucleation/depletion of histone H1. The gypsy MAR/SAR could then be responsible for the repressing effect observed in the mod(mdg4) mutants, as well as in the present assay within heterologous cells (assuming further that appropriate D-like proteins are absent in those cells). Taking into account, in addition, that mutations in the mod(mdg4) or the su(Hw) genes modify position-effect variegation, it could be further hypothesized that the su(Hw)/mod(Mdg4) complex acts as the D-like proteins and modifies the nucleation processes to allow the switch from a repressing to an active state. Accordingly, a model in which the su(Hw) binding sites and the associated su(Hw)/mod(Mdg4) complex modulate the effects of the MAR/SAR DNA sequence could rather simply account for the biological effects of the gypsy insulator in both the wild type and su(Hw)/mod(mdg4) mutants. The proposed model would then reconcile the two previous models for gypsy insulation, i.e. the heterochromatinization and the looping models (Nabirochkin, 1998 and references).

Histone H1 is ubiquitinated by TAFII250

Ubiquitination of histones has been linked to the complex processes that regulate the activation of eukaryotic transcription. However, the cellular factors that interpose this histone modification during the processes of transcriptional activation are not well characterized. A biochemical approach has identified the Drosophila coactivator TAFII250, the central subunit within the general transcription factor TFIID, as a histone-specific ubiquitin-activating/conjugating enzyme (ubac). TAFII250 mediates monoubiquitination of histone H1 in vitro. Point mutations within the putative ubac domain of TAFII250 abolish H1-specific ubiquitination in vitro. In the Drosophila embryo, inactivation of the TAFII250 ubac activity reduces the cellular level of monoubiquitinated histone H1 and the expression of genes targeted by the maternal activator Dorsal. Thus, coactivator-mediated ubiquitination of proteins within the transactivation pathway may contribute to the processes directing activation of eukaryotic transcription (Pham, 2000).

Polyubiquitination represents a mark on proteins that identifies them for degradation and requires the involvement of three enzymes: (1) ubiquitin-activating enzymes (E1), which mediate the adenosine triphosphate (ATP)-dependent conjugation of E1 with ubiquitin via a covalent thioester linkage; (2) ubiquitin-conjugating enzymes (E2), which mediate the transfer of ubiquitin from E1 to E2, conjugate ubiquitin via thioester bonds and, (3) together with ubiquitin-protein ligase (E3), link ubiquitin to target proteins via isopeptide bonds. Polyubiquitination requires all three enzymes, whereas monoubiquitination of proteins requires E1 and E2 activities only. Unlike polyubiquitination, monoubiquitination of histones has been correlated with activation of gene expression. However, the functional connections between histone ubiquitination and activation of gene expression remain unknown. Thus, as a first step toward understanding the role of histone ubiquitination for transcriptional regulation, attempts were made to identify enzymes that ubiquitinate histones in Drosophila embryonic nuclear extract using an activity gel assay (Pham, 2000).

Nuclear extract was separated in SDS-polyacrylamide gels containing histones. After electrophoresis (SDS-PAGE), gel-bound proteins were subsequently denatured, renatured, and, to monitor enzymatic activities, incubated with 32P-labeled ubiquitin. By using this assay, a protein was identified with a molecular mass of approximately 200 kD that mediates ubiquitination of histones. The 200-kD activity coincides with TAFII250, suggesting that TAFII250 may ubiquitinate histones (Pham, 2000).

TAFII250 most likely does not interact with E1, E2, or E3 enzymes. Since mono-ubiquitination requires at least E1 and E2 activities, these results imply that TAFII250 may have intrinsic E1 and E2 activities. The ubiquitin/H1 conjugates resisted reducing agents, suggesting that TAFII250 may mediate a covalent bond between ubiquitin and H1 by means of isopeptide linkages. Since this enzymatic reaction is characteristic for E2 enzymes, TAFII250 may have intrinsic E2 activity. The E1 enzyme requires ATP to conjugate with ubiquitin by means of thioester bonds. Therefore, to explore whether TAFII250 has E1 activity, the capability of TAFII250 for conjugating with ubiquitin by means of thioester bonds was investigated. TAFII250 conjugated with ubiquitin in an ATP-dependent manner in the absence, but not in the presence, of reducing agents, suggesting that TAFII250 and ubiquitin form a covalent bond by means of a thioester linkage. Thus, TAFII250 may have both E1 and E2 activities and may therefore be a ubac (Pham, 2000).

To provide supporting evidence that TAFII250 mediates ubiquitination of H1, solution assays were used. Reactions containing TAFII250, 32P-labeled ubiquitin, H1, and ATP mediate the formation of a 39-kD protein that is recognized by antibodies to both ubiquitin and H1. These results suggest that the 39-kD protein represents a conjugate composed of one ubiquitin moiety (7 kD) and H1 (32 kD). By contrast, TAFII250 does not ubiquitinate other histones, H2A/H2B dimers, H3/H4 tetramers, or core nucleosomes. Thus, TAFII250 mediates monoubiquitination of H1 (Pham, 2000).

To determine the portion of TAFII250 that mediates monoubiquitination of H1, TAFII250 mutants truncated at the COOH-terminal were used. Membrane assays indicate that full-length TAFII250 and 250deltaC850 (lacking the 850 amino acids closest to the COOH-terminal), but not 250deltaC1300 (lacking the 1300 amino acids closest to the COOH-terminal) ubiquitinate H1. Thus, the H1-specific ubac activity is likely to reside between amino acids 768 and 1218 (Pham, 2000).

Two Drosophila TAF250 alleles, TAF250XS-2232 and TAF250S-625, have been described that contain single-amino acid point mutations that reside within the putative TAFII250 ubac domain. TAF250XS-2232 contains a valine-1072 to aspartic acid change, and TAF250S-625 an arginine-1096 to proline change. To investigate the effect of these mutations on TAFII250 ubac activity, the middle region of TAFII250 containing amino acids 612 to 1140 (TAF250-M), TAFII250-M-V1072D (containing the V1072 to D mutation), and TAFII250-M-R1096P (containing the R1096 to P mutation) were subjected to membrane assays. Although TAF250-M ubiquitinates H1, the mutants do not. Wild-type and mutant TAFII250-M proteins have histone acetyltransferase activity. The TAF250-M proteins (used for the membrane assays) acetylate histones; this suggests that the lack of ubac activity seen with TAF250-M-V1072D and TAFII250-M-R1096P is most likely not due to a general functional inactivity of the mutant proteins (Pham, 2000).

In Drosophila, TFIID mediates transcriptional activation by the maternal activator Dorsal. Dorsal activates the expression of the mesoderm-determining genes twist (twi) and snail (sna), which are transcribed in 20 and 18 of the ventral-most cells of cellularizing embryos, respectively. To investigate the functional relevance of TAFII250 ubac activity for Dorsal-dependent transcriptional activation in vivo, in situ hybridization was used to monitor twi and sna expression in Drosophila embryos containing reduced levels of Dorsal and expressing TAFII250XS-2232 or TAFII250S-625, which lack ubac activity in vitro. Both twi and sna expression are severely reduced in dl-sensitized, TAF250XS-2232 embryos and dl-sensitized, TAF250S-625 embryos, but not in control embryos. Weak twi mRNA levels were detectable in 10 to 12 cells, and sna expression was restricted to 4 to 12 ventral-most cells and disrupted by gaps. Analyses of cuticular preparations revealed that dl-sensitized TAF250XS-2232 mutants or dl-sensitized TAF250S-625 mutants, but not control embryos exhibit a dorsalized and twisted body pattern. These results indicate that Dorsal-dependent activation of transcription is impaired in embryos lacking TAFII250 ubac activity (Pham, 2000).

To investigate whether H1 may represent a target for TAFII250 ubac activity in Drosophila, H1 was purified from nuclei prepared from 0- to 3-hour-old wild-type and TAFII250 mutant embryos. Western blot analyses indicate that antibodies to both H1 and ubiquitin detect a monoubiquitin/H1 conjugate. This result indicates that at least a fraction of H1 present in early Drosophila embryos is monoubiquitinated. Moreover, Western blot analyses indicate that compared with wild-type embryos, mutant embryos that lack TAFII250 ubac activity contain a significantly reduced level of monoubiquitinated H1. These results suggest that TAFII250 ubac activities may contribute to monoubiquitination of H1 in Drosophila (Pham, 2000).

How coactivators convert activation signals from activation domains of transcription factors into enhanced levels of mRNA synthesis lies at the heart of transcriptional regulation. These results suggest that one coactivator, TAFII250, may use intrinsic ubiquitin-activating/conjugating activities to mediate activation of transcription. Multiple-alignment analysis and comparison with protein database sequences reveal that TAF250-M exhibits similarities to E1 and E2 enzymes. Thus, the result that TAFII250 mediates monoubiquitination of H1 in vitro is in agreement with other results suggesting that E1 and E2 activities are sufficient to mediate monoubiquitination of proteins. As point mutations that abrogate TAFII250 ubac activity in vitro also reduce gene expression in the Drosophila embryo, TAFII250 ubac activity may play an important role for the activation of gene expression in Drosophila. Although the in vivo targets of TAFII250 ubac activity remain unknown, the results that H1 is monoubiquitinated in Drosophila and that the level of monoubiquitinated H1 is significantly reduced in embryos lacking TAFII250 ubac activity imply that H1 may represent one in vivo target of TAFII250. Thus, ubiquitination of H1 or other proteins within the transcription machinery, or both, by TAFII250 may constitute an important coactivator function of TAFII250 and, hence, may allow TFIID to direct events during the processes of transcriptional activation (Pham, 2000).

Ash2 maintains active transcription by binding Skittles, a producer of nuclear phosphoinositides, and downregulating histone H1 hyperphosphorylation

The products of trithorax group (trxG) genes maintain active transcription of many important developmental regulatory genes, including homeotic genes. Several trxG proteins have been shown to act in multimeric protein complexes that modify chromatin structure. Ash2, the product of the Drosophila trxG gene absent, small, or homeotic discs 2 (ash2) is a component of a 500-kD complex. ASH2 binds directly to Skittles (Sktl), a predicted phosphatidylinositol 4-phosphate 5-kinase, and the association of these proteins is functionally significant. Histone H1 hyperphosphorylation is dramatically increased in both ash2 and sktl mutant polytene chromosomes. These results suggest that Ash2 maintains active transcription by binding a producer of nuclear phosphoinositides and downregulating histone H1 hyperphosphorylation (Cheng, 2004).

The Drosophila gene skittles encodes a putative PIP5KI, which is required for cell viability and germline and bristle development; sktl mutations affect the ovary, dorsal appendage, egg, and wing. Ash2 and Sktl bind directly to each other in vitro and in vivo and sktl mutations enhance the homeotic transformation phenotype of ash2 mutations. This study also shows that histone H1 hyperphosphorylation within euchromatin is dramatically increased on ash2 and sktl mutant polytene chromosomes. These results support a model in which PIP2 plays a role in maintaining transcriptionally active chromatin via histone H1 modification (Cheng, 2004 and references therein).

A result that shows functional significance of the physical association between Ash2 and Sktl is a similar dramatic increase in histone H1 hyperphosphorylation on both ash2 and sktl mutant chromosomes compared to wild-type chromosomes. Histone H1 is thought to be a general repressor of transcription by RNA polymerase II. The presence of histone H1 affects the ability of transcription factors to interact with DNA and is associated with transcription repression, while the removal of histone H1 is associated with transcriptional activation. Studies in mammals and Tetrahymena have found a correlation between transcriptional activation and increased histone H1 phosphorylation. Dephosphorylated histone H1 bound to chromatin over the mouse mammary tumor virus promoter is thought to restrict chromatin remodeling and transcription factor access. Phosphorylation of histone H1 has also been shown to regulate ATP-dependent chromatin-remodeling enzymes. The effect of phosphorylation is to create a region of negative charge, which may displace histone H1 from chromatin, allowing the binding of specific regulating factors. Alternatively, proteins that regulate transcription may recognize the phosphorylated residues (Cheng, 2004 and references therein).

However, histone H1 hyperphosphorylation has the opposite effect and is linked to high chromatin condensation, possibly by allowing the binding of accessory factors. During mitosis, histone H1 becomes hyperphosphorylated, which may facilitate the interaction with the DNA minor groove and factors involved in metaphase chromosome condensation. Therefore, increased histone H1 hyperphosphorylation as observed in ash2 and sktl mutants implies increased chromosome condensation and reduced transcription (Cheng, 2004).

Ash1 has been shown to be able to methylate K4 of histone H3 and ash1 mutant chromosomes show complete loss of histone H3 K4 methylation. This result suggests that Ash1 is required for all of the histone H3 K4 methylation that occurs in vivo. The S. cerevisiae SET1 complex, which contains two subunits that are thought to represent a bipartite functional homolog of Ash2, has also been shown to methylate K4 of histone H3. In Drosophila, if Ash2 was also in a complex that could methylate histone H3 K4, then it would be predicted that ash2 mutant chromosomes would show a decrease in histone H3 K4 methylation. Indeed, a decrease is seen in histone H3 K4 methylation on ash2 mutant chromosomes (Cheng, 2004 and references therein).

During the assembly of nucleosomes, histone acetylation regulates the binding of histone H1 and chromatin condensation. Displacement of histone H1 is required prior to acetylation of target genes and activation of transcription, because histone H1 inhibits histone H3 acetylation by hindering the access of histone acetyltransferases to the histone H3 tail. It has been predicted that chromatin-remodeling complexes would contain components that modify the interaction of histone H1 with chromatin. Ash2 and Sktl may represent such components. The results suggest that Ash2 and Sktl are direct binding partners that are associated in a complex. When the Ash2-Sktl complex binds to chromatin, a source of PIP2 (Sktl) is brought to the chromatin. PIP2 can bind to and displace histone H1 and/or be metabolized to IP3 and phosphorylated derivatives. The displacement of histone H1 would prevent its hyperphosphorylation and allow for chromatin decondensation, histone acetylation, and eventually, transcription activation. The presence of IP4 and IP5 would also stimulate transcription (Cheng, 2004).

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).

Structural insights into the histone H1-nucleosome complex

Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. This study investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. The globular domain of H1 was found to bridge the nucleosome core and one 10-base pair linker DNA asymmetrically, with its alpha3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1-nucleosome complex. These results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo (Zhou, 2013).

A small number of residues can determine if linker histones are bound on or off dyad in the chromatosome

Linker histones bind to the nucleosome and regulate the structure and function of chromatin. Previous studies have shown that the globular domains of chicken H5 and Drosophila H1 linker histones bind to the nucleosome with on- or off-dyad modes, respectively. To explore the determinant for the distinct binding modes, the binding of a mutant globular domain of H5 to the nucleosome was examined. This mutant, termed GH5_pMut, includes substitutions of five globular domain residues of H5 with the corresponding residues in the globular domain of Drosophila H1. The residues at these five positions play important roles in nucleosome binding by either H5 or Drosophila H1. NMR and spin-labeling experiments showed that GH5_pMut bound to the nucleosome off the dyad. It was further found that the nucleosome array condensed by either GH5_pMut or the globular domain of Drosophila H1 displayed a similar sedimentation coefficient, whereas the same nucleosome array condensed by the wild type globular domain of H5 showed a much larger sedimentation coefficient. Moreover, NMR and spin-labeling results from the study of the nucleosome in complex with the full-length human linker histone H1.0, whose globular domain shares high sequence conservation with the corresponding globular domain of H5, are consistent with an on-dyad binding mode. Taken together, these results suggest that a small number of residues in the globular domain of a linker histone can control its binding location on the nucleosome and higher-order chromatin structure (Zhou, 2016).

Histone H1: Biological Overview | Evolutionary Homologs | Developmental Biology | References

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