Histone H3

Transcriptional Regulation

Since purified GAGA factor and TFIID interact similarly with the hsp70 and histone H3 promoters, the architecture of the endogenous H3 promoter has been analyzed to determine what interactions might be needed to establish a potentiated state containing a pause as seen in HSP70 promoter. Despite the detection of TFIID and GAGA on the H3 promoter, no paused polymerase as seen in the HSP70 promoter is evident. In addition, no proteins appear to interact with the transcription start. These results suggest that the GAGA factor and TFIID are not sufficient to establish a potentiated state containing paused polymerase and that TFIID interactions downstream from the TATA element could be important for pausing (Weber, 1995).

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

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

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

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

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

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

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

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

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

The assembly of chromatin during DNA synthesis

The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To gain a better understanding of the assembly of chromatin during DNA synthesis, the Caf1-180 and Caf1-105 subunits of Drosophila chromatin assembly factor 1 (dCAF-1: see Drosophila Chromatin assembly factor 1 subunit) have been identified, cloned, and characterized. The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled chromatin assembly. Furthermore, the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant dCAF-1 revealed an interaction between dCAF-1 and the Drosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1 is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, dASF1 and dCAF-1 p105 colocalize in vivo in Drosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin (Tyler, 2001).

The analysis of factors that are required in addition to CAF-1 for DNA replication-coupled chromatin assembly led to the identification of RCAF. RCAF comprises the Drosophila homolog of the yeast anti-silencing function 1 protein (dASF1) and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones that are assembled onto newly replicated DNA. RCAF functions synergistically with CAF-1 in the assembly of chromatin in DNA replication-chromatin assembly reactions. The study of yeast strains that are lacking CAF-1 and/or RCAF further suggested that CAF-1 and RCAF have both common and unique functions in the cell. RCAF-mediated chromatin assembly appears to be essential for normal progression through the cell cycle, gene expression, DNA replication, and DNA repair. Furthermore, it appears that the checkpoint kinase Rad53 may regulate the chromatin assembly function of ASF1 during DNA replication and repair (Tyler, 2001 and references therein).

To analyze the biochemical properties of dCAF-1, the p180, p105, and p55 proteins were synthesized in Sf9 cells by using baculovirus expression vectors. The p180 subunit contained a C-terminal FLAG epitope tag and was thus designated as p180-FLAG. The p105 subunit contained a C-terminal His6 tag and was therefore termed p105-His6. Different combinations of dCAF-1 subunits were synthesized and purified by either anti-FLAG or Ni(II) affinity chromatography. When p180-FLAG, p105-His6, and p55 were cosynthesized and subjected to anti-FLAG immunoaffinity chromatography, the purified p180+p105+p55 dCAF-1 complex was obtained. Similarly, cosynthesis of p180-FLAG with either p105-His6 or p55 yielded p180+p105 and p180+p55 subcomplexes. Although the three-subunit p180+p105+p55 complex can be purified by Ni(II) affinity chromatography via p105-His6, cosynthesis of p105-His6 and p55 and subsequent Ni(II) affinity chromatography yielded only p105. Hence, these findings indicate that dCAF-1 p180 interacts with both p105 and p55, but that p105 and p55 do not interact with one another (Tyler, 2001).

To test whether the p180, p105, and p55 subunits are required for chromatin assembly, DNA replication-chromatin assembly reactions were performed with partial and complete (i.e., p180+p105+p55) dCAF-1 complexes. These experiments revealed that the purified recombinant p180+p105+p55 dCAF-1 complex possesses a specific activity for DNA replication-coupled chromatin assembly that is comparable to that of native dCAF-1, as demonstrated by plasmid supercoiling analysis. It was further confirmed that dCAF-1-mediated plasmid supercoiling is a consequence of chromatin assembly by using micrococcal nuclease digestion analysis. In addition, the two-subunit p180+p105 subcomplex is fully active for chromatin assembly. In contrast, neither the p180 subunit alone nor the p105 subunit alone is sufficient for chromatin assembly. These results thus indicate that the p180 and p105 subunits are each essential for DNA replication-coupled chromatin assembly by dCAF-1 (Tyler, 2001).

It is relevant that the DNA replication extract used in these experiments contains significant amounts of hCAF-1 p60 and hCAF-1 p48 (also known as RbAp48: Drosophila homolog Caf1), which are homologous to dCAF-1 p105 and dCAF-1 p55, respectively. Based on the requirement of dCAF-1 p105 for chromatin assembly, it appears that the hCAF-1 p60 subunit cannot function with the Drosophila CAF-1 polypeptides. However, the lack of a requirement for dCAF-1 p55 may be due to the ability of the hCAF-1 p48 subunit, which is about 87% identical to dCAF-1 p55, to function with the dCAF-1 p180 and p105 subunits in lieu of dCAF-1 p55. It is also possible, however, that the dCAF-1 p180+p105 subcomplex has the intrinsic ability to mediate chromatin assembly. It has not been possible to immunodeplete the hCAF-1 p48 protein from the DNA replication extract to differentiate between these possibilities. It is noteworthy, however, that the Arabidopsis equivalent of dCAF-1 p55 is required for DNA replication-coupled chromatin assembly with the same assay (Tyler, 2001).

The assembly of newly replicated DNA into chromatin requires both dCAF-1 and the RCAF chromatin assembly factor, which comprises Drosophila ASF1 (dASF1) and specifically acetylates histones H3 and H4. To investigate this effect further, coimmunoprecipitation analyses was performed with a crude Drosophila embryo extract. In these experiments, it was observed that immunoprecipitation with anti-dASF1 results in the coimmunoprecipitation of dCAF1 p180, p105, and p55, but not dCAF-1 p75. Conversely, immunoprecipitation with anti-p105 or with anti-p55 results in the coimmunoprecipitation of dASF1. Thus, these findings indicate that native dASF1 interacts with the native p180+p105+p55 form of dCAF-1 but not with the p75-containing form of dCAF-1. Immunoprecipitation of dCAF-1 with anti-p105+p75 does not result in the coimmunoprecipitation of dASF1, which suggests that the anti-p105+p75 antibodies destabilize the interaction between dASF1 and the p180+p105+p55 form of dCAF-1 (Tyler, 2001).

To summarize, the p75 subunit of dCAF-1 appears to be a C-terminally truncated form of p105 and there are distinct forms of dCAF-1 that contain either the p105 subunit or the p75 subunit. The p105-containing form of dCAF-1 comprises the p180, p105, and p55 proteins. The purified recombinant p180+p105+p55 dCAF-1 complex is as active for DNA replication-coupled chromatin assembly as native dCAF-1. Both the p180 and p105 subunits are essential for chromatin assembly. A preexisting interaction between dCAF-1 and the dASF1 chromatin assembly factor has been discovered in crude extracts. This dCAF-1-ASF1 interaction occurs via the dCAF-1 p105 subunit, and this interaction appears to be direct. dASF1 and dCAF-1 p105 colocalize in vivo in Drosophila polytene chromosomes. These results suggest that there is physical cooperation between dCAF-1 and dASF1 during chromatin assembly. The p105 and p75 subunits of dCAF-1 are closely related, and dCAF-1 is not a single four-subunit complex but rather a three-subunit p180+p105+p55 complex and a presumed p180+p75+p55 complex. Thus, the basic three-subunit structure of CAF-1 is conserved among yeast, Drosophila, and humans. The presence of multiple forms of dCAF-1 is of particular interest. Because dCAF-1 was isolated from whole embryos instead of a specific cell line, there is potential for considerable diversity in the range of functions that may be performed by the different forms of dCAF-1. It is possible, for instance, that the p105-containing form of dCAF-1 functions in ASF1-dependent processes, whereas the p75-containing form of dCAF-1 may function in ASF1-independent processes. Alternatively, the activity of dCAF-1 may be regulated during embryogenesis by processing the p105 polypeptide into p75 (Tyler, 2001).

This physical interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin. The coupling of DNA synthesis and chromatin assembly appears to require a specific interaction between CAF-1 and PCNA. The results presented in this work further extend this model to include the binding of ASF1 to CAF-1. It is possible, for instance, that a complex of RCAF and CAF-1 is recruited to sites of DNA synthesis via the interaction of CAF-1 with PCNA. In the future, it will be interesting to study how RCAF and CAF-1 mediate the formation of nucleosomes in conjunction with the other components of the chromatin assembly machinery (Tyler, 2001).

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

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

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

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

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

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

Aurora kinase and phosphorylation of Histone H3 during mitosis

Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. The segregation of chromosomes with high fidelity requires exquisite coordination of cellular processes. The mechanisms that coordinate the cycle of chromosome condensation and decondensation with the assembly, function, and subsequent disassembly of the mitotic spindle are poorly understood. Highly conserved genes essential for chromosome condensation have been found through genetic screens in yeasts and Drosophila. For example, five members of a protein complex known as condensin, have been identified that are functionally and structurally conserved. Mutants exhibit incomplete chromosome condensation associated with failure of segregation and the stretching of chromatin upon the spindle. Biochemical approaches also identified the protein complex in Xenopus and showed that it can promote chromatin condensation by directing the supercoiling of the DNA in an ATP-dependent manner. Chromosome condensation is also accompanied by phosphorylation of histones H1 and H3. Indeed, mutation of the mitotic phosphorylation site of histone H3 of Tetrahymena leads to both chromosome condensation and segregation defects. A direct link between histone H3 phosphorylation and condensin recruitment onto chromosomes has recently been suggested by the colocalization of members of the condensin complex with phosphorylated histone H3 during the early stages of mitotic chromosome condensation. However, the generality of the requirement for the phosphorylation of histone H3 for chromosome condensation and segregation must be questioned by the finding that budding yeast cells in which serine 10 of histone H3 is replaced with alanine show no apparent defects in cell cycle progression or chromosome transmission. Nevertheless, maximal chromosome condensation in meiosis does correlate with maximal levels of phospho-histone H3 in wild-type cells. The enzyme required for histone H3 phosphorylation in Saccharomyces cerevisiae is the aurora-related protein kinase Ipl1p (Hsu, 2000). Moreover, one of its two counterparts from Caenorhabditis elegans, the air-2 protein kinase, has been shown to have the same function (Giet, 2001 and references therein).

One striking effect of aurora B RNAi is to permit progression through mitosis with improperly condensed chromosomes. It was possible to account for these condensation defects by a diminution of the phosphorylation of serine 10 of histone H3 and a failure to localize condensin on the chromosomes. The former finding is consistent with several studies that now implicate a requirement for the phosphorylation of the NH₂-terminal region of histone H3 at this residue for chromosome condensation. Not only does the formation of mitotic chromosomes in a Xenopus cell-free extract by a nucleosome-associated kinase correlate with histone H3 phosphorylation, but when the serine 10 residue is mutated to alanine it results in abnormal segregation and chromosome loss during mitosis and meiosis in Tetrahymena. One enzyme credited with the ability to phosphorylate histone H3 at mitosis is the NIMA kinase of Aspergillus. However, the finding that levels of histone H3 phosphorylation are reduced after aurB RNAi in Drosophila cells is more in keeping with the report that the Aurora-like kinase homologs, Ipl1 of yeast and Air-2 (but not Air-1) of C. elegans, are required for histone H3 phosphorylation in these organisms (Hsu, 2000). The finding of some residual histone H3 phosphorylation either could reflect the incomplete elimination of Aurora B by RNAi, or could indicate that an alternative kinase has this capability, offering an explanation of the partial chromosome condensation seen in the RNAi-treated cells. The current data are important in emphasizing the importance of histone H3 phosphorylation for chromosome transmission and as such are in line with the findings in Tetrahymena. This differs from the effects seen in budding yeast cells that continue through division cycles in the absence of histone H3 phosphorylation without showing defects in chromosome transmission. As an explanation, it has been suggested that other histones could be phosphorylated in addition to the histone H3 in the yeast cell and that such phosphorylation events could be sufficient to ensure normal chromosome dynamics. A major role of the yeast enzyme Ipl1p is to regulate the function of the kinetochore-associated protein Ndc10p through its phosphorylation. Therefore, the increase in ploidy reported in ipl1 mutant cells has been attributed more to inappropriate kinetochore function, and consequently the effects of Air-2 depletion upon chromosome condensation in C. elegans have been a little overshadowed. It seems likely that the abnormal chromosome segregation in Drosophila cells after aurB RNAi is due to incomplete condensation, since a similar phenotype is seen in mutants of the condensin subunit Barren. Of course, this does not exclude the possibility that defects in the organization of the centromeric regions and kinetochores arise directly as a result of aurB RNAi or as either a direct or indirect consequence of condensation defects. The increase in ploidy seen after aurora B RNAi is reminiscent of the Ipl1 phenotype in budding yeast, but differs in that it arises from both chromosome segregation and cytokinesis defects (Giet, 2001).

The resemblance of the mitotic phenotype of cells after RNAi with aurB to that previously reported for Drosophila barren mutants (Bhat, 1996) can be further explained by the failure of Barren protein to be recruited to the mitotic chromosomes after aurB RNAi. Originally recognized through this mutant defect, it was later realized that Barren is the fly homolog of a member of the pentameric complex, condensin, first shown to be required for mitotic chromosome condensation in Xenopus. It is possible that Barren or other members of the condensin complex could themselves be directly phosphorylated by Aurora B during chromosome condensation. However, the process seems likely to involve a plethora of phosphorylation events: the nuclear A-kinase anchoring protein (AKAP95) appears to target the human hCAP-D2 condensin to chromosomes and phosphorylation of condensin subunits by cdk1 has been associated both with their nuclear accumulation and activation. It has been proposed that phosphorylation of the NH₂ terminus of histone H3 leads to the recruitment or the activation of the condensin complex to the chromosome, where it can modify DNA topology. The data presented here indicate that phosphorylation of histone H3 by the Aurora B kinase and the localization of Barren onto chromosomes are associated events in mitosis. They support and extend a recent observation that human condensin proteins hCAP-E, hCAP-C, and hCAP-D2 colocalize with phosphorylated histone H3 in clusters in partially condensed regions of chromosomes in early prophase. The similarity of the effects seen on chromosome condensation resulting from loss of either aurora B or barren function is striking and points to the value of studying these processes in a single model organism amenable to both genetic manipulation and RNAi. It is perhaps surprising that in both cases partial chromosome condensation is achieved and that there can be some degree of segregation of chromatin to the poles (Giet, 2001 and references therein).

The second major mitotic abnormality observed after aurB RNAi in Drosophila cells is a failure of cytokinesis. Thus, like its mammalian and nematode counterparts AIM-1 and AIR-2, the enzyme encoded by aurora B appears essential for this process. Two proteins that play a role in cytokinesis have recently been shown to associate with the Aurora B-like kinases: Incenp, as discussed above (Adams, 2000; Kaitna, 2000), and the Zen-4 kinesin-like protein of C. elegans (Kaitna, 2000; Severson, 2000). The localization of the latter is disrupted after disruption of air-2 function using RNAi or conditional mutant alleles. Zen-4 is the C. elegans homolog of the Pavarotti KLP of Drosophila, which likewise is mislocalized on the central spindle from anaphase onwards after aurB RNAi. Pav-KLP also cooperates with Polo kinase to achieve its localization and function in Drosophila, suggesting that multiple mitotic kinases may be required to coordinate central spindle formation before cytokinesis, just as several kinases appear to be required for centrosome maturation and separation and chromosome condensation (Giet, 2001).

It is striking that aurB RNAi cells are not arrested by a mitotic checkpoint, given the abnormalities that they show in chromosome alignment at metaphase and the subsequent disorganization of the later mitotic spindle. However, the treated cells do undergo multiple cell cycles, as is clearly demonstrated in this cell culture system in which one can monitor the shift in ploidy by FACS analysis and the increase in chromosome and centrosome complements by immunocytology. It is possible that these abnormalities arise too late in the mitotic cycle to trigger checkpoint arrest, although this seems unlikely for the chromosome segregation defect. Although it is possible that Aurora B is itself required for checkpoint functions, it could also be that the kinetochore regions of chromosomes are insufficiently well organized after aurB RNAi to promote the checkpoint activity of the complex of Bub/Mad proteins that associate with unaligned centromeres. It is noteworthy that the C. elegans baculovirus inhibitor of apoptosis (IAP)-related repeat protein Bir-1 appears to be required for the localization of Air-2. Bir-1 localizes to chromosomes and then the spindle midzone and Air-2 fails to localize to these same sites in the absence of Bir-1 (Speliotes, 2000). These IAP proteins, also known as survivin, are caspase inhibitors and as such counteract apoptosis. Is it possible that B-type Aurora kinases might play a role alongside survivin in an apoptotic checkpoint to promote mitosis? (Giet, 2001).

It is of considerable interest to know the multiple substrates of Aurora B kinase and to understand its mode of regulation in mitotic progression. It seems that subcellular localization of the enzyme could be one critical means of controlling access to its substrates. The enzyme localizes throughout condensing chromosomes when phosphorylation of histone H3 is required. Aurora B's subsequent concentration at centromeres could direct enzyme activity toward specific chromosomal proteins at these sites, but may be instrumental in its movement onto the central spindle at anaphase, thereby providing an effective way of removing the enzyme from the chromatin to facilitate chromosome decondensation at telophase. Understanding the intricacies of these processes will be a future challenge (Giet, 2001).

The availability of mitotic cells containing chromosomes with a range of levels of H3 phosphorylated on serine¹⁰ has enabled an assessment of the widely held hypothesis that H3 phosphorylation is correlated with the degree of chromatin condensation. When phospho-H3 levels and the degree of chromatin compaction were compared by quantitative fluorescence microscopy, only a weak correlation between the two values was observed. Instead, interference with Incenp and Aurora B function appears to correlate much more strongly with difficulties in assembling mitotic chromosomes of normal morphology. Mitotic chromosomes deficient in phospho-H3 have a characteristic dumpy morphology, with no evidence of resolved sister chromatids. This resembles the defects seen in Drosophila mutants in the SMC4 subunit of condensin and also those of a ts mutant in C. elegans aurora B/AIR-2 when it enters mitosis at nonpermissive temperature. Phosphorylation of histone H3 or another chromosomal substrate by Aurora B might be required for the binding of condensins or other chromosomal proteins that give mitotic chromosomes their characteristic morphology (Adams, 2001).

The chromosomal passenger complex (CPC) is a key regulator of mitosis in many organisms, including yeast and mammals. Its components co-localise at the equator of the mitotic spindle and function interdependently to control multiple mitotic events such as assembly and stability of bipolar spindles, and faithful chromosome segregation into daughter cells. This study reports the first detailed characterisation of a CPC mutation in Drosophila, using a loss-of-function allele of borealin (borr). Like its mammalian counterpart, Borr colocalises with the CPC components Aurora B kinase and Incenp in mitotic Drosophila cells, and is required for their localisation to the mitotic spindle. borr mutant cells show multiple mitotic defects that are consistent with loss of CPC function. These include a drastic reduction of histone H3 phosphorylation at serine 10 (a target of Aurora B kinase), and a pronounced attenuation at prometaphase and multipolar spindles. The evidence suggests that borr mutant cells undergo multiple consecutive abnormal mitoses, producing large cells with giant nuclei and high ploidy that eventually apoptose. The delayed apoptosis of borr mutant cells in the developing wing disc appears to cause non-autonomous repair responses in the neighbouring wild-type epithelium. These responses involve Wingless signalling, which ultimately perturbs the tissue architecture of adult flies. Unexpectedly, during late larval development, cells survive loss of borr and develop giant bristles that may reflect their high degree of ploidy (Hanson, 2005).

One crucial role of the CPC during mitosis is to mediate the H3 phosphorylation of serine 10 (P-H3) by Aurora B, as has been demonstrated in budding yeast, C. elegans and Drosophila. The numbers of P-H3-positive (dividing) cells are reduced in the VNC of borr mutant embryos. Furthermore, the P-H3 levels of individual borr mitotic nuclei are typically reduced compared with those of wild-type nuclei. Often, they exhibit blotchy P-H3 staining rather than the more 'structured' staining outlining condensed chromosomes as observed in the wild type. A similar loss of P-H3 staining has also been observed in borr RNAi-depleted Kc167 cells. This reduction of the P-H3 levels in borr mutant cells is consistent with a loss of Aurora B kinase activity and, thus, with a disruption of CPC function (Hanson, 2005).

Despite the strong reduction of the P-H3 levels in mitotic VNC cells of borr mutant embryos, these cells display only a slight undercondensation of their chromatin, although the degree of undercondensation is somewhat variable from cell to cell. These results suggest that borr may not be essential for chromatin condensation (Hanson, 2005).

To examine the effects of borr loss on actively dividing epithelial cells, FRT-FLP-mediated recombination was used to generate borr mutant clones in imaginal discs whose cells undergo cell divisions throughout larval development. If borr mutant clones are induced during early larval stages and examined in fully grown larval discs, these clones are rare and are much smaller than the corresponding wild-type twin spots, suggesting that a large fraction of the mutant cells die. Hoechst staining revealed that many of the surviving borr mutant cells are large, with giant but well-formed nuclei that appear healthy, and well integrated into the epithelial tissue (Hanson, 2005).

Imaginal discs bearing borr mutant clones were stained with antibodies against Incenp and Aurora B, to assess the effect of borr loss on these CPC components during mitosis. Wild-type cells in metaphase show characteristic well-ordered mitotic spindles, with distinct staining of Aurora B and Incenp at specific sites along condensed chromatin. By contrast, borr mutant cells invariably show abnormal mitotic spindles, including multipolar ones. Most of these mutant spindles do not show any chromatin-associated Incenp or Aurora B staining, although occasionally patches of Incenp staining can still be observed, but they do not seem to be associated with any of the spindle components. These staining patterns suggest that these CPC components fail to localise properly to mitotic spindles in the absence of borr (and their levels may also be reduced, though the low frequency of surviving borr mutant cells does not allow quantitative assessment of this). Therefore, as in mammalian cells, the correct localisation of Incenp and Aurora B to mitotic spindles of dividing imaginal disc cells depends on Borr. This is further evidence that Borr is a CPC protein, and that it interacts functionally with other known CPC components (Hanson, 2005).

The chromosomal passenger protein complex has emerged as a key player in mitosis, with important roles in chromatin modifications, kinetochore-microtubule interactions, chromosome bi-orientation and stability of the bipolar spindle, mitotic checkpoint function, assembly of the central spindle and cytokinesis. The inner centromere protein (Incenp; a subunit of this complex) is thought to regulate the Aurora B kinase and target it to its substrates. To explore the roles of the passenger complex in a developing multicellular organism, a genetic screen was performed looking for new alleles and interactors of Drosophila Incenp. A new null allele of Incenp has been isolated that has allowed a study of the functions of the chromosomal passengers during development. Homozygous incenp^EC3747 embryos show absence of phosphorylation of histone H3 in mitosis, failure of cytokinesis and polyploidy, and defects in peripheral nervous system development. These defects are consistent with depletion of Aurora B kinase activity. In addition, the segregation of the cell-fate determinant Prospero in asymmetric neuroblast division is abnormal, suggesting a role for the chromosomal passenger complex in the regulation of this process (Chang, 2006).

By embryonic stage 13, Drosophila Incenp was no longer detectable by immunostaining in incenp^EC3747 embryos. Consistent with this, phosphorylation of histone H3 on Ser10 (a known Aurora B kinase substrate) was also no longer detected. This phenotype confirms that Incenp is required for Aurora B kinase to function as a histone H3 Ser10 kinase. At this stage, cells of the central nervous system (CNS) in incenp^EC3747 embryos showed enlarged nuclei compared with wild-type as observed by DAPI staining for DNA. Staining with anti-gamma-tubulin antibody showed that these enlarged cells contain bigger than normal centrosomes or multiple centrosomes. This phenotype, detectable in mutant embryos following the complete disappearance of the Drosophila Incenp, can be explained as the result of failure of cytokinesis in the previous division and is consistent with a lack of Aurora B kinase function (Adams, 2001c; Oegema, 2001) in the early development of the CNS (Chang, 2006).

JIL-1 targets Histone H3 during dosage compensation in Drosophila

JIL-1 is part of the dosage compensation apparatus. JIL-1 colocalizes and physically interacts with male specific lethal (MSL) dosage compensation complex proteins. Ectopic expression of the MSL complex directed by MSL2 in females causes a concomitant upregulation of JIL-1 to the female X that is abolished in msl mutants unable to assemble the complex. Thus, these results strongly indicate JIL-1 associates with the MSL complex and further suggest that JIL-1 functions in signal transduction pathways regulating chromatin structure (Jin, 1999 and 2000).

To analyze the function of the chromosomal kinase JIL-1, an allelic series of hypomorphic and null mutations was generated. JIL-1 is an essential kinase for viability, and reduced levels of JIL-1 kinase activity led to a global change in chromatin structure. In JIL-1 hypomorphs, euchromatic regions of polytene chromosomes are severely reduced and the chromosome arms condensed. This is correlated with decreased levels of histone H3 Ser10 phosphorylation. These levels can be restored by a JIL-1 transgene placing JIL-1 directly in the pathway mediating histone H3 phosphorylation. A model is proposed where JIL-1 kinase activity is required for maintaining the structure of the more open chromatin regions that facilitate gene transcription (Wang, 2001).

The original EP(3)3657 line in conjunction with the newly generated JIL-1^z60 and JIL-1^z2 lines constitute an allelic series of JIL-1 hypomorphic and null mutations. This allows an anaysis of the effects of decreasing levels of JIL-1 protein on viability and male to female sex ratios. In order to measure and compare viability, homozygous pupae were collected from heterozygous crosses of the three alleles and their eclosion rates were determined. The homozygous mutant pupae could be readily identified because they did not display the Tubby marker carried on the balancer third chromosome. It was found that homozygous EP(3)3657 larvae, which have only one tenth of the level of JIL-1 protein normally found in wild-type, have an eclosion rate of 81%. However, as the level of JIL-1 protein further decreases to about 3% in JIL-1^z60/JIL-1^z60 larvae, the eclosion rate reduces to only 0.5%. Moreover, the few homozygous JIL-1^z60/JIL-1^z60 animals surviving to adulthood are unable to produce offspring and most die shortly after eclosion. The eclosion rate for the null allele JIL-1^z2/JIL-1^z2 larvae was 0%. These results strongly suggest that JIL-1 is an essential kinase for viability (Wang, 2001).

Eclosed EP(3)3657/EP(3)3657 adults are fertile and able to produce offspring, and thus embryos from homozygous parents can be analyzed for the effect of reduced levels of JIL-1 on embryonic development. The hatch rate of such embryos is only 4%, showing a significant decrease below the 83% observed in wild-type. Whereas reduced eclosion levels are observed for both males and females, male viability is more severely affected in all cases of lowered JIL-1 expression. For example, in EP(3)3657/EP(3)3657 homozygous offspring from heterozygous mothers that provide maternal levels of JIL-1 protein during early development due to the mother's wild-type allele, the number of males eclosing was 73% that of females. In adults eclosing from crosses of homozygous parents and thus developing without the increased maternal levels of JIL-1, the percentage of males relative to females was 48%. Further reduction is observed in the severe JIL-1^z60/JIL-1^z60 hypomorph, which gives rise to only 32% the expected number of males relative to females. The male to female sex ratio in EP(3)3657 flies can be rescued to near wild-type ratio by the JIL-1- GFP transgene. When this transgene is introduced into these flies, the male to female sex ratio recovers from 48% to 97% (Wang, 2001).

The phenotypic consequences of reduced levels of JIL-1 kinase were investigated in embryos from EP(3)3657 homozygous parents by labeling chromatin with Hoechst and microtubules with anti-tubulin antibody. The average expression level of JIL-1 kinase in these mutant animals is reduced to about one tenth that of wild-type. A range of phenotypes was observed from embryos appearing wild-type with regularly spaced nuclei to embryos where chromatin structure had completely disintegrated. In intermediate phenotypes, nuclei in various stages of fragmentation were still discernible. The variable penetrance and range of phenotypes are likely to be a result of different levels of JIL-1 expression in individual embryos. Some embryos have enough JIL-1 to carry them through embryogenesis as reflected in the 4% hatching rate, whereas others are below the threshold for maintaining JIL-1 kinase function. In embryos double labeled with Hoechst and anti-tubulin antibody, centrosomes were often observed to be separated from the nuclear remnants, and in other cases, the nuclear fragmentation would lead to aberrant and misaligned tubulin spindles. These data suggest that reduced levels of JIL-1 kinase lead to a disintegration of nuclear and chromatin structure during embryonic development (Wang, 2001).

The consequences of loss of JIL-1 on polytene chromosome structure in interphase nuclei was assessed. Chromosomal squashes prepared from either wild-type or homozygous hypomorphic EP(3)3657, JIL-1^z60, or null JIL-1^z2 larvae were fixed and labeled with Hoechst to visualize the DNA, anti-MSL2 antibody to identify the male X chromosome, and, in some cases, with anti-JIL-1 antibody. Labeling with anti-MSL2 antibody revealed that MSL2 protein still localizes to the X chromosome in all three JIL-1 mutant alleles, indicating that JIL-1 is not necessary for targeting of the MSL complex to the male X chromosome. Identical results were obtained using anti-MSL1, -MSL3, or histone H4Ac16 antibodies, confirming this observation. However, chromosome morphology in both males and females is markedly affected. Whereas wild-type polytene chromosomes show extended arms with a regular pattern of Hoechst-stained bands, this pattern, while relatively normal in the hypomorphic EP(3)3657 mutant animals, becomes severely perturbed in strong JIL-1^z60 hypomorphs and the null JIL-1^z2 larvae. In these latter preparations, the euchromatic interband regions are largely absent and the chromosome arms are highly condensed. Thus, these results suggest that the JIL-1 kinase is involved in both males and females in establishing or maintaining the more open chromatin structure found in the gene-active interband regions that comprise less tightly packed euchromatin (Wang, 2001).

Although all of the chromosomes from JIL-1 mutant animals display abnormalities, perturbation of the male X chromosome is relatively more severe than that of the autosomes. This can be observed in the weaker hypomorphic phenotype from EP(3)3657 preparations where although the autosomes are only subtly affected, the male X chromosome is significantly shorter and has lost a large degree of its banding pattern. In the strong JIL-1^z60 hypomorph or the null JIL-1^z2 mutant, the male X chromosome is even more condensed with no remaining observable banding pattern or structure. Further support for the fact that the reduction of JIL-1 protein level is responsible for the defects observed in the homozygous animals comes from rescue experiments in which transgenic JIL-1-GFP is introduced into JIL-1^z2/JIL-1^z2 animals. Chromosomes from these animals now appear essentially wild-type including the male X chromosome; JIL-1 antigenicity is restored and upregulated on the X chromosome as detected by JIL-1 antibody. Identical results were observed in rescue experiments employing a full-length JIL-1 transgene which did not contain the GFP moiety (Wang, 2001).

The upregulation of JIL-1 on the male X chromosome in conjunction with its ability to phosphorylate histone H3 Ser10 in vitro led to an examination of the question of whether higher levels of phosphorylated histone H3 Ser10 (pH3S10) are also present on the male X. Conventional polytene chromosome fixation and squash techniques led to inconsistent banding patterns. It was reasoned that the highly acidic fixation conditions of the conventional squash protocol might be interfering with either antibody performance or antigen stabilization during fixation. Therefore, a modified whole-mount staining technique was developed for salivary glands that gently compress nuclei beneath a coverslip before fixation in a standard paraformaldehyde/PBS solution with a physiological pH. Although the overall resolution of the bands is inferior to the normal squash technique, it does allow visualization of the chromosomes suitable for analysis. Such salivary gland preparations were double labeled with antibodies to JIL-1 and pH3S10 as well as with antibodies to JIL-1 and phosphoacetylated histone H3 (pH3S10Ac14). JIL-1 protein is upregulated on the male X chromosome. This upregulation is concomitant with an upregulation of both pH3S10 and pH3S10Ac14 labeling on the male X chromosome as compared to the autosomes. Furthermore, it is evident that the staining pattern of the antibodies in wild-type animals overlap as indicated by a predominantly yellow banding pattern. This labeling pattern was consistently observed in different experiments using different lots of pH3S10 antibody from two different companies. Such an upregulation was not observed in the female, and in homozygous JIL-1^z2/JIL-1^z2 polytene chromosomes; neither JIL-1 nor pH3S10 or pH3S10Ac14 labeling was detectable. These data suggest that levels of phosphorylated histone H3 Ser10 are increased on the male X chromosome in a pattern overlapping with that found for the JIL-1 kinase. However, it is of interest to note that Western blot analysis does not indicate higher overall levels of pH3S10 in males than females (Wang, 2001).

Recent studies have revealed a tight correlation between histone H3 Ser10 phosphorylation and proper chromosome condensation and segregation during mitosis. This mitotic phosphorylation of histone H3 is governed by the lpl1/aurora kinase in budding yeast and nematodes and by the NIMA kinase in Aspergillus. Thus, different kinases or more than one kinase may serve this function in different organisms. This raises the question whether JIL-1 regulates mitotic histone H3 Ser10 phosphorylation in Drosophila. To address this issue, pH3S10 levels were analyzed in null JIL-1^z2/JIL-1^z2 larval neuroblast mitotic chromosomes. In the null JIL-1 background, pH3S10 is not observed in interphase nuclei, but is enriched on the mitotic chromosomes at a level comparable to wild-type. Therefore, loss of JIL-1 activity does not appear to alter the mitotic phosphorylation of histone H3 Ser10 in larval neuroblasts (Wang, 2001).

Since the interphase upregulation of pH3S10 phosphorylation levels correlates with JIL-1 kinase localization and since the majority of larval cells are in interphase at any one given time, whether pH3S10 phosphorylation levels were decreased in JIL-1 hypomorphs was examined, as would be predicted if JIL-1 were involved in this process. Levels of phosphorylated histone H3 Ser10 were determined by immunoblot analysis of larval protein lysates from wild-type or homozygous JIL-1 mutant (EP(3)3657, JIL-1^z60, JIL-1^z2) animals. Lysates were fractionated and probed with anti-pH3S10, anti-tubulin, anti-lamin, and anti-histone H3 antibodies. All of the JIL-1 mutants showed lower levels of pH3S10 than observed in wild-type larvae. Furthermore, the level of reduction of pH3S10 corresponded directly to the severity of the JIL-1 allele, with the null JIL-1^z2/JIL-1^z2 allele showing the lowest level of pH3S10 phosphorylation and EP(3)3657/EP(3)3657, the weaker of the two hypomorphs showing higher pH3S10 levels than the strong JIL-1^z60/JIL-1^z60 hypomorph. In contrast, the levels of control proteins such as histone H3, tubulin, and lamin were roughly equivalent to wild-type levels in all three mutant lines. Since introduction of the JIL-1-GFP transgene on the second chromosome of JIL-1^z2/JIL-1^z2 animals rescued the chromosomal defects, it was of interest to determine whether there was also a corresponding restoration of pH3S10 levels in these animals. Western blots of larval protein lysates from wild-type, JIL-1^z2/JIL-1^z2, or JIL-1^z2/JIL-1^z2 larvae carrying the JIL-1-GFP transgene were probed with anti-pH3S10 antibody or anti-histone H3 total protein antibody. In the presence of the JIL-1-GFP transgene, pH3S10 levels are restored to essentially wild-type levels (Wang, 2001).

Thus, in a JIL-1 null mutant allele that shows no detectable JIL-1 kinase, the level of histone H3 Ser10 phosphorylation is reduced to about 5% of wild-type levels. These results suggest the existence of another kinase that can phosphorylate histone H3 Ser10 and are consistent with the recent identification of Aurora B kinase as the likely mitotic H3 Ser10 kinase in Drosophila (Giet, 2001). However, these results suggest that JIL-1 is the predominant kinase regulating the phosphorylation state of this residue at interphase. This is further supported by findings that histone H3 Ser10 phosphorylation is upregulated on the male X chromosome in a pattern similar to that of the JIL-1 kinase and that the loss of histone H3 Ser10 phosphorylation as well as aberrant chromosome structure found in JIL-1 mutants can be rescued by the presence of a JIL-1 transgene. Thus, taken together these results demonstrate that JIL-1 is in the pathway mediating histone H3 Ser10 phosphorylation and that JIL-1 kinase activity is required for the maintenance of normal chromosome architecture in Drosophila at interphase (Wang, 2001).

In embryos with dividing nuclei, a reduction in JIL-1 kinase activity leads to nuclear fragmentation and to dispersion of centrosomes and deformed mitotic spindles. However, this phenotype is thought to be a consequence of altered chromatin structure that occurs during interphase, such as that observed in polytene chromosomes. The aberrant chromosome structure interferes with proper mitotic condensation and segregation and ultimately leads to chromatin disintegration. The findings that a low level of histone H3 Ser10 phosphorylation persists in JIL-1^z2 homozygous flies, that mitotic chromosomes in neuroblasts from JIL-1 null mutant larvae show high levels of histone H3 Ser10 phosphorylation, and that the Drosophila Aurora B kinase phosphorylates histone H3 Ser10 during mitosis (Giet, 2001) further support the notion that JIL-1 is not a mitotic histone H3 Ser10 kinase in Drosophila, but rather is important for maintenance of chromatin structure at interphase. It is becoming clear that multiple kinases can phosphorylate the Ser10 residue on histone H3 and that this single histone modification can elicit diverse cellular responses. What determines which of multiple pathways are activated may be influenced by the presence of additional histone tail modifications that, used in a combinatorial fashion, mediate context-dependent signaling (Wang, 2001 and references therein).

The significant decrease in male viability beyond that observed in females argues strongly for a specific role of JIL-1 in dosage compensation in males that may be separate from its function in maintenance of global chromatin structure. Dosage compensation results in a 2-fold hypertranscription of the male's single X chromosome relative to the female's two X chromosomes. The level of H4Ac16 is increased on the male X chromosome as a consequence of MSL chromatin remodeling complex activity, and this targeted acetylation has been directly linked to transcriptional activation. Notably, levels of phosphorylated histone H3 Ser10 as well as the double H3 Ser10Ac14 modifications are also upregulated on the male X, in agreement with the model that enhanced transcription may require a combined signaling via both acetylation and phosphorylation motifs. However, that the male X continues to be hyperacetylated at histone H4Ac16 in the absence of JIL-1 indicates that JIL-1's role in dosage compensation is not likely to be effected via regulation of the MSL complex's histone acetyltransferase activity (Wang, 2001).

MSL complex is attracted to genes marked by H3K36 trimethylation using a sequence-independent mechanism

In Drosophila, X chromosome dosage compensation requires the male-specific lethal (MSL) complex, which associates with actively transcribed genes on the single male X chromosome to upregulate transcription 2-fold. On the male X chromosome, or when MSL complex is ectopically localized to an autosome, histone H3K36 trimethylation (H3K36me3) is a strong predictor of MSL binding. Mutants lacking Set2, the H3K36me3 methyltransferase, were isolated, and it was found that Set2 is an essential gene in both sexes of Drosophila. In set2 mutant males, MSL complex maintains X specificity but exhibits reduced binding to target genes. Furthermore, recombinant MSL3 protein preferentially binds nucleosomes marked by H3K36me3 in vitro. These results support a model in which MSL complex uses high-affinity sites to initially recognize the X chromosome and then associates with many of its targets through sequence-independent features of transcribed genes (Larschan, 2007).

MSL complex colocalizes with H3K36 trimethylation on X-linked genes: To investigate the relationship between MSL complex recruitment and histone methylation, ChIP-on-chip analysis of SL2 cells was performed with antibodies that recognize H3 trimethylated at K36 (H3K36me3) or dimethylated at K4 (H3K4me2). The SL2 cell line exhibits a male phenotype with respect to dosage compensation. NimbleGen tiling arrays were used; these contain the entire X chromosome and left arm of chromosome 2, tiled at 100 bp resolution. A general histone H3 antibody was used as a control for histone occupancy, and three biological replicates for tiling arrays indicated a high degree of reproducibility. As expected, the H3K36me3 and H3K4me2 modifications were associated with the 3' and 5' ends of transcribed genes, respectively, as previously reported for S. cerevisiae, mammals, and chicken. Close to 100% of transcribed genes on the X and 2L chromosomes were methylated at H3K36 and H3K4, largely independent of transcript level as previously reported for other organisms. Similar results were observed for MSL3-TAP, specifically on the X chromosome, but a lower fraction of transcribed genes on the X was bound (approximately 80%). With improved computational analysis, 1014 genes on the X chromosome scored positive for MSL binding in SL2 cells (up from previous estimate of 675 genes). 67% of the newly scored MSL-bound genes in SL2 cells were identified previouslyw was clearly bound in at least one cell type (Larschan, 2007).

To determine whether MSL binding colocalizes with H3K36me3 or H3K4me2, the correlation was examined between the data sets at the gene level. Of the 1014 MSL-bound genes in SL2 cells, 93% were positive for H3K36me3, and 83% were positive for H3K4me2. Interestingly, it was previously reported that a small percentage of untranscribed genes were bound by MSL3-TAP (7%), and the current study found that these genes also carried the H3K36me3 histone modification. In addition, untranscribed genes bound by MSL have significantly higher levels of H3K36me3 than untranscribed genes that are unbound by MSL complex. A likely explanation is that some nontranscribed genes are located near transcribed genes with very extensive H3K36me3 and MSL signals or within domains that have continuous strong signal over many kilobases. Specifically, 82% of MSL3-TAP-bound genes are transcribed, while 93% percent of MSL3-TAP-bound genes carry the H3K36me3 modification. Therefore, H3K36me3 is an even better predictor of MSL binding on the X than transcription state as defined by Affymetrix expression arrays. Similar results were observed for clone 8 cells, a Drosophila cell line derived from the wing disc (Larschan, 2007).

Colocalization in terms of whole genes could occur without coincident binding along the gene. It was previously reported that MSL3-TAP binds over the body of transcribed genes specifically on the X chromosome with a bias toward the 3' end. To determine whether H3K36me3 on the X chromosome and MSL complex colocalize spatially within transcription units, average gene profiles were compared for H3 methylation modifications and MSL3-TAP. It was found that H3K36me3 and MSL3-TAP exhibit a similar 3' biased profile, whereas H3 lysine 4 dimethylation is associated with the 5' end of transcription units, as reported in other organisms. Furthermore, at the probe level, a strong positive correlation is observed between MSL binding and H3K36me3 association. In contrast, a weaker correlation is observed with H3K4me2 that associates with the 5' ends of genes. These results demonstrate that H3K36 trimethylation is a 3' biased mark associated generally with active transcription units and that it is a very strong predictor of MSL binding on the X chromosome (Larschan, 2007).

MSL complex attracted to chromosome 2L by a roX2 transgene binds neighboring 2L genes marked by transcription and H3K36me3: When either a roX1 or a roX2 genomic transgene is inserted on an autosome, it attracts MSL complex to its site of insertion, with occasional signs of additional binding to neighboring regions along the autosome. Ectopic binding along the autosome is greatly increased when the X chromosome in the same nucleus is deleted for both roX1 and roX2. Such binding generally extends >1 Mb bidirectionally from the site of the roX transgene insertion, as measured by immunofluorescence for the MSL proteins. One interpretation is that nascent roX RNAs compete for attraction of the MSL proteins for assembly at their site of synthesis and that, after local assembly, MSL complex becomes competent to search for targets in its new chromosome environment. To determine whether ectopic binding on a normally untargeted chromosome would provide clues to the specificity of MSL binding, ChIP-on-chip analysis was performed on MSL3-TAP male larvae mutant for both roX1 and roX2 on the X chromosome and containing a roX2 transgene inserted at position 26D8-9 (near the CG9537 gene) on chromosome 2L. When assayed by immunostaining of polytene chromosomes, such males consistently show MSL binding in interbands along chromosome 2L, surrounding the site of the transgene insertion. At the level of genomic tiling arrays, ChIP results map this binding at high resolution. As a control, an additional array was used that contains the 3R chromosome and the entire X. It was found that the domain of MSL binding extends greater than 2 Mb in each direction from the insertion site on 2L, while binding to 3R was undetected. Importantly, the targets of binding are transcribed 2L genes, with the averaged binding profile showing enrichment over the bodies of genes, with a bias toward 3'ends. Each of these characteristics is typical of target genes on the X chromosome in wild-type larvae, cells, and embryos. Furthermore, when the 2L pattern of ectopic MSL binding in larvae was compared to the wild-type distribution of H3K36 trimethylation in tissue culture cells, a strong correlation was found between MSL binding and K36me3 within 1 Mb of the site of the roX transgenic insertion. Interestingly, although MSL-bound genes are consistently marked with H3K36me3, at greater than 1 Mb distances from the transgene insertion site, MSL complex increasingly skips some H3K36me3-bound genes while binding others. Overall, it was found that MSL targets selected on 2L were transcribed genes enriched for H3K36 trimethylation and that MSL binding showed a 3′ bias analogous to that normally found on X chromosome targets. These results raise the strong possibility that, once targeted to a chromosomal domain by a high-affinity site, MSL complex recognizes general marks for transcription such as H3K36me3 or other 3′-associated features rather than an X-specific sequence element at each individual target (Larschan, 2007).

Set2 is required for H3K36 trimethylation and for viability in both males and females in Drosophila : To investigate whether H3K36me3 plays a functional role in MSL complex targeting, a genetic approach was taken to inactivate the methyltransferase responsible for H3K36me3 in Drosophila. In S. cerevisiae, the Set2 histone methyltransferase is responsible for di- and trimethylation of H3K36. The CG1716-encoded protein has been identified as the likely functional homolog of ySet2 in Drosophila based on the presence of SRI and SET domains. Two initial tests were pursued to examine CG1716 function, the first in yeast and the second in Drosophila tissue culture cells. To test the function of CG1716 in yeast, an inducible CG1716 expression vector was transformed into set2Δ mutant S. cerevisiae that lack detectable H3K36me3. When CG1716 was induced by growth in media containing galactose, H3K36me3 (and some H3K36me2) was restored, demonstrating that a CG1716 cDNA functionally complements the yeast set2Δ. Also, the CG1716-encoded protein can interact with the RNA Pol II CTD as observed for S. cerevisiae Set2, further confirming the identity of CG1716 as the functional homolog of the S. cerevisiae SET2 gene. To test the function of CG1716 in Drosophila tissue culture cells, RNAi was used to target CG1716. A strong reduction of CG1716 mRNA was found to correlate with a significant loss of H3K36me3 by Western blot, immunostaining, and ChIP analysis. H3K4me2, a distinct chromatin mark for transcribed genes, was largely unaffected. ChIP analysis allowed quantification of a 3- to 5-fold reduction in H3K36me3 and only very small changes in H3K4me2. Based on these results, a Drosophila mutant was isolated that disrupts the CG1716 gene, henceforth referred to as the Set2 gene (Larschan, 2007).

Imprecise excision of a P element upstream of the Set2 gene was induced to create a series of Set2 deletion strains, and Set2¹ was selected for further analysis. dSet2¹ eliminates most of the coding region including the catalytic SET domain without extending bidirectionally into the neighboring CG1998 gene. Since the Set2 gene is located on the X chromosome, hemizygous males were initially isolated, and they were found to die as late third-instar larvae. To demonstrate that this lethality was due to loss of Set2, and not to any additional defects that might have been induced during P element excision, a transgene was constructed encompassing only the genomic region of Set2; it was able to fully rescue the Set2¹ mutants. Using the rescued males as fathers, homozygous mutant females were subsequently examined, and the Set2¹ mutation was found to cause late larval lethality in both sexes. To further analyze the viability of Set2 mutants at the cellular level, homozygous mutant Set2 eyes were created in the context of heterozygous mutant adult females, using the GMR-hid system. set2 mutant eyes were diminished in size and rough compared to wild-type eyes, which is a qualitative assay suggesting that Set2 is important for normal cell proliferation (Larschan, 2007).

To determine whether or not H3K36me3 was affected in the set2 mutant, polytene chromosome squashes of mutant larvae were were immunostained. H3K36me3 was significantly depleted in the Set2¹ mutant when compared to wild-type. As a control for the specificity of this defect, the same nuclei were immunostained for the interband protein Z4, which showed similar staining in wild-type and mutant. Set2¹ mutant larvae were further analyzed by ChIP to quantify the H3K36me3 levels in wild-type and Set2¹ mutants. H3K36me3 in the Set2¹ mutant was found to be dramatically decreased at the transcribed genes tested, to levels comparable to an untranscribed gene (CG15570). Changes in H3K4me2 varied from slight to none. Thus, Set2 is required for viability and methylation of H3K36 in Drosophila (Larschan, 2007).

Set2 contributes to optimal MSL complex targeting at transcribed genes, but not at high-affinity sites: To examine whether MSL complex targeting requires H3K36me3, polytene chromosomes of Set2¹ mutant larvae were immunostained with antibodies directed against MSL complex, but no difference in MSL pattern or intensity was detected at this level of resolution. Upon initial consideration, this result would appear to rule out a requirement for H3K36me3 in MSL targeting. However, when attempts were made to validate this observation with ChIP assays conducted with two independent fly stocks and ChIP protocols (both anti-MSL2 and MSL3-TAP IPs), it was found that wild-type and Set2¹ mutant larvae showed significant differences at many specific gene targets. Nine genes with high, medium, or low levels of MSL complex binding were assayed for recruitment of MSL2 and MSL3-TAP in wild-type and Set2¹ mutant third-instar larvae by ChIP analysis. Highly reproducible 2- to 10-fold decreases were observed in MSL2 and MSL3-TAP association at all nine genes assayed. In contrast, MSL complex association with previously reported 'high-affinity sites', such as roX1, roX2, and 18D11, was largely unaffected in the Set2¹ mutant (Larschan, 2007).

Such a result might be attributed to indirect effects in Set2¹ mutant larvae as opposed to specific defects in MSL targeting. To address this, roX RNA and msl2 mRNA levels were measured, and it was found that they were not affected significantly in the Set2¹ mutant, suggesting that H3K36me3 does not affect MSL complex recruitment indirectly by affecting expression of MSL components. Western and polytene staining analysis of Msl1 and Msl2 also indicate that protein levels are largely unchanged. It was also found that ChIP for H3K4me2 and RNA polymerase II were not significantly affected in set2 mutants, further supporting a direct role for H3K36me3 in stabilization of MSL complex at target genes (Larschan, 2007).

To address the functional role of H3K36me3 in transcription of genes bound by MSL complex, the transcript levels of MSL complex target genes were compared in wild-type and Set2¹ mutant larvae. Transcription of MSL target genes is not strongly affected in Set2¹ mutant larvae, although genes that exhibit the strongest loss of MSL complex binding (CG13316, CG12690, CG32555, and CG32575) exhibit decreases in transcript level. Dosage compensation involves a 2-fold upregulation of transcription, limiting the expected transcriptional changes to a 50% decrease in transcript. Furthermore, when H4K16 acetylation at these genes was examined, significant residual levels were found (10-fold over autosomal controls or untranscribed genes), even when very small amounts of MSL complex remain. Thus, residual MSL complex function may be largely sufficient for transcriptional upregulation in the Set2¹ mutant, yet MSL complex targeting is significantly reduced (Larschan, 2007).

Together, these results suggest that a subset of MSL binding sites is particularly sensitive to H3K36me3 levels, while others, including three previously defined high-affinity sites are not. Since MSL binding is diminished significantly but not ablated in the Set2¹ mutant, these results support a model in which recognition of H3K36me3 is one contributing factor to MSL complex targeting that functions with additional features of transcribed genes (Larschan, 2007).

An important caveat to the conclusion that H3K36me3 functions together with other recognition features is that the heterozygous mothers of hemizygous Set2¹ mutants carry a functional Set2 gene and thus could provide a maternal supply of wild-type Set2 mRNA or protein to the mutant embryos. This maternal contribution of H3K36me3 could be sufficient to initially establish MSL binding, which might be maintained through development, independent of the initial recognition mark. Thus, if the maternal contribution of H3K36me3 could be eliminated, it was hypothesized that an even more significant defect would be observed in MSL complex recruitment. To address this possibility genetically, a stock designed to create homozygous set2 mutant germline clones was constructed using FLP-FRT-mediated recombination in an ovo^D dominant female sterile mutant. After recombination, the set2 mutant germ cells would no longer carry ovo^D and thus should produce oocytes that would lack any maternal Set2 mRNA or protein. Despite recombination to remove ovo^D from germ cells, no functional oocytes were produced, demonstrating that Set2 is essential for oogenesis. Therefore, the maternal contribution of Set2 remains in these studies; its elimination might reveal an even more significant role or H3K36me3 in MSL recruitment than has been reported (Larschan, 2007).

Recombinant MSL3 binds preferentially to nucleosomes trimethylated at H3K36: Eaf3, the yeast member of the conserved MSL3/MRG family of proteins, has been implicated in a physical and functional interaction of Rpd3(S) complexes with H3K36me3, raising the attractive hypothesis that MSL3 plays an analogous function in MSL complex. Furthermore, the distinction between high-affinity MSL binding sites such as roX1, roX2, and 18D11 and the majority of MSL targets is that high-affinity sites are MSL3 independent. Therefore, sensitivity to loss of H3K36me3 might be a specific characteristic of MSL3-dependent targets. To test the idea that MSL3 contributes to specific recognition of H3K36me3-modified nucleosomes, gel shift analyses was performed with recombinant MSL3 protein produced in baculovirus using nucleosomes assembled in vitro. Using an EMSA assay system where specifically modified recombinant nucleosomes were assembled, it was found that purified MSL3 protein showed increased affinity to nucleosomes pretreated with active Set2, and thus marked with H3K36 methylation, as opposed to nucleosomes that were unmodified at H3K36. This preferential binding was only detected in nucleosomes bearing linker DNA, suggesting that affinity for free DNA may be contributing to the binding of MSL3 to the nucleosomes methylated at H3K36. Titrations were performed to measure the relative affinity of MSL3 association with methylated compared to unmethylated nucleosomes. The increased affinity of MSL3 for methylated nucleosomes is best observed at the 4.4 nM concentration. These results provide additional evidence supporting a model in which H3K36me3 is a 3' chromatin mark required for the robust, wild-type MSL binding pattern on the X chromosome (Larschan, 2007).

This study has found that ectopic spreading of MSL complex to the 3' ends of transcribed genes on autosomes indicates that a sequence-independent mechanism can define MSL complex target genes. Furthermore, trimethylation of H3K36 is required for optimal MSL complex targeting to transcribed genes on the male X chromosome subsequent to initial recognition of the X. In the absence of H3K36me3, MSL complex can associate with high-affinity sites on the X chromosome but exhibits reduced binding to target genes. Since MSL binding is reduced but is not eliminated, a model if favored in which association with H3K36me3 is a contributing factor that functions with recognition of one or more additional 3' features of transcribed genes such as nascent mRNAs or RNA Pol II CTD phosphorylation (Larschan, 2007).

In addition to a function for Set2 in MSL complex targeting, this study demonstrates that Set2 is essential for viability of both sexes in Drosophila. Conservation of the Set2 H3K36 methyltransferase function from S. cerevisiae to Drosophila was observed, as predicted by sequence conservation. A variety of roles have been reported for Set2 in several organisms. In Neurospora, S. pombe, and NIH 3T3 cells, Set2 is required for optimal growth rate. The S. cerevisiae set2Δ mutant suppresses the loss of positive elongation factors. In Drosophila, mutants lacking zygotic Set2 function fail to proceed through the developmental transitions from late larval to adult stages. The cause(s) of inviability in Drosophila set2 mutants remains to be determined, but eyes composed entirely of homozygous set2 mutant tissue were small and rough, indicating defects in cell proliferation (Larschan, 2007).

In vitro studies using recombinant MSL3 produced in baculovirus revealed preferential interaction with nucleosomes that were trimethylated at H3K36, suggesting that a direct interaction may occur between MSL complex and H3K36me3 chromatin on the X chromosome. In S. cerevisiae, an MSL3 homolog, Eaf3, mediates an interaction between the Rpd3(S) complex and H3K36me3 at active genes. If conserved, this function in Drosophila presumably would be played by another MSL3 family member, MRG15. In S. cerevisiae, Rpd3(S) is thought to deacetylate histones in the wake of RNA polymerase II to prevent uncontrolled activation and transcription initiation from cryptic start sites within genes. This raises the possibility that, on the X chromosome, MSL complex might compete for binding to H3K36me3 with the repressive deacetylation function of Rpd3(S). Alternatively, H3K36me3 may simply be a mark utilized by MSL complex to regulate target genes by a mechanism independent of Rpd3(S) (Larschan, 2007).

H3K36me3 marks transcribed genes independent of transcript level but is a weak modulator of endogenous transcript and RNA polymerase II levels. In S. cerevisiae, where its role is best understood, Set2 functions to suppress formation of aberrant internal transcripts by facilitating histone deacetylation yet has only small effects on endogenous transcript levels. In Drosophila, small but reproducible changes were detected in transcript levels at MSL complex target genes in set2 mutant larvae. Also, minimal changes were observed in RNA Pol II levels as previously reported for the set2Δ mutant in S. cerevisiae. Also, changes in transcription level due to loss of dosage compensation are small, with a maximal 50% decrease predicted. Thus, the combined loss of the Set2 protein and reduction in MSL complex recruitment did not cause dramatic changes in transcript level. Furthermore, levels of H4Ac16 were decreased but not eliminated at target genes, consistent with residual MSL function that can explain why more dramatic changes in transcription of MSL complex target genes were not observed (Larschan, 2007).

A defined mechanism for MSL complex targeting to hundreds of sites along the male X chromosome has remained elusive. Previous reports have posited two highly related models for MSL complex recruitment: a 'spreading' model and an 'affinities' model. Both models are based on the idea that specific MSL interaction occurs at high-affinity sites that mark the X chromosome. These sites have been mapped on polytene chromosomes, but most are not yet defined at the molecular level. roX genes and other high-affinity sites are thought to concentrate MSL complex within an X chromosome domain. In the spreading model, MSL complex creates the full MSL binding pattern by searching the X chromosome for general characteristics of active genes without necessarily requiring a specific DNA sequence at each gene. This could occur either by scanning along the chromosome in a linear manner or by releasing and rebinding chromosomal regions in close physical proximity. It has been demonstrated that roX RNAs can move in trans from one DNA molecule to another, so linear scanning is possible but not obligatory. The affinities model proposes that there is a continuum of affinity sites for MSL complex, ranging from high to low. Only when high-affinity sites are locally concentrated can low-affinity sites be recognized, similar to the spreading model. The major difference is that even low-affinity sites are predicted to contain sequence elements that direct MSL binding. It is thought that the results documenting the pattern of ectopic MSL binding on chromosome 2L surrounding a roX transgene make the existence of sequence elements at every MSL binding site on the X chromosome unlikely. That the 2L pattern was analogous to that normally found on the X chromosome, targeting transcribed genes marked by H3K36me3 and binding with a 3' bias, is strong evidence that MSL complex recognizes target genes marked by transcription. This does not exclude the possibility that transcribed genes carry common sequence elements but makes it unlikely that such sequence elements differ between autosomal genes and the majority of MSL target genes on the X chromosome (Larschan, 2007).

In summary, the data are consistent with a model in which MSL complex first recognizes nascent roX transcripts and a series of high-affinity sequences along the male X chromosome and then scans the X for target genes that exhibit H3K36 trimethylation and other marks of active transcription. Recognition may involve the MSL3 chromodomain and additional factors. Trimethylation of H3K36 marks the middle and 3' ends of transcription units, independent of absolute transcript levels in Drosophila, consistent with S. cerevisiae and mammalian systems. Thus, MSL complex recognition of H3K36me3 provides an important mechanism for identification of transcribed genes and avoidance of silenced regions (Larschan, 2007).

Drosophila dSet2 functions in H3-K36 methylation and is required for development

Drosophila Set2 encodes a developmentally essential histone H3 lysine 36 (K36) methyltransferase. Larvae subjected to RNA interference-mediated (RNAi) suppression of Set2 lack Set2 expression and H3-K36 methylation, indicating that Set2 is the sole enzyme responsible for this modification in Drosophila. Set2 RNAi blocks puparium formation and adult development, and causes partial (blister) separation of the dorsal and ventral wing epithelia, defects suggesting a failure of the ecdysone-controlled genetic program. A transheterozygous EcR null mutation/Set2 RNAi combination produces a complete (balloon) separation of the wing surfaces, revealing a genetic interaction between the Ecdysone receptor (EcR) and Set2. Immunoprecipitation studies demonstrated that Set2 associates with the hyperphosphorylated form of RNA polymerase II (RNAPII) (Stabell, 2007).

Ash1 targets H3 and H4

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

ASH1 is required for methylation of lysine 4 residues on histone H3

Covalent modifications of histone tails modulate gene expression via chromatin organization. As examples, methylation of lysine 9 residues of histone H3 (H3) (H3-K9) is believed to repress transcription by compacting chromatin, whereas methylation of lysine 4 residues of H3 (H3-K4) is believed to activate transcription by relaxing chromatin. The Drosophila trithorax group protein Absent, small, or homeotic discs 1 (Ash1) is involved in maintaining active transcription of many genes. In extreme ash1 mutants, no H3-K4 methylation is detectable. This lack of detectable H3-K4 methylation implies that Ash1 is required for essentially all H3-K4 methylation that occurs in vivo. The 149-aa SET domain of ASH1 is sufficient for H3-K4 methylation in vitro. These findings support a model in which ASH1 is directly involved in maintaining active transcription by conferring a relaxed chromatin structure (Byrd, 2003).

The extent of H3-K4 methylation, observed by immunofluorescence on polytene chromosomes from ash1 mutants, correlates extremely well with that observed by immunoblotting of salivary gland extracts. Immunofluorescence on polytene chromosomes provides qualitative information about the genomic distribution of H3-K4 methylation in addition to quantitative information about the extent of H3-K4 methylation. By using immunofluorescence on polytene chromosomes from salivary glands and immunoblotting of salivary gland extracts, it has been shown that detectable H3-K4 methylation is essentially eliminated by strong ash1 mutations. This lack of detectable H3-K4 methylation in ash 1 mutants indicates that Ash1 is required for H3-K4 methylation, but it does not indicate whether that requirement is direct. One possibility is that histone integrity is destroyed in ash1 mutants, and the failure to detect H3-K4 methylation is only a secondary consequence. This possibility was ruled out by showing that in those same mutants histone acetylation and methylation of other residues is not affected. Ash1 is a component of a multimeric protein complex; another possibility is that some other component of the complex is responsible for histone methylation. In the antimorphic alleles where no full-length Ash1 protein can accumulate, the complex might not form and thus prevent another component from functioning. One argument against this possibility is that in the missense mutant ash1¹⁰, no methylation of H3-K4 is detected despite the accumulation of normal amounts of full-length Ash1 protein on polytene chromosomes. However, an even stronger argument against this possibility is that fragments of Ash1 can methylate H3-K4 in vitro, so the evidence is that Ash1 is required directly for virtually all of the H3-K4 methylation that occurs in vivo (Byrd, 2003).

In wild-type polytene chromosomes the vast majority of H3 methylated on lysine 9 residues is located in the chromocenter. In Su(var)3-9 mutants, there is strongly reduced accumulation of methylated H3-K9 in the chromocenter. It is evident, however, that Su(var)3-9 is not the sole HMTase with specificity for H3-K9 in Drosophila, because there is still some H3-K9 methylation on chromosomes from Su(var)3-9 null mutants. Su(var)3-9 null mutants are viable, affecting only position effect variegation, suggesting that the catalytic activity of Su(var)3-9 alone is not sufficient for global gene silencing. There is also significantly reduced accumulation of methylated H3-K9 at the chromocenter of ash1 mutants. This was surprising, because Ash1 is not known to play any role in heterochromatic gene silencing. Perhaps Ash1 plays a role in gene silencing in combination with SU(VAR)3-9 that has yet to be discovered. In any case, this means that Ash1 is one of at least two enzymes that can catalyze methylation of H3-K9. This is consistent the observation that a 588-aa fragment of Ash1 can methylate both K4 and K9 residues of H3 in vitro. However, little H3-K9 methylation is detected outside of the chromocenter on wild-type chromosomes. The pattern of H3-K9 methylation observed is completely different from the pattern of H3-K4 methylation, which is not consistent with the idea that Ash1 catalyzes the methylation of both K4 and K9 on the same H3 molecules. One possible explanation of this discrepancy is that Ash1 catalyzes only a small fraction of the H3-K9 that occurs in vivo, and that the amount of H3-K9 methylation in the chromocenter catalyzed by SU(VAR)3-9, Ash1, and possibly other enzymes overwhelms the ability to detect any on the chromosome arms. A widespread distribution of acetylated H3-K9 is detected on chromosome arms. If Ash1 methylates H3-K9 along chromosome arms, it might have been expected that the level of H3-K9 acetylation would increase in ash1 mutants, because the absence of methylation would increase the availability of free H3-K9 residues. However, no such increase was observed. The 588-aa Ash1 fragment can also catalyze a low level of methylation of H4-K20. A widespread distribution of methylated H4-K20 along the chromosome arms is observed. However, no loss of H4-K20 methylation was observed even in the strongest ash1 mutants. If Ash1 catalyzes H4-K20 methylation in vivo, it must catalyze only a small fraction of the total H4-K20 methylation that occurs (Byrd, 2003).

The SET domain of Ash1 is important for HMTase activity. The ash1¹⁰ allele that has a substitution within the SET domain (N1385I) causes absence of H3-K4 methylation. The significance of this conserved asparagine for the HMTase activity of the Ash1 SET domain is underscored by evidence that H3-K4 methylase activity of a 588-aa Ash1 fragment is lost when this same substitution is introduced. The finding that the ash1²¹ allele that has a substitution within the preSET domain (E1248K) causes reduction but not elimination of H3-K4 methylation suggests that the preSET domain may affect the efficiency of methylation but is not essential for activity. This conclusion is supported by data showing that the SET domain by itself (residues 1300-1448) can methylate H3-K4 in vitro. However, this conclusion is not supported by a report that the H3-K4 methylase activity of a 588-aa Ash1 fragment (1032-1619) is lost when this same substitution (E1248K) is introduced. It has also been reported that the 588-aa fragment of Ash1 can methylate H3-K9 residues. The 149-aa SET domain contained within that 588-aa fragment cannot methylate H3-K9 residues, suggesting that the preSET and postSET domains add functionality required for H3-K9 methylation (Byrd, 2003).

Lysine residues can be mono-, di-, or tri-methylated. Recent evidence suggests that, at least in Saccharomyces cerevisiae, active genes are trimethylated. The antibody used to detect lysine 4 methylation of H3 was made against a peptide with a dimethylated lysine. It has the highest specificity for peptides with dimethylated lysine residues, but it can also detect peptides with mono- and tri-methylated lysine residues. Although the 149-aa SET domain of Ash1 can methylate H3-K4 in vitro, the methylation state of the product detected is not known. The in vivo function of Ash1 may be to mono- and/or dimethylate H3-K4 residues; di- and/or trimethylation may be the function of some other HMTase. If this is the case, then the absence of H3-K4 methylation found in extreme ash1 mutants means that the methylation function of Ash1 creates a substrate essential for subsequent methylation. Support for this idea comes from data showing that in ash2 null mutants, the extent of H3-K4 methylation is reduced, but the distribution is like wild-type. The human homologue of ASH2 is in a multimeric complex with SET1, a protein shown to have H3-K4 methylase activity. If Drosophila ASH2 is also in a complex with a SET1 homologue, then such a complex may be responsible for subsequent methylation of H3-K4 residues initially methylated by Ash1 (Byrd, 2003).

Genetic evidence has indicated that Ash1 is a member of the Trx group of proteins that is involved in maintaining active transcription of many genes. The activities of Ash1 and Trx are functionally related. Mutations in ash1 and trx exhibit intergenic noncomplementation; Ash1 and Trx colocalize at multiple sites on polytene chromosomes, and Ash1 can be coimmunoprecipitated from embryonic nuclear extracts by antibodies that recognize Trx. Moreover, Trx accumulation on polytene chromosomes is reduced in an ash1 mutant and to associate with a histone acetyltransferase, dCBP. These results, together with the results showing that Ash1 functions as an HMTase, suggest a model for the sequential order to Ash1 and Trx association and in turn histone methylation and acetylation. According to this model, Ash1 binds to H3 via its SET domain and methylates K4 residues. The SET domain of Trx recognizes these methylated H3-K4 residues, which could explain the loss of Trx on chromosomes from an ash1 mutant. Trx recruits dCBP, which can then acetylate nearby lysine residues. If this model were correct, one would expect that loss of Ash1 catalyzed methylation would secondarily cause loss of acetylation. The data, however, do not fulfill this expectation. In ash1 mutants, where the levels of H3-K4 methylation are barely detectable, the levels of acetylation on both H3 and histone H4 are unchanged as compared with wild type. Thus, site-specific changes in histone methylation due to disruption of ash1 have no apparent effect on histone acetylation. It could be, however, that only acetylation of residues that do not depend on methylation of H3-K4 were examined. Moreover, ash1¹⁴, the mutant that showed reduced chromosomal Trx, has a molecular defect at nearly the same location as ash1¹⁶. It is likely that ash1¹⁴ also has a normal level of HMTase activity, which suggests that Trx requires the C-terminal domain of Ash1 rather than its HMTase activity to bind to chromosomes. Further work will be required to understand the molecular basis of the functional relationship between Ash1 and Trx and the roles of other components of the 2MDa Ash1 complex (Byrd, 2003).

Drosophila Paf1 modulates chromatin structure at actively transcribed genes

The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. The Drosophila homologs of three subunits of the yeast Paf1 complex have been identified and characterized and striking differences were found between the yeast and Drosophila complexes. Although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure (Adelman, 2006; full text of article).

Characterization of the Drosophila homologs of yeast Paf1 subunits has revealed several features in common and several critical differences between the yeast and Drosophila Paf1 complexes. The most striking similarities between the yeast and Drosophila Paf1 complexes are their association with elongating RNA Pol II and their roles in gene activation, while the nature of the Pol II association and the composition of the Paf1 complex reflect marked differences between the species (Adelman, 2006).

The global view provided by Drosophila polytene chromosomes shows that the chromosome-associated Paf1 and Rtf1 proteins colocalize with active Pol II. This result supports the idea that these proteins participate in most, if not all, Pol II transcription. Remarkably, Paf1 and Rtf1 do appear to be separable from actively elongating Pol II under conditions of heat shock. Although Paf1 and Rtf1 are recruited actively to heat shock loci upon heat stress, these factors also remain associated with a number of additional sites on the chromosome, while Pol II is localized almost exclusively at heat shock loci under these conditions. These data suggest that Paf1 and Rtf1 may remain bound to the chromosome at activated genes through interactions with additional proteins (Adelman, 2006).

It has been suggested that, in yeast, while the Paf1 complex is entirely nuclear in its localization, it has cellular functions that are independent of elongating Pol II. The nucleolar association of Paf1 and Rtf1 observed on Drosophila polytene chromosomes could possibly represent such a function. At the nucleolar organizer, Paf1 shows broad labeling while the Rtf1 signal is restricted to the nucleolar periphery in a manner that is largely nonoverlapping. Interestingly, although the yeast Paf1 complex does not show strong nucleolar association normally (Porter, 2005), in an Rtf1 mutant, the Paf1 complex shows a strong association that is postulated to be a manifestation of its normal role in nuclear processing or export (Adelman, 2006).

By using ChIP experiments, this study obtained a higher-resolution view of the localization of Paf1, Rtf1, and Cdc73 at the Hsp70 gene. The lack of a ChIP signal at Hsp70 under uninduced conditions demonstrates that the presence of engaged Ser-5-P Pol II or the associated elongation factors such as Spt5 and TFIIS is not sufficient to recruit Paf1, Rtf1, or Cdc73. Upon heat induction, recruitment of all three proteins was observe primarily within the coding regions of active Drosophila genes, rather than regions upstream of the promoter, or downstream of the site for cleavage and polyadenylation. The reduction in the Paf1 signal downstream of the polyadenylation site, which accompanies a decrease in the Pol II signal, likely signifies that Paf1 dissociates from chromatin within this region, consistent with recent results obtained with yeast. However, it is noted that the absence of a significant Paf1 signal obtained with a given primer pair may simply indicate that the interactions of Paf1 with a particular region are transient (Adelman, 2006).

The Paf1 complex in S. cerevisiae has been reported to be required for full Ser-2 phosphorylation of the Pol II CTD. This role of Paf1 in CTD phosphorylation regulation also appears consistent with the fact that rtf1Delta mutants show synthetic lethality with CTD kinase and phosphatase mutants in CTK1 and FCP1. The lack of a Ser-2-P Pol II signal detected in yeast Paf1 mutants resulted in reduced recruitment of cleavage and polyadenylation factors, causing a defect in the polyadenylation of nascent transcripts. However, although depletion of Drosophila Paf1 or Rtf1 has a marked effect on induced Hsp70 RNA levels, no change was seen in the levels of Ser2-P Pol II on the Hsp70 gene in Paf1 or Rtf1 RNAi-treated cells, indicating a difference between the functions of Paf1 in yeast and a metazoan system (Adelman, 2006).

Another fundamental difference that observed between Drosophila and yeast Paf1 complexes is the relationship of the Paf1 and Rtf1 subunits in providing anchorage of the complex to Pol II. In yeast, it has shown that the association of Paf1 with Pol II and active chromatin depends on the presence of Rtf1. In contrast, this study found that the recruitment of Paf1 to activated Drosophila Hsp70 is independent of Rtf1, while Rtf1 recruitment is dependent on Paf1. These results may reflect the evolution of a more important role for the Paf1 protein in metazoans in providing affinity of the complex for Pol II, while Rtf1 became a more loosely bound component of the complex (Adelman, 2006).

The role was investigated of Drosophila Paf1 in the modification of histones within actively transcribed regions. Whereas yeast Paf1 has been implicated in regulating the bulk levels of methylation of histone H3 at lysine residues 4 and 79, an effect was observed of Paf1 depletion on the trimethylation of H3-K4, but not on di- or trimethylation of H3-K79. Similarly, it was observed that trimethylation of H3-K4 occurred within the promoter-proximal region of Hsp70 and Hsp26 upon heat shock and could be seen to increase from 2.5 to 10 min after heat induction, but no significant levels of H3-K79 dimethylation were observed within the active Hsp70 gene. The latter result differs from results from other systems which link H3-K79 dimethylation with active transcription. However, it is consistent with recent data suggesting that both Grappa, the Drosophila H3-K79 methyltransferase, and the signal corresponding to H3-K79 dimethylation are localized to both active and intergenic regions of Drosophila polytene chromosomes. An alternative possibility is that the apparent differences between yeast and Drosophila result from the experimental systems used; RNAi treatments in Drosophila decrease, but do not completely abolish, their target, and thus the small amount of remaining protein may be sufficient to carry out certain functions. Conversely, the deletion mutants used to investigate yeast Paf1 entirely remove an important protein for many generations of cell growth, raising the possibility that some observed effects are indirect or secondary in nature (Adelman, 2006).

It is interesting that although H3-K4 trimethylation depends upon Paf1 and the recruitment of Paf1 is temporally similar to H3-K4 methylation, the distribution of Paf1 appears to be spatially distinct from the promoter region where the strongest trimethylated H3-K4 signals are observed. Thus, the results suggest that the effects of Paf1 mutants on the modification of promoter-proximal nucleosomes (including the ubiquitination of H2B-K123) may occur through indirect mechanisms. These data are consistent with reports on yeast that indicate that the distribution of Paf1 subunits does not strictly correlate with the patterns of ubiquitinated H2B or methylated histone H3 (Ng, 2003). The localization of H3-K4 trimethylation reported in this study is in agreement with the recently described distribution of Trithorax, a Drosophila H3-K4 methyltransferase (Smith, 2004). Furthermore, recent studies employing a Drosophila Trithorax mutant fly line suggest that a multiprotein complex that contains Trithorax plays a role in Hsp70 gene activation. However, whether the role of Trithorax in Hsp70 activation is direct or indirect remains to be established. It is noted that no effect of Paf1 depletion is observed on the rates of Pol II recruitment, or distribution over the gene, suggesting that H3-K4 trimethylation may serve as a mark of transcription activation rather than a prerequisite for gene activation (Adelman, 2006).

These studies have provided new insights into the increased importance of the Paf1 complex in a metazoan system. It is significant that Paf1 is recruited in a manner that is spatially and temporally identical to that of chromatin-associated factors Spt6 and FACT. In agreement with the strong colocalization of Paf1 with these nucleosome-associated factors, it was shown that depletion of Paf1 significantly reduces the recruitment of both Spt6 and the FACT subunit SSRP1. A relationship among Paf1, Rtf1, and FACT is consistent with findings that an rtf1Delta mutation shows synthetic lethality with POB3, a subunit of the yeast FACT complex. Moreover, the FACT complex has been shown to interact with the Paf1 complex and the chromodomain-containing Chd1 protein at actively transcribed genes. In vitro, FACT has been shown to function optimally to facilitate transcription through nucleosomes when it is present at approximately one molecule of FACT per two nucleosomes; the effectiveness of FACT in promoting elongation is decreased dramatically below this threshold. If these results reflect the situation in vivo, the greater than 50% decrease in FACT levels at the active Hsp70 gene in Paf1-depleted cells would result in a rather pronounced effect on transcription through nucleosomes (Adelman, 2006).

Furthermore, recent evidence obtained with yeast has shown that mutations of Spt6 or the FACT subunit Spt16 lead to aberrant chromatin architecture in the wake of elongating Pol II, presumably due to defects in reassembly of nucleosome structure. The failure to efficiently repackage transcribed DNA results in transcription initiation from cryptic sites and a reduction in levels of properly initiated and processed RNA. If a primary role of Drosophila Paf1 is to help stably recruit factors like Spt6 and FACT, then loss of Paf1 activity could also lead to the accumulation of nonfunctional or improperly processed RNA species. In support of this idea, a paper that was published during the preparation of this report states that mutations in yeast Spt6 alter the recruitment of Paf1 subunit Ctr9 and lead to defects in 3'-end processing of nascent RNA (Kaplan, 2005). It is thus tempting to speculate that the vast array of transcription elongation and RNA processing and export defects reported in yeast Paf1 mutant strains could result from perturbation of the nucleosome structure along actively transcribed genes. Moreover, it may be these chromatin and processing defects that account for the decrease in the amount of Hsp70 mRNA that accumulates in response to heat shock in Paf1- or Rtf1-depleted cells (Adelman, 2006).

Finally, the Paf1 gene in yeast is nonessential while the Paf1 gene in Drosophila is essential. This may reflect the more varied and demanding requirements of the transcription machinery in higher eukaryotes, where chromatin frequently plays a greater and more stringent role in regulation. This, in turn, may place a greater demand on the Paf1 complex, which appears to function at the interface betwe