InteractiveFly: GeneBrief

Set2: Biological Overview | References


Gene name - Set2

Synonyms -

Cytological map position - 11F4-11F5

Function - chromatin, enzyme

Keywords - histone H3 K36 methyltransferase, dosage compensation, ecdysone-controlled program

Symbol - Set2

FlyBase ID: FBgn0030486

Genetic map position - X:13,307,311..13,315,061 [+]

Classification - Set2-Rpb1 interacting, SET domain

Cellular location - nuclear



NCBI link: EntrezGene
Set2 orthologs: Biolitmine
Recent literature
Mitra, R., Richhariya, S., Jayakumar, S., Notani, D. and Hasan, G. (2021). IP3-mediated Ca2+ signals regulate larval to pupal transition under nutrient stress through the H3K36 methyltransferase Set2. Development 148(11). PubMed ID: 34117888
Summary:
dPersistent loss of dietary protein usually signals a shutdown of key metabolic pathways. In Drosophila larvae that have reached a 'critical weight' and can pupariate to form viable adults, such a metabolic shutdown would needlessly lead to death. Inositol 1,4,5-trisphosphate-mediated calcium (IP3/Ca2+) release in some interneurons (vGlutVGN6341) allows Drosophila larvae to pupariate on a protein-deficient diet by partially circumventing this shutdown through upregulation of neuropeptide signaling and the expression of ecdysone synthesis genes. This study shows that IP3/Ca2+ signals in vGlutVGN6341 neurons drive expression of Set2, a gene encoding Drosophila Histone 3 Lysine 36 methyltransferase. Furthermore, Set2 expression is required for larvae to pupariate in the absence of dietary protein. IP3/Ca2+ signal-driven Set2 expression upregulates key Ca2+-signaling genes through a novel positive-feedback loop. Transcriptomic studies, coupled with analysis of existing ChIP-seq datasets, identified genes from larval and pupal stages that normally exhibit robust H3K36 trimethyl marks on their gene bodies and concomitantly undergo stronger downregulation by knockdown of either the intracellular Ca2+ release channel IP3R or Set2. IP3/Ca2+ signals thus regulate gene expression through Set2-mediated H3K36 marks on select neuronal genes for the larval to pupal transition.
McCarthy, A., Sarkar, K., Martin, E. T., Upadhyay, M., Jang, S., Williams, N. D., Forni, P. E., Buszczak, M. and Rangan, P. (2022). Msl3 promotes germline stem cell differentiation in female Drosophila. Development 149(1). PubMed ID: 34878097
Summary:
Gamete formation from germline stem cells (GSCs) is essential for sexual reproduction. However, the regulation of GSC differentiation is incompletely understood. Set2, which deposits H3K36me3 modifications, is required for GSC differentiation during Drosophila oogenesis. The H3K36me3 reader Male-specific lethal 3 (Msl3) and histone acetyltransferase complex Ada2a-containing (ATAC) cooperate with Set2 to regulate GSC differentiation in female Drosophila. Msl3, acting independently of the rest of the male-specific lethal complex, promotes transcription of genes, including a germline-enriched ribosomal protein S19 paralog RpS19b. RpS19b upregulation is required for translation of RNA-binding Fox protein 1 (Rbfox1), a known meiotic cell cycle entry factor. Thus, Msl3 regulates GSC differentiation by modulating translation of a key factor that promotes transition to an oocyte fate.
Jauregui-Lozano, J., McGovern, S. E., Bakhle, K. M., Hagins, A. C. and Weake, V. M. (2023). Establishing the contribution of active histone methylation marks to the aging transcriptional landscape of Drosophila photoreceptors. Sci Rep 13(1): 5105. PubMed ID: 36991154
Summary:
Studies in multiple organisms have shown that aging is accompanied by several molecular phenotypes that include dysregulation of chromatin. Since chromatin regulates DNA-based processes such as transcription, alterations in chromatin modifications could impact the transcriptome and function of aging cells. In flies, as in mammals, the aging eye undergoes changes in gene expression that correlate with declining visual function and increased risk of retinal degeneration. However, the causes of these transcriptome changes are poorly understood. This study profiled chromatin marks associated with active transcription in the aging Drosophila eye to understand how chromatin modulates transcriptional outputs. Both H3K4me3 and H3K36me3 globally decrease across all actively expressed genes with age. However, no correlation was found with changes in differential gene expression. Downregulation of the H3K36me3 methyltransferase Set2 in young photoreceptors revealed significant changes in splicing events that overlapped significantly with those observed in aging photoreceptors. These overlapping splicing events impacted multiple genes involved in phototransduction and neuronal function. Since proper splicing is essential for visual behavior, and because aging Drosophila undergo a decrease in visual function, these data suggest that H3K36me3 could play a role in maintaining visual function in the aging eye through regulating alternative splicing.
BIOLOGICAL OVERVIEW

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).

A second study further describes the effects of Set2 inactivation (Stabell, 2007). 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).

Located on the X chromosome, dSet2 (Flybase CG1716) has an ORF of 7086 bp predicting a 2362-amino-acid protein with a domain structure similar to that of Saccharomyces cerevisiae Set2p, NSD1 and HYPB in mammalia, and SDG8 (set domain group 8) in Arabidopsis, including a SET domain with the adjacent AWS (associated with SET), and post-SET domains. The SET domain contains the R(H)FFNHSC motif (where F denotes a hydrophobic residue) that is correlated with HKMT activity. Drosophila Set2 also contains the conserved WW domain and SRI domains near and at the very end of the C-terminus, respectively; the former is involved in protein-protein interactions, and the latter is associated with the elongating RNAPII. In addition, Drosophila Set2 also contains coiled-coil domains, two AT-hooks, and a putative dead-box helicase domain. The identification of several putative single and bipartite nuclear localization signals implies that this protein exerts its function(s) in the nucleus. Immunostaining of polytene chromosomes showed that Set2 indeed binds to DNA and gives a distinct banding pattern. There is no staining in the chromocenter, indicating that Set2 is mainly associating with euchromatin. It should be noted, however, that the telomeric regions of chromosome 2L and 4 were stained consistently. A similar staining pattern has, with regards to the tip of 2L and 4, been reported for SU(VAR)2-10 that belongs to the PIAS protein family. Members of this family act, for instance, as nuclear receptor coregulators for the androgen, glucocorticoid, and progesterone receptors (Stabell, 2007 and references therein).

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

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 (Bannister, 2005; Pokholok, 2005; Vakoc, 2006). 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 (Rao, 2005; Kim, 2007). 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 previously (Alekseyenko, 2006) as 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 (Alekseyenko, 2006). 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 (Rao, 2005; Strahl, 2002). 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 (Morris, 2005). 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 (Vojnic, 2006). 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 ovoD dominant female sterile mutant. After recombination, the set2 mutant germ cells would no longer carry ovoD and thus should produce oocytes that would lack any maternal Set2 mRNA or protein. Despite recombination to remove ovoD 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 (Morris, 2005). 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 (Morris, 2005; Adhvaryu, 2005; Brown, 2006). The S. cerevisiae set2Δ mutant suppresses the loss of positive elongation factors (Biswas, 2006; Chu, 2006; Keogh, 2005). 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 (Carrozza, 2005; Keogh, 2005). 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 (Carrozza, 2005). 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 (Krogan, 2003; Rao, 2005; Kim, 2007; Li, 2007b). 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 (Carrozza, 2005; Li, 2007a). 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 (Krogan, 2003). Also, changes in transcription level due to loss of dosage compensation are small, with a maximal 50% decrease predicted (Hamada, 2005). 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 (Rao, 2005; Kim, 2007). Thus, MSL complex recognition of H3K36me3 provides an important mechanism for identification of transcribed genes and avoidance of silenced regions (Larschan, 2007).

Transcription-coupled methylation of histone H3 at lysine 36 regulates dosage compensation by enhancing recruitment of the MSL complex in Drosophila melanogaster

In Drosophila melanogaster, dosage compensation relies on the targeting of the male-specific lethal (MSL) complex to hundreds of sites along the male X chromosome. Transcription-coupled methylation of histone H3 lysine 36 is enriched toward the 3' end of active genes, similar to the MSL proteins. This paper reports a study of the link between histone H3 methylation and MSL complex targeting using RNA interference and chromatin immunoprecipitation. Trimethylation of histone H3 at lysine 36 (H3K36me3) relies on the histone methyltransferase Hypb (Set2) and is localized promoter distal at dosage-compensated genes, similar to active genes on autosomes. However, H3K36me3 has an X-specific function; reduction specifically decreases acetylation of histone H4 lysine 16 on the male X chromosome. This hypoacetylation is caused by compromised MSL binding and results in a failure to increase expression twofold. Thus, H3K36me3 marks the body of all active genes yet is utilized in a chromosome-specific manner to enhance histone acetylation at sites of dosage compensation (Bell, 2008).

This study reports that trimethylation of histone H3 lysine 36 is required for high levels of H4K16ac at dosage-compensated genes on the male X chromosome. This function does not reflect an X-specific methylation signature, since both H3K36 methylation states have similar localization patterns at autosomal genes: dimethylation peaks promoter-proximal, and trimethylation shows a 3' bias. Furthermore, the regulation of H3K36me3 depends on the activity of Hypb, which is equally targeted to autosomal and X-linked loci, indicating a common mode of regulation (Bell, 2008).

Nevertheless, downregulation of H3K36me3 in Drosophila SL2 cells resulted in reduced levels of H4K16 hyperacetylation at X-linked genes but simultaneously increased levels at autosomal genes in the same cells. This differential effect on acetylation suggests a context-dependent readout of lysine 36 methylation. In Saccharomyces cerevisiae, H3K36me signals binding of the chromodomain-containing protein Eaf3, which in turn recruits an Rpd3 complex to deacetylate the 3' end of transcribed genes. Evidence is provided that the X-specific reduction of histone acetylation in Hypb-depleted Drosophila SL2 cells reflects compromised recruitment of MSL1 and MOF at dosage-compensated genes. This is in full agreement with reduced binding of MSL3 upon Hypb knockdown, which was recently reported by Larschan and colleagues. MSL3 is one of the Drosophila homologues of yeast Eaf3 and localizes together with MOF and MSL1 to the 3' end of dosage-compensated genes. Thus, in analogy to yeast, MSL3 is likely to associate with H3K36me3 at the 3' end of X-linked genes, leading to robust complex binding and enhanced H4K16 hyperacetylation. This is supported by evidence showing that MSL3 preferentially interacts with Set2-methylated nucleosomes in vitro. Moreover, the observation of Hypb localizing to active sites on polytene chromosomes provides further evidence for a direct role of H3K36me3 in MSL recruitment. However, not all sites enriched for Hypb were also bound by MSL1, suggesting that H3K36me3 is necessary but not sufficient for MSL complex recruitment (Bell, 2008).

Whereas proper binding of the MSL complex to Par-6, CG8173, and Ucp4A relies on the presence of H3K36me3, Hypb knockdown has been shown to not significantly decrease MSL1 recruitment at the roX2 gene. This is similar to the binding of MSL1 and MSL2 to high-affinity sites in msl3 or mof mutant flies, suggesting that strong sequence affinity can target partial MSL complexes independent of H3K36me3. Importantly, despite its presence at the roX2 locus in Hypb knockdown cells, MSL1 was insufficient for adequate MOF recruitment and transcriptional upregulation. Thus, these data indicate that H3K36me3 is necessary at high-affinity sites to facilitate robust MOF interaction and the subsequent hyperacetylation needed to double transcription (Bell, 2008).

Interestingly, roX2 transcription was unaffected by Hypb RNAi when expressed from a plasmid model system. Since the consequence of reduced H3K36me3 on H4K16ac on the roX2 plasmid was not determined in this study, it is possible that a less pronounced reduction in acetylation might account for this effect (Bell, 2008).

In contrast to the roX2 gene, H3K36me3 was required for MSL1 binding to lower-affinity genes. At these genes, transcription-dependent methylation might facilitate DNA accessibility in the 3' end by enhancing the recruitment of MOF and the hyperacetylation of H4K16 (Bell, 2008).

At autosomal genes, reduced trimethylation caused the opposite effect on H4 lysine 16 acetylation. Thus, one modification may signal two different outcomes in the same cell in a chromosome-specific fashion. It is conceivable that such differential readouts involve interaction with either distinct methyl-binding proteins or alternative subunit compositions (Bell, 2008).

The presence of antagonistic activities in the same nucleus, which are targeted to the same modification, requires spatial restriction of individual protein complexes to avoid deregulation by improper acetylation or deacetylation. Thus, the preferential interaction of MSL proteins with H3K36me3 on the X chromosome might be favored by locally accumulating MSL proteins at high-affinity sites. MSL interactions with nuclear pore proteins suggest a possible role of nuclear organization in X chromosome dosage compensation, which may further contribute to a preferential binding of MSL proteins to H3K36me3. Conversely, while this confines histone acetyltransferase activity to dosage-compensated genes on the X chromosome, it might also ensure that the same activity is not mistargeted to autosomal genes (Bell, 2008).

H3K36 trimethylation-mediated epigenetic regulation is activated by Bam and promotes germ cell differentiation during early oogenesis in Drosophila

Epigenetic silencing is critical for maintaining germline stem cells in Drosophila ovaries. However, it remains unclear how the differentiation factor, Bag-of-marbles (Bam), counteracts transcriptional silencing. This study found that the trimethylation of lysine 36 on histone H3 (H3K36me3), a modification that is associated with gene activation, is enhanced in Bam-expressing cells. H3K36me3 levels were reduced in flies deficient in Bam. Inactivation of the Set2 methyltransferase, which confers the H3K36me3 modification, in germline cells markedly reduced H3K36me3 and impaired differentiation. Genetic analyses revealed that Set2 acts downstream of Bam. Furthermore, orb expression, which is required for germ cell differentiation, was activated by Set2, probably through direct H3K36me3 modification of the orb locus. These data indicate that H3K36me3-mediated epigenetic regulation is activated by bam, and that this modification facilitates germ cell differentiation, probably through transcriptional activation. This work provides a novel link between Bam and epigenetic transcriptional control (Mukai, 2015).

To examine histone modifications in differentiating germ cells, wild-type ovaries were stained using monoclonal antibodies specific for histone modifications. The H3K36me3 histone modification, associated with active genes, accumulated in differentiating cystoblasts. H3K36me3 signals were increased in the differentiating cystoblasts that expressed the bam reporter gene (bam-GFP). By contrast, the H3K27me3 modification associated with gene repression accumulated in early germ cells, and its signals decreased as the cells differentiated. These results suggest that the H3K36me3 levels were upregulated in differentiating cystoblasts. Next, H3K36me3 levels were examined in the ovaries of the third instar larvae and bam86 mutant adult females, both of which contain undifferentiated germ cells. Although H3K27me3 signals were detected in these undifferentiated germ cells, strong H3K36me3 signals were not detected. Taken together, these data supported the idea that H3K36me3-mediated epigenetic regulation may be involved in germ cell differentiation. (Mukai, 2015).

Set2 methyltransferase is responsible for the H3K36me3 modification. Immunostaining revealed that, in the germarium region, Set2 was expressed in most of the germline cells, and that nuclear Set2 levels increased in differentiating cystoblasts. To determine whether Set2 participates in H3K36me3 accumulation and differentiation, Set2 expression was inhibited by using an UAS-Set2.IR line. Set2 levels in germ cells were reduced by the expression of Set2 RNAi. Specifically, while Set2 signals in differentiating cystoblasts were detected in 100% of control (nanos-Gal4/+) germaria, the Set2 signals in the cystoblasts were significantly reduced in 57% of the germaria, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver. Next, H3K36me3 levels were investigated in the ovaries expressing Set2 RNAi. As expected, H3K36me3 levels were reduced as a consequence of Set2 RNAi treatment. In control ovaries, H3K36me3 signals in differentiating cystoblasts were detected in 97% of germaria. By contrast, when Set2 RNAi was expressed in germ cells under the control of the nanos-Gal4 driver, H3K36me3 signals in cystoblasts were severely reduced in 41% of the germaria. Moreover, germ cell differentiation was impaired because of the expression of Set2 RNAi. In 96% of the control germaria, cysts with branched fusomes were observed. However, fragmented fusomes were detected in 34% of the germaria expressing Set2 RNAi. These results indicate that Set2 was required for both H3K36me3 accumulation and cyst formation. Mosaic analysis was performed by using a Set2 null allele Set21. Strong H3K36me3 signals were observed in 80% of the control germline clones. By contrast, H3K36me3 levels were considerably reduced in 74% of the Set2- cystoblasts. Furthermore, a differentiation defect was observed that was similar to that induced by Set2 RNAi treatment in 84% of Set2- mutant cysts. These results suggest that Set2 is intrinsically required both for H3K36me3 accumulation in cystoblasts and for differentiation (Mukai, 2015).

To investigate the potential regulatory link between Set2 and Bam, their genetic interaction was analyzed. Reduction in Set2 activity by introduction of a single copy of Set21 dominantly increased the number of germaria with the differentiation defect in bam86/+ flies. Fragmented fusomes were observed in 26% of germaria from the Set21/+; bam86/+ females , as compared to 5% in bam86/+ and 3% in Set21/+ females. These results indicated that Set2 cooperates with bam to promote cyst formation. To determine whether bam expression requires Set2 activity, Bam expression in Set2- germline clones by immunostaining. Indeed, Set2 activity in germ cells was dispensable for bam expression. Conversely, nuclear Set2 expression in the germ cells was significantly reduced by bam mutation, suggesting that bam is involved in the regulation of Set2 in these cells. This result is consistent with the observation that H3K36me3 levels were reduced by bam mutation. Moreover, reducing of bam activity by introducing of a single copy of bam86 dominantly increased the number of germaria with weaker H3K36me3 signals in Set21/+ flies. Decreased H3K36me3 signals in the cystoblasts were observed in 29% of germaria from the Set21/+; bam86/+ females, as compared to 3% in Set21/+ and 2% in bam86/+ females. These data prompted an exploration of the mechanism of regulation of Set2 activity by bam (Mukai, 2015).

To address whether bambam is sufficient for H3K36me3 accumulation, H3K36me3 levels were examined in the ovaries carrying the hs-bam transgene, which is used to ectopically express bam+ by heat shock treatment (Ohlstein and McKearin, 1997). No GSCs with a strong H3K36me3 signal were observed in germaria from wild-type females 1 hour post-heat shock (PHS; nā€Š=ā€Š42). However, H3K36me3 levels in the GSCs were significantly increased in 51% of the germaria from hs-bam females 1 hour PHS (nā€Š=ā€Š65), indicating that ectopic bam expression is sufficient for H3K36me3 accumulation. Because Set2 is responsible for H3K36me3, it is speculated that bam may regulate Set2 activity to control H3K36me3 accumulation and GSC differentiation. To determine whether Set2 activity is required for these bam-mediated processes, the effect was studied of a reduction in Set2 activity on the GSC differentiation induced by bam. When bam+ was ectopically expressed by heat shock, GSC differentiation was induced as previously reported. In 71% of ovaries from hs-bam flies dissected 24 hours PHS, it was found that differentiating cysts, instead of GSCs, occupied the tip of germaria. By contrast, when both bam and Set2 RNAi were ectopically expressed, GSC loss was significantly suppressed. These data suggest that Set2 activity is regulated by Bam, and that Set2 acts downstream of bam and promotes differentiation (Mukai, 2015).

Nuclear Set2 levels were increased in differentiating cystoblasts. Furthermore, nuclear Set2 levels in germ cells were reduced by bam mutation. It is speculated that bam may regulate Set2 nuclear localization. Therefore, whether bam expression is sufficient for Set2 nuclear accumulation was examined. The subcellular localization of Set2 was examined in hs-bam flies cultured at 30°C. First, H3K36me3 levels were examined in the GSCs. H3K36me3 levels in GSCs were increased in 36% of the germaria from the hs-bam females, as compared to 6% in wild-type females. This result suggests that the ectopic expression of bam is sufficient for H3K36me3 accumulation. Next, Set2 subcellular localization was examined in GSCs of hs-bam females cultured at 30°C. Nuclear Set2 levels in GSCs were increased in 54% of the germaria from the hs-bam females, as compared to 12% in wild-type females. These results suggest that bam promotes the nuclear accumulation of Set2 (Mukai, 2015).

To understand the mechanism by which Set2 regulates germ cell differentiation, the genetic interaction between Set2 and the differentiation genes A2BP1 and orb, both of which are required for cyst differentiation, were examined. Reduction of Set2 activity by introduction of a single dose of Set21 dominantly increased the number of germaria exhibiting a differentiation defect in orbdec/+ flies. In 24% of germaria from the Set21/+; orbdec/+ females, fragmented fusomes were observed, as compared with 4% in orbdec/+ and 7% in Set21/+ females. By contrast, the reduction of Set2 activity did not significantly affect cyst formation in A2BP1KG06463/+ ovaries). These results implied that Set2 function is required to specifically regulate orb expression and promote cyst formation. To confirm this, orb expression was examined in Set2- cyst clones. Deletion of Set2 led to the delayed activation of orb. Although 74% of the control cyst clones located at the boundary of germarium regions 1 and 2a initiated orb expression, only 31% of Set2- cyst clones expressed orb. Most (61%) of the Set2- cyst clones in germarium region 2b recovered orb expression. These observations suggest that Set2 was required for the proper activation of orb in differentiating cysts. Next, the H3K36me3 state of the orb locus was investigated in the ovaries. ChIP assays demonstrated that the H3K36me3 enrichment in the 3'-UTR region of orb was significantly higher than in the 5'-UTR region. It has been reported that the H3K36me3 modification exhibits a 3'-bias, such that H3K36me3 is preferentially enriched at the 3' regions of actively transcribed genes. These results support the idea that orb expression in differentiating cysts is controlled in part by H3K36me3-mediated epigenetic regulation (Mukai, 2015).

Next, the H3K36me3 status was investigated in the orb gene in bam86 mutant ovaries. ChIP assays showed that bam mutation reduced the amount of H3K36me3 in the 3'-UTR region of the orb gene. The H3K36me3 modification is linked to transcriptional elongation. Therefore, the results suggested that bam activates orb expression through the epigenetic control. Additionally, H3K4me3 and RNA polymerase II levels in the 5'-UTR region of the orb gene were also reduced by bam mutation, implying a role for bam in transcriptional initiation. To investigate this possibility, further investigation will be needed in order to identify the enzymes responsible for H3K4me3 and exploring the interactions between bam and those enzymes (Mukai, 2015).

These results have shown that H3K36me3 levels are regulated by bam. As a cytoplasmic protein, Bam may indirectly regulate Set2 nuclear localization. Set2 exerts its functions through the interactions with cofactors. Understanding the mechanism by which Bam regulates Set2 will require the identification of the cofactors that mediate the nuclear transport of Set2. These data suggest a link between Bam and epigenetic transcriptional control. Bam may counteract epigenetic silencing in GSCs through H3K36me3-mediated epigenetic regulation. This study showed that orb expression is activated by epigenetic regulation. Because orb encodes a cytoplasmic polyadenylation element-binding protein, Orb may control translation in differentiating cysts in a polyadenylation-associated manner. Bam antagonizes the Nanos/Pumilio complex, which suppresses the translation of target mRNAs that encode differentiation factors . However, the ientity of the target mRNAs and the mechanisms for transcriptional activation have not yet been elucidated. Because Set2 is required for bam-induced GSC differentiation, studies focused on identifying the genes marked by H3K36me3 and on their epigenetic regulation will aid in the identification of the differentiation genes. Because Set2 is linked to transcriptional elongation, differentiation genes in GSCs might be poised for expression, but may be kept awaiting bam expression for full activation. It is anticipated that these results will facilitate a better understanding of the epigenetic mechanisms that regulate gametogenesis (Mukai, 2015).

Drosophila Set2 functions in H3-K36 methylation and is required for development

The expression level of Set2 during development was determined by RT-PCR. Set2 is expressed throughout the life cycle of Drosophila. Whereas the level of expression is rather low in 0-6 h embryos it increases to an apparently steady level after that time-point, with a noticeable peak in late third instar larvae. It is interesting to note that the major expression of Set2 in adults is in the gonads (Stabell, 2007).

To investigate the function of Set2, RNA interference (RNAi) was used to lower Set2 mRNA and protein levels. Fly stocks were generated carrying the responder transposon UAS-dSet2.IR, in which a Gal4-dependent promoter expresses the inverted repeat of Set2 to yield a double-stranded RNA molecule. The pUdsGFP vector used for this purpose has an independent UAS-GFP marker so that a tissue exposed to RNAi will simultaneously show GFP expression. Different driver stocks, each expressing Gal4 in a temporally and spatially distinct developmental domain, were selected, and the stocks were crossed to yield hybrid progeny with targeted UAS-dSet2.IR expression (Stabell, 2007).

Initially, a salivary gland-specific driver, Sgs3-Gal4 was used. This allowed immunostaining to be used against Set2 and H3-K36Me, and at the same time visualization of nuclei with DAPI staining and GFP fluorescence. Immunostaining of wild type salivary glands with anti-Set2 and anti-H3K36Me, respectively, showed that both Set2 and dimethylated of H3-K36 are present in the cells. The effect of RNAi knockdown of Set2 by the Sgs3-Gal4 driver was analyzed by immunostaining. Salivary glands from UAS-dSet2/Sgs3-Gal4 larvae were dissected at 3 ± 1 h before puparium formation and immunostained with anti-Set2, anti-dimethyl H3-K4, anti-dimethyl H3-K27, anti-dimethyl H3-K36, anti-H3-9Me, and anti-acetyl H3-K9. Immunostaining with anti-Set2 reveals the absence of Set2 protein in salivary gland cells where UAS-dSet2.IR is expressed by the Sgs3-Gal4 driver. During this work two nuclei in one salivary gland lobe (out of 10 pairs) were found that did not express GFP but were clearly detectable by DAPI staining. Immunostaining with anti-H3-K36Me reveals the absence of H3-K36 dimethylation in salivary gland cells where UAS-dSet2.IR is expressed. Most importantly, however, UAS-dSet2.IR is not induced in the two salivary gland cells not expressing GFP, and here the H3-K36 dimethylation is clearly observable. Furthermore, fat body cells, where the Sgs3-Gal4 driver is not expressed, also show H3-K36 dimethylation in all nuclei. The reason to why the two cells have lost the ability to express the GFP-tagged UAS-dSet2.IR construct may be either loss of UAS or Gal4 sequences or the whole construct(s) prior to the onset of polyploidization. Methylation and acetylation of H3-K9, respectively, are unaffected by the absence of Set2. This also is true for H3-K4 and H3-K27, thus showing the specificity of Set2 RNAi. It is concluded that the Set2 gene encodes a HKMT responsible for all detectable H3-K36 dimethylation in Drosophila (Stabell, 2007).

Set2 interacts genetically with the ecdysone receptor: To test whether a knock down of Set2 affects the fly, the ubiquitously expressed da-Gal4 driver was crossed with UAS-dSet2.IR flies. All progeny are da-Gal4/UAS-dSet2.IR and develop normally until the end of the third larval instar. However, these RNAi larvae do not form their puparium and crawling larvae are found after 10-12 days. The larvae finally stop moving, and in a few cases melanin-less 'pseudo-prepupae' are formed, which maintain the elongated larval form and fail to evert the anterior spiracles. These observations may indicate a defect in ecdysone responses at puparium formation, similar to those reported for mutants in the ecdysone pathways, and thus implicates Set2 in this regulatory cascade (Stabell, 2007).

The defects in puparium formation seen in Set2 RNAi animals could result from either a decrease in the ecdysone titer or a decrease in the ability of the ecdysone signal to be transduced. To distinguish between these possibilities the effects were examined of feeding ecdysone to Set2 RNAi larvae. This method has been shown to effectively rescue phenotypes associated with ecdysone-deficient mutations. Mid- and late-third instar larvae were transferred to food either with or without 20-hydroxyecdysone (20E) for 6-8 h and scored on a 12 h basis. Feeding 20E to Set2 RNAi larvae did not rescue them to puparium formation. Therefore, it is concluded that ecdysone is not limiting in the da-Gal4/UAS-dSet2.IR animals and that Set2 functions downstream of ecdysone biosynthesis and release (Stabell, 2007).

The development of a flat bilayered wing from an imaginal disc monolayer involves four steps that occur twice during metamorphosis. The timing of these two rounds of wing morphogenesis correlates with the two main ecdysone peaks during metamorphosis; namely during mid- and late prepupal stages, and from 24 to 72 h after puparium formation, and it has been shown that ecdysone controls wing morphogenesis and cell adhesion by regulating integrin expression during metamorphosis. The effects of EcR mutations on integrin transcription and the essential role for integrins during wing morphogenesis are both principally restricted to the pupal stages. To investigate if Set2 might be involved in wing morphogenesis, Set2 RNAi was triggered in the wing discs by the ap-Gal4 driver. Induction of Set2 RNAi by the ap-Gal4 driver generates a blister in the proximal (towards thorax) part of the wings. The transparent blisters are initially large balloons filled with air and hemolymph that deflate after a while, leaving a scar that may cause the wing to bend up. This phenotype shows 100% penetrance (Stabell, 2007).

Ecdysone exerts its effects on development through a heterodimer of two nuclear receptors, encoded by EcR and usp. The ecdysone/EcR/USP complex then directly activates cascades of gene expression. To assess if Set2 and EcR genetically interacts in a common pathway controlling wing morphogenesis, whether the EcR null mutation EcRM554fs could dominantly affect the blistered wing phenotype of ap-Gal4/+;UAS-dSet2.IR/+ flies described above was tested. Therefore, the stock y w;EcRM554fs/CyO;UAS-dSet2.IR was synthesized and crossed reciprocally to y w;ap-Gal4/CyO flies. EcRM554fs dramatically enhances the blistered wing phenotype in all EcRM554fs/ap-Gal4;UAS-dSet2.IR/+ flies. The wing layers were completely separated (ballooned) and often one or both wings were held in a Dichaete-like fashion. The balloon soon becomes blackened and remains inflated for 1-2 days. Again, all flies of the genotype ap-Gal4/CyO;UAS-dSet2.IR/+ have the blistered wing phenotype. It should also be mentioned that heterozygous EcRM554fs/CyO;UAS-dSet2.IR/+ flies have normal wings. Taken together, these results suggest that Set2 and EcR may function in a common ecdysone signaling pathway during wing morphogenesis, and that this interaction may be dose sensitive (Stabell, 2007).

These results prompted an analysis by RT-PCR of the ecdysone receptor genes DHR3, DHR4 and reaper (rpr) in dSet2.IR animals around pupariation. While an apparently normal expression pattern for DHR3, DHR4 and rpr was observed at the same time points in control salivary glands, these genes show a clear decline in mRNA levels in Set2 RNAi salivary glands. Both DHR3 and DHR4 execute essential functions during development, including at the onset of metamorphosis. The parallel functions of these two nuclear receptors suggest that they act together to direct the switch from late-larval to prepupal genetic programmes. Although these results were obtained from Set2 RNAi in salivary glands it is believed that these genes, and probably several others, also are affected in da-Gal4/UAS-dSet2.IR larvae, thus explaining the failure of these larvae to pupariate. The fact that none of the studied genes is fully down regulated also suggests that the non-pupariating phenotype may be ascribed to a collective down regulation of essential genes involved in the larval-to-pupal transition. It is also interesting to note the decreased level of rpr mRNA, which is significant at +2 h. Although rpr is not involved in metamorphosis other than in programmed cell death, its down regulation supports the notion that Set2 is linked to the ecdysone regulatory hierarchy (Stabell, 2007).

Set2 associates with hyperphosphorylated RNAPII: Recent reports have shown that Set2 from various organisms binds to the hyperphosphorylated CTD of RNAPII, implying that K36 methylation plays an important role in the transcription elongation process. The presence of both the WW and SRI domains suggested that Set2 may associate with RNAPII also in Drosophila. To address this issue, extracts prepared from control or Set2 RNAi embryos were immunoprecipitated with antibodies directed against Ser5-phosphorylated CTD, followed by immunoblotting with antibodies directed against Set2 and Ser5-phosphorylated CTD form of RNAPII. Immunoprecipitation of Ser5-phosphorylated CTD resulted in strong immunoreactivity of both phosphorylated CTD and Set2 in control embryos whereas no Set2 is detected in extracts from RNAi embryos. This result was corroborated by showing co-localization of Set2 and elongating RNAPII on salivary gland chromosomes. While these results demonstrate that Set2 is associated with the elongating form of RNAPII in Drosophila, the precise role of this association is currently unclear. However, the fact that a loss of Set2/K36 methylation results in mutant phenotypes associated with defects in the ecdysone response indicates that Set2/K36 methylation plays an important role in the ecdysone regulatory hierarchy (Stabell, 2007).

Histone H3 K36 methylation is mediated by a trans-histone methylation pathway involving an interaction between Set2 and histone H4

Set2-mediated H3 K36 methylation is an important histone modification on chromatin during transcription elongation. Although Set2 associates with the phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), the mechanism of Set2 binding to chromatin and subsequent exertion of its methyltransferase activity is relatively uncharacterized. This study identified a critical lysine residue in histone H4 that is needed for interaction with Set2 and proper H3 K36 di- and trimethylation. It was also determined that the N terminus of Set2 contains a histone H4 interaction motif that allows Set2 to bind histone H4 and nucleosomes. A Set2 mutant lacking the histone H4 interaction motif is able to bind to the phosphorylated CTD of RNAPII and associate with gene-specific loci but is defective for H3 K36 di- and trimethylation. In addition, this Set2 mutant shows increased H4 acetylation and resistance to 6-Azauracil. Overall, this study defines a new interaction between Set2 and histone H4 that mediates trans-histone regulation of H3 K36 methylation, which is needed for the preventative maintenance and integrity of the genome (Du, 2008).


REFERENCES

Search PubMed for articles about Drosophila Set2

Adhvaryu, K. K., et al. (2005). S.A. Morris, B.D. Strahl and E.U. Selker, Methylation of histone H3 lysine 36 is required for normal development in Neurospora crassa. Eukaryot. Cell 4: 1455-1464. PubMed ID: PubMed ID; Online text

Alekseyenko A. A., et al. (2006). High-resolution ChIP-chip analysis reveals that the Drosophila MSL complex selectively identifies active genes on the male X chromosome. Genes Dev. 20: 848-857. PubMed ID: PubMed ID; Online text

Bannister, A. J., et al. (2005). Spatial distribution of di- and tri-methyl lysine 36 of histone H3 at active genes. J. Biol. Chem. 280: 17732-17736. PubMed ID: PubMed ID; Online text

Bell, O., et al. (2008). Transcription-coupled methylation of histone H3 at lysine 36 regulates dosage compensation by enhancing recruitment of the MSL complex in Drosophila melanogaster. Mol. Cell Biol. 28(10): 3401-9. PubMed ID: 18347056

Biswas, D., et al. (2006). Opposing roles for Set2 and yFACT in regulating TBP binding at promoters. EMBO J. 25: 4479-4489. PubMed ID: PubMed ID; Online text

Brown, M. A., et al. (2006). Identification and characterization of Smyd2: a split SET/MYND domain-containing histone H3 lysine 36-specific methyltransferase that interacts with the Sin3 histone deacetylase complex. Mol. Cancer 5: 26. PubMed ID: PubMed ID; Online text

Carrozza, M. J., et al. (2005). Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription. Cell 123: 581-592. PubMed ID: 16286007

Chu, Y., et al. (2006). The BUR1 cyclin-dependent protein kinase is required for the normal pattern of histone methylation by SET2. Mol. Cell. Biol. 26: 3029-3038. PubMed ID: 16581778

Du, H. N., Fingerman, I. M. and Briggs, S. D. (2008). Histone H3 K36 methylation is mediated by a trans-histone methylation pathway involving an interaction between Set2 and histone H4. Genes Dev. 22(20): 2786-98. PubMed ID: 18923077

Hamada, F. N., et al. (2005). Global regulation of X chromosomal genes by the MSL complex in Drosophila melanogaster. Genes Dev. 19: 2289-2294. PubMed ID: 16204180

Keogh, M. C., et al. (2005). Cotranscriptional set2 methylation of histone H3 lysine 36 recruits a repressive Rpd3 complex. Cell 123: 593-605. PubMed ID: 16286008

Kim, A., et al. (2007). Distinctive signatures of histone methylation in transcribed coding and noncoding human beta-globin sequences. Mol. Cell. Biol. 27: 1271-1279. PubMed ID: 17158930

Krogan, N. J., et al. (2003). Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 23: 4207-4218. PubMed ID: 12773564

Larschan, E., et al. (2007). MSL complex is attracted to genes marked by H3K36 trimethylation using a sequence-independent mechanism. Mol. Cell 28(1): 121-33. PubMed ID: 17936709

Li, B., et al. (2007a). Combinatorial action of the Rco1 PHD domain and the Eaf3 chromodomain targets the Rpd3S complex to deacetylate transcribed chromatin. Science 316: 1050-1054. PubMed ID: 17510366

Li, B., et al. (2007b). Infrequently transcribed long genes depend on the Set2/Rpd3S pathway for accurate transcription. Genes Dev. 21: 1422-1430. PubMed ID: 17545470

Morris, S. A., et al. (2005). Histone H3 K36 methylation is associated with transcription elongation in Schizosaccharomyces pombe. Eukaryot. Cell 4: 1446-1454. PubMed ID: 16087749

Mukai, M., Hira, S., Nakamura, K., Nakamura, S., Kimura, H., Sato, M. and Kobayashi, S. (2015). H3K36 trimethylation-mediated epigenetic regulation is activated by Bam and promotes germ cell differentiation during early oogenesis in Drosophila. Biol Open 4(2):119-24. PubMed ID: 25572421

Pokholok, D. E., et al. (2005). Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122: 517-527. PubMed ID: 16122420

Rao, B., et al. (2005). Dimethylation of histone H3 at lysine 36 demarcates regulatory and nonregulatory chromatin genome-wide. Mol. Cell. Biol. 25: 9447-9459. PubMed ID: 16227595

Stabell, M., Larsson, J., Aalen, R. B. and Lambertsson, A. (2007). Drosophila dSet2 functions in H3-K36 methylation and is required for development. Biochem. Biophys. Res. Commun. 359(3): 784-9. PubMed ID: 17560546

Strahl, B. D., et al. (2002). Set2 is a nucleosomal histone H3-selective methyltransferase that mediates transcriptional repression. Mol. Cell. Biol. 22: 1298-1306. PubMed ID: 11839797

Vakoc, C. R., et al. (2006). Profile of histone lysine methylation across transcribed mammalian chromatin. Mol. Cell. Biol. 26: 9185-9195. PubMed ID: 17030614

Vojnic, E., et al. (2006). Structure and carboxyl-terminal domain (CTD) binding of the Set2 SRI domain that couples histone H3 Lys36 methylation to transcription. J. Biol. Chem. 281: 13-15. PubMed ID: 16286474


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