Localized H3K36 methylation states define histone H4K16 acetylation during transcriptional elongation in Drosophila
2007; Springer Nature; Volume: 26; Issue: 24 Linguagem: Inglês
10.1038/sj.emboj.7601926
ISSN1460-2075
AutoresOliver Bell, C Wirbelauer, Marc Hild, Annette Scharf, Michaela Schwaiger, David M. MacAlpine, Frédéric Zilbermann, Fred van Leeuwen, Stephen P. Bell, Axel Imhof, Dan Garza, Antoine H.F.M. Peters, Dirk Schübeler,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle15 November 2007free access Localized H3K36 methylation states define histone H4K16 acetylation during transcriptional elongation in Drosophila Oliver Bell Oliver Bell Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author Christiane Wirbelauer Christiane Wirbelauer Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author Marc Hild Marc Hild Novartis Institutes for Biomedical Research, Cambridge, MA, USA Search for more papers by this author Annette ND Scharf Annette ND Scharf Adolf-Butenandt Institute, University of Munich, Munich, Germany Search for more papers by this author Michaela Schwaiger Michaela Schwaiger Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author David M MacAlpine David M MacAlpine Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USAPresent address: Duke University Medical Center, Department of Pharmacology and Cancer Biology, Durham, NC 27710, USA Search for more papers by this author Frédéric Zilbermann Frédéric Zilbermann Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author Fred van Leeuwen Fred van Leeuwen Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Stephen P Bell Stephen P Bell Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Axel Imhof Axel Imhof Adolf-Butenandt Institute, University of Munich, Munich, Germany Search for more papers by this author Dan Garza Dan Garza Novartis Institutes for Biomedical Research, Cambridge, MA, USA Search for more papers by this author Antoine HFM Peters Antoine HFM Peters Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author Dirk Schübeler Corresponding Author Dirk Schübeler Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author Oliver Bell Oliver Bell Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author Christiane Wirbelauer Christiane Wirbelauer Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author Marc Hild Marc Hild Novartis Institutes for Biomedical Research, Cambridge, MA, USA Search for more papers by this author Annette ND Scharf Annette ND Scharf Adolf-Butenandt Institute, University of Munich, Munich, Germany Search for more papers by this author Michaela Schwaiger Michaela Schwaiger Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author David M MacAlpine David M MacAlpine Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USAPresent address: Duke University Medical Center, Department of Pharmacology and Cancer Biology, Durham, NC 27710, USA Search for more papers by this author Frédéric Zilbermann Frédéric Zilbermann Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author Fred van Leeuwen Fred van Leeuwen Netherlands Cancer Institute, Amsterdam, The Netherlands Search for more papers by this author Stephen P Bell Stephen P Bell Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Axel Imhof Axel Imhof Adolf-Butenandt Institute, University of Munich, Munich, Germany Search for more papers by this author Dan Garza Dan Garza Novartis Institutes for Biomedical Research, Cambridge, MA, USA Search for more papers by this author Antoine HFM Peters Antoine HFM Peters Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author Dirk Schübeler Corresponding Author Dirk Schübeler Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland Search for more papers by this author Author Information Oliver Bell1, Christiane Wirbelauer1, Marc Hild2, Annette ND Scharf3, Michaela Schwaiger1, David M MacAlpine4, Frédéric Zilbermann1, Fred van Leeuwen5, Stephen P Bell4, Axel Imhof3, Dan Garza2, Antoine HFM Peters1 and Dirk Schübeler 1 1Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland 2Novartis Institutes for Biomedical Research, Cambridge, MA, USA 3Adolf-Butenandt Institute, University of Munich, Munich, Germany 4Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA 5Netherlands Cancer Institute, Amsterdam, The Netherlands *Corresponding author. Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel 4058, Switzerland. Tel.: +41 61 69 78269; Fax: +41 61 69 73976; E-mail: [email protected] The EMBO Journal (2007)26:4974-4984https://doi.org/10.1038/sj.emboj.7601926 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Post-translational modifications of histones are involved in transcript initiation and elongation. Methylation of lysine 36 of histone H3 (H3K36me) resides promoter distal at transcribed regions in Saccharomyces cerevisiae and is thought to prevent spurious initiation through recruitment of histone-deacetylase activity. Here, we report surprising complexity in distribution, regulation and readout of H3K36me in Drosophila involving two histone methyltransferases (HMTases). Dimethylation of H3K36 peaks adjacent to promoters and requires dMes-4, whereas trimethylation accumulates toward the 3′ end of genes and relies on dHypb. Reduction of H3K36me3 is lethal in Drosophila larvae and leads to elevated levels of acetylation, specifically at lysine 16 of histone H4 (H4K16ac). In contrast, reduction of both di- and trimethylation decreases lysine 16 acetylation. Thus di- and trimethylation of H3K36 have opposite effects on H4K16 acetylation, which we propose enable dynamic changes in chromatin compaction during transcript elongation. Introduction Nucleosomal packaging provides a barrier for protein binding to DNA and for the processivity of DNA and RNA polymerases. Changes in chromatin structure involve ATP-dependent remodeling of nucleosomes and numerous post-translational modifications of histones (Felsenfeld and Groudine, 2003). Depending on the modification and the targeted residue, these alterations can affect sequence accessibility for DNA-binding proteins or create recognition modules for effector proteins with defined functions (Jenuwein and Allis, 2001; Peters and Schubeler, 2005). Indeed, chromatin modifications appear involved in all steps of transcription, such as initiation, elongation and termination (Sims et al, 2004). A large body of work links histone H3 modifications, such as lysine 4 methylation and lysine 9 and 14 acetylation to RNA polymerase recruitment at the promoter. Recent genome-wide studies revealed that these active modifications are present at transcribed genes, yet occur preferentially at the promoter and adjacent downstream regions. This suggests a shared chromatin profile consisting of several histone tail modifications involved in early events of transcription (Robert et al, 2004; Schubeler et al, 2004; Pokholok et al, 2005; Barski et al, 2007). Although extensive knowledge exists on promoter-proximal events, less is known about the cross talk between the elongating polymerase and chromatin. Several chromatin-associated proteins are implicated in aiding transcriptional elongation. Some affect histone acetylation (Wittschieben et al, 1999; Winkler et al, 2002), whereas others show nucleosomal remodeling or histone chaperone activity (Belotserkovskaya et al, 2003; Kaplan et al, 2003; Mason and Struhl, 2003; Morillon et al, 2003). These findings suggest that nucleosomes are acetylated and mobilized during transcriptional elongation. Moreover, high levels of transcription cause nucleosomal displacement, leading to reduced nucleosomal density, which in metazoa appears to be compensated in part by depositing nucleosomes that contain the variant histone H3.3 (Lee et al, 2004; Mito et al, 2005; Schwartz and Ahmad, 2005; Wirbelauer et al, 2005). Thus, although chromatin opening is required for gene activation at promoters, transcription itself causes chromatin disruption, which appears to be compensated by reestablishing a compact chromatin state. Methylation of lysine 36 of histone H3 (H3K36me) is the only covalent histone modification reported to be enriched in the 3′ end of active genes (Bannister et al, 2005; Kizer et al, 2005; Pokholok et al, 2005; Rao et al, 2005; Barski et al, 2007; Mikkelsen et al, 2007). Therefore, understanding the regulation of this modification is likely to shed light on the interplay between chromatin and transcriptional elongation. In S. cerevisiae, all lysine 36 methylation is mediated by Set2 histone methyltransferase (HMTase), which is directed to active genes through interaction with elongation-competent RNA polymerase II (Krogan et al, 2003; Li et al, 2003; Xiao et al, 2003; Kizer et al, 2005). In turn, Set2-methylated nucleosomes signal for cooperative binding of two subunits of the Rpd3S complex (Li et al, 2007a), resulting in recruitment of histone-deacetylase activity to the body of active genes (Carrozza et al, 2005; Joshi and Struhl, 2005; Keogh et al, 2005). Thus, H3K36 methylation has been proposed to be involved in the maintenance of repressive chromatin structure. Indeed, loss of Rpd3 recruitment by deletion of SET2 results in an increase of spurious intragenic initiation events (Carrozza et al, 2005; Li et al, 2007b). This supports a function for H3K36me in compensating for transcription-coupled disruption and hyperacetylation of chromatin, which otherwise could unmask cryptic promoters. However, because deacetylation interferes with transcriptional initiation, mechanisms need to be in place to ensure that H3K36me-mediated HDAC recruitment does not occur in vicinity of active promoters. Little is known about potentially different chromosomal distributions or functions of the mono-, di- or trimethylated states of H3K36. Interestingly, two recently identified histone demethylases in the human genome have been shown to demethylate either preferentially H3K36me2 (Tsukada et al, 2005) or H3K36me3 (Klose et al, 2006; Whetstine et al, 2006) opening the possibility of enzymatically regulated differential turnover of H3K36 methylation states. To investigate function and regulation of this residue in a higher eukaryote, we have characterized H3K36 methylation in Drosophila melanogaster. We show that dimethylation and trimethylation of H3K36 have distinct chromosomal localization, and we suggest that these methylation states rely on separate HMTases. Importantly, we find that H3K36 methylation states show antagonistic cross talk to H4 acetylation at lysine 16 (H4K16ac), which has been shown to directly influence packaging of higher-order chromatin (Dorigo et al, 2003; Shogren-Knaak et al, 2006). These findings suggest opposing functions for H3K36 methylation states in Drosophila to regulate chromatin acetylation and presumably compaction during transcriptional elongation in higher eukaryotes. Results Global distribution of H3K36 di- and trimethylation in Drosophila cells To address if H3K36me associates with repressive or permissive chromatin in metazoa, we determined the nuclear localization of H3K36me2 and H3K36me3 in Drosophila Kc cells. Similar to other euchromatic marks such as H3K4 methylation (Wirbelauer et al, 2005), both H3K36 methylation states are largely excluded from the transcriptionally inert heterochromatin but are highly enriched in the transcriptionally active euchromatic regions of the nucleus (Figure 1A). Figure 1.Nuclear and chromosomal localization of H3K36me2 and H3K36me3. (A) Drosophila Kc cells were stained with DAPI in combination with antibodies specific to H3K36me2 and H3K36me3. Merged pictures are pseudo-colored with antibody staining in green and DAPI staining in blue. The heterochromatic chromocenter is visible in these cells as a region with high signal for DAPI, whereas the euchromatin shows weaker DAPI staining. Both methylation states of H3K36 strongly stain euchromatin and are excluded from the transcriptionally inert chromocenter. (B) H3K36me2 and H3K36me3 are enriched at the variant histone H3.3. Histones were isolated from Kc cells expressing either epitope-tagged H3.1 or H3.3 (Wirbelauer et al, 2005) and analyzed by western blot. This analysis finds H3K36me2 and H3K36me3 enriched at H3.3 compared to canonical H3.1. Detection of the V5-epitope tag serves as a loading control. (C) Chromosome-wide analysis of the distribution of H3K4me3 and H3K36me2/me3 relative to the 5′ or 3′ end of coding regions. Following ChIP, DNA from input and bound fraction were co-hybridized to a microarray representing the complete Drosophila chromosome 2L (see Materials and methods). To determine the relative enrichment for either the 5′or 3′ end of genes, we focused on tiles that were within genes and either contained the 5′ or 3′ end. We ignored those that were intergenic or mid-genic. The 5′ and 3′ end tiles (2105 for H3K4me3, 2041 for H3K36me2, 2123 for H3K36me3) were ranked according to their ChIP enrichment, and then we asked if tiles that are highly enriched are biased toward the 5′ or 3′ end. Note that enrichment of 3′ end tiles implies absence from 5′ tiles. Shown is a moving average (%, n=100) of tiles in the 3′ end relative to enrichment. This illustrates a preferential 5′ position of tiles enriched for H3K4me3 (85% of enriched regions reside at a 5′ end of a gene) and a 3′ bias for tiles enriched for H3K36me3 (67% of enriched tiles are at the 3′ end). H3K36me2 shows a 5′ bias in this analysis similar to H3K4me3, yet less pronounced. (D) Summary of chromosomal array with 2 kb resolution. The bar chart shows the abundance in 5′ or 3′ positions for those sequences with the highest enrichment in each modification (upper 10% of tiles, illustrated with a gray background in C). Download figure Download PowerPoint This nuclear localization reflects the presence of both H3K36 methylation states at active genes, as we find both enriched at the ectopically expressed histone variant H3.3 (Figure 1B). H3.3 is deposited at active genes, which are subject to transcription-coupled nucleosomal displacement events (Mito et al, 2005; Schwartz and Ahmad, 2005; Wirbelauer et al, 2005). This confirms previous observations by mass spectrometry of endogenous H3.3 in Drosophila Kc cells (McKittrick et al, 2004) and suggests that both H3K36 methylation states are enriched at sites of active transcription. To investigate the chromosomal distribution of lysine 36 methylation, we performed chromatin-immunoprecipitation (ChIP) using antisera specific for H3K36me2 or H3K36me3. DNA from enriched chromatin was compared to input chromatin by comparative hybridization to a DNA microarray representing chromosome 2L of the Drosophila genome in a 2 kb tiling resolution (MacAlpine et al, 2004; Schubeler et al, 2004). In addition, we determined the chromosomal distribution of H3K4me3, which we and others have previously shown to occur promoter proximal at active genes (Bernstein et al, 2005; Pokholok et al, 2005; Wirbelauer et al, 2005). The resulting distribution on chromosome 2L confirms these previous studies, as we find preferential enrichment of H3K4me3 at the 5′ end of active genes (Figure 1C and D). H3K36me3, in contrast, shows a different localization, as it is highly enriched toward the 3′ end of active genes as previously reported for S. cerevisiae (Pokholok et al, 2005). The distribution of H3K36me2 with preferential distribution toward the 5′ end is remarkably different, although this distribution is less pronounced than that of H3K4me3 (Figure 1C and D). Together, this chromosome-wide analysis revealed different distributions of di- and trimethylated lysine 36, with trimethylation localizing toward the 3′ end and dimethylation being adjacent to the promoter. Before proceeding with further analysis, we confirmed the specificity of the antibodies. We did not detect cross-reactivity against different methylation states of H3K36 when tested against peptides, suggesting that both antibodies are selective for either the di- or trimethylated state of this residue (Supplementary Figure 1A). We note that not all tested commercial antibodies showed equally high levels of discrimination in this assay (Supplementary Figure 1A), which might explain why a differential distribution of di- and trimethylation of H3K36 has not been reported previously. To test for potential cross-reactivity to regions of histone H3 outside of the peptides used, we tested both antibodies against ectopically expressed H3.3 in which H3K36 had been mutated to alanine. This point mutation leads to a loss of detection for each antibody (Supplementary Figure 1B), indicating that both are specific for defined H3K36 methylation states in the context of full-length histone H3. Next, we determined the distribution of H3K36 methylation states at a subset of genes using real-time PCR (RT–PCR) and previously published amplicons with a spatial resolution of approximately 750 bp (as compared to over 2000 bp of the microarray) (Wirbelauer et al, 2005). This enabled us to relate the observed enrichments to our existing datasets of other histone tail modifications, RNA polymerase II (RNA-Pol II) and the replacement histone H3.3 (Wirbelauer et al, 2005). This analysis confirmed that H3K36me3 is biased toward the 3′ end of active genes (Figure 2A and B). It also verified a different distribution for H3K36me2, as this mark was most abundant at a region between the 5′ peak of H3K4me3 and the 3′ peak of H3K36me3 (Figure 2B). The observed distribution is not cell line-specific, as similar results were obtained at the same set of genes in a second Drosophila cell line, SL2 (data not shown). Figure 2.High-resolution analysis of di- and trimethylation of H3K36 at individual genes. (A) ChIP analysis in Drosophila Kc cells along the body of several genes using antibodies specific for H3K36me2 or H3K36me3 and quantification by RT–PCR. Enrichments were normalized to nucleosomal abundance determined with an antibody against the C-terminus of H3. Shown is average and standard deviation from at least three independent repeats starting with cells at different passages. X-axis reflects the base-pair position relative to the transcriptional start site. Y-axis reflects enrichment (bound/input normalized to an intergenic control). H3K36me2 (orange line, scale on the left), H3K36me3 (blue line, scale on the right). Numbers in graphs are gene IDs according to Flybase. (B) Summary of individual genes. Probes at active genes shown in A were grouped according to the distance from the transcriptional start site (promoter n=5, 200–1200 bp n=8, 1200–2200 bp n=4, 2200-end bp n=7). All probes at inactive genes were grouped separately (inactive n=7). For each group, the average enrichment for each modification was calculated. Values for H3K4me3 and H3K36me3 are according to the left scale, values for H3K36me2 are according to the right scale. This representation illustrates a strong 5′ bias for H3K4me3, an intermediate 5′ bias for H3K36me2 and a strong 3′ bias for H3K36me3. Download figure Download PowerPoint We conclude that three chromatin signatures can be distinguished along active genes based on H3 tail modifications. A promoter-proximal region of high H3K4 methylation, an intermediate region characterized by a peak in H3K36me2 and a further 3′ region characterized by high H3K36me3. Thus, different K36 methylation states mark discrete regions of transcribed genes. Two proteins mediate H3K36 methylation in Drosophila To gain further insights into the underlying enzymatic regulation and function, we sought to identify the proteins responsible for H3K36 methylation based on homology to the SET domain sequence of the single H3K36 HMTase in S. cerevisiae (Set2) (Supplementary Figure 2A). We performed an RNAi screen against putative HMTases and used bulk analysis of H3K36me2 and H3K36me3 levels by western blot as a readout for loss of function. This identified two SET domain-containing proteins (CG4976 and CG1716) (Figure 3A) that upon knockdown showed reduced levels of H3K36 methylation (Figure 3B and D). Thus, we find that at least two putative HMTases are involved in H3K36 methylation in flies. To ensure specificity of the RNAi, we raised specific antibodies against both proteins (see Material and methods), which confirmed efficient protein reduction upon addition of dsRNA (Figure 3C). We named CG1716 as ‘Drosophila Hypb’ (dHypb) based on homology to the human HMTase HYPB (Sun et al, 2005). CG4976, in contrast, shows homology to the nuclear-receptor-binding SET-domain-containing protein (NSD) family of SET domain proteins (Supplementary Figure 2B) and has previously been annotated as Drosophila Mes-4 (dMes-4) based on its similarity to a SET domain-containing protein in the C. elegans genome. In worms, Mes-4 is required for H3K36 methylation at autosomes in early embryo and is necessary for germline viability (Bender et al, 2006). Figure 3.Identification of Drosophila SET domain proteins involved in H3K36 methylation. (A) Domain structure of full-length HMTase proteins as predicted by the SMART software (EMBL). DEAD=ATP dependant helicase domain, SRI=Set2 Rpb1 interacting domain. The gray bar indicates protein fragments tested for HMTase activity in vitro. (B) Validation of mRNA knockdown in cultured Kc cells. RT–PCR of target message in the absence (−) or presence (+) of dsRNA reveals efficient mRNA knockdown. The gene expression of CG6388 is unaffected in both knockdowns and serves as a loading control. (C) Validation of protein reduction. Western blot using antibodies specific for dMes-4 and dHypb in the absence (−) or presence (+) of dsRNA reveals efficient reduction of the targeted proteins in Kc cells. dHsp70 and dMOF are unaffected by RNAi knockdown and serve as loading control. (D) Reductions of dMes-4 and dHypb have specific effects on global levels of H3K36 methylation. Loss of dHypb results in the reduction of H3K36me3 and coinciding increase of H3K36me2. Knockdown of dMes-4 leads to a reduction in both H3K36me2 and H3K36me3. H2A serves as loading control. Global levels of H3 and H4 were unaffected (Supplementary Figure 5A). (E) Mass spectrometry analysis of H3K36-methylated peptides. MS-MS analysis of mono-, di- or trimethylated H3K36 moieties following knockdown of putative HMTases in Drosophila Kc cells. The bar chart displays fold changes in the abundance of H3K36 methylation states relative to untreated control cells. (F) Knockdown of putative Drosophila H3K36 HMTases in vivo. RT–PCR from larvae uninduced (−) or induced (+) for targeted knockdown of either dMes-4 or dHypb mRNA in vivo. Reduced transcript abundance is detected in the presence of GAL4 driver under the control of a ubiquitously expressed (tubulin) promoter. CG6388 mRNA levels serve as loading control. (G) Western blot analysis of H3K36 methylation states in fly larvae mirror the observations in cultured cells. Reduction of dMes-4 message results in the reduction of H3K36 di- and trimethylation, whereas dHypb RNAi specifically downregulates H3K36me3. Levels of total H4 serve as loading control. Download figure Download PowerPoint When we compared the levels of di- versus trimethylation of H3K36 upon reduction of dHypb or dMes-4, we noted a striking difference. RNAi against dMes-4 reduces di- and trimethylation, suggesting that the activity of this enzyme is required for both methylation states (Figure 3D). Knockdown of dHypb, in contrast, results in the downregulation of trimethylation alone, whereas levels of dimethylation slightly increase. This result is in disagreement with the recent study that reported reduction of dimethylation following RNAi knockdown of CG1716 in flies (Stabell et al, 2007). However, we point out that the authors relied for detection on an antibody that we found cross-reactive with trimethylated lysine 36 peptide (Supplementary Figure 1A), possibly accounting for this discrepancy. To validate the differential effects on lysine 36 methylation by an antibody-independent approach, we analyzed histones isolated from either control or RNAi-treated cells by mass spectrometry. We compared the levels of H3K27 and H3K36 methylation within the peptides comprising amino acids 27–40 of histone H3. To determine the changes in methylation of H3K36, we analyzed the mono-, di- and trimethylated isoforms and measured the levels of H3K36 methylation relative to the methylation at H3K27 by nanospray MS/MS (see Materials and methods). This confirmed the downregulation of both di- and trimethylation of H3K36 upon loss of dMes-4, whereas loss of dHypb reduced trimethylation alone (Figure 3E). In addition, we observed a modest increase of K36me2 in the dHypb knockdown in line with the results obtained by western blot; however, the low abundance of K36me2 relative to K27me2 precludes a robust quantification. dHypb is essential for fly development To examine whether changes in H3K36 methylation states would influence organismal development, we generated transgenic fly lines harboring an RNAi construct complementary to either dMes-4 or dHypb message under the control of a GAL4-inducible promoter (see Materials and methods). Transcription of the respective RNAi construct was triggered by crossing in a fly strain that expresses the GAL4 activator ubiquitously under the control of the tubulin promoter. Induction of the dMes-4 RNAi construct led to a detectable reduction of target mRNA (Figure 3F), yet only in a subset of fly lines. Those with reduced expression of dMes-4 mRNA showed a decrease of both K36me2 and K36me3 at larval stages, confirming the results from cell culture (Figure 3G). In the case of dHypb, induction of the RNAi transgene efficiently reduced dHypb mRNA (Figure 3F) and led to decreased levels of H3K36me3, confirming the results in cultured cells (Figure 3G and D). Moreover, postzygotic depletion of dHypb levels was lethal at the larvae–pupae transition with 100% penetrance observed in multiple independent integration sites of the RNAi construct (see Materials and methods) in agreement with a recent report (Stabell et al, 2007). We conclude that reducing HMTase levels in flies mirror the chromatin effects seen in cultured cells and that a strong reduction of dHypb is lethal, suggesting that dHypb-mediated H3K36me3 is essential for development. dHypb shows HMTase activity at histone H3 in vitro To test the enzymatic activity of both enzymes in vitro, we expressed fragments containing pre-SET, SET and post-SET domains as GST-tagged fusion proteins using Baculovirus infection of insect cells (Supplementary Figure 3B). Recombinant proteins were purified and incubated with radioactively labeled SAM as methyldonor and histones purified from calf thymus as a substrate. HMTase activity was reproducibly detected for dHypb by measuring radioactive incorporation into histones. Subsequent gel separation of labeled products revealed that dHypb methylates preferentially histone H3 (Figure 4A). Unlike dHypb, dMes-4 displayed only weak activity under various conditions tested (data not shown). Although this might reflect the lack of necessary cofactors, it precluded us from further defining dMes-4 activity in vitro. Figure 4.dHypb methylates lysine 36 in vitro and colocalizes with dMes-4 at active genes. (A) dHypb shows histone-methyltransferase activity in vitro. Recombinant protein fragments containing pre- and post-SET domain (dHypb: aa 1351–1553, shown as gray bar in Figure 3A) were incubated with radioactive SAM and histones from calf thymus as substrates. Shown in the upper panel is the reaction product that was separated by SDS–PAGE and incorporated radioactivity measured by exposure to film. The lower panel displays Coomassie-stained SDS–PAGE gel and serves as loading control. Recombinant dHypb-SET shows HMTase activity to histone H3 in this assay. (B) dHypb methylates H3K36 in vitro. Western blot analysis displays H3K36 di- and trimethylation levels of HMTase assay with recombinant full-length dHypb using mutant yeast nuclear extracts deficient for H3K4me, K36me and K79me (ΔSET2, ΔSET1, ΔDOT1) or K4me and K79me (wt SET2, Δset1, Δdot1) as a substrate. A dHypb-dependent increase of di- and trimethylation is obtained only with chromatin substrate from wt SET2 strain, suggesting that dHypb requires premethylated lysine 36 substrate for its activity. (C) Western blot analysis of Drosophila Kc-overexpressing dHypb shows a specific increase in trimethylation. A similar experiment with full-length dMes-4 in Kc cells did not reveal robust changes in H3K36 methylation (data not shown). (D) ChIP analysis using antibodies generated against endogenous dMes-4 and dHypb along the body of two active genes (CG6137 and CG5686) and one inactive gene (CG3324). Shown is average and standard deviation from at least three independent repeats. X-axis reflects the base-pair positi
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