Drosophila Set1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription
2011; Springer Nature; Volume: 30; Issue: 14 Linguagem: Inglês
10.1038/emboj.2011.194
ISSN1460-2075
AutoresM. Behfar Ardehali, Amanda Mei, Katie L. Zobeck, Matthieu Caron, John T. Lis, T Kusch,
Tópico(s)Invertebrate Immune Response Mechanisms
ResumoArticle21 June 2011free access Drosophila Set1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription M Behfar Ardehali M Behfar Ardehali Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USAPresent addresses: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA Search for more papers by this author Amanda Mei Amanda Mei Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Katie L Zobeck Katie L Zobeck Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Search for more papers by this author Matthieu Caron Matthieu Caron Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author John T Lis Corresponding Author John T Lis Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Search for more papers by this author Thomas Kusch Corresponding Author Thomas Kusch Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author M Behfar Ardehali M Behfar Ardehali Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USAPresent addresses: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA Search for more papers by this author Amanda Mei Amanda Mei Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Katie L Zobeck Katie L Zobeck Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Search for more papers by this author Matthieu Caron Matthieu Caron Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author John T Lis Corresponding Author John T Lis Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Search for more papers by this author Thomas Kusch Corresponding Author Thomas Kusch Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Author Information M Behfar Ardehali1,‡, Amanda Mei2,‡, Katie L Zobeck1, Matthieu Caron2, John T Lis 1 and Thomas Kusch 2 1Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA 2Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA ‡These authors contributed equally to this work *Corresponding authors: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA. Tel.: +1 607 255 2442; Fax: +1 607 255 6249; E-mail: [email protected] of Molecular Biology and Biochemistry, Rutgers University, Nelson Biological Labs, Room A123, Piscataway, NJ 08854, USA. Tel.: +1 732 445 6895; Fax: +1 732 445 6186; E-mail: [email protected] The EMBO Journal (2011)30:2817-2828https://doi.org/10.1038/emboj.2011.194 Present addresses: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. Here, we report that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax; Trithorax-related protein), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. Our data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation. Introduction Genomic DNA of eukaryotic cells is organized into a nucleoprotein structure called chromatin. The fundamental building block of chromatin is the nucleosome core particle, which consists of 147 base pairs of DNA wrapped around an octamer of the histones H2A, H2B, H3, and H4. Nucleosomes can form an obstacle to processes requiring access to the DNA such as transcription. Covalent modifications of histones at particular residues can alter the properties of nucleosomes by both changing chromatin's compactness and accessibility and by specifying new interactions of histones with transcription factors. One of these modifications, histone H3 lysine 4 trimethylation (H3K4me3), has been shown to be a major conserved mark of chromatin at nucleosomes immediately downstream of promoters of transcribed genes in yeast, Drosophila, and mammals (Pokholok et al, 2005; Barski et al, 2007; Guenther et al, 2007; Schuettengruber et al, 2009). Although nucleosomes carrying this modification are targeted by a number of transcription and chromatin regulators (Martin et al, 2006; Shi et al, 2006; Taverna et al, 2006; Sims et al, 2007; Vermeulen et al, 2007, 2010), its contribution to transcription remains mysterious. In baker's yeast, the mutation in the gene coding for the H3K4 methyltransferase, SET1, does not cause obvious transcription defects during vegetative growth, while in human cells, the functional study of this mark is hampered by the existence of several genes encoding H3K4 trimethyltransferases (Eissenberg and Shilatifard, 2010). H3K4 methylation is introduced into nucleosomes by the type-2 histone lysine methyltransferases (KMT2s; Allis et al, 2007). The prototypic KMTs from Drosophila are Suppressor of variegation 3–9, Enhancer of zeste, and Trithorax (Trx), and they share similarity in their catalytic SET domain. Of these, only Trx is H3K4-specific, and has been proposed to be the major KMT2 in flies (Shilatifard, 2008; Eissenberg and Shilatifard, 2010). Flies possess a second, Trithorax-related protein (Trr), which functions in the regulation of hormone-response gene expression (Sedkov et al, 1999, 2003). The two Drosophila Trx relatives are related to the mammalian Mixed lineage leukaemia (Mll)1–4 KMT2s (Eissenberg and Shilatifard, 2010). While some Mll complexes contain subunits found in the yeast Set1 complex (COMPASS; Miller et al, 2001; Roguev et al, 2001; Nagy et al, 2002), Trx/Mll relatives have also been shown to assemble into complexes that are unrelated to COMPASS and contain histone acetyltransferases (Ernst et al, 2001; Petruk et al, 2001; Smith et al, 2004; Dou et al, 2005). These findings suggest a functional diversification of Trx/Mll complexes. Surprisingly, genome-wide studies in flies and mammals revealed that Trx/Mll relatives do not localize to over 97% of H3K4me3 domains, including those adjacent to transcription start sites (TSSs) of most expressed genes (Schuettengruber et al, 2009; Wang et al, 2009; Eissenberg and Shilatifard, 2010; Schwartz et al, 2010). Recent studies led to the identification of two Set1 orthologues in humans and showed that they assemble into complexes similar to yCOMPASS (Lee and Skalnik, 2005; Lee et al, 2007a); however, their contribution to overall H3K4me3 is still obscure. Since a Set1 homologue was not found in Drosophila, the role of Set1 versus Trx/Mll relatives in global, transcription-linked H3K4 trimethylation at promoters is still unclear in multicellular organisms. Here, we report the identification and characterization of a Set1 homologue from Drosophila, CG40351/dSet1. Proteomics and functional studies revealed that the dSet1 complex is identical in its composition to its human counterpart and has strong H3K4 trimethyltransferase activity towards recombinant nucleosomes. RNAi-mediated knockdown (KD) studies demonstrated that dSet1 is responsible for bulk H3K4 di- and trimethylation, while the KD of Trx or Trr had less pronounced effects on H3K4me2/3. dSet1 co-localizes with H3K4me3 and transcribing Pol II on polytene chromosome, and the loss of the dSet1-complex subunit, dCfp1, diminishes dSet1 and H3K4me3 at transcription puffs. The KD of dSet1 causes reduced mRNA levels at all tested genes, including heat shock (HS) genes. Live cell imaging studies also revealed that EGFP–dSet1 is rapidly recruited to the Hsp70 HS loci upon activation. Photobleaching/recovery assays demonstrated that EGFP–dSet1 is continuously exchanged at the activated hsp70 loci. Moreover, time course HS experiments showed that KD of dSet1 caused increased Pol II levels at the hsp70 promoter during extended HS periods. Our data supports a model in which dSet1-dependent H3K4me3 regulates chromatin changes at promoter-proximal nucleosomes that positively influence the release of Pol II into productive elongation, thereby contributing to optimal mRNA levels. Results Characterization of the Drosophila dSet1 complex The structural conservation of yeast and human Set1 complexes suggested that Drosophila also might possess a Set1-type protein. Database searches identified CG40351 as the closest match of yeast SET1 and human Set1B. Phylogenetic comparisons of Set1- and Trx/Mll-KMT2s from humans, flies, and yeast indicated that these factors form three distinct subfamilies (Figure 1A). CG40351 falls into the same group with the human and yeast Set1 relatives, while Trx has the highest similarity to MLL1 and MLL4. A third subgroup consists of Trr, MLL2, and MLL3. A comparison of the domain structure of these proteins indicated that the CG40351 protein contains an RNA recognition motif (Tresaugues et al, 2006; Lee et al, 2007a) in the N-terminus (Supplementary Figure S1). This domain is characteristic for Set1-type KMT2s and missing in Trx/Mll-type proteins. These comparative studies also revealed that a variety of domains present in distinct Trx/Mll relatives are absent in CG40351. In summary, the CG40351 protein is structurally very similar to the yeast or human Set1 relatives, but does not have features typical for Trx/Mll-type KMT2s. Figure 1.Characterization of the Drosophila dSet1 complex. (A) Phylogenetic comparison of the Set1 and Mll1-4 homologues from yeast, flies, and humans generated with Phylip (bootstrap value 1000, 15 repeats). (B) Purification scheme for FH-dSet1 or dSet1-HF complexes. (C) Identified polypeptides from purified dSet1 complexes compared with human and yeast complexes. #N, #C: unique peptides from N- or C-terminally tagged dSet1. (D) Immunoblot analysis of purified dSet1 complexes. The asterisk marks a non-specific band. (E) Western blot analysis of nuclear extract fractionated by gel filtration. Arrowheads, calibration marker sizes in kDa. Download figure Download PowerPoint With the exception of a study where CG40351 was knocked down by RNAi to test its involvement in dosage compensation in flies (Yokoyama et al, 2007), this factor has remained uncharacterized. To further confirm that CG40351 codes for a bona fide Set1 homologue, we characterized its interaction partners by mass spectrometry. For this purpose, we generated cell lines expressing an N- or C-terminally FLAG/HA-tagged CG40351. Matching data from both cell lines would ensure that intact complexes were isolated. CG40351 and associated factors were affinity purified from nuclear extracts followed by glycerol gradient centrifugation and compared by Silverstaining (Supplementary Figure S2). CG40351-peak fractions were then analysed by mass spectrometry (Figure 1B). The data from both purifications revealed that CG40351 is a component of a complex similar to human and yeast COMPASS (Figure 1C; Miller et al, 2001; Lee et al, 2007a), and that the tagging of dSet1 in the N- or C-terminus had no major effect on complex assembly and stability. Antisera against CG40351 recognized the same band that was labelled by anti-Flag antibodies confirming that these antisera were specific in immunoblotting experiments (Figure 1D; Supplementary Figure S3). Western blots on fractions from size exclusion chromatography on nuclear extracts showed that dSet1 complexes eluted in a single peak near a 670-kDa marker protein (Figure 1E). The combined theoretical mass of all dSet1-complex subunits in single copy number is about 632.8 kDa. Immunoblotting experiments confirmed that an identified subunit, Host cell factor (dHcf), was present in Drosophila COMPASS (Figure 1D and E). Its human homologue is a component of hCOMPASS (Lee et al, 2007a). Since dHcf associates with other chromatin complexes (Kusch et al, 2003; Wysocka et al, 2003; Guelman et al, 2006), it eluted in a broader profile from sizing columns. In summary, our data indicates that CG40531 is a component of a conserved dCOMPASS complex and the bona fide Set1 homologue in Drosophila. We, therefore, will refer to CG40351 in the following as dSet1. dSet1 is responsible for bulk H3K4 trimethylation We next conducted RNAi-mediated KD studies to determine the contribution of Trx, Trr, and dSet1 to genome-wide H3K4me1, -me2, and -me3 (Figure 2A and B; Supplementary Figure S4). As determined by signal intensity measurements, the KD efficiency in all cases appears to be higher than 85%. The KD of each KMT2 had no discernable effect on the protein levels of the other factors, and each had a different impact on H3K4 methylation. The loss of Trr most strongly affected H3K4me1, and to a lesser extent H3K4me2 and H3K4me3, relative to lacZ-KD samples (Figure 2B; Supplementary Figure S4), while the KD of Trx had the least influence on H3K4me1, -2, and -3. In dSet1-depleted cells, H3K4me1 was nearly unaffected, while H3K4me2 and -3 showed the most prominent decrease (Figure 2B; Supplementary Figure S4). Note that changes in H3K4me1 or -2 levels in these experiments need to be interpreted cautiously, because flies possess two H3K4-specific demethylases, Su(var)3-3 and Lid, which, respectively, use H3K4me2 or -3 as substrate (Gildea et al, 2000; Eissenberg et al, 2007; Lee et al, 2007b; Rudolph et al, 2007; Secombe et al, 2007; Di Stefano et al, 2011). The results suggested that these KMT2s might in part functionally interact in H3K4 methylation. Combinatorial KDs of Trx/Trr, Trx/dSet1, Trr/dSet1, and triple KDs only showed additive effects, suggesting that these KMT2s act rather independently in H3K4 methylation (not shown). Figure 2.dSet1 is required for bulk H3K4 di- and trimethylation. (A) Western blot analysis of nuclear extracts from RNAi-treated cells (top). lacZ: control from cells treated with dsRNA for E. coli lacZ. Tubulin served as loading control. (B) H3K4 methylation changes upon KD of dSet1, Trr, or Trx. Immunoblots of histone extracts from RNAi-treated cells probed with antibodies against methylated H3K4 (me1, me2, me3) or H3K9me3. Values below each lane represent the relative intensity of each band in comparison with the respective lacZ lane as quantified by ImageJ. (C) Purified dSet1 complexes methylate H3K4 in vitro. Immunoblots of KMT assays with the purified dSet1 complex (FHdSet1). Mock, purifications from mock-transfected cells. nCH, native fly histones served as positive control; rH3, recombinant H3; rCH, core histones; rNuc, nucleosomes. Coomassie, loading control. Download figure Download PowerPoint Using recombinant histone H3, core histones, and nucleosomes as substrates, we next tested if the purified dSet1 complex was capable of trimethylating H3K4 in vitro (Figure 2C). The relative levels of H3K4me1–3 were determined by immunoblots using specific antibodies. To reduce the frequently observed cross-reactivity of anti-methyl-lysine antibodies, competitor H3K4 peptides were added to the reactions (see Materials and methods for details). While mock-purified samples had no discernable H3K4 methylation activity, the purified dSet1 complex was capable of mono-, di-, and trimethylating H3K4 of all three substrates in a processive manner. In vitro studies on other KMT2 complexes revealed that these complexes usually do not fully trimethylate their substrates, but generate substantial quantities of mono- or dimethylated H3K4. The partial methylation of H3K4 in these reactions is not necessarily indicative of a limited KMT activity of the dSet1 complex in vivo, because H2B ubiquitination is known to enhance H3K4 trimethylation by Set1 complexes (Kim et al, 2009; Takahashi et al, 2009; Chandrasekharan et al, 2010). Moreover, other KMT2 enzymes have been found to be rather inefficient in their methylation activity in vitro (Takahashi and Shilatifard, 2009; Cosgrove and Patel, 2010). GST fusions of the SET domains of dSet1 (or Trx) showed no KMT activity on H3 and core histones, in contrast to GST-Trr, which had H3K4 monomethyltransferase activity towards H3 and core histones (Supplementary Figure S5). This indicates the catalytic SET domain of dSet, unlike the complete complex, is ineffective as KMT. This was expected, since most SET domain fusions were incapable of trimethylating nucleosomes in vitro (with few exceptions; e.g. HypB; Supplementary Figure S5; Sun et al, 2005). In summary, our studies indicate that the dSet1 complex has a major role in H3K4me3 in Drosophila and suggest that it might be responsible for deposition of this mark at active promoters. The KD of the two Trx relatives also had some impact on H3K4me3 levels, but their overall contribution is substantially less compared with dSet1. dSet1 extensively overlaps with H3K4 trimethylation on polytene chromosomes The immunological analyses from RNAi-treated cells indicated that dSet1 has a major role in H3K4 trimethylation in Drosophila S2 cells in immunoblotting experiments (Figure 2B). After confirming that the antibodies against dSet1 were also specific in immunofluorescence staining experiments (Supplementary Figure S6), we next assessed the extent to which dSet1 regulates H3K4 methylation in distinct chromosomal regions during later developmental stages. For this purpose, we co-labelled polytene chromosomes from third-instar larvae with antibodies against dSet1 and H3K4me1, -me2, or -me3 in the presence of competitor peptides (Figure 3A–C). As shown in Figure 3A, the signals of dSet1 and H3K4me3 nearly fully overlapped on polytene chromosomes. H3K4me2 and dSet1 also co-localized at a large number of transcription puffs; however, many H3K4me2 signals were also found in dSet1-negative and DAPI-positive chromatin regions lacking dSet1 (Figure 3B; Supplementary Figure S7). H3K4me1 and dSet1 only overlapped in a few transcription puffs, while the majority of H3K4me1 was found in compacted chromosomal regions (Figure 3C; Supplementary Figure S7). Figure 3.dSet1 co-localizes with and is required for H3K4 trimethylation at transcription sites. (A) Chromosomes co-stained with antibodies against H3K4me3 (red) and dSet1 (green). (B) Chromosomes labelled with anti-H3K4me2 (red) and anti-dSet1 (green) antibodies. (C) Chromosomes stained for H3K4me1 (red) and dSet1 (green). (D) Chromosomes co-stained with antibodies against Pol IIo (S5P; red) and dSet1 (green). (E) Chromosomes double labelled with antibodies against Pol IIo (S2P; red) and dSet1 (green). (F) Chromosomes co-labelled for Trr (red) and dSet1 (green). (G) Chromosome spread stained for Trx (red) and dSet1 (green). Yellow/orange signals in the channel merges indicate co-localization. Boxed areas in (A–E) are shown in magnification to the right. Magnifications in (D) and (E) were enhanced for the red channels for better visualization of Pol IIo. Download figure Download PowerPoint Since H3K4me3 is universally found at the promoters of transcriptionally active genes, we hypothesized that dSet1 may be involved in global transcription regulation. To examine the extent of dSet1 involvement in H3K4 methylation at sites of transcription, we next compared the distributions of dSet1 and transcriptionally engaged Pol II on polytene chromosomes by indirect immunofluorescence microscopy. We examined the co-localization of dSet1 with Pol II phosphorylated at serine 5 of its CTD (a mark of paused and productively elongating Pol II in metazoans; Boehm et al, 2003; Gomes et al, 2006) and elongating serine 2-phosphorylated Pol II (Figure 3D and E), respectively. Our results revealed considerable co-localization between dSet1 and Pol IIo. At most puffs, the dSet1 signals were narrower than Pol IIo bands, and usually concentrated at the edge of transcription puffs (Supplementary Figure S8). While many dSet1-positive regions did not appear to contain easily discernable levels of Pol IIo, a careful inspection of these sites revealed that most contain low levels of Pol IIo (Figure 3D and E, magnifications). Although we cannot exclude that some regions are only positive for dSet1, adjustments of the Pol IIo channel intensity suggested that the vast majority of dSet1-positive bands also labelled for Pol IIo. To assess the relative abundance and potential contribution of the Trx relatives to H3K4 methylation on polytene chromosomes, we next compared their distributions to that of dSet1. These studies revealed that dSet1-positive regions were far more abundant than Trr or Trx signals (Figure 3F and G). We also noticed that Trr-containing bands are more abundant than Trx-positive ones, which is consistent with previous reports (Kuzin et al, 1994; Chinwalla et al, 1995; Sedkov et al, 2003). In summary, dSet1 appears to be the most abundant of the three KMT2s on polytene chromosomes, and nearly completely overlaps with H3K4me3. This provides further support for the notion that dSet1 is the principal histone H3K4 trimethyltransferase with possible involvement in transcription at the global level. dCfp1 is required for chromatin association of dSet1 and global H3K4 trimethylation Recent studies showed that Cfp1, a subunit of hCOMPASS, is required for the restriction of Set1 to euchromatin and directs it to unmethylated CpG islands near active genes (Tate et al, 2010; Thomson et al, 2010). Our mass-spectrometric data indicated that the dSet1 complex contains the fly homologue of this factor, dCfp1 (Figure 1C); however, the lack of CpG islands in flies suggested that the protein might have a different role in the regulation of dSet1-dependent H3K4 methylation. To get a first insight into the role of dCfp1, we decided to analyse polytene chromosomes from flies carrying a P element insertion immediately upstream of the TSS of the dCfp1 gene, which leads to about a 98% reduction of dCfp1 mRNA levels in salivary glands of third-instar larvae as assessed by reverse transcription/quantitative PCR (RT/qPCR; Figure 4A). In these animals, dSet1 expression is not reduced; however, the protein is not detectable on polytene chromosomes, while Pol IIo distribution appeared essentially normal (Figure 4B). Furthermore, H3K4me3, which normally co-localizes extensively with Pol IIo (Figure 4C), was not detectable at transcription puffs in these mutants (Figure 4D). We conclude that dCfp1 is crucial for dSet1 association with chromatin and H3K4 trimethylation at transcription puffs. Additionally, and in contrast to human Cpf1, which prevented the mislocalization of Set1A to heterochromatin, dCpf1 was critical for general chromosomal association of dSet1. Figure 4.Loss of dCfp1 abolishes chromosomal association of dSet1 and H3K4me3. (A) RT/qPCR data for dCfp1 (blue) or dSet1 (red) using total RNA from salivary glands of wild-type (+/+), dCfp1 heterozygotes (+/−), or dCfp1 homozygous mutant (−/−) larvae. All values were normalized against rp49. Error bars represent the s.e.m. of three independent RNA preparations. (B) Polytene chromosomes from dCfp1 homozygous larvae co-stained with antibodies against dSet1 (red) and Pol IIo (green). (C) Polytene chromosomes of wild-type larvae co-labelled with antibodies against Pol IIo (red) and H3K4me3 (green). (D) Polytene chromosomes of dCfp1 homozygous mutants co-stained with the same antibodies as in (C). (B–D) DNA was counterstained with DAPI. Yellow-orange signals in the merged channels (right panels) indicate co-localization. Download figure Download PowerPoint dSet1 is required for optimal transcription from tested genes To test whether dSet1-dependent H3K4me3 has a role in transcription, we studied the expression changes on seven genes upon the KD of dSet1 by RT/qPCR. These genes were chosen by their relative expression levels as previously published (Muse et al, 2007). As shown in Figure 5A, the expression of all genes showed a significant drop (40–90% reduction) compared with cells treated with lacZ dsRNA (P<0.005, t-test). Notably, highly expressed genes (R, sda, α-tubulin) showed the strongest reduction, while lower level expressed genes (e.g. CG5629, pum, β-tubulin) were less dependent on dSet1. Figure 5.dSet1 is required for transcription and promoter-proximal H3K4 methylation. (A) RT/qPCR values from seven transcribed genes, expressed as ratio of mRNA levels from dSet1 KD versus lacZ-KD samples (=1). All values were normalized against levels of rp49. (B) ChIP/qPCR values of H3K4me3 at promoters versus ends of transcribed genes in dSet1 KD and lacZ-KD cells. The values are plotted as relative enrichment of IP chromatin compared with inputs after normalization using antibodies against H3. (C) ChIP assays evaluating the levels of dSet1 at promoters and ends of transcribed genes, plotted as percent IP versus input chromatin. Error bars represent the s.e.m. of three independent RNAi treatments (*P<0.01, t-test). Download figure Download PowerPoint We next tested whether the KD of Set1 would affect H3K4me3 at the promoters versus the ends of one strongly (R), two moderately (β-tubulin, Df31), as well as one low-level expressed gene (CG5629) by competitive chromatin immunoprecipitation/qPCR assays (cChIP/qPCR; Figure 5B). In order to account for differences in nucleosome density, the data was normalized using antibodies against H3. While the TSSs of all four genes were enriched for H3K4me3 in controls, the KD of dSet1 significantly diminished H3K4me3 here (P<0.005, t-test). At the 3′ ends, H3K4 methylation levels were low and unchanged in the KD samples. ChIP/qPCR experiments confirmed that dSet1 is mainly enriched at the promoters of these genes (Figure 5C). We also looked at a subset of transcriptionally active genes that are modulated by diverse sets of regulatory elements and tested the RNAi KD effect of all putative histone H3K4 methyltransferases on the levels of histone H3K4me3 at the 5′-end region of these genes. In almost all cases, the KD of dSet1, not Trr or Trx, resulted in a notable decrease in the H3K4me3 levels (Supplementary Figure S9). dSet1 is rapidly recruited to activated hsp70 loci Previous studies showed that hsp loci become trimethylated at H3K4 as early as 2.5 min after HS (Adelman et al, 2006). Therefore, we sought to more closely examine the association and recruitment kinetics of dSet1 to the Hsp70 genes at the 87A and 87C loci. We generated fly lines carrying a transgene allowing for the GAL4-dependent expression (Rorth, 1996) of an EGFP–dSet1 fusion protein and confirmed by immunoblotting experiments that EGFP–dSet1 is detectable in the salivary gland extracts of third-instar larvae (Supplementary Figure S10). Confocal reflection microscopy in combination with laser-scanning confocal microscopy (LSCM) of EGFP confirmed that EGFP–dSet1 predominantly accumulated in the nuclei of the salivary glands (Supplementary Figure S11). Polytene immunostainings using antibodies against GFP showed that EGFP–dSet1 does not localize to DAPI-intense heterochromatic regions (Supplementary Figure S12), and a co-staining using antibodies against dSet1 confirmed that EGFP–dSet1 protein is targeted to the same chromosomal regions as the endogenous protein (Figure 6A). The two showed essentially a complete overlap, indicating that the N-terminal tagging of dSet1 did not negatively affect its chromosomal targeting. Staining with anti-GFP antibodies on polytene chromosomes from wild-type flies further confirmed that the anti-GFP antibodies did not exhibit any unspecific cross-reactivity to chromosomal proteins, including Pol IIo (Supplementary Figure S13). Like untagged dSet1, the EGFP–dSet1 extensively co-localizes with transcriptionally active Pol II (Figures 3D and 6B). Figure 6.EGFP–dSet1 is rapidly recruited to the activated Hsp70 loci. Polytene chromosomes from transgenic third-instar larvae expressing EGFP–dSet1 double labelled with antibodies against (A) GFP (green) and dSet1 (red); (B) GFP (green) and Pol IIo (red); (C) laser-scanning microscopy (maximum intensity projections) shows co-localization of EGFP–dSet1 and mRFP–Rpb3 prior and after a 10-min HS in living salivary gland ce
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