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Additional sex combs interacts with enhancer of zeste and trithorax and modulates levels of trimethylation… Li, Taosui; Hodgson, Jacob W; Petruk, Svetlana; Mazo, Alexander; Brock, Hugh W Sep 19, 2017

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Li et al. Epigenetics & Chromatin  (2017) 10:43 DOI 10.1186/s13072-017-0151-3RESEARCHAdditional sex combs interacts with enhancer of zeste and trithorax and modulates levels of trimethylation on histone H3K4 and H3K27 during transcription of hsp70Taosui Li1, Jacob W. Hodgson1, Svetlana Petruk2, Alexander Mazo2 and Hugh W. Brock1* Abstract Background: Maintenance of cell fate determination requires the Polycomb group for repression; the trithorax group for gene activation; and the enhancer of trithorax and Polycomb (ETP) group for both repression and activation. Additional sex combs (Asx) is a genetically identified ETP for the Hox loci, but the molecular basis of its dual function is unclear.Results: We show that in vitro, Asx binds directly to the SET domains of the histone methyltransferases (HMT) enhancer of zeste [E(z)] (H3K27me3) and Trx (H3K4me3) through a bipartite interaction site separated by 846 amino acid residues. In Drosophila S2 cell nuclei, Asx interacts with E(z) and Trx in vivo. Drosophila Asx is required for repres-sion of heat-shock gene hsp70 and is recruited downstream of the hsp70 promoter. Changes in the levels of H3K4me3 and H3K27me3 downstream of the hsp70 promoter in Asx mutants relative to wild type show that Asx regulates H3K4 and H3K27 trimethylation.Conclusions: We propose that during transcription Asx modulates the ratio of H3K4me3 to H3K27me3 by selectively recruiting the antagonistic HMTs, E(z) and Trx or other nucleosome-modifying enzymes to hsp70.Keywords: Additional sex combs, SET domain, Trithorax, Enhancer of zeste, hsp70 transcriptional elongation, Histone trimethylation© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.BackgroundPolycomb group (PcG) and trithorax group (trxG) proteins maintain gene repression and activation, respectively, during metazoan development [1–3]. In Drosophila melanogaster, Asx was originally identified as a PcG mutant because of prominent posterior trans-formations caused by derepression of Hox genes [4–6]. Subsequently, it was observed that embryos mutant for Asx exhibit both anterior and posterior transformations, because Hox genes are improperly activated and dere-pressed, respectively [6–8]. Consistent with this model, Asx mutants enhance the homeotic transformation of trxG [8] and PcG [9, 10] mutations. Genes with these characteristics have been termed enhancers of trithorax and Polycomb (ETP) [11, 12]. Genetic analysis suggests that Asx is required for both trxG and PcG function.Various enzymatic activities are associated with trxG and PcG proteins, including trimethylation of his-tone H3 lysine 4 (H3K4) and H3K27 [13, 14]. Thus, one model to explain the ETP function of Asx is that it inter-acts directly with E(z) and Trx to regulate H3K4 and H3K27 methylation. An alternative model is that Asx affects trimethylation of H3K4 and H3K27 indirectly by Open AccessEpigenetics & Chromatin*Correspondence:  hugh.brock@ubc.ca 1 Department of Zoology, Life Sciences Institute, University of British Columbia, 2350 Health Science Mall, Vancouver, BC V6T 1Z4, CanadaFull list of author information is available at the end of the articlePage 2 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 regulating histone demethylases or acetyltransferases. In either model, Asx should be required to regulate levels of H3K4 and H3K27 methylation in vivo. To our knowl-edge, neither of these models has been tested on Asx or its mammalian homologs, perhaps because of difficulty of identifying a single locus at which both PcG and trxG proteins act at the same time in the same cell.The hsp70 gene is well characterized. Before heat-shock induction, the hsp70 promoter region is maintained in a nucleosome-free conformation by the GAGA factor [15], with a paused Pol II located approximately 25 nucleotides downstream of the transcription starting site [16, 17]. The paused Pol II is phosphorylated at serine 5 (Ser-5) but not Ser-2 of the C-terminal domain (CTD) [18], showing that transcriptional elongation has not begun. In Drosophila, these events occur 2–4 h after egg deposition. Heat stress leads to recruitment of heat-shock factor (HSF) [19], pos-itive transcription elongation factor b (P-TEFb), media-tor and various elongation factors including Spt5, Spt6 and facilitates chromatin transcription (FACT) complex that contains Spt16 for synthesis of full-length transcripts [20–22]. P-TEFb contains Cdk9 that is required for Pol II CTD Ser-2 phosphorylation and transcription elongation [18].Any temporal analysis of the heat-shock response in Drosophila later in development than the first 4  h of embryogenesis will have three phases: (1) an early phase corresponding to the switch from a promoter-paused state to elongation; (2) an intermediate phase that com-bines transcriptional initiation, promoter clearance and elongation; and (3) a phase in which transcription of heat-shock genes is terminated. Recruitment of the trithorax (Trx) protein complex, TAC1, is required to maintain high levels of transcriptional elongation and of H3K4 trimethylation at the hsp70 promoter region [23]. The PcG gene pleiohomeotic (pho) is required to repress hsp70 transcription after heat shock during termination phase [24]. Maternal deposition of Asx mRNA or Asx protein prevents analysis of transcriptional initiation or promoter clearance at hsp70 in Asx mutants in early embryos (first 4  h of embryogenesis). In later embryos, when initiation, clearance and elongation are occurring simultaneously, it is difficult to distinguish these phases of transcription in chromatin immunoprecipitation experiments.Here, we show that Asx interacts directly in vitro and associates in vivo with E(z) and Trx, suggesting a recruit-ment mechanism for modulation of trimethylation of H3K4 and H3K27 at hsp70. We also show that hsp70 is an excellent target to investigate the molecular basis of Asx function as an ETP after 10 min of heat-shock induction. We show that at the hsp70 locus, Asx represses hsp70 transcription because Asx mutants induce induction of the heat-shock response, but unlike the PcG gene pho, it is not required in the termination phase. Asx is recruited downstream of the promoter following heat stress induc-tion, but during the first 10  min of heat-shock induc-tion, Asx repression of hsp70 is independent of changes in levels of H3K4me3 and H3K27me3. Subsequently, Asx modulates levels of H3K4me3 and H3K27me3, notably at transition from elongation to termination during the heat-shock cycle. This, however, does not exclude the modulation of trimethylation by recruitment of other histone methyltransferases.MethodsFly culture, transgenic lines, embryo imaging and cell cultureFlies were maintained at 22  °C on standard cornmeal-sucrose medium containing Tegosept as a mold inhibitor. The Asx3 allele was maintained over a CyO twist-GAL4, UAS-eGFP balancer chromosome. The Asx3 mutant is a null mutation with 1.3-kb deletion in the middle of cod-ing region that produces a truncated protein product of approximately 800 N-terminal amino acids [4, 25] (Hodg-son, unpublished).AntibodiesThe following antibodies were employed: sheep poly-clonal anti-Asx (aa 75–95) IgG [25]; rabbit polyclonal anti-trimethyl histone H3K4 antibody (Active Motif, Cat.# 39159; 1:1000 dilution); rabbit polyclonal anti-trimethyl histone H3K27 (Millipore, Cat.# 07-449; 1:100 dilution); rabbit polyclonal IgG antibody (Abcam, Cat.# ab27478; 1:200 dilution) used as a negative control; rab-bit polyclonal anti-E(z) (Santa Cruz, Cat.# sc-98265); rat polyclonal anti-Trx antisera and purified rabbit anti-Trx IgG (Mazo Lab). Rabbit anti-Asx antibody was raised against Drosophila Asx (aa 200–356) (Additional file  1: Text S1). The specificity of rabbit anti-Asx antibody was tested (Additional file 2: Fig. S1).Construction of Asx full length and deletion mutants for cell‑free expressionThe DNA fragment corresponding to the full-length Asx (1669 amino acid residues) was subcloned from pBS(KS+)–Asx(1–1669) as an NdeI–KpnI fragment into the NdeI–SmaI sites of the vector pTβSTOP (kindly provided by Robert Tjian) downstream of the T7 promoter and β globin leader sequence to generate pTβSTOP-A(1–1669). An Asx COOH terminal dele-tion mutant A(1–1200) was generated by replacing the wild-type 1.59-kb SpHI–KpnI sequence in pBS(KS+)–Asx with a 0.495-kb PCR fragment produced using the following primer pairs: forward: 5′-ccggattccttgg GCA AGA CAT TAC CAG TGG CT-3′ and reverse: Page 3 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 5′-ccggagtggtacc TCA CAT ATT ACT GTT GTG-3′. The pBS(KS+)–Asx(1–1200) was subsequently digested with KpnI, end-repaired with T4 DNA polymerase and digested with NdeI. The truncated 3.6-kb Asx fragment was subcloned into the NdeI–SmaI site of pTβSTOP to generate pTβSTOP-A(1–1200). Four additional COOH terminal deletion fragments as well as four NH2 ter-minal deletion fragments of Asx were amplified from the pTβSTOP-A(1–1669) by PCR (Additional file  3: Table S1), subcloned into the NdeI–SmaI/EcoRV site of pTβSTOP and transformed into DH5α cells (Thermo Fisher). All plasmid constructs were expressed in the TNT-coupled T7 transcription/translation system (Pro-mega) using rabbit reticulocyte lysate (IVT–RRL) for GST pull-down assays.Construction of GST fusions of AsxETSI‑2 and the SET domains of E(z) and TrxThe SET domain of E(z) (aa residues 626–740) was amplified by PCR using the primer pair shown in Addi-tional file 3: Table S1 and subcloned into the EcoRI–XhoI sites of pGEX-6P1 to generate pGEX-6P1-E(z)SET. The SET domain of Trx (aa 3608-3759) (kindly provided by Michael Kyba) was subcloned into the EcoRI–XhoI sites of pGEX-6P1 to generate pGEX-6P1-TrxSET. The E(z)/Trx SET domain interaction site 2 of Asx (AsxETSI-2), residues 1200–150  l, was amplified using primer pair in Additional file 3: Table S1 and subcloned into the EcoRI–XhoI sites of pGEX-6P-1 to generate pGEX-6P1-Asx-ETSI-2. The three GST fusion constructs were each transformed into the E. coli Rosetta 2(DE3) strain (Nova-gen) for expression.Expression and purification of GST‑TrxSET and GST‑AsxETSI‑2 fusion proteinsOvernight cultures of 10  ml of cells transformed with either pGEX-6P-1, pGEX-6P1-TrxSET or pGEX-6P1-AsxETSI-2 were diluted into 240  ml Luria–Bertani (LB)/100 μg  ml−1 Amp media and induced at an  A600 of 0.8 units with 1  mM isopropyl-β-D-thio-galactoside (IPTG)/100 μM  ZnSO4 for 14 h at 23 °C. The cells were centrifuged, washed with PBS, lysed in 20  ml buffer TEEZMG—0.5 M KCl, pH 7.9, supplemented with pro-tease inhibitors and treated with 4  mg/ml lysozyme (Sigma) for 30  min at 4  °C. Each lysate was sonicated, diluted twofold with buffer TEEZMG—0.5  M KCl/pro-tease inhibitors and centrifuged at 14,000 rpm for 20 min at 4 °C. Extracts of GST, GST-TrxSET or GST-AsxETSI-2 were recovered in the supernatant as soluble fractions named S1.Twenty ml of each S1 fraction was rotated with 0.5 ml of GSH-agarose pre-equilibrated with buffer TEMZG—0.3  M KCl, pH 7.9, for 3  h at 4  °C. The protein-bound resin was washed three times with buffer PBSMG—0.3 M NaCl, pH 7.2, three times with buffer PBSMG—0.8  M NaCl, pH 7.2, three times with buffer TEMZG—0.5  M KCl, pH 7.9, and once with buffer TEMZ—0.1  M KCl, pH 9.0, at 4 °C. Proteins were eluted from a column with 6  ml of buffer Elut-TEMZ—0.1  M KCl, pH 9.0/10  mM reduced glutathione–NaOH and collected in 0.5 ml frac-tions. Peak fractions were pooled and dialyzed into buffer Dyl-TEEMZG—0.1 M KCl, pH 7.9, for 18 h at 4  °C and stored at −80  °C. Buffers used in these experiments are described in Additional file 4: Text S2.Expression and purification of GST‑E(z)SET fusion proteinA 10-ml overnight culture of pGEX-6P1-E(z)SET was diluted into 240  ml LB/100  μg/ml Amp media and induced at an  A600 of 0.8 units with 1 mM IPTG/100 μM  ZnSO4 for 3  h at 23  °C. The cells were washed in PBS, lysed in 20 ml buffer TEEZG—0.5 M KCl, pH 7.9, supple-mented with protease inhibitors and treated with 4 mg/ml lysozyme (Sigma) for 30 min at 4  °C. The lysate was sonicated, diluted twofold with buffer TEEZG—0.5  M KCl, pH 7.9/protease inhibitors and centrifuged at 14,000 rpm for 20 min at 4 °C.The pellet was resuspended in 15  ml buffer TZS—0.3  M NaCl, pH 7.9 using a Dounce homogenizer and mixed on a nutator for 2 h at 4 °C to solubilize GST-E(z)SET. The homogenate was centrifuged at 14,000 rpm for 20 min at 4 °C, and the supernatant (PI-Ext) was diluted fivefold with buffer TZD—0.3 M NaCl, pH 7.9, to reduce the sarkosyl concentration to 2% in the presence of Triton X-100 and CHAPS [26]. Fifty ml of diluted P1-Ext was mixed with 0.5 ml of GSH-agarose and pre-equilibrated with buffer TZXSC—0.3 M NaCl, pH 7.9, for 3 h at 4 °C on a rotator. The protein-bound resin was washed three times with buffer TZGXSC—0.3  M KCl, pH 7.9, three times with buffer TZGXSC—0.6 M KCl, pH 7.9, and two times with buffer TZXSGC—0.1 M NaCl, pH 9.0, at 4 °C. Proteins were eluted from a column with 6 ml of buffer Elut-TZGXSC—0.1  M NaCl, pH 9.0/10  mM reduced glutathione–NaOH, pH 9.0, and collected in 0.5 ml frac-tions. Peak fractions were pooled and dialyzed into 1 l buffer Dyl-TZXS—0.1  M NaCl/15% glycerol for 18  h at 4 °C and stored at −80 °C.GST pull‑down assay of 35S‑Asx interaction with SET domains of Trx and E(z)For each reaction, 60  μl of packed GSH-agarose equili-brated with immobilization buffer IM-A pH 7.9 was resuspended in 400  μl buffer IM-A and mixed with 6  μg of purified GST for 120  min at 4  °C on a nutator. The resin was pelleted at 6000  rpm for 2  min, washed two times and resuspended with 300  μl buffer IM-A on ice. For each fragment tested, the immobilized GST Page 4 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 (GST-agarose) was split into a 250-μl aliquot to pre-clear the Asx in  vitro translation mixture and a 50-μl aliquot for a control GST pull-down assay (30). Asx protein fragments were produced by in vitro trans-lation in rabbit reticulocyte lysate (RRL; Promega). Briefly, 1 μg supercoiled pTβSTOP plasmids containing Asx DNA fragments 1.8 kb to 5 kb long (Additional file 3: Table S1) were denatured at 80  °C, chilled on ice, expressed and labeled with 35S using the 25-μl reaction TNT T7-cou-pled transcription/translation rabbit reticulocyte lysate kit (Promega). The lysates were subsequently adjusted to 5 mM Mg acetate and treated with DNase I and RNase A/RNase T1 (Fermentas) for 15 min at 25 °C, and the Roche protease inhibitor cocktail was added. The lysate was pre-cleared by mixing with 100 μl of buffer 2× PDB-P5 and 25 μl of GST-agarose for 30 min at 4 °C. To assay interac-tions, 65  μl of pre-cleared 35S-Asx-containing lysate was mixed with either immobilized 1 μg GST, GST-E(z)SET or GST-TrxSET for 2  h on a nutator at 4  °C. Lysate-bound agarose beads were washed once with 300  μl buffer 1× PDB-P5, three times with buffer WB—0.6  M NaCl, pH 7.9, and once with buffer WB—0.1 M NaCl, pH 7.9. The protein-bound agarose pellet was mixed with 15  μl 2× SDS sample buffer, resolved by SDS–PAGE and analyzed by autoradiography.GST pull‑down western assays of embryo nuclear extractAsx fragments indentified as interaction sites for SET domains of either Trx (TSI), E(z) (ESI) or both (ETSI) were subcloned into pGEX-6P-1, expressed and purified from Rosetta 2(DE3) cells as described above for GST-AsxETSI-2. Detailed methods are described in Addi-tional file 5: Text S3.Co‑immunoprecipitation western assays of embryo nuclear extractFor co-immunoprecipitation experiments, 800 micro-grams of nuclear extract Bio-Rex 70 fraction was diluted into 300  μl of 1× IP buffer containing 5% polyethylene glycol 10,000 in place of Ficoll and mixed with 1:100 dilution of purified sheep anti-Asx IgG at 4  °C for 3  h [25]. Immune complexes were precipitated with protein G-Sepharose at 25  °C for 15  min and washed six times with buffer TELG containing 0.22 and 0.26  M KCl and two times with buffer TELG containing 0.05 M KCl. The samples were subsequently resolved on SDS–polyacryla-mide gels and transferred onto nitrocellulose membranes for western analysis as described above. Blots were probed with 1:3000 dilution of purified rabbit anti-Trx IgG or 1:200 dilution of rabbit anti-E(z) IgG (Santa Cruz Biotech, cat# sc-98265).Demonstrating protein–protein interaction in situ by PLADrosophila S2 cells were cultured at room temperature in chamber slides, heat shocked at 37 °C for 15 min and allowed to recover 60 or 180  min at room tempera-ture. Cells were fixed with 2% formaldehyde in culture medium for 20  min, washed with PBS, blocked and incubated overnight at 4  °C with either sheep anti-Asx and rat anti-Trx or sheep anti-Asx and rabbit anti-E(z). Proximity ligation assays (PLAs) were performed as described [27, 28] with several modifications. Second-ary antibodies (Jackson Immuno Research) were conju-gated to 5′-amino-modified MTPX oligonucleotides in Additional file 6: Table S2 using the Thunder-Link oligo conjugation systems (Innova Biosciences 420-0300) and stored at 4  °C. Circularization PLA 5′-phosphorylated oligonucleotides as well as detector PLA oligonucleo-tides were synthesized (Additional file 6: Table S2). For each reaction, 40 μl secondary antibodies with conju-gated oligonucleotides were incubated on a shaker for 1  h in a humidity chamber at 37  °C. Three circulariza-tion PLA oligos were annealed to two corresponding PLA probes (Additional file 6: Table S2) and ligated with T4 DNA ligase (Thermo) for 30 min at 37  °C. A closed circle forms if proteins are in close proximity [29]. Roll-ing-circle amplification by phi28 polymerase (Thermo) was carried out in the presence of fluorescent-labeled detector oligonucleotides (Additional file 6: Table S2) for 100 min in the dark at 37 °C.Immunostaining of salivary gland polytene chromosomesPreparation and immunostaining of chromosomes have been described [23]. For immunostaining, sheep anti-Asx IgG and FITC- or Texas-Red-conjugated secondary anti-bodies (Jackson Immunoresearch, PA) were used at dilu-tions of 1:100 [25]. Images of labeled chromosomes were acquired with a Zeiss microscope equipped with a digital camera, and processed using Adobe Photoshop.Embryo collectionFlies were acclimated to the laying chamber for 2 days before 10–14-h AEL embryos from about 300 flies were collected at 22  °C on 2% agar (supplemented with 1% sucrose/3.5% ethanol/1.5% apple cider vinegar). Embryos on laying plates were immediately washed onto a nylon sieve to remove excess yeast, dechorionated with 50% bleach and washed twice with 120  mM NaCl; 0.02% Triton X-100, followed by two washes in 1x PBS; 0.05% Triton X-100. Asx3 homozygous mutant embryos were identified by the absence of GFP expression under a wide-field GFP fluorescence microscope. The wild-type strain Oregon R was used as a control.Page 5 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 Heat‑shock induction and recovery in embryosWild-type or homozygous Asx3 mutant embryos were collected in 200 μl of 1× PBT into plastic microcentri-fuge tubes that were incubated in a 37 °C water bath for 5, 10 or 15 min. To study recovery, embryos heat shocked for 15 min were transferred onto a small piece of moist filter paper placed in a moist chamber and incubated for up to 180 min at room temperature, and transferred into a new tube with 200 μl of 1× PBT for further analysis.Determining hsp70 mRNA level in embryosRNA preparation and first-strand cDNA synthesis were done as previously described [30]. A dilution series of Drosophila genomic DNA was used to generate standard curves for hsp70 and Ahcy89E. The relative mRNA levels of genes were measured with comparison to the standard curve. All quantitative PCRs (qPCRs) were performed on the Step-One Plus Real-Time PCR system (ABI). The hsp70 expression level in different samples was normal-ized to the expression of Ahcy89E, whose expression does not change during heat shock.Chromatin immunoprecipitation (ChIP)ChIP was carried out essentially as described [31] except as follows. Exactly 200 embryos (in 200 μl buffer) were sonicated for five pulses of 10  s at 30% power at room temperature with an ultrasonic processor (CPX 130 PB, Cole Parmer), followed by 50  s on ice to yield 500-bp fragments. Samples were mixed with 200 μl of 6 M urea and incubated for 10 min on ice, and insoluble material was removed by centrifugation at 12,000×g for 10  min at 4  °C. The supernatant was divided into 4  ×  100 μl aliquots, and ChIP experiments were performed with H3K4, H3K27 and IgG antibodies. After the final wash-ing step, 100 μl of 10% Chelex 100 resin (BioRad) was added to protein G beads with vortexing, and samples were incubated at 95 °C for 10 min. Samples were depro-teinized with proteinase K (Sigma) at 55 °C, incubated for a further 10 min at 95 °C and centrifuged to recover the beads [32]. Approximately 3% of the immunoprecipitated material was assayed by qPCR using primers specific to sequences at the hsp70 promoter downstream, bxd PRE and Ubx promoter (Additional file 7: Text S4).ResultsAsx contains a bipartite site for interaction with SET domains of both E(z) and TrxGenetic analysis suggests that Asx is required for both trxG and PcG function. However, no genetic experi-ment can show that Asx has a direct effect on the histone methyltransferases (HMT) responsible for trimethylation of H3K4 and H3K27. Therefore, we looked for evidence of direct association of Asx with Trx, a key HMT for H3K4, and E(z), the HMT for H3K27. Alignment of the protein sequences of Trx and E(z) revealed a SET domain catalytic site [33] with a 35% amino acid identity between both proteins (Fig. 1a). To determine whether association between Asx and both HMTs can occur through the SET domains of Trx and E(z), we developed a GST pull-down autoradiography (GST pull-down) assay [34]. The SET domains of Trx and E(z) were fused to GST, expressed in E. coli Rosetta 2 (DE3) cells, purified by GSH-agarose affinity chromatography and immobilized on GSH-agarose (Fig.  1b). A preparation of 35S-Met-labeled Asx produced using coupled in  vitro transcription/transla-tion in rabbit reticulocyte lysate (IVT–RRL) (Promega) was mixed with immobilized GST-TrxSET, GST-E(z)SET and GST in control experiments. Full-length 35S-Asx interacted specifically with both GST-E(z)SET and GST-TrxSET (Fig.  1c) and exhibited a difference in the salt sensitivity of association with GST-TrxSET relative to GST-E(z). Higher salt concentrations increased the association of Asx with GST-E(z)SET but decreased the association with GST-TrxSET, which may reflect ionic or hydrophobic effects on interaction [35].To determine the strength of association of Asx with E(z) or Trx in nuclear extracts, given that in  vitro, the Asx-Trx(SET) interaction is about 5× weaker than the Asx-E(z)(SET) interaction, the Bio-Rex 70 fractions were further analyzed by anti-Asx co-immunoprecipitation coupled with α-Trx or α-E(z) western blotting (Fig.  2). E(z) and Trx were resolved into two distinct salt fractions of 0.1 and 0.85 M, respectively, by Bio-Rex 70 (Fig.  2b). Asx was co-eluted with Trx in the BR70-0.85 fraction and weakly co-immunoprecipitated at 0.26 M NaCl (Fig. 2c). By contrast, there was no detectable co-elution nor co-immunoprecipitation of Asx with E(z) in either the BR70-0.1 or BR70-0.85 fractions (Fig. 2d, e). This suggests that in nuclear extracts, the interaction between Asx and E(z) is weakly ionic or transient, which may be readily dis-rupted on the Bio-Rex 70 resin by a salt gradient. Alter-natively, the amount of Asx in association with E(z) is below the limits of detection of immunoprecipitation. A third untested explanation is that interactions between Asx and Trx complexes are more stable than interactions of the individual proteins, and vice versa for Asx and E(z).The interaction sites of GST-E(z)SET and GST-Trx-SET on full-length Asx were mapped by determining the association of 35S-Asx COOH terminal or  NH2 terminal deletion fragments (Fig. 3a) with GST-E(z)SET and GST-TrxSET using the GST pull-down assay (Figs. 3b, 4). Two interaction sites were identified on Asx for both GST-E(z)SET and GST-TrxSET (Fig. 3b): (1) at  NH2 terminal residues 1–354 termed E(z)/Trx SET domain interaction site 1 (AsxETSI-1) and (2) at COOH terminal residues 1200–1501 termed (AsxETSI-2) (Fig.  4). In addition, a Page 6 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 weak but specific COOH interaction site at terminal resi-dues 1501–1669 was mapped for Trx (Fig.  3b), termed Trx SET domain interaction site (AsxTSI) (Fig. 4). These results suggest that Asx contains a bipartite E(z)/Trx SET domain interaction (ETSI-1 and ETSI-2) site that has low sequence identity (14.97%) and is separated by 846 amino acids (Additional file 8: Fig. S2). It is interesting that the Asx  NH2 terminal residues 1–1200 which contain ETSI-1 did not show any significant binding with GST-E(z)SET and GST-TrxSET. In together with other pull-down results, it is possible that the Asx 610–1200 fragment which contains HR2–HR6 domain plays a negative role on ETSI-1 and GST-E(z)SET/GST-TrxSET interaction.Alignment of the amino acid sequences of Asx3 mutants and wild type [25] indicated a deletion of the COOH terminal ETSI-2 and TSI in Asx3 mutants (Fig.  5a). To test for interaction between E(z) and Asx-ETSI-2, we developed a GST pull-down western assay using purified GST-ETSI-2 (Fig. 5b) and chromatography fractionated embryo nuclear extracts as a source of E(z) (Fig.  5c). Similar experiments were not performed with AsxETSI-1 because of the instability of purified GST-ETSI-1. The E(z)-enriched BR70-0.1  M chromatography fraction (Fig.  5c) was mixed with immobilized GST-ETSI-2 and resolved by SDS–PAGE coupled with west-ern blotting using anti-E(z) antibody. Two bands were specifically detected on the blot corresponding to E(z) and a breakdown fragment (Fig. 5d) consistent with the interaction of GST-E(z) with AsxETSI-2. It is possible that E(z) was fragmented due to the sub-optimized pull-down condition. These results indicate that Asx ETSI-2 (which is deleted in Asx3) can associate with E(z) in vivo.Asx associates with E(z) and Trx after induction and recovery from heat shock in vivoTo further investigate whether Asx weakly or transiently associates with Trx and E(z) in vivo, we performed prox-imity ligation assay (PLA) in situ following heat shock on Trx-SET         ---VGVFRSHIHGRGLYCTKDIEAGEMVIEYAGELIRSTLTDKRERYYDSRGIGCYMFKI 57E(z)-SET        HKHLLMAPSDIAGWGIFLKEGAQKNEFISEYCGEIISQDEADRRGKVYD-KYMCSFLFNL 59: :  *.* * *:: .:. : .*:: **.**:* .  :*:* : ** : : .::*::Trx-SET         DDNLVVDATMRGNAARFINHCCEPNCYSKVVDILGHKHIIIFALRRIVQGEELTYDYKFP 117E(z)-SET        NNDFVVDATRKGNKIRFANHSINPNCYAKVMMVTGDHRIGIFAKRAIQPGEELFFDYRY- 118::::***** :**  ** **. :****:**: : *.::* *** * *  **** :**:: Trx-SET         FEDE 121E(z)-SET        ----AdoMet K substrateCatalytic sitea b37755025Pseudoknot1% Input/MWr1.20.6 1.20.6 1.20.9 M NaCl0.9 0.60.925075150100GST GST-EzSET GST-TrxSET35S-Asxc1      2       3      4       5       6     7      8       9     10   (1)    (1)    (1)  (4.0) (4.6) (6.7) (1.7) (2.3) (1.1)*Fig. 1 Full-length Asx interacts directly with SET domains of trxG activator, Trx and PcG repressor, E(z) in a GST pull-down assay. a Clustal Omega sequence alignment of SET domains of Drosophila Trx and E(z), showing 35% sequence identity. b SDS–PAGE (13%) analysis of affinity purified E. coli-expressed GST fusions of SET domains of E(z) and Trx. c GST pull-down analysis of 35S-methionine-labeled rabbit reticulocyte lysate-in vitro translated (RRL-IVT) Asx with GST fusions of SET domains of E(z) and Trx. The stringency of the NaCl washes indicates stronger interaction between Asx and GST-E(z)SET than GST-TrxSET. Proteins were analyzed on 13% SDS–polyacrylamide reducing gels. ()* Estimated fold binding of Asx with GST fusions relative to GST (determined by densitometry) is shown beneath each lanePage 7 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 Drosophila S2 cells. This assay allows very sensitive (com-pared to immunoprecipitation) detection of protein–pro-tein interactions either in solution or in situ at single-cell resolution [36]. Association between Asx with Trx was only observed after heat-shock induction, and the level of association becomes significant after 60–180  min of recovery period (Fig.  6a). The association between Asx with E(z) was observed before and after heat-shock induction (Fig. 6b). These results suggest Asx associates with E(z) and Trx in  vivo during heat-shock recovery. Taken together, these three protein assays indicate that Asx associates both in vitro and in vivo with E(z) and Trx.Asx binds to hsp70 promoter downstream region upon heat‑shock induction and is required for hsp70 repression during induction and recoveryThe foregoing results suggest a mechanism whereby Asx modulates the ratio of H3K4 and H3K27 trimethyla-tion by associating with the antagonistic HMTs during heat shock and recovery. If so, then mutations in Asx should affect levels of trimethylation at histone H3K4 and H3K27 during transcription. To determine whether Asx was recruited to heat-shock loci on chromatin, poly-tene chromosomes were prepared from salivary glands subjected to 20 min of heat shock at 37  °C, and stained with antibodies to Asx. These were compared to prepara-tions from glands that were not heat shocked. Asx was recruited to hsp70 region (at 87AC) and other heat-shock loci following 20 min of heat shock (Fig. 7a). In contrast, in control polytene chromosomes that were not heat shocked, Asx was recruited to region 89E (Fig. 7a) which includes Ultrabithorax (Ubx) thus serving as a positive control for the polytene staining [25].Pol II recruitment and initiation of hsp70 transcription to generate the paused hsp70 promoter occur early in embryogenesis (2–4 h after egg deposition) when mater-nally deposited Asx protein or Asx mRNA is still present. To allow enough time for maternal levels of Asx mRNA or protein to drop, and thus allow us to detect the embry-onic effect of Asx mutations, heat shock was induced in MWr/kDa10% INPUTα-Asxd10070E(z)IgG10% INPUTα-AsxIgGBR70-0.85/ α-Asx Co-IPP10% INPUTα-AsxIgGα -Trx-Western10070Trxe250ab250AsxTrx0.1 0.18 0.850.3 0.6 M KCl250MWr/kDaBio-Rex 70  FractionsTrxBR70-0.1/ α -Asx Co-IPPMWr/kDa BR70-0.85/ α-Asx Co-IPPMWr/kDaα-E(z)-Westernα-E(z)-Westernc0.1 0.85 M KClBio-Rex 700.3 0.60.18α-Asx Co-IPP/WesternDrosophila Embryo Nuclei9%-56% AmSO4Western blot1        2      3       4      5                      E(z)10070Fig. 2 Asx co-fractionates and co-immunoprecipitates with Trx in embryo nuclear extracts. a Schematic of Bio-Rex-70 fractionation of embryo nuclear extracts and coupled co-immunoprecipitation–western blot analysis. b Western blot analysis of Bio-Rex 70 fractions with anti-Asx (upper panel), anti-Trx (middle panel) and anti-E(z) (lower panel). c Coupled anti-Asx immunoprecipitation and anti-Trx western blot analysis of the BR70-0.85 fraction, showing Asx association with Trx. d Coupled anti-Asx immunoprecipitation and anti-E(z) western blot analysis of the BR70-0.1 fraction, showing lack of Asx association with E(z) in the enriched E(z) fraction. e Coupled anti-Asx immunoprecipitation and anti-E(z) western blot analysis of the BR70-0.85 fraction, showing lack of Asx association with E(z) in the enriched Trx fractionPage 8 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 14–16-h embryos. Using this protocol, we are unable to assay the role of Asx in establishment of transcrip-tional initiation and promoter clearance of hsp70 in early embryos. Our studies in later embryos do not attempt to distinguish the role of Asx in elongation between roles at the promoter in re-initiation and promoter clearance, although we biased the results toward elongation by selection of a primer downstream of the promoter (+218 to +392 bp) previously identified as a site of recruitment for Trx upon heat shock [23] for the ChIP experiments below.The hsp70 gene is induced in all cells of the embryo in response to thermal stress, allowing us to use whole embryos for this study [37]. To determine whether Asx binds downstream of the hsp70 promoter downstream region, we performed ChIP and quantitative PCR (qPCR) using the anti-Asx antibody (Fig.  7b, c). In control embryos that were not subject to thermal stress (0 min of heat shock), Asx did not significantly bind at the hsp70 promoter downstream region. After 15  min of heat shock, the level of Asx binding was threefold higher than with no heat shock. At 30-min recovery after 15-min heat shock, Asx binding to the hsp70 gene increased seven-fold relative to no heat shock. The level of Asx binding at hsp70 gradually decreased from 60-min recovery to 180-min recovery (Fig. 7b). As a positive control for Asx binding in our assay conditions, we tested Asx binding to selected regions within Ubx promoter and bithoraxoid (bxd) Polycomb response element (PRE) in Drosophila embryos with the our Asx antibody in our ChIP assay [10]. The results were consistent with previous observa-tions that Asx binds chromatin at these locations without heat-shock induction (Additional file  9: Fig. S3). These results confirm that the hsp70 locus is a direct binding target of Asx upon heat shock, with peak binding occur-ring between 15-min induction/30-min recovery and 15-min induction/120-min recovery.To investigate the effect of Asx on transcription dur-ing induction and recovery from heat shock, we com-pared the steady-state hsp70 mRNA levels during heat shock and recovery of homozygous Asx3 null mutants to wild-type (Oregon R) embryos (see “Methods” section). The mRNA level difference between the homozygous Asx3 mutant and wild type was at least twofold during 35S-Asx-COOH-terminaldeletion Fragments2501501007550372520151       2        3       4       5        6       7       8       9   a25A(1 - 354)A(1200 –1669)1     2     3    4A(1 - 1200)A(1200 –1501)A(1501 –1669)A(1 - 610)50757510037503725035S-Asx-NH2-terminaldeletion FragmentsbGST3% InputA(1 - 1669)MWr/kDa25035S- Asxfragments150GST-E(z)SETGST-TrxSET150(24.7) (4.8)*(15.4) (4.5)(6.8) (1.6)(2.1) (3.0)(4.5) (4.1)(5.0)Lanes(1)(1) (1)(1)(1)(1)(1)(1)(1)(1)Fig. 3 Asx contains two shared interaction sites for GST-TrxSET and GST-E(z)SET. a SDS–PAGE analysis of expression of 35S-labeled RRL-IVT dele-tion fragments of Asx (3% of the input for GST pull-down assay in Fig. 2b) and exposed for an hour at −70 °C (lanes 1–9). b Identification of two shared interaction sites for GST-E(z)SET and GST-TrxSET (1–354 and 1200–1500) and one unique, weak site for GST-TrxSET (1501–1669) on Asx. GST pull-down analysis of the Asx fragments in A) with GST-E(z)SET (lane 3) and GST-TrxSET (lane 4) compared to GST controls (lane 1). ()* Estimated fold binding of Asx with GST fusions relative to GST (determined by densitometry) is shown beneath each lane. Unbound fragments, as shown for A(1–1200), are listed in Fig. 3bPage 9 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 heat-shock induction and recovery, and at 90-min recov-ery, the difference reached a maximum level at 2.7-fold (Fig.  8a). The steady-state Ahcy89E mRNA levels dur-ing heat shock and recovery were stable and the mRNA level was comparable between Asx3 mutant and wild-type embryos, indicating that Ahcy89E is suitable for normalizing the hsp70 mRNA levels (Fig. 8b). Together, these data show that Asx represses the hsp70 locus dur-ing heat-shock induction and recovery. After 180-min recovery, the hsp70 mRNA level in both wild type and homozygous Asx3 mutant decreased to the level before heat-shock induction, showing that Asx is not required for terminating transcription after heat-shock induction. In addition, peak transcription observed between 15-min induction/30-min recovery and 15-min induction/90-min recovery correlates with Asx binding within this period (Figs. 7b, 8a).Asx transiently reduces H3K4 trimethylation at hsp70 during heat‑shock inductionAsx represses hsp70 during heat shock, so in Asx3 mutant embryos, we expected to observe increased levels of H3K4me3 downstream of the hsp70 promoter after heat shock compared to wild type. As shown in Fig.  9a, in ChIP-qPCR experiments, the level of H3K4 trimethyla-tion in wild type and Asx3 mutant did not differ in the first 10 min of heat-shock induction. However, at 15-min induction and 15-min/30-min recovery (Fig.  9a), the level of H3K4 trimethylation in Asx3 mutants was 1.8-fold higher than in wild-type (significant at P  <  0.05), suggesting that in wild-type embryos, Asx reduces the level of histone H3K4me3 at these time points. Interest-ingly, in the later recovery phase, Asx3 mutants showed less H3K4me3 than wild-type embryos after 120 min of recovery from 15-min heat-shock induction (significant 1 354 610 1200 1669(HR2 –HR6)AsxH PHD1501200ETSI-135420035420011161012001 16691200 1669166915011200 1501COOH terminal deletion fragments NH2 terminal deletion fragmentsA(1-1200)A(1-610)A(1-354)A(200-354)A(1-200)A(1-1669)1A(1200 – 1669)A(1200 – 1501)A(1501 -1669)++E(z)SET TrxSET+1381 1501A(1381 – 1501)- -+ +/-- -Asx Interaction with GST-SET after 0.6M NaCl wash :HR1++ +- -++- -ETSI-2-+++abTSICalypso-BS2 3381 354 120016301669(24.7)(15.4)(6.8)(2.1)(4.5)(4.8)(4.5)(1.6)(3.0)(4.1)(5.0)Fig. 4 Map of Asx summarizing interaction sites of E(z) and Trx SET domains indentified using a GST pull-down assay. a Linear map of Asx showing a bipartite recognition site for both E(z)SET and TrxSET domains. The NH2 terminal recognition site (residues 1–354) termed E(z)-Trx SET domain interaction site 1 (ETSI-1) and the COOH terminal site (residues 1200–1501) termed ETSI-2 are marked above in blue. The single weak recognition site for TrxSET domain (residues 1501–1669), termed TSI, is marked in orange. The ETSI-1 site overlaps both the conserved AsxH domain (residues 200–354) and the Calypso binding site (marked below in green). b Summary of the GST pull-down analysis of the interaction between 35S-labeled Asx deletion fragments with GST fusion proteins of E(z) SET domain or Trx SET domain after a 0.6 M NaCl wash. Non-binding Asx fragments A(1–1200), A(1–200), A(200–354) and A(1381–1501) are indicated by (−). Scouring of the interactions of the Asx deletion fragments with GST-E(z) SET or GST-Trx SET is based on the densitometry of band intensity relative to GST [(++; > tenfold),; +; (twofold–tenfold)]Page 10 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 at P  <  0.05), consistent with the rapid drop of hsp70 mRNA level during 120-min recovery after heat-shock induction. At this time, Asx could transiently modulate the rate of transcription by governing the rate of tran-script decay. We suggest that Asx directly or indirectly regulates H3K4me3 to an appropriate level during heat shock and recovery.Asx regulates the ratio of histone H3K4 to H3K27 trimethylation at hsp70 during heat‑shock induction and recoveryIf Asx represses hsp70 by acting as a PcG protein, then we expect to observe reduced H3K27me3 levels in Asx3 mutants compared to wild type during heat-shock induc-tion and recovery. We performed ChIP-qPCR experi-ments with anti-H3K27me3 antibody on wild-type and Asx3 mutant embryos during heat shock and recovery. The level of H3K27me3 in wild type and Asx3 mutant did not differ markedly at 10 min of heat-shock induction. At subsequent time points from 15-min heat-shock induc-tion to 120-min recovery after heat shock except the 30-min recovery, the level of H3K27me3 in Asx3 mutants was 1.5-fold lower than in wild type (Fig. 9b).Thus, the data in Fig. 9a, b show that mutation of Asx alters the levels of H3K4me3 and H3K27me3 in the expected way at one target gene during the same regu-latory event. Interestingly, the significant increase in the ratio of H3K4me3/H3K27me3 from 15-min induction to 15-min induction/30-min recovery from heat shock in Asx3 compared to wild type (Fig.  9c) occurs close to the peak of transcription (Fig.  8a) that coincides with the transition from promoter clearance to elongation. As MWr/kDa2507013010055d10 % BR70-0.1BR70-0.1/α-E(z) WesternGST GST-ETSI-2 Pull-Down/α-E(z)WesternETSI-21669AsxwtAsx3aE(z) E(z)∆cDrosophila Embryo Nuclei9%-56% AmSO4 Pptn0.1 0.18 0.85 M KClBio-Rex 70 (BR70)0.3 0.60.10.3α-E(z) Western blottingSDS ElutionGST-Asx(ETSI-2)Pull-DownM NaCl1 354 610 1200 16691630(HR2 –HR6)ASXH PHD1501200ETSI-1HR11 354 610 1270200HR1836TSI1        2             3      4ASXH372507515010050GST-ETSI-2b1    2      3     4     5E(z) Fig. 5 GST-fused AsxETSI-2 interacts with E(z) in embryo nuclear extracts. a Alignment of the amino acid sequence of wt and  Asx3 mutant indicates deletion of ETSI-2 and TSI sites in the COOH terminal region, which may disturb the levels of H3K4me3 and H3K27me3. b SDS–PAGE (13%) analysis of purification of E. coli-expressed GST fusions of ETSI-2 domains. c The interaction of ETSI-2 with endogenous E(z) was assayed using extracts prepared from 6- to 18-h-old embryo nuclei. Fractionation of extracts on a Bio-Rex70 column by a step gradient from 0.1 to 0.85 M NaCl elutes E(z) at 0.1 M NaCl fraction as opposed to Trx which elutes at 0.85 M NaCl. d GST-fused ETSI-2 was mixed with the 0.1 M NaCl-containing E(z) fraction in a pull-down assay, using GST as a negative control. The pull-down products were analyzed by 7.5% SDS–PAGE and probed by anti-E(z) antibody in a western blot assay. Lanes (1) pre-marked molecular weight markers (BioRad); (2) 10% input of the pull-down fraction; (3) GST/BR70-0.1 fraction pull down; (4) GST-ETSI-2/BR70-0.1 fraction pull down. The lower band in lane 4 is likely a breakdown product of E(z)Page 11 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 positive and negative controls for the levels of H3K4me3 and H3K27me3 in our assay conditions, we compared the highest and lowest levels of recovery of H3K4me3 and H3K27me3 observed at hsp70 in ChIP experiments to levels obtained with a fragment from within the bxd PRE as a positive control, and a region upstream of the DRP12 gene as a negative control, in Drosophila embryos (Additional file 10: Fig. S4). The results show that signifi-cantly more H3K4me3 is recovered at the hsp70 locus compared to bxd, consistent with hsp70 being actively transcribed in all cells, whereas bxd is expressed only in a subset of cells. During the recovery phase from heat shock, when H3K27me3 levels are highest, they are equivalent to those observed at bxd. Even at the lowest levels of H3K27me3 observed at hsp70 (15  min of heat shock, 120  min of recovery), the levels observed are significantly higher than the negative or IgG controls. Thus, we are confident that the changes we observe in H3K4me3 and K3K27me3 we observe at hsp70 are bio-logically important.DiscussionOur experiments on the role of Asx in regulation of the relative levels of H3K4me3 to H3K27me3 suggest that Asx recruits Trx and E(z) at different temporal stages via interaction with their SET domains (Figs. 9, 6). The map-ping data shown in Figs. 1 and 2 confirm the hypothesis that Asx interacts directly with E(z) and Trx in vitro. The mapping data also show that there are two non-overlap-ping Asx domains that interact with E(z) and Trx SET domains, termed ETSI-1 and ETSI-2 (Fig.  4). Sequence comparison of ETSI-1 and 2 reveals 15% sequence iden-tity (Additional file  8: Fig. S2), suggesting that either there is conservation of 2D structure with clustered amino acid sequence conservation, or that ETSI-1 and 2 domains have a unique interaction mechanism. The third Asx interaction site, TSI (Figs.  3, 4), is a distinct Trx SET domain interaction site, which overlaps the con-served C-terminal atypical PHD motif, consistent with the suggestion that the conserved atypical PHD motif lacks structural features which restricts its binding to the N-terminal tail of H3 [38].The identification of Asx interaction sites for the SET domains of E(z) and Trx (ETSI-1/2 and TSI) (Fig.  4) is consistent with Asx association with equivalent levels of E(z) and Trx in vivo at 60 to 180 min of recovery (Fig. 6). These findings support the hypothesis that Asx directly regulates H3K4me3 and H3K27me3 levels downstream of the hsp70 promoter (Fig. 9). However, the mapping of 2 SET domain interaction sites suggests that it is unlikely a bcd0 min 15/180 minDAPIPLAMergeAsx-Trx PLA Asx-E(z) PLA15/60 min 0 min 15/180 min15/60 minRed-Sp20 merFig. 6 Asx interacts with E(z) and Trx in Drosophila S2 cells in vivo. a Proximity ligation assay (PLA) between Asx and Trx with Drosophila S2 cells before and after heat-shock induction. Nuclei were labeled with DAPI in blue and PLA signals in green for Asx-Trx and in red for Asx-E(z). b PLA between Asx and E(z) with S2 cells before and after heat-shock induction. c, d Schematic diagram of PLA reaction. Oligonucleotides and antibod-ies used in PLA are listed in “Methods” section. A closed circle between connector and splints forms if proteins are in close proximity. Rolling-circle amplification (RCA) was carried out in the presence of fluorescent-labeled oligonucleotides. Signals would be detected only when proteins were in distance between 10 and 80 nmPage 12 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 that Trx and E(z) compete to bind Asx. This conclusion is supported by the data in Fig. 2 showing that E(z) and Trx do not co-fractionate after chromatography of nuclear extracts on Bio-Rex 70. These results suggest that Asx association with Trx or E(z) in vivo may be influenced by the different temporal stages of heat shock and recovery. These experiments do not rule out interactions of Asx with domains outside the SET domains of Trx and E(z), but this possibility was not tested.Comparison of existing structure function analysis of Asx homologs in Drosophila and mammals with our new data allows us to speculate that Asx acts as an ETP by interacting with multiple nucleosome-modifying enzymes to modulate histone modifications. Asx and its mammalian homolog ASXL1 interact with Calypso/BAP1 to form the PR-DUB complex, which deubiqui-tinates histone H2AK118Ub/H2AK119Ub at the Ubx PRE and promoter [10]. The ASXH domain is conserved between all mammalian ASXL and Drosophila Asx and directly binds the deubiquitinase BAP1 [10, 39]. Interest-ingly, in mammals the ASXL1 DEUBAD domain (amino acid 238–290) located within the ASXH (amino acid 249–368) domain is required to activate the BAP1 enzy-matic function on deubiquinating H2AK119Ub [40, 41]. Unlike Asx, mutation of calypso did not have a signifi-cant effect on the relative hsp70 mRNA levels in calypso2 mutant and wild-type embryos at selected time points of heat shock or recovery (Additional file 11: Fig. S5). Thus, calypso is not required for the activity of Asx at hsp70 during heat-shock induction and recovery.Both our results on Asx and recent studies on ASXL1 suggest that ETSI-1 site alone is insufficient to maintain the normal H3K27me3 level at target promoters [42]. We therefore suggest that both the ETSI-1 and ETSI-2 regions are required to recruit and/or retain the func-tion of E(z)/EZH2 at target promoters. Consistent with this view, ASXL1 co-immunoprecipitates with the PRC2 components EZH2, SUZ12 and EED [43]. The C-terminal abcInduction Recovery87AC87AC+1Fig. 7 Asx binds to the hsp70 promoter downstream upon heat-shock induction. a Asx antibody staining of the central part of wild-type 3R polytene chromosomes before and after 20-min heat shock at 37 °C. The major heat-shock loci at 87AC, 93D and 95D are labeled. After 20-min heat shock, Asx is recruited to major heat-shock loci indicated. The bithorax complex located at 89E serves as a control for constitutive binding of Asx. b Chromatin immunoprecipitation with anti-Asx and IgG control antibodies from wild-type embryos during heat-shock induction at 37 °C and recov-ery. The DNA from ChIP samples at hsp70 promoter downstream region was analyzed by qPCR and is shown along the y-axis, and times of heat shock or recovery are shown in the x-axis. Duration of heat shock/recovery is denoted as 15/30, 15/60, 15/90, 15/120 and 15/180 min. The signals are represented as mean ± SEM with N = 3. c Primer map showing the location of primers 218- and 392-bp downstream of hsp70 transcription start site used in ChIP experimentsPage 13 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 region of ASXL1 aligns to the Asx ETSI-2 with 15% sequence identity (Additional file 8: Fig. S2B), suggesting conservation of function. It is possible that ETSI-1 and ETSI-2 interact independently or cooperatively. In the future, it will be interesting to determine whether each of these domains simultaneously recruits Trx and E(z), whether binding of Trx or E(z) occurs preferentially to ETSI-1 or 2 and whether histone demethylases or histone acetyl transferases also associate with these domains.One major finding in this study is that Asx is required to repress the transcription of hsp70 during heat stress and recovery. In the first 10 min of heat stress, when ab00.010.020.030.040.050.060.070.08WTAsx[3]Relative mRNA levelhsp70Ahcy89EFig. 8 Asx is required for hsp70 repression during heat-shock induction and recovery. a The relative mRNA levels in wild-type and Asx3 homozygous null mutant embryos were measured by RT-qPCR. Asx null mutations are embryonic lethal at a late embryonic stage [4], so homozygous 12–15-h Asx3 mutants were collected using absence of expression of the GFP-marked balancer chromosome as a criterion. The duration of heat shock/recov-ery is as described in Fig. 7. The x-axis shows the heat-shock induction times at 37 °C and heat-shock recovery times after 15 min of 37 °C heat-shock induction. The y-axis indicates the hsp70 mRNA level normalized to the control gene Ahcy89E mRNA level. The signals are represented as mean ± SEM with N = 3. b The relative mRNA levels of the control gene Ahcy98E are compared between Asx3 mutant and wild-type embryos throughout the heat-shock induction and recovery. The x-axis shows the heat-shock induction times at 37 °C, and heat-shock recovery times after 15 min of 37 °C heat-shock induction. The y-axis shows the relative mRNA level of the control gene Ahcy89E mRNA, as mean ± SEM with N = 3Page 14 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 transcriptional elongation should predominate over re-initiation, Asx mutants exhibit higher levels of hsp70 transcription compared to controls (Fig. 8), so Asx acts as a governor to prevent rapid increase in the rate of hsp70 transcription. Interestingly, we do not detect any signifi-cant change in H3K4me3 levels during this time (Fig. 9), implying that the Asx effect in the first 10  min is Trx independent [23]. Given previous observations that Asx is not recruited to polytene chromosomes in trx mutants and vice versa [44] and that trx mutations abolish induc-tion of heat shock [23], one would predict no heat-shock response in Asx mutants. As noted in “Background,” we would not detect a Trx-dependent effect in our experi-ments immediately after heat-shock induction because recruitment of Trx and Pol II and initiation of transcrip-tion occur many hours earlier in the presence of mater-nal Asx. We suggest that changes in H3K4me3 levels detected after 15 min of heat stress reflect re-initiation of hsp70 transcription.Asx may have a previously unreported role in tran-scriptional elongation of hsp70 that is independent of the catalytic activity of Trx or other H3K4 HMT because Asx mutants have higher transcription compared to wild type in the first 10 min of heat stress that cannot be attributed to changes in levels of H3K4me3. Asx may regulate fac-tors required for hsp70 transcriptional elongation includ-ing Mediator, elongation factors (such as Spt5, Spt6 and FACT) or the H3K36 histone methyltransferase activity ab Ratio of H3K4me3/H3K27me3 after heat shock c0246810121416182010  min 15 min 15/30 min 15/60 min 15/120 minArbitrary unitInduction Recovery**Fig. 9 Asx regulates H3K4me3 and H3K27me3 levels at hsp70. Both panels show ChIP-qPCR analysis comparing control rabbit IgG to trimethylated histones in wild-type (blue) and Asx3 (red) embryos at different times of heat-shock induction and recovery as indicated in the x-axis. The y-axis indicates recovery after ChIP as a percentage of input DNA. The notation for the duration of heat shock/recovery is described in Fig. 7. All data are represented as mean ± SEM with N = 3. (*) P < 0.05. The standard deviation (SD) of each data point is presented in Additional file 12: Table S3 and Additional file 13: Fig. S6. a ChIP-qPCR analysis with trimethylated histone H3K4 antibody. During late heat-shock induction and the first 30 min of recovery, H3K4me3 levels are higher in Asx mutants than wild type. In late recovery, H3K4me3 levels are lower in mutants than wild type. b ChIP-qPCR analysis with trimethylated histone H3K27 antibody. Levels of H3K27me3 are essentially constant in Asx mutants and are significantly lower than wild type at the end of induction and the recovery phases. c Ratio of histone H3K4me3 to H3K27me3 during heat shock and recovery, expressed in arbitrary units, and derived from the data in a, bPage 15 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 [18, 20, 22, 45]. Alternatively, Asx may participate in Pol II pausing and retention or release during the 0–10-min heat-shock phase before a Trx-dependent step [46].From 15  min of heat-shock induction up to 60  min of recovery, Asx is required to govern the rate of hsp70 transcription and changes the ratio of H3K4me3 to H3K27me3. As shown in Fig. 9c, the ratio of H3K4me3/H3K27me3 levels is consistently higher in Asx mutants compared to wild type during this period suggesting that the relative levels of H3K4 and H3K27 trimethylation regulate hsp70 transcription. Alternatively, the ratio of H3K4me3/H3K27me3 may reflect consequences of other events regulated by Asx that lead to changes in the ratio.From 90 to 180 min after recovery from heat shock, tran-scription of hsp70 falls in both control and Asx3 mutant embryos, showing that Asx is not required for repression of hsp70 transcription at this phase (Fig. 8a). By contrast, the PcG gene pho is required for this repression because the level of hsp70 mRNA in pho1 mutant larvae was sig-nificantly higher than in wild-type embryos after 30-min induction/180-min or 300-min recovery, but no significant difference was observed after 30-min induction/60-min recovery after heat shock [24]. Therefore, the timing of Asx regulation of hsp70 transcription does not significantly overlap with the requirement for pho.Asx was identified first as a PcG gene [4], and subse-quent experiments in Drosophila supported this conclu-sion [5, 6]. To our knowledge, our observations provide the first example where Asx mutations cause simultaneous changes in both H3K4me3 and H3K27me3 at the same locus at the same time. Protein nulls such as Asx22P4 have no change in global H3K27 trimethylation and show very slight reduction in global H3K4 trimethylation [10]. The increased sensitivity of gene-specific analysis or particular features of hsp70 regulation may allow us to detect clear effects of Asx mutations on relative levels of H3K4me3 and H3K27me3 that are not detected in bulk chromatin. Our data do not rule out alternative models could account for the regulation of the H3K4me3 to H3K27me3 levels: (1) Asx acts indirectly via CBP-mediated H3K27 acetyla-tion to block methylation [47, 48]; (2) a role for two other H3K4me3 HMTs, SET1 and Trr, cannot be excluded since both contain SET domains with 50% amino acid sequence identity to the SET domain of Trx (NCBI protein sequences, see web refence below); (3) Asx affects demeth-ylation of H3K27me3 by Jarid2 by recruiting E(z), and its HMT PRC2 subunits Su(z)12, Esc and Jarid2 [49].ConclusionsThe major finding in this study is that Asx interacts directly with E(z) and Trx in  vitro and in  vivo during heat-shock recovery. Asx is required to repress tran-scription of the hsp70 locus during heat stress and the first 60 min of recovery by regulating the relative H3K4 and H3K27 trimethylation levels at the promoter. These results are consistent with genetic identification of Asx as an ETP required for both trxG and PcG function.Additional filesAdditional file 1: Text S1. Rabbit anti-Asx antibody.Additional file 2: Fig. S1. Validation of Asx antibody. Western blots with Drosophila wild-type and Df(2R)trix mutant embryo extract showing the rabbit anti-Asx antibody (aa. 200–356) generated for this study binds specifically to Asx. Df(2R)trix mutant contains deletion of entire Asx. The binding level was significantly reduced in Df(2R)trix mutant embryo extract compared to wild-type embryo extract.Additional file 3: Table S1. PCR primer pairs for construction of E(z) SET and Asx expression vectors.Additional file 4: Text S2. Buffers.Additional file 5: Text S3. GST pull-down western assays of embryo nuclear extract.Additional file 6: Table S2. Oligonucleotides for in situ PLA in Drosophila S2 cells.Additional file 7: Text S4. Primers for hsp70, Ahcy89E, bxd PRE and Ubx promoters.Additional file 8: Fig. S2. Alignment of amino acid sequences of AsxETSI. (A) Clustal Omega alignment of AsxETSI-1 and AsxETSI-2 showing 14.97 % sequence identity. (B) Clustal Omega alignment of AsxETSI-2 and ASXL1 (943–1307) showing 15.1% sequence identity.Additional file 9: Fig. S3. Asx binds to Ultrabithorax (Ubx) promoter and bithoraxoid (bxd) PRE region. (A) Primer map showing the location of prim-ers used in ChIP experiments. L2, L7 and L8 primers are located within the bxd PRE region, 12.5-kb upstream of the Ubx promoter. U2 and U3 primers are located downstream of the Ubx promoter. (B, C) ChIP-qPCR analysis of anti-Asx and control rabbit IgG antibodies from wild-type embryos. The DNA recovered from ChIP samples was analyzed by qPCR and is shown along the y-axis. The signals are represented as mean ± SEM with N = 3.Additional file 10: Fig. S4. H3K4me3 and H3K27me3 levels at bithorax-oid (bxd) Polycomb response element (PRE) and DPR12 genes compared to highest and lowest levels observed at hsp70. ChIP-qPCR analysis of H3K4me3 and H3K27me3 and control rabbit IgG antibodies from wild-type embryos. The DNA recovered from ChIP samples was analyzed by qPCR, and percent recovery is shown along the y-axis. The data for hsp70 are taken from Fig 9. The signals are represented as mean ± SEM with N = 3. The bxd PRE primers are located between BX-C 218839 and 218959. C1 is located at +39kb to the DPR12 gene.Additional file 11: Fig. S5. calypso is not required for hsp70 repression during heat-shock induction and recovery. The relative mRNA levels in wild-type and calpyso2 homozygous null mutant embryos were measured by RT-qPCR. The x-axis shows the heat-shock induction times at 37 °C, and heat-shock recovery times after 15 min of 37 °C heat-shock induction. The y-axis indicates the hsp70 mRNA level normalized to the control gene Ahcy89E mRNA level. The signals are represented as mean ± SEM with N = 3.Additional file 12: Table S3. Standard deviation (SD) table for ChIP experiments.Additional file 13: Fig. S6. Asx regulates H3K4me3 and H3K27me3 levels at hsp70. Both panels show ChIP-qPCR analysis comparing control rabbit IgG to trimethylated histones in wild-type (blue) and Asx3 (red) embryos at different times of heat-shock induction and recovery as indi-cated in the x-axis. The y-axis indicates recovery after ChIP as a percent-age of input DNA. The notation for the duration of heat shock/recovery is described in Fig. 7. All data are represented as mean ± SD. There was minimal difference when compared with the error bars using SEM as the error source in Fig. 9.Page 16 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 AbbreviationsPcG: Polycomb group; TrxG: trithorax group; ETP: enhancer of trithorax and Polycomb; Asx: additional sex combs; HMT: histone methyl transferase; E(z): enhancer of zeste; Trx: trithorax; hsp70: 70-kDa heat-shock proteins; SET: Su(var)3–9, enhancer of zeste and trithorax; CTD: C-terminal domain; HSF: heat-shock factor; ChIP: chromatin immunoprecipitation; ETSI: E(z)/Trx SET domain interaction site; TSI: Trx SET domain interaction site; PLA: proximity ligation assay; Ahcy89E: adenosylhomocysteinase 89E; PR-DUB: Polycomb repressive deubiquitinase.Authors’ contributionsHWB and AM conceived the study; TL, JWH and SP performed experiments. All authors discussed the results, analyzed the data and commented on drafts of the manuscript. TL, JWH and HWB wrote the manuscript. All authors read and approved the final manuscript.Author details1 Department of Zoology, Life Sciences Institute, University of British Colum-bia, 2350 Health Science Mall, Vancouver, BC V6T 1Z4, Canada. 2 Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadel-phia, PA 19107, USA. AcknowledgementsWe thank Bloomington Stock Center for fly stocks. We acknowledge the assis-tance of Dr. Sheryl T. Smith and Maya Kupczyk during the initial phase of this project. We thank Drs. Robert Tjian and Michael Kyba for plasmid vectors.Competing interestsThe authors declare that they have no competing interests.Availability of data and materialsThe datasets used and/or analyzed during the current study are available form the corresponding author on reasonable request.Consent for publicationNot applicable.Ethics approval and consent to participateNot applicable.FundingSP was supported by a training program from the National Cancer Institute (NCI) (CA009678). This study was supported by grants from the Natural Sciences and Engineering Research Council and the Canadian Institutes of Health Research to HWB. The funders had no role in study design, collec-tion, analysis or interpretation of data, in the writing or in the decision to submit.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Received: 26 April 2017   Accepted: 13 September 2017References 1. Paro R. Imprinting a determined state into the chromatin of Drosophila. Trends Genet. 1990;6(12):416–21. 2. Kennison JA. Transcriptional activation of Drosophila homeotic genes from distant regulatory elements. Trends Genet. 1993;9(3):75–9. 3. Brock HW, Fisher CL. Maintenance of gene expression patterns. Dev Dyn. 2005;232(3):633–55. 4. Jurgens G. A group of genes controlling the spatial expression of the bithorax complex in Drosophila. Nature. 1985;316(6024):153–5. 5. Simon J, Peifer M, Bender W, O’Connor M. Regulatory elements of the bithorax complex that control expression along the anterior-posterior axis. EMBO J. 1990;9(12):3945–56. 6. Sinclair DA, Campbell RB, Nicholls F, Slade E, Brock HW. Genetic analysis of the additional sex combs locus of Drosophila melanogaster. Genetics. 1992;130(4):817–25. 7. Duncan I. The bithorax complex. Annu Rev Genet. 1987;21:285–319. 8. Milne TA, Sinclair DA, Brock HW. The Additional sex combs gene of Drosophila is required for activation and repression of homeotic loci, and interacts specifically with Polycomb and super sex combs. Mol Gen Genet. 1999;261(4–5):753–61. 9. Campbell RB, Sinclair DA, Couling M, Brock HW. Genetic interactions and dosage effects of Polycomb group genes of Drosophila. Mol Gen Genet. 1995;246(3):291–300. 10. Scheuermann JC, de Ayala Alonso AG, Oktaba K, Ly-Hartig N, McGinty RK, Fraterman S, Wilm M, Muir TW, Muller J. Histone H2A deubiquit-inase activity of the Polycomb repressive complex PR-DUB. Nature. 2010;465(7295):243–7. 11. Gildea JJ, Lopez R, Shearn A. A screen for new trithorax group genes identified little imaginal discs, the Drosophila melanogaster homologue of human retinoblastoma binding protein 2. Genetics. 2000;156(2):645–63. 12. Brock HW, van Lohuizen M. The Polycomb group-no longer an exclusive club? Curr Opin Genet Dev. 2001;11(2):175–81. 13. Muller J, Verrijzer P. Biochemical mechanisms of gene regulation by Poly-comb group protein complexes. Curr Opin Genet Dev. 2009;19(2):150–8. 14. Schuettengruber B, Martinez AM, Iovino N, Cavalli G. Trithorax group proteins: switching genes on and keeping them active. Nat Rev Mol Cell Biol. 2011;12(12):799–814. 15. Tsukiyama T, Becker PB, Wu C. ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor. Nature. 1994;367(6463):525–32. 16. Rasmussen EB, Lis JT. In vivo transcriptional pausing and cap forma-tion on three Drosophila heat shock genes. Proc Natl Acad Sci USA. 1993;90(17):7923–7. 17. Rougvie AE, Lis JT. The RNA polymerase II molecule at the 5′ end of the uninduced hsp70 gene of D. Melanogaster is transcriptionally engaged. Cell. 1988;54(6):795–804. 18. Boehm AK, Saunders A, Werner J, Lis JT. Transcription factor and polymer-ase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock. Mol Cell Biol. 2003;23(21):7628–37. 19. Westwood JT, Wu C. Activation of Drosophila heat shock factor: confor-mational change associated with a monomer-to-trimer transition. Mol Cell Biol. 1993;13(6):3481–6. 20. Andrulis ED, Guzman E, Doring P, Werner J, Lis JT. High-resolution localiza-tion of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation. Genes Dev. 2000;14(20):2635–49. 21. Kaplan CD, Morris JR, Wu C, Winston F. Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. Melanogaster. Genes Dev. 2000;14(20):2623–34. 22. Saunders A, Werner J, Andrulis ED, Nakayama T, Hirose S, Reinberg D, Lis JT. Tracking FACT and the RNA polymerase II elongation complex through chromatin in vivo. Science. 2003;301(5636):1094–6. 23. Smith ST, Petruk S, Sedkov Y, Cho E, Tillib S, Canaani E, Mazo A. Modula-tion of heat shock gene expression by the TAC1 chromatin-modifying complex. Nat Cell Biol. 2004;6(2):162–7. 24. Beisel C, Buness A, Roustan-Espinosa IM, Koch B, Schmitt S, Haas SA, Hild M, Katsuyama T, Paro R. Comparing active and repressed expression states of genes controlled by the Polycomb/Trithorax group proteins. Proc Natl Acad Sci USA. 2007;104(42):16615–20. 25. Sinclair DA, Milne TA, Hodgson JW, Shellard J, Salinas CA, Kyba M, Ran-dazzo F, Brock HW. The Additional sex combs gene of Drosophila encodes a chromatin protein that binds to shared and unique Polycomb group sites on polytene chromosomes. Development. 1998;125(7):1207–16. 26. Tao H, Liu W, Simmons BN, Harris HK, Cox TC, Massiah MA. Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS. Biotechniques. 2010;48(1):61–4. 27. Leuchowius KJ, Clausson CM, Grannas K, Erbilgin Y, Botling J, Zieba A, Lande-gren U, Soderberg O. Parallel visualization of multiple protein complexes in individual cells in tumor tissue. Mol Cell Proteom. 2013;12(6):1563–71. 28. Soderberg O, Leuchowius KJ, Gullberg M, Jarvius M, Weibrecht I, Lars-son LG, Landegren U. Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods. 2008;45(3):227–32.Page 17 of 17Li et al. Epigenetics & Chromatin  (2017) 10:43 •  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step: 29. Klasener K, Maity PC, Hobeika E, Yang J, Reth M. B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk. Elife. 2014;3:e02069. 30. Beck SA, Falconer E, Catching A, Hodgson JW, Brock HW. Cell cycle defects in polyhomeotic mutants are caused by abrogation of the DNA damage checkpoint. Dev Biol. 2010;339(2):320–8. 31. Petruk S, Sedkov Y, Riley KM, Hodgson J, Schweisguth F, Hirose S, Jaynes JB, Brock HW, Mazo A. Transcription of bxd noncoding RNAs promoted by trithorax represses Ubx in cis by transcriptional interference. Cell. 2006;127(6):1209–21. 32. Nelson JD, Denisenko O, Bomsztyk K. Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat Protoc. 2006;1(1):179–85. 33. Dillon SC, Zhang X, Trievel RC, Cheng X. The SET-domain protein super-family: protein lysine methyltransferases. Genome Biol. 2005;6(8):227. 34. Frangioni JV, Neel BG. Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem. 1993;210(1):179–87. 35. Kauzmann W. Some factors in the interpretation of protein denaturation. Adv Protein Chem. 1959;14:1–63. 36. Greenwood C, Ruff D, Kirvell S, Johnson G, Dhillon HS, Bustin SA. Proxim-ity assays for sensitive quantification of proteins. Biomol Detect Quantif. 2015;4:10–6. 37. Lakhotia SC, Prasanth KV. Tissue- and development-specific induction and turnover of hsp70 transcripts from loci 87A and 87C after heat shock and during recovery in Drosophila melanogaster. J Exp Biol. 2002;205(Pt 3):345–58. 38. Aravind L, Iyer LM. The HARE-HTH and associated domains: novel mod-ules in the coordination of epigenetic DNA and protein modifications. Cell Cycle. 2012;11(1):119–31. 39. Fisher CL, Berger J, Randazzo F, Brock HW. A human homolog of Addi-tional sex combs, ADDITIONAL SEX COMBS-LIKE 1, maps to chromosome 20q11. Gene. 2003;306:115–26. 40. Sahtoe DD, van Dijk WJ, Ekkebus R, Ovaa H, Sixma TK. BAP1/ASXL1 recruitment and activation for H2A deubiquitination. Nat Commun. 2016;7:10292. 41. Katoh M. Functional and cancer genomics of ASXL family members. Br J Cancer. 2013;109:299. 42. Balasubramani A, Larjo A, Bassein JA, Chang X, Hastie RB, Togher SM, Lahdesmaki H, Rao A. Cancer-associated ASXL1 mutations may act as gain-of-function mutations of the ASXL1-BAP1 complex. Nat Commun. 2015;6:7307. 43. Abdel-Wahab O, Adli M, LaFave LM, Gao J, Hricik T, Shih AH, Pandey S, Patel JP, Chung YR, Koche R, Perna F, Zhao X, Taylor JE, Park CY, Carroll M, Melnick A, Nimer SD, Jaffe JD, Aifantis I, Bernstein BE, Levine RL. ASXL1 mutations promote myeloid transformation through loss of PRC2-medi-ated gene repression. Cancer Cell. 2012;22(2):180–93. 44. Petruk S, Smith ST, Sedkov Y, Mazo A. Association of trxG and PcG proteins with the bxd maintenance element depends on transcriptional activity. Development. 2008;135(14):2383–90. 45. Park JM, Werner J, Kim JM, Lis JT, Kim YJ. Mediator, not holoenzyme, is directly recruited to the heat shock promoter by HSF upon heat shock. Mol Cell. 2001;8(1):9–19. 46. Samarakkody A, Abbas A, Scheidegger A, Warns J, Nnoli O, Jokinen B, Zarns K, Kubat B, Dhasarathy A, Nechaev S. RNA polymerase II paus-ing can be retained or acquired during activation of genes involved in the epithelial to mesenchymal transition. Nucleic Acids Res. 2015;43(8):3938–49. 47. Tie F, Banerjee R, Stratton CA, Prasad-Sinha J, Stepanik V, Zlobin A, Diaz MO, Scacheri PC, Harte PJ. CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing. Development. 2009;136(18):3131–41. 48. Petruk S, Sedkov Y, Smith S, Tillib S, Kraevski V, Nakamura T, Canaani E, Croce CM, Mazo A. Trithorax and dCBP acting in a complex to maintain expression of a homeotic gene. Science. 2001;294(5545):1331–4. 49. Herz HM, Mohan M, Garrett AS, Miller C, Casto D, Zhang Y, Seidel C, Haug JS, Florens L, Washburn MP, Yamaguchi M, Shiekhattar R, Shilatifard A. Polycomb repressive complex 2-dependent and -independent func-tions of Jarid2 in transcriptional regulation in Drosophila. Mol Cell Biol. 2012;32(9):1683–93.Web References 50. Trr; http://www.ncbi.nlm.nih.gov/protein/NP_726773.2. Accessed 17 June 2016. 51. SET1; http://www.ncbi.nlm.nih.gov/protein/NP_001015221.1. Accessed 17 June 2016. 52. Trx; http://www.ncbi.nlm.nih.gov/protein/AAA29025.1. Accessed 17 June 2016.

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