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A feed-forward repression mechanism anchors the Sin3/histone deacetylase and N-CoR/SMRT corepressors on chromatin.
Vermeulen M
,
Walter W
,
Le Guezennec X
,
Kim J
,
Edayathumangalam RS
,
Lasonder E
,
Luger K
,
Roeder RG
,
Logie C
,
Berger SL
,
Stunnenberg HG
.
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Transcription in eukaryotes is governed in part by histone acetyltransferase (HAT)- and histone deacetylase (HDAC)-containing complexes that are recruited via activators and repressors, respectively. Here, we show that the Sin3/HDAC and N-CoR/SMRT corepressor complexes repress transcription from histone H3- and/or H4-acetylated nucleosomal templates in vitro. Repression of histone H3-acetylated templates was completely dependent on the histone deacetylase activity of the corepressor complexes, whereas this activity was not required to repress H4-acetylated templates. Following deacetylation, both complexes become stably anchored in a repressor-independent manner to nucleosomal templates containing hypoacetylated histone H3, but not H4, resulting in dominance of repression over activation. The observed stable anchoring of corepressor complexes casts doubt on the view of a dynamic balance between readily exchangeable HAT and HDAC activities regulating transcription and implies that pathways need to be in place to actively remove HDAC complexes from hypoacetylated promoters to switch on silent genes.
FIG. 1. (A) Schematic representation of the transcription template used for all the experiments in this study. The primer pairs used for the in vitro ChIPs that were performed in this study are also indicated with arrows. (B) Gal4-VP16-dependent in vitro transcription reactions from nucleosomal templates under conditions favoring either HAT or HDAC activity as indicated. prom., promoter; NE, nuclear extract; rNTPs, ribonucleoside triphosphates; AcCoA, acetyl-CoA; but., butyrate; TXN, transcription.
FIG. 2. In vitro transcriptional repression by the Sin3/HDAC complex. (A) Timeline describing the order of events of the experiment. (C) Following recruitment of the Sin3/HDAC complex to histone H3- or H4-acetylated templates in the presence of TSA, templates were washed and subsequently incubated with Gal4-VP16. Templates were washed again, and the amount of Gal4-VP16 retained on the templates was determined by Western blotting using a Gal4 antibody. As a loading control, the blot was probed with an antibody against the globular part of histone H3. (B) Nucleosomal templates reconstituted with recombinant Xenopus octamers were acetylated (Ac) on histone H3 (lanes 1 to 4), histone H4 (lanes 5 to 8), or histones H3 and H4 (lanes 9 to 12), incubated with LexA-Mad or a LexA-Mad mutant, and subsequently incubated in a large excess of competitor oligonucleosomes in the presence or absence of Sin3/HDAC complex and TSA. Templates were then washed and incubated with Gal4-VP16 and HeLa nuclear extract (NE) for 20 min to allow the formation of preinitiation complexes. Ribonucleotides were then added, and templates were transcribed for 20 min. The amount of transcription was visualized by primer extension analysis.
FIG. 3. In vitro transcriptional repression by the N-CoR/SMRT complex. (A) Timeline describing the order of events of the experiment. (B) Nucleosomal templates were acetylated (Ac) and washed as described for Fig. 2 and subsequently incubated with or without LexA-TR(DE) in the presence or absence of 5 μM T3. Templates were then washed and incubated in a large excess of competitor oligonucleosomes in the presence or absence of the N-CoR/SMRT complex, TSA, and T3. After another washing step, transcription reactions were performed as described for Fig. 2. rNTPs, ribonucleoside triphosphates.
FIG. 4. Results of in vitro ChIP to assess nucleosomal histone H3 deacetylation by the Sin3/HDAC (A) and N-CoR/SMRT (B) complexes. Corepressor recruitment reactions were performed as described for Fig. 2 and 3, but instead of proceeding with the transcription reactions, templates were cross-linked, digested with a cocktail of restriction enzymes, and subjected to immunoprecipitation using an antibody that recognizes diacetyl histone H3. Real-time PCR analyses were performed using promoter (Prom) and upstream region (Up) primer sets. Error bars represent standard deviations between at least three independent experiments. Ac, acetylated.
FIG. 5. Results of in vitro ChIP to assess promoter occupancy by the Sin3/HDAC and N-CoR/SMRT complexes after recruitment by LexA-Mad and LexA-TR(DE), respectively. Following recruitment reactions, templates were subjected to immunoprecipatation using antibodies specific for the Sin3/HDAC complex (HDAC2) (A) or for the N-CoR/SMRT complex (HDAC3) (B). Error bars represent standard deviations between at least three independent experiments. Up, upstream region. Prom, promoter.
FIG. 6. Results of LexA binding site competition experiments to study promoter anchoring of the Sin3/HDAC and N-CoR/SMRT complexes. (A) Timeline describing the order of events of the experiment. After recruitment of the Sin3/HDAC complex (B) or the N-CoR/SMRT complex (C) to the E4 promoter, templates were washed (200 mM KCl, 0.25% Triton X-100) and then incubated with control DNA (gray bars) or DNA containing LexA binding sites (black bars). Templates were then cross-linked, digested with restriction enzymes, and subsequently subjected to ChIP using an antibody against HDAC2 (B) or HDAC3 (C). Real-time PCR analyses were then performed to assess promoter occupancy of the Sin3/HDAC and N-CoR/SMRT complexes following LexA binding site competition. Error bars represent standard deviations between at least three independent experiments. (D) Schematic representation of the results obtained in the LexA binding site competition experiments. Ac, acetylated.
FIG. 7. Results of LexA binding site competition experiments with octamers lacking the H3 or H4 N terminus and histone peptide pulldowns in nuclear extracts. After recruitment of the Sin3/HDAC (HDAC2 ChIPs) and N-CoR (HDAC3 ChIPs) complexes to templates lacking the H4 (A) or H3 (B) N terminus, binding site competition experiments and ChIP analyses were performed as described for Fig. 6. Error bars represent standard deviations between at least three independent experiments. (C) H3 and H4 histone peptide tail pulldowns in HeLa nuclear extract were performed, and silver staining (left) as well as Western blotting (right) against Sin3a, HDAC1, and HDAC2 was performed to determine binding of these corepressors to the unmodified histone H3 and histone H4 N termini.
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