In vitro targeting reveals intrinsic histone tail specificity of the Sin3/histone deacetylase and N-CoR/SMRT corepressor complexes

Abstract

The histone code is among others established via differential acetylation catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). To unambiguously determine the histone tail specificity of HDAC-containing complexes, we have established an in vitro system consisting of nucleosomal templates reconstituted with hyperacetylated histones or recombinant histones followed by acetylation with native SAGA or NuA4. Selective targeting of the mammalian Sin3/HDAC and N-CoR/SMRT corepressor complexes by using specific chimeric repressors created a near physiological setting to assess their histone tail specificity. Recruitment of the Sin3/HDAC complex to nucleosomal templates preacetylated with SAGA or NuA4 resulted in deacetylation of histones H3 and H4, whereas recruitment of N-CoR/SMRT resulted in deacetylation of histone H3 only. These results provide solid evidence that HDAC-containing complexes display distinct, intrinsic histone tail specificities and hence may function differently to regulate chromatin structure and transcription.

FIG.1.

Purification of the Sin3/HDAC and N-CoR/SMRT complexes and recruitment to DNA by using chimeric repressor molecules. (A) Conventional purification of the Sin3/HDAC and N-CoR/SMRT complexes. HeLa cell nuclear extract was fractionated as shown. The Sin3/HDAC and N-CoR/SMRT complexes were monitored throughout the purification by Western analysis with antibodies against HDAC2, mSin3a, SAP30, N-CoR, and HDAC3. (B) Elution profile of the Sin3/HDAC complex on a Superose 6 column analyzed by Western blotting against Sin3a, HDAC2, and SAP30. (C) As in panel B, using the N-CoR/SMRT fractions and antibodies against N-CoR, HDAC2, HDAC3, and Sin3a. (D) Recruitment of the Sin3/HDAC and N-CoR/SMRT complexes by the LexA-Mad and LexA-TR(DE) fusion proteins, respectively. Fractions 9 to 13 (Fig. 1B) and 7 to 11 (Fig. 1C) from the Superose 6 columns containing the Sin3/HDAC and N-CoR/SMRT complexes were used for the LexA-Mad and LexA-TR(DE) pull-down on paramagnetic streptavidin-conjugated Dynal Dynabeads containing multimerized LexA oligonucleotides. Bound proteins were eluted from DNA with 1 M NaCl, loaded on a sodium dodecyl sulfate-polyacrylamide (8%) gel, and analyzed by Western blotting with the indicated antibodies. Input fractions used for the pull-down are also shown.

FIG. 2.

Analysis of the affinity-purified Sin3/HDAC complex by silver stain and MS. (A) Recruitment of the Sin3/HDAC complex by the LexA-Mad fusion protein. Fractions 9 to 13 (Fig. 1B) from the Superose 6 column containing the Sin3/HDAC complex were used for the LexA-Mad pull-down on paramagnetic streptavidin-conjugated Dynal Dynabeads containing multimerized LexA oligonucleotides. Bound proteins were eluted from DNA with 1 M NaCl, loaded onto an SDS-polyacrylamide (8%) gel, and analyzed by silver staining. (B) A pull-down similar to that described for panel A was performed. After elution of the recruited proteins with 1 M NaCl, they were analyzed by nLC-MS/MS.

FIG. 3.

Reconstitution and analysis of the nucleosomal template. (A) Schematic representation of the DNA template containing eight LexA binding sites and a 5S nucleosome positioning element. (B) Analysis of purified recombinant (Rec.) Xenopus octamers and hyperacetylated (Hyperac.) core histones purified from HeLa cells on SDS-polyacrylamide (15%) gel electrophoresis gel stained with Coomassie brilliant blue. (C) Partial micrococcal nuclease digestion. Nucleosomal templates were incubated with 10 mU micrococcal nuclease at 37°C for 0, 20, 40, 60, and 180 s. Reactions were stopped by adding 10 mM EGTA. DNA was phenol chloroform extracted, precipitated, and loaded onto a 1.5% agarose gel. DNA size markers are indicated on the left. An arrow indicates mononucleosomal DNA.

FIG. 4.

Deacetylase activity of the Sin3/HDAC complex. (A) Nucleosomal templates reconstituted with hyperacetylated (Hyperac.) histones were incubated with the Sin3/HDAC complex in the absence or presence of TSA. The amount of H3 deacetylation was determined by Western blotting with an antibody that recognizes diacetylated histone H3 Lys 9,14. (B) Nucleosomal templates reconstituted with recombinant (Rec.) Xenopus octamers were incubated with the S. cerevisiae SAGA complex, washed, and subsequently incubated with Sin3/HDAC complex in the presence or absence of TSA, after which the amount of H3 acetylation (Ac) was determined as described for panel A.

FIG. 5.

The Sin3/HDAC complex can deacetylate histones H3 and H4 upon targeting to a nucleosomal template. Nucleosomal templates reconstituted with recombinant Xenopus octamers were incubated with the S. cerevisiae SAGA complex (A and C) or NuA4 complex (B), washed, incubated with or without LexA-Mad (A and B) or a LexA-Mad mutant (C), and subsequently incubated with the Sin3/HDAC complex in the presence or absence of competitor oligonucleosomes, in the presence or absence of TSA. The amount of H4 acetylation (Ac) was determined by Western blotting with an antibody that recognizes tetra-acetylated H4.

FIG. 6.

The N-CoR/SMRT complex deacetylates histone H3 upon targeting to a nucleosomal template. Nucleosomal templates reconstituted with recombinant Xenopus octamers were incubated with the S. cerevisiae SAGA complex (A and C) or NuA4 complex (B), washed, incubated with or without LexA-TR(DE) (A and B) or LexA-TR(DE) in the presence of 5 μM T3 (C), and subsequently incubated with the N-CoR/SMRT complex in the presence or absence of competitor oligonucleosomes, in the presence or absence of TSA. Ac, acetylation.