The MTA family proteins as novel histone H3 binding proteins

Abstract

Background: The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined.

Results: In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells.

Conclusions: Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail.

Figure 1

Unique binding of H3 tail peptide by NURD but not other class I HDAC complexes. A. Coomassie blue staining revealed the H3 tail peptide-binding proteins isolated from HeLa nuclear extracts. The identities of the protein bands were determined by mass spectrometry. ‘-‘, the beads only control. B. Among several class I HDAC complexes only NURD exhibited a substantial H3 tail-binding activity in vitro. The HeLa nuclear extracts were incubated with various immobilized histone tails in vitro and the binding of various class I HDAC complexes was examined by western blot analysis using antibodies against complex-specific subunits. Sin3A, the unique subunit of the Sin3A/HDAC1/2 complex; CoREST, the unique subunit of CoREST complex; MTA1/2, the unique subunits of NURD; NCoR, the unique subunit of NCoR/SMRT/HDAC3 complex. C. The binding specificity of NURD complexes toward histone H3 tail peptides. The HeLa nuclear extracts were incubated with the immobilized histone tail peptides as indicated and the binding of NURD complexes was examined by subsequent western blot analysis using antibodies against CHD3, CHD4, MTA1 and HDAC1. PHF8 served as a positive control for H3K4me2/3-binding proteins.

Figure 2

The MTA family subunits bind directly the H3 tail peptide. A. the H3 tail peptide-binding specificity of individual subunits of NURD complex. Each subunit of NURD complex was synthesized and ³⁵S-met labeled via in vitro coupled transcription and translation. The proteins were then subjected to in vitro pulldown with immobilized H3 tail peptides as indicated. The binding was visualized by autoradiography. Note that the MTA1 family proteins, CHD3 and RbAp48 bound H3 tail in a H3K4me3-sensitive manner, whereas p66/68 and HDAC1 were not. B. UV-induced cross-linking demonstrated a direct association of MTA family proteins with H3 tail peptide. The H3(Bpa) peptide contained at K9 position a Bpa moiety that mediated a UV-induced cross-linking with associated protein(s) in close proximity. Note that once cross-linked to the H3(Bpa) peptide, the associated protein(s) became resistant to wash with 0.2% SDS buffer.

Figure 3

The H3BD of MTA1 and MTA2 mediates the in vitro binding of H3 tail peptide by NURD. A. Mammalian expressed MTA1(1–460) but not MTA1(454–715) was incorporated into the core HDAC1/2 and RbAp46/48 containing complex. The MTA1 and its N- and C-terminal regions were expressed in 293T cells and assayed for association with other subunits of NURD complex by IP-western analysis. Note that the N-terminal but not the C-terminal region of MTA1 associated with HDAC1/2 and RbAp48 and that no association with CHD4 was detected for MTA1. Input, 10%. B. The MTA1 C-terminal region but not the N-terminal region that was incorporated into the HDAC1/2-RbAp46/48 core complex bound the H3 tail peptide in in vitro pulldown assay. The samples in A were subjected to in vitro pulldown assay. Note that no binding was detected for Flag-MTA1(1–460), whereas Flag-MTA1(454–715) bound the H3 tail peptide as efficient as Flag-MTA1. Input, 10%. C. The C-terminal region of MTA1 binds H3 tail peptide independent of HDAC1/2 and RbAp48. The 293T-expressed, Flag-tagged MTA1, MTA1(1–460) and MTA1(454–715) were purified as illustrated in the top panel and then subjected to in vitro pulldown assay for binding of H3 tail peptide. D. Mammalian expressed MTA2(1–434) but not MTA2(427–668) was incorporated into the endogenous NURD complex. The MTA2 and its N- and C-terminal regions were expressed in 293T cells and assayed for association with other subunits of NURD complex by IP-western analysis. Note that the N-terminal but not the C-terminal region of MTA2 associated with HDAC1/2 and CHD4. Input, 10%. E. The C-terminal region but not the N-terminal region that was incorporated into the endogenous NURD complex bound the H3 tail peptide in in vitro pulldown assay. The samples in D were subjected to in vitro pulldown assay. Input, 10%.

Figure 4

The H3 -binding domain (H3BD) resides within the C-terminal regions of MTA proteins. A. The binding of various MTA1 deletion mutants to H3 tail peptide in vitro. The left panel illustrates the primary structures of MTA1 and various mutants. Also indicated are four structural motifs in the N-terminal regions of MTA proteins, namely BAH, ELM2, SANT and ZnF and the C-terminal H3BD determined here. The proteins were synthesized and ³⁵S-met labeled and subjected to in vitro pulldown assay with and without the immobilized H3 peptide. B. The C-terminal region of MTA1 exhibits the same H3 peptide-binding specificity as the full-length MTA1. The experiment was performed as in B except additional immobilized H3 peptides were included. C. The recombinant MTA1 and MTA2 C-terminal regions are sufficient for binding H3 tail peptide. The 6xHis-tagged MTA1(454–715) and 6xHis-MTA2(427–668) were expressed and purified from E.coli and subjected to in vitro pulldown assay with immobilized H3 tail peptide. D. The H3-binding activity is also mapped to the C-terminal region of MTA2. The MTA2 and its N- and C- terminal regions were expressed as ³⁵S-Met labeled proteins and subjected to in vitro pulldown assay with immobilized H3 tail peptide.

Figure 5

The H3-MTA interaction independent mechanism exists for NURD chromatin association. A. Pulldown assay using GFP-tagged MTA1 proteins confirmed the MTA1 C-terminal H3 binding activity. Note that a very weak H3 binding activity was detected for N-terminal MTA1 with intensive exposure. B. Both the N-terminal and C-terminal GFP-MTA1 were chromatin associated in 293T cells. The 293T cells were transfected with GFP-MTA1, GFP-MTA1(1–460) and GFP-MTA1(454–715) and the resulting cells were subjected to cellular fractionation of soluble cellular fraction and insoluble chromatin fraction as described in Materials and Methods. The fractions were further extracted with buffer containing different concentration of NaCl as indicated to generate different fractions. Note that while DNMT1 was gradually stripped from chromatin by an increasing salt concentration, all three MTA1 proteins essentially remained to be associated with chromatin. C. Fractionation of soluble chromatin fragments derived from MNase digestion by a 5–30% sucrose gradient centrifugation. The soluble chromatin was prepared from the 293T cells expressing both GFP-MTA1(1–460) and GFP-MTA1(454–715). After sucrose gradient centrifugation, consecutive fractions (300 μl each) were collected from top. Half of the samples were used for preparation of DNA and analyzed by agarose gel electrophoresis. D. The remaining half of the sucrose gradient fractionation samples was subjected to protein precipitation by TCA and resolved by SDS-PAGE. After transfer to nitrocellulose membrane, the proteins were revealed by Ponceau S staining (top panel) and then analyzed by western blot. Note that the majority of GFP-MTA1(1–460) and GFP-MTA1(454–715) were present in the chromatin fractions.