Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Oct;22(20):7302-12.
doi: 10.1128/MCB.22.20.7302-7312.2002.

Association of class II histone deacetylases with heterochromatin protein 1: potential role for histone methylation in control of muscle differentiation

Chun Li Zhang  1 , Timothy A McKinsey, Eric N Olson
Affiliations

Association of class II histone deacetylases with heterochromatin protein 1: potential role for histone methylation in control of muscle differentiation

Chun Li Zhang et al. Mol Cell Biol. 2002 Oct.

Abstract

Class II histone deacetylases (HDACs) 4, 5, 7, and 9 repress muscle differentiation through associations with the myocyte enhancer factor 2 (MEF2) transcription factor. MEF2-interacting transcription repressor (MITR) is an amino-terminal splice variant of HDAC9 that also potently inhibits MEF2 transcriptional activity despite lacking a catalytic domain. Here we report that MITR, HDAC4, and HDAC5 associate with heterochromatin protein 1 (HP1), an adaptor protein that recognizes methylated lysines within histone tails and mediates transcriptional repression by recruiting histone methyltransferase. Promyogenic signals provided by calcium/calmodulin-dependent kinase (CaMK) disrupt the interaction of MITR and HDACs with HP1. Since the histone methyl-lysine residues recognized by HP1 also serve as substrates for deacetylation by HDACs, the interaction of MITR and HDACs with HP1 provides an efficient mechanism for silencing MEF2 target genes by coupling histone deacetylation and methylation. Indeed, nucleosomal histones surrounding a MEF2-binding site in the myogenin gene promoter are highly methylated in undifferentiated myoblasts, when the gene is silent, and become acetylated during muscle differentiation, when the myogenin gene is expressed at high levels. The ability of MEF2 to recruit a histone methyltransferase to target gene promoters via HP1-MITR and HP1-HDAC interactions and of CaMK signaling to disrupt these interactions provides an efficient mechanism for signal-dependent regulation of the epigenetic events controlling muscle differentiation.

PubMed Disclaimer

Figures

FIG.1.
FIG.1.
Association of HP1α with MITR and class II HDACs. (A) Interaction of HP1α and MITR in yeast. A mutant of MITR containing alanines in place of serines 218 and 448 (MITR S218/448A) was fused to the GAL4 DNA-binding domain and used as bait in a yeast two-hybrid screen (see Materials and Methods). A positive clone encoding full-length HP1α was rescued as a prey. The relative association of HP1α with the indicated MITR baits was determined by measuring the activity of a β-galactosidase reporter in yeast filter lift or liquid culture assays. Values from the liquid culture assay are expressed as activity relative to that seen with MITR S218/448A and HP1α. (B) Schematic diagrams of MITR and HDACs. The MEF2-binding region and nuclear localization signals (NLS) of MITR, HDAC4, and HDAC5 are indicated by the blue and green boxes, respectively. The red boxes indicate the HDAC catalytic domain. The number of amino acids in each protein is shown at the right. (C) GST pull-down assays. GST-HP1α (top panel) or GST alone (middle panel) was expressed in E. coli, conjugated to glutathione-agarose beads, and incubated with [35S]methionine-labeled MITR and HDACs, as described in Materials and Methods. Associated proteins were resolved by SDS-PAGE and analyzed by autoradiography (top and middle panels). In the bottom panel, 10% of the [35S]methionine-labeled protein was applied directly to the gel to control for input. (D) COS cells were cotransfected with expression vectors encoding Myc-tagged HP1α and the indicated FLAG-tagged proteins (1 μg each). FLAG-tagged proteins were immunoprecipitated from cell lysates with a monoclonal anti-FLAG antibody, and coimmunoprecipitating Myc-tagged proteins were detected by immunoblotting with a polyclonal anti-Myc antibody (upper panel). The positions of Myc-HP1α and the light chain of immunoglobulin (IgL) are indicated. The membrane was reprobed with anti-FLAG antibody to reveal total immunoprecipitated FLAG-tagged protein (bottom panel). (E) 293T cells were transiently transfected with expression vectors encoding Myc-tagged HP1α or GFP. At 48 h posttransfection, cell extracts were immunoprecipitated with anti-Myc antibody and immune complexes were assayed for HDAC activity as described in Materials and Methods. Values represent means ± standard deviations.
FIG.2.
FIG.2.
Mapping of the HP1α-binding region of MITR. (A) Association of GST-HP1α with amino- and carboxy-terminal MITR deletions. GST-HP1α (top panel) or GST alone (middle panel) was expressed in E. coli, conjugated to glutathione-agarose beads, and incubated with the indicated [35S]methionine-labeled MITR deletion mutants. Associated proteins were resolved by SDS-PAGE and analyzed by autoradiography (top and middle panels). The only MITR mutant that failed to interact with HP1α in this assay (mutant 1-300) is indicated with an asterisk. In the bottom panel, 20% of the [35S]methionine-labeled protein was applied directly to the gel to control for input. (B) Association of GST-HP1α with internal deletion mutants of MITR. The ability of GST-HP1α (top panel) to associate with the indicated MITR proteins was determined as described for panel A. In the bottom panel, 10% of the 35S-labeled MITR was applied directly to the gel to control for input. None of the MITR proteins exhibited significant binding to GST alone (data not shown). (C) COS cells were cotransfected with expression vectors encoding FLAG-tagged HP1α and the indicated Myc-tagged MITR proteins or CtBP (1 μg each). Myc-tagged proteins were immunoprecipitated from cell lysates with a polyclonal anti-Myc antibody, and coimmunoprecipitating FLAG-tagged proteins were detected by immunoblotting with a monoclonal anti-FLAG antibody (top panel). The positions of FLAG-HP1α and the light chain of immunoglobulin (IgL) are indicated. The membrane was reprobed with anti-Myc antibody to reveal the total amount of immunoprecipitated Myc-tagged protein (bottom panel). (D) Schematic representations of MITR proteins and their interactions with HP1α. There are two adjacent domains that are predicted to form α-helices (I and II); each is sufficient to mediate HP1α-binding. NLS, nuclear localization signal.
FIG. 3.
FIG. 3.
Mapping of the MITR-binding region of HP1α. (A) Binding of [35S]methionine-labeled MITR to GST-HP1α deletion mutants. HP1α and a series of HP1α deletion mutants, as shown in panel B, were fused to GST, expressed in E. coli, and conjugated to glutathione-agarose beads. [35S]methionine-labeled MITR was mixed with GST-HP1α-bound beads, and associated MITR was resolved by SDS-PAGE. Following electrophoresis, the GST-HP1 fusion proteins were stained with Coomassie blue dye (bottom panel), and then the amount of HP1-associated MITR was assessed by autoradiography (top panel). In lane 1 (top panel), 10% of the input [35S]methionine-labeled MITR was applied directly to the gel. Lane 2 shows only a low level of MITR associated with GST alone. (B) Schematic representations of full-length HP1α (FL) and mutants containing or lacking (Δ) the CD (green box), the CSD (blue box), or the hinge region (red box) of the protein. A summary of the GST pull-down data is shown to the right (+, association with MITR; −, no significant association with MITR). (C) Bacterially expressed GST-HP1β and GST-HP1γ were used in GST pull-down assays with 35S-labeled HDAC4, HDAC5, MITR, HDAC1, HDAC3, or CtBP as described for Fig. 1C. (D) COS cells were cotransfected with expression vectors encoding Myc-tagged MITR and FLAG-tagged HP1α, -β, or -γ. Ectopic HP1 proteins were immunoprecipitated from cell lysates with anti-FLAG antibodies, and associated MITR was detected with a polyclonal anti-Myc antibody (top panel). The membrane was reprobed with anti-FLAG antibody to reveal immunoprecipitated HP1 (bottom panel).
FIG. 4.
FIG. 4.
Association of MITR and class II HDACs with SUV39H1 and HP1α-mediated repression of MEF2C. (A) Coimmunoprecipitation of MITR and HDAC4 and -5 with SUV39H1. COS cells were cotransfected with expression vectors encoding HA-tagged SUV39H1 and the indicated FLAG-tagged proteins. FLAG-tagged proteins were immunoprecipitated from cell lysates with a monoclonal anti-FLAG antibody, and coimmunoprecipitating SUV39H1 was detected by immunoblotting with an anti-HA antibody (top panel). The membrane was reprobed with anti-FLAG antibody to reveal immunoprecipitated FLAG-tagged proteins (bottom panel). Arrows indicate the positions of full-length CtBP, HP1α, HDAC4, HDAC5, and MITR. (B) Association of HP1α and SUV39H1 with MITR by GST pull-down assays. Residues 389 to 506 of MITR were fused to GST and expressed in bacteria. The GST-MITR fusion protein was then conjugated to glutathione agarose beads and used in pull-down assays with 35S-labeled HP1α and SUV39H1. The associated proteins were resolved by SDS-PAGE and visualized using a phosphorimager. GST alone was used as negative control. Ten percent of the 35S-labeled protein was also directly applied to the gel to control for protein input. The positions of labeled HP1α and SUV39H1, which appears as a doublet, are indicated with arrows. (C) Inhibition of MEF2 transcriptional activity by HP1α. COS cells were transiently transfected with expression vectors for HP1α (0.1 to 0.8 μg), MEF2C (0.2 μg), MITR (5 ng), a MEF2-dependent reporter plasmid (3XMEF2-luciferase; 0.1 μg), and a CMV-lacZ reporter (0.1 μg) to control for differences in transfection efficiency. Luciferase activity was determined as described in Materials and Methods. Values represent means ± standard deviations.
FIG. 5.
FIG. 5.
Effects of promyogenic signals on histone H3 methylation and MITR or HDAC-HP1 complexes. (A) Methylation and acetylation states of histone H3 lysine 9 on a MEF2-binding element during myogenesis. Soluble chromatin from C2 myoblasts in growth medium (GM) or from confluent myotubes in differentiation medium (DM) was subjected to immunoprecipitation with antibodies specific for dimethylated lysine 9 of histone H3 [α-MeH3(K9)] or acetylated lysine 9 and 14 of histone H3 [α-AcH3(K9/K14)]. Precipitated DNA was used as a template for PCR with primers spanning either the MEF2-binding site on the myogenin gene promoter or the transcription start site of the GAPDH gene promoter. The positions of the primer-binding sites (numbers) relative to the transcription start sites (arrows) are indicated. The position of the MEF2-binding element within the myogenin promoter is shown. PCR was also performed using genomic DNA prior to immunoprecipitation to control for equal input of chromatin (bottom panel). (B) CaMK-mediated dissociation of HP1α from MITR and HDAC5. COS cells were cotransfected with expression vectors encoding FLAG-tagged HP1α and Myc-tagged HDAC5 or Myc-tagged versions of wild-type MITR (MITR) or a mutant of MITR containing alanines in place of serines 218 and 448 (MITR S218/448A) in the absence or presence of an expression vector encoding activated CaMKI. Myc-tagged proteins were immunoprecipitated from cell lysates with a polyclonal anti-Myc antibody, and coimmunoprecipitating FLAG-tagged HP1α was detected by immunoblotting with a monoclonal anti-FLAG antibody (top panel). The membrane was reprobed with anti-Myc antibody to reveal immunoprecipitated HDAC5 and MITR (bottom panel).
FIG. 6.
FIG. 6.
Regulation of gene expression by HDAC and MITR-HP1 interactions. (A) Acetylation (Ac) of lysine 9 (K9) on nucleosomal histone H3 by HATs relaxes chromatin structure, resulting in gene activation. In contrast, removal of the acetyl group from K9 by HDACs results in chromatin condensation and gene repression. Deacetylated K9 is a substrate for the HMTase SUV39H1. Methylation of K9 by SUV39H1 further enhances the repressive state of chromatin. (B) Schematic diagram of human HDAC5 showing functional domains and binding regions for cofactors. These functional domains and binding sites are conserved in HDAC4, HDAC7, HDAC9, and MITR, although MITR lacks an HDAC catalytic domain and a nuclear export sequence (NES). NLS, nuclear localization signal.(C) Model for the assembly of a corepressor complex by a class II HDAC or MITR and its disassembly by CaMK signaling. HDAC or MITR, HP1 and the HMTase SUV39H1 form an efficient corepressor complex. Deacetylation of histone tails by HDAC creates substrates for SUV39H1, which is recruited to the target site by the HDAC- or MITR-HP1 complex. Methylation of histone tails by SUV39H1 generates a binding signature for HP1, which further recruits HDACs and SUV39H1 to propagate the repressive effect on the target gene. CaMK phosphorylates class II HDACs and MITR, which results in disruption of MEF2-HDAC or MEF2-MITR interactions and allows p300, which possesses HAT activity, to associate with MEF2 and acetylate regional histones. These events lead to the activation of MEF2 target gene expression. CaMK also dissociates HP1-HDAC and HP1-MITR complexes through an unknown mechanism, providing an efficient means to control histone methylation in response to extracellular cues.
See this image and copyright information in PMC

References

    1. Aagaard, L., G. Laible, P. Selenko, M. Schmid, R. Dorn, G. Schotta, S. Kuhfittig, A. Wolf, A. Lebersorger, P. B. Singh, G. Reuter, and T. Jenuwein. 1999. Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31. EMBO J. 18:1923-1938. - PMC - PubMed
    1. Ainsztein, A. M., S. E. Kandels-Lewis, A. M. Mackay, and W. C. Earnshaw. 1998. INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1. J. Cell Biol. 143:1763-1774. - PMC - PubMed
    1. Andres, V., and K. Walsh. 1996. Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. J. Cell Biol. 132:657-666. - PMC - PubMed
    1. Bannister, A. J., P. Zegerman, J. F. Partridge, E. A. Miska, J. O. Thomas, R. C. Allshire, and T. Kouzarides. 2001. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410:120-124. - PubMed
    1. Czermin, B., G. Schotta, B. B. Hulsmann, A. Brehm, P. B. Becker, G. Reuter, and A. Imhof. 2001. Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila. EMBO Rep. 2:915-919. - PMC - PubMed

Publication types

MeSH terms