Post-translational modifications of histones H3 and H4 associated with the histone methyltransferases Suv39h1 and G9a

Mass spectrometry analysis of the post-transcriptional modifications of histones H3 and H4 that were co-purified with histone methyltransferases Suv39h1 and G9a shows that, in HeLa cells, histone methyltransferases can be physically associated with acetylated histones, which normally mark transcriptionally active chromatin

The Core Binding Factor CBF Negatively Regulates Skeletal Muscle Terminal Differentiation

BACKGROUND: Core Binding Factor or CBF is a transcription factor composed of two subunits, Runx1/AML-1 and CBF beta or CBFbeta. CBF was originally described as a regulator of hematopoiesis. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that CBF is involved in the control of skeletal muscle terminal differentiation. Indeed, downregulation of either Runx1 or CBFbeta protein level accelerates cell cycle exit and muscle terminal differentiation. Conversely, overexpression of CBFbeta in myoblasts slows terminal differentiation. CBF interacts directly with the master myogenic transcription factor MyoD, preferentially in proliferating myoblasts, via Runx1 subunit. In addition, we show a preferential recruitment of Runx1 protein to MyoD target genes in proliferating myoblasts. The MyoD/CBF complex contains several chromatin modifying enzymes that inhibits MyoD activity, such as HDACs, Suv39h1 and HP1beta. When overexpressed, CBFbeta induced an inhibition of activating histone modification marks concomitant with an increase in repressive modifications at MyoD target promoters. CONCLUSIONS/SIGNIFICANCE: Taken together, our data show a new role for Runx1/CBFbeta in the control of the proliferation/differentiation in skeletal myoblasts

Coopération entre le facteur de transcription CBF (Runx1-CBFß) et le facteur myogénique MyoD dans la régulation de l'équilibre prolifération-différenciation musculaire

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

The SWI/SNF Subunit/Tumor Suppressor BAF47/INI1 Is Essential in Cell Cycle Arrest upon Skeletal Muscle Terminal Differentiation

<div><p>Myogenic terminal differentiation is a well-orchestrated process starting with permanent cell cycle exit followed by muscle-specific genetic program activation. Individual SWI/SNF components have been involved in muscle differentiation. Here, we show that the master myogenic differentiation factor MyoD interacts with more than one SWI/SNF subunit, including the catalytic subunit BRG1, BAF53a and the tumor suppressor BAF47/INI1. Downregulation of each of these SWI/SNF subunits inhibits skeletal muscle terminal differentiation but, interestingly, at different differentiation steps and extents. BAF53a downregulation inhibits myotube formation but not the expression of early muscle-specific genes. BRG1 or BAF47 downregulation disrupt both proliferation and differentiation genetic programs expression. Interestingly, BRG1 and BAF47 are part of the SWI/SNF remodeling complex as well as the N-CoR-1 repressor complex in proliferating myoblasts. However, our data show that, upon myogenic differentiation, BAF47 shifts in favor of N-CoR-1 complex. Finally, BRG1 and BAF47 are well-known tumor suppressors but, strikingly, only BAF47 seems essential in the myoblasts irreversible cell cycle exit. Together, our data unravel differential roles for SWI/SNF subunits in muscle differentiation, with BAF47 playing a dual role both in the permanent cell cycle exit and in the regulation of muscle-specific genes.</p></div

Downregulation of BAF47 alters muscle terminal differentiation and cell cycle exit.

<p><b>A.</b> BAF47 and BRG1 occupancy at <i>cyclin D1</i> promoter. ChIP-qPCR analyses of BAF47 and BRG1 in myoblast in proliferating C2C12 cells and at 24 h of differentiation. The immunoprecipitated material was quantified by qPCR, and results are expressed as fold enrichment of the % of Input of BAF47 or BRG1 ChIP over % of Input of the IgG average. Data are represented as mean ±SEM, n = 3. <b>B.</b> Scheme of the timing for samples collection. C2C12 myoblasts were transfected with control, BRG1, BAF53a or BAF47 siRNAs and samples prepared either 24 h after transfection (0) or after 72 h in DM (72) or 10 h after the switch back to GM (72+10 h GM). <b>C.</b> Total protein extracts were analyzed by WB with the indicated antibodies. <b>D.</b> Quantification of cyD1 levels from 3 to 8 independent WB. Data are expressed compared to control 0 h. Error bars represent SEM. For each time point, statistics were calculated compare to the same time point from control. Only significative p-values (Student T-test, two tailed, unpaired) are indicated ** = <0.01. <b>E</b>. Total RNA was isolated, reverse transcribed using random primers and used as templates for PCR amplification with cyclin D1-specific primers and normalized to cyclophilin A specific primers. Data are expressed compared to control 0 h.</p

BRG1 and BAF47 interact with SWI/SNF and N-CoR-1 complex components in a differentiation-dependent manner.

<p><b>A.</b> Nuclear extracts from proliferating (GM) or differentiating C2C12 myoblasts (24 h DM) were used for immunoprecipitation (IP) with antibodies against BRG1 or BAF47, or with normal rabbit IgG as a negative control. The resulting precipitates were analyzed by WB with the indicated antibodies. Nuclear extracts (Inputs, 1% of input extracts) were loaded to assess endogenous protein levels. *: non-specific IgG band. <b>B.</b> Nuclear extracts from proliferating (GM) or differentiating C2C12 myoblasts (24 h DM) were fractionated on glycerol gradient ranging from 11% (fraction 1) to 33% (fraction 11); (-) empty lane. Fractions were collected and analyzed by WB using the indicated antibodies. Nuclear extracts (Inputs, 2.5% of input extracts) were loaded to assess endogenous protein levels.</p