Comparative in silico analysis identifies bona fide MyoD binding sites within the Myocyte Stress 1 gene promoter

<p>Abstract</p> <p>Background</p> <p>Myocyte stress 1 (MS1) is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathway. To date, little is known about the molecular mechanisms that govern skeletal muscle specific expression of MS1. Such mechanisms are likely to play a major role in modulating SRF activity and therefore muscle determination, differentiation and regeneration. In this study we employed a comparative <it>in silico </it>analysis coupled with an experimental promoter characterisation to delineate these mechanisms.</p> <p>Results</p> <p>Analysis of MS1 expression in differentiating C2C12 muscle cells demonstrated a temporal differentiation dependent up-regulation in <it>ms1 </it>mRNA. An <it>in silico </it>comparative sequence analysis identified two conserved putative myogenic regulatory domains within the proximal 1.5 kbp of 5' upstream sequence. Co-transfecting C2C12 myoblasts with <it>ms1 </it>promoter/luciferase reporters and myogenic regulatory factor (MRF) over-expression plasmids revealed specific sensitivity of the <it>ms1 </it>promoter to MyoD. Subsequent mutagenesis and EMSA analysis demonstrated specific targeting of MyoD at two distinct E-Boxes (E1 and E2) within identified evolutionary conserved regions (ECRs, α and β). Chromatin immunoprecipitation (ChIP) analysis indicates that co-ordinated binding of MyoD at E-Boxes located within ECRs α and β correlates with the temporal induction in <it>ms1 </it>mRNA.</p> <p>Conclusion</p> <p>These findings suggest that the tissue specific and differentiation dependent up-regulation in <it>ms1 </it>mRNA is mediated by temporal binding of MyoD at distinct evolutionary conserved E-Boxes within the <it>ms1 </it>5' upstream sequence. We believe, through its activation of <it>ms1</it>, this is the first study to demonstrate a direct link between MyoD activity and SRF transcriptional signalling, with clear implications for the understanding of muscle determination, differentiation and regeneration.</p

Phosphorylation of linker histones regulates ATP-dependent chromatin remodeling enzymes.

Item does not contain fulltextMembers of the ATP-dependent family of chromatin remodeling enzymes play key roles in the regulation of transcription, development, DNA repair and cell cycle control. We find that the remodeling activities of the ySWI/SNF, hSWI/SNF, xMi-2 and xACF complexes are nearly abolished by incorporation of linker histones into nucleosomal array substrates. Much of this inhibition is independent of linker histone-induced folding of the arrays. We also find that phosphorylation of the linker histone by Cdc2/Cyclin B kinase can rescue remodeling by ySWI/SNF. These results suggest that linker histones exert a global, genome-wide control over remodeling activities, implicating a new, obligatory coupling between linker histone kinases and ATP-dependent remodeling enzymes

Functional delineation of three groups of the ATP-dependent family of chromatin remodeling enzymes.

Contains fulltext : 129327.pdf (Publisher’s version ) (Open Access)ATP-dependent chromatin remodeling enzymes antagonize the inhibitory effects of chromatin. We compare six different remodeling complexes: ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, and dNURF. We find that each complex uses similar amounts of ATP to remodel nucleosomal arrays at nearly identical rates. We also perform assays with arrays reconstituted with hyperacetylated or trypsinized histones and isolated histone (H3/H4)(2) tetramers. The results define three groups of the ATP-dependent family of remodeling enzymes. In addition we investigate the ability of an acidic activator to recruit remodeling complexes to nucleosomal arrays. We propose that ATP-dependent chromatin remodeling enzymes share a common reaction mechanism and that a key distinction between complexes is in their mode of regulation or recruitment