CLP-1 associates with MyoD and HDAC to restore skeletal muscle cell regeneration.
ABSTRACT Emerging evidence suggests that eukaryotic gene transcription is regulated primarily at the elongation stage by association and dissociation of the inhibitory protein cardiac lineage protein 1 (CLP-1/HEXIM1) from the positive transcription elongation factor b (P-TEFb) complex. It was reported recently that P-TEFb interacts with skeletal muscle-specific regulatory factor, MyoD, suggesting a linkage between CLP-1-mediated control of transcription and skeletal myogenesis. To examine this, we produced CLP-1 knockdown skeletal muscle C2C12 cells by homologous recombination, and demonstrated that the C2C12 CLP-1 +/- cells failed to differentiate when challenged by low serum in the medium. We also showed that CLP-1 interacts with both MyoD and histone deacetylases (HDACs) maximally at the early stage of differentiation of C2C12 cells. This led us to hypothesize that the association might be crucial to inhibition of MyoD-target proliferative genes. Chromatin immunoprecipitation analysis revealed that the CLP-1/MyoD/HDAC complex binds to the promoter of the cyclin D1 gene, which is downregulated in differentiated muscle cells. These findings suggest a novel transcriptional paradigm whereby CLP-1, in conjunction with MyoD and HDAC, acts to inhibit growth-related gene expression, a requirement for myoblasts to exit the cell cycle and transit to myotubes.
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ABSTRACT: Many common causes of blindness involve the death of retinal photoreceptors followed by progressive inner retinal cell remodelling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine tri-phosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterise the chronic effects of ATP on retinal integrity. Five week old, dark agouti rats were administered 50mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within one day and loss of 50% of the photoreceptors within 1 week. At three months, 80% of photoreceptor nuclei were lost, while total photoreceptor loss occurred by six months. The degeneration and remodelling was similar to that found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration and formation of new synapses; Müller cell gliosis, migration and scarring; blood vessel loss and; retinal pigment epithelium migration. In addition, extreme degeneration and remodelling events such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells were observed. These extreme changes were also observed in the two year old P23H Rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for the development of pharmaceutical therapies or for the testing of electronic implants aimed at restoring vision. J. Comp. Neurol., 2014. © 2014 Wiley Periodicals, Inc.The Journal of Comparative Neurology 02/2014; 522(13). · 3.51 Impact Factor
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ABSTRACT: Hypoxia is associated with a variety of physiological and pathological conditions and elicits specific transcriptional responses. The elongation competence of RNA Polymerase II is regulated by the positive transcription elongation factor b (P-TEFb)-dependent phosphorylation of Ser2 residues on its C-terminal domain. Here, we report that hypoxia inhibits transcription at the level of elongation. The mechanism involves enhanced formation of inactive complex of P-TEFb with its inhibitor HEXIM1 in an HDAC3-dependent manner. Microarray transcriptome profiling of hypoxia primary response genes identified ∼79% of these genes being HEXIM1-dependent. Hypoxic repression of P-TEFb was associated with reduced acetylation of its Cdk9 and Cyclin T1 subunits. Hypoxia caused nuclear translocation and co-localization of the Cdk9 and HDAC3/N-CoR repressor complex. We demonstrated that the described mechanism is involved in hypoxic repression of the monocyte chemoattractant protein-1 (MCP-1) gene. Thus, HEXIM1 and HDAC-dependent deacetylation of Cdk9 and Cyclin T1 in response to hypoxia signalling alters the P-TEFb functional equilibrium, resulting in repression of transcription.Nucleic Acids Research 07/2014; 42(14). · 8.81 Impact Factor
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ABSTRACT: PCAF (KAT2B) belongs to the GNAT family of lysine acetyltransferases (KAT) and specifically acetylates the histone H3K9 residue and several nonhistone proteins. PCAF is also a transcriptional coactivator. Due to the lack of a PCAF KAT-specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here, we report that a natural compound of the hydroxybenzoquinone class, embelin, specifically inhibits H3Lys9 acetylation in mice and inhibits recombinant PCAF-mediated acetylation with near complete specificity in vitro. Furthermore, using embelin, we have identified the gene networks that are regulated by PCAF during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation in addition to the regulation via MyoD acetylation.ACS Chemical Biology 05/2013; 8(6):1311–1323. · 5.36 Impact Factor