Differential binding modes of the bromodomains of CREB-binding protein (CBP) and p300 with acetylated MyoD

We investigated whether blocking of monocyte chemoattractant-1 (MCP-1) function would inhibit recruitment of tumor-associated macrophages (TAMs) and prevent tumor angiogenesis and tumor growth of human malignant melanoma. B16-F1 melanoma cells were implanted onto the back of C57BL/6 mice (Day 0). At Day 7, a dominant negative MCP-1 mutant (7ND) gene was transfected in the thigh muscle to make overexpressed 7ND protein secreted into systemic circulation. 7ND treatment inhibited TAM recruitment and partially reduced tumor angiogenesis and tumor growth. Also, 7ND treatment attenuated inductions of tumor necrosis factor-α (TNFα), interleukin-1α (IL-1α), and vascular endothelial growth factor (VEGF) in the stroma and tumor. Melanoma cells expressed not only MCP-1 but also its receptor CCR2. Accordingly, it was suggested that MCP-1 would enhance tumor angiogenesis and early tumor growth in the early stages by inducing TNFα, IL-1α, and VEGF through TAM recruitment and probably the direct autocrine/paracrine effects on melanoma cells.     

Myf5 and MyoD activation define independent myogenic compartments during embryonic development

Gene targeting has indicated that Myf5 and MyoD are required for myogenic determination because skeletal myoblasts and myofibers are missing in mouse embryos lacking both Myf5 and MyoD. To investigate the fate of Myf5:MyoD-deficient myogenic precursor cells during embryogenesis, we examined the sites of epaxial, hypaxial, and cephalic myogenesis at different developmental stages. In newborn mice, excessive amounts of adipose tissue were found in the place of muscles whose progenitor cells have undergone long-range migrations as mesenchymal cells. Analysis of the expression pattern of Myogenin-lacZ transgene and muscle proteins revealed that myogenic precursor cells were not able to acquire a myogenic fate in the trunk (myotome) nor at sites of MyoD induction in the limb buds. Importantly, the Myf5-dependent precursors, as defined by Myf5(nlacZ)-expression, deficient for both Myf5 and MyoD, were observed early in development to assume nonmuscle fates (e.g., cartilage) and, later in development, to extensively proliferate without cell death. Their fate appeared to significantly differ from the fate of MyoD-dependent precursors, as defined by 258/-2.5lacZ-expression (-20 kb enhancer of MyoD), of which a significant proportion failed to proliferate and underwent apoptosis. Taken together, these data strongly suggest that Myf5 and MyoD regulatory elements respond differentially in different compartments.     

A role for the myogenic determination gene Myf5 in adult regenerative myogenesis

The myogenic determination genes Myf5, Myod and Mrf4 direct skeletal muscle cell fate prenatally. In adult myogenesis, Myod has been shown to regulate myoblast differentiation, however, our understanding of satellite cell regulation is incomplete since the roles of Myf5 and Mrf4 had not been clearly defined. Here we examine the function of Myf5 and Mrf4 in the adult using recently generated alleles. Mrf4 is not expressed in normal or Myf5 null satellite cells and myoblasts, therefore excluding a role for this determination gene in adult muscle progenitors. Skeletal muscles of adult Myf5 null mice exhibit a subtle progressive myopathy. Crucially, adult Myf5 null mice exhibit perturbed muscle regeneration with a significant increase in muscle fibre hypertrophy, delayed differentiation, adipocyte accumulation, and fibrosis after freeze-injury. Satellite cell numbers are not significantly altered in Myf5 null animals and they show a modest impaired proliferation under some conditions in vitro. Mice double mutant for Myf5 and Dystrophin were more severely affected than single mutants, with enhanced necrosis and regeneration. Therefore, we show that Myf5 is a regulator of regenerative myogenesis and homeostasis, with functions distinct from those of Myod and Mrf4.     

Functional analysis of the p300 acetyltransferase domain: the PHD finger of p300 but not of CBP is dispensable for enzymatic activity

Acetylation of nucleosomal histones is a major regulatory step during activation of eukaryotic gene expression. Among the known acetyltransferase (AT) families, the structure–function relationship of the GNAT superfamily is the most well understood. In contrast, less information is available regarding mechanistic and regulatory aspects of p300/CBP AT function. In this paper, we investigate in closer detail the structure and sequence requirements for p300/CBP enzymatic activity. Unexpectedly, we find that the PHD finger of p300, but not of CBP, is dispensable for AT activity. In order to identify residues involved in substrate or acetyl-coenzyme A (acetyl-CoA) recognition, we have introduced 19 different amino acid substitutions in segments that are highly conserved between animal and plant p300/CBP proteins. By performing acetylation reactions with histones, a p53 peptide or the AT domain itself, we define several residues required for histone and p53 substrate recruitment but not for acetyl-CoA binding. Finally, we show that identical mutations in the p300 and CBP AT domain impair AT activity differently. This latter result combined with the finding of a differential requirement for the PHD finger provides evidence for structural differences between p300 and CBP that may in part underlie a previously reported functional specialization of the two proteins.     

p300/CBP及其相关因子PCAF与转录调控

p300/CBP及相关因子PCAF具有乙酰转移酶活性,能通过乙酰化组蛋白和非组蛋白的方式参与基因的转录调控.同时,它们能在转录因子和基本转录复合物之间起到桥梁作用,而且也能为整合多种转录因子提供支架,是一种典型的转录辅激活子. p300/CBP与细胞周期调控、细胞凋亡以及癌症的发生等过程之间有着直接的联系。本文概括了p300/CBP与PCAF的基本特性,并简要介绍它们与其他蛋白之间的相互作用,特别是E1A的最新研究进展。