Regulation Of Myod Post-transcriptional Modifications

MyoD是肌肉特异性基因表达所需的转录激活因子。外源性MyoD在许多终末分化细胞系（神经元、脂肪细胞、皮肤细胞、软骨细胞等）中的表达将其命运转向骨骼肌表型。此外，MyoD和相关的Myf-5蛋白对动物骨骼肌的形成至关重要。为了激活转录，MyoD需要其他相互作用伙伴的帮助，如p300/CBP和PCAf共激活子。我们之前报道过p300和PCAF是肌肉特异性转录因子MyoD的节点共激活因子。MyoD被PCAF乙酰化，这导致dna结合和转录活性增加。乙酰化和去乙酰化处于动态平衡状态，我们现在已经开始探索去乙酰化酶HDAC-1在控制肌肉分化中的作用。我实验室产生的数据表明，去乙酰化酶HDAC-1抵消了MyoD将原始成纤维细胞转化为肌肉细胞的能力，并阻碍了培养的小鼠成肌细胞的分化。这种现象的分子基础与HDAC-1在未分化的成肌细胞中物理相互作用并使MyoD和染色质MyoD结合位点周围的组蛋白去乙酰化的能力有关。在诱导细胞分化后，肿瘤抑制蛋白pRb被去磷酸化，参与HDAC-1并阻断由转录因子E2F控制的基因的转录，这些基因是G1进展所必需的。在这一过程中，HDAC-1在物理上从MyoD转移到pRB，允许MyoD被乙酰化转移酶PCAF乙酰化。PRb对肌肉分化至关重要，事实表明，PRb -/-无合子小鼠的肌肉细胞不能分化。我们利用从pRb-/-动物中提取的成纤维细胞，用编码MyoD的逆转录病毒转导，证明外源pRb的重新引入消除了MyoD与HDAC-1的相互作用。因此，这些细胞现在可以退出细胞周期，并开始表达骨骼肌球蛋白重链（MHC）和肌酸激酶（MCK）等终末肌肉分化标志物。我们的数据支持一个模型，即HDAC-1从MyoD转运到pRb调节转录并促进细胞分化。我们利用特异性去乙酰化酶抑制剂trichostatin A （TSA）进一步评估了肌肉分化过程中全局乙酰化的影响。我们的研究结果表明，暴露于TSA的小鼠成肌细胞表现出增生性反应，通过MHC表达增加、多核和纤维直径增加来判断。

MyoD is a transcriptional activator required for muscle-specific gene expression. Expression of exogenous MyoD in numerous terminally differentiated cell lineages ( neurons, adipocytes, skin cells, chondrocytes adn others) redirect their fates towards the skeletal muscle phenotype. Furthermore, MyoD - and the related Myf-5 protein- is essential for the formation of skeletal muscles in the animal. In order to activate transcription, MyoD requires the assistance of other interacting partners such as the p300/CBP and PCAf coactivators. We have previously reported that p300 and PCAF are nodal coactivators of the muscle specific transcription factor MyoD. MyoD is acetylated by PCAF and this results in an increased DNA-binding and transcriptional activity. Acetylation and deacetylation are in a dynamic equilibrium and we have now begun to explore the role of the deacetylase HDAC-1 in controlling muscle differentiation. Data generated in my laboratory indicate that the deacetylase HDAC-1 counteracts the ability of MyoD to convert naive fibroblasts to muscle cells and impedes differentiation of cultured mouse myoblasts. The molecular basis of this phenomenon correlates with the ability of HDAC-1 to physically interact and deacetylate both MyoD and histones surrounding chromatin MyoD-binding sites in undifferentiated myoblasts. Upon induction of cellular differentiation, the tumor suppressor protein pRb is dephosphorylated, engages HDAC-1 and blocks transcription of genes controlled by the transcription factor E2F and required for G1 progression. During this process, HDAC-1 is physically displaced from MyoD to pRB allowing acetylation of MyoD by the acetyltrasferases PCAF. PRb is essential for muscle differentiation as indicated by the fact that muscle cells derived from pRb-/- nullizygous mice fail to differentiate. Using fibroblasts derived from pRb-/- animals and transduced with a retrovirus coding for MyoD, we have documented that reintroduction of exogenous pRb abrogates the interaction of MyoD with HDAC-1. Consequently, these cells can now withdraw from the cell cycle and start expressing markers of terminal muscle differentiation such as skeletal myosin heavy chain (MHC) and muscle creatine kinase (MCK). Our data support a model in which shuttling of HDAC-1 from MyoD to pRb regulates transcription and promotes cellular differentiation. We have further evaluated the effects of global acetylation during muscle differentiation by employing the specific deacetylase inhibitor trichostatin A (TSA). Our results indicate that mouse myoblasts exposed to TSA display a hypertrophic response as judged by an increase d expression of MHC, multinucleation and increased fiber diameter.