MECHANISMS AFFECTING MUSCLE ACTIN GENE EXPRESSION AND MYOGENIC CAPACITY

影响肌肉肌动蛋白基因表达和生肌能力的机制

• 批准号: 6240013
• 负责人: SANDRA B SHARP
• 金额: $ 13.94万
• 依托单位: CALIFORNIA STATE UNIVERSITY LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240013
• 关键词: actins; developmental genetics; gel mobility shift assay; gene expression; genetic regulatory element; immunocytochemistry; messenger RNA; myogenesis; northern blottings; nucleic acid sequence; polymerase chain reaction; recombinant DNA; regulatory gene; retinoate; striated muscles; tissue /cell culture; transfection

Myogenesis is the result of a complex interplay among multiple regulatory factors which determines where, when, and how far differentiation may proceed. Molecular regulatory differences will be detected in two cell lines which have different myogenic capacities and resemble myogenic cells from different locations and developmental stages in vivo. C2C12s form multinucleated myotubes, exit the cell cycle irreversibly, and express a broad array of muscle-specific genes at high levels. BC3H1s remain mononucleated, differentiate reversibly, fail to express some muscle specific genes and underexpress others, such as the skeletal muscle actin gene. C2C12s came from skeletal muscle and are mesodermal. BC3H1 cells came from a cranial tumor; the contribution of lineage to BC3H1 myogenic capacity is unclear. C2C12s express all four myogenesis determination genes; BC3H1s express only two. Initial work will establish baselines of differentiation: the % of nuclei which are expressing myogenin in differentiated cultures of BC3H1 and C2C12 will be determined by immunocytochemistry and the level of accumulation of myogenin mRNA relative to the level of accumulation of skeletal muscle actin mRNA will be determined and compared between lines. Skeletal muscle actin mRNA stability will be compared in the two lines using pulse-chase and pharmacological methods. MEF2s and the members of the thyroid hormone receptor family are important influences in myogenesis. MEF2 gene expression in the two lines will be compared by Northern and RT-PCR analysis and binding activity will be compared by gel shift analysis using probes from the skeletal muscle actin gene promoter. Proteins bound to DNA will be further characterized. The effect of specific MEF2 isoforms on BC3H1 myogenic capacity and the effect of forced BC3H1 terminal differentiation on MEF2 expression will be addressed with gene transfer studies. The hypothesis that in BC3H1 cells retinoic acid will inhibit rather than enhance myogenesis will be tested. The lineage of BC3H1 cells will be characterized with assays for Mhox and beta-tubulin Ill, mesodermal and neural markers, respectively. If appropriate, the effect of added Mhox expression on BC3H1 myogenic capacity will be tested. Results of this work will aid in understanding the balance of factors required for successful myogenesis and will thus contribute to the understanding of problems of abnormal development or loss of the differentiated state.

肌发生是多个调节的复杂相互作用的结果 决定差异可以在何处，何时和多远的因素 继续。分子调节差异将在两个细胞中检测到 具有不同肌源能力和类似肌原细胞的线条 来自不同位置和体内发展阶段。 C2C12S形式 多核肌管，不可逆地退出细胞周期，并表达一个 高水平的广泛肌肉特异性基因。仍然存在BC3H1 单核，可逆地分化，无法表达一些肌肉 特定的基因和其他其他基因，例如骨骼肌肌动蛋白 基因。 C2C12来自骨骼肌，是中胚层的。 BC3H1细胞 来自颅肿瘤；谱系对BC3H1肌原性的贡献 容量尚不清楚。 C2C12S表达所有四个肌发生测定 基因； BC3H1仅表示两个。 最初的工作将建立 分化：表达肌蛋白蛋白的核的百分比 BC3H1和C2C12的分化培养物将由 免疫细胞化学和肌蛋白mRNA的积累水平 相对于骨骼肌肌动蛋白mRNA的积累水平 确定并在线之间进行比较。骨骼肌肌动蛋白mRNA 使用脉冲练习和 药理方法。 MEF2S和甲状腺激素的成员 受体家族是肌发生的重要影响。 MEF2基因 北部和RT-PCR将比较两条线中的表达 分析和结合活性将通过使用凝胶移位分析进行比较 来自骨骼肌肌动蛋白基因启动子的探针。与DNA结合的蛋白质 将进一步表征。特定MEF2同工型对 BC3H1肌原性和强制BC3H1终端的影响 MEF2表达的分化将通过基因转移来解决 研究。 在BC3H1细胞中，视黄酸会抑制的假设 而不是增强肌发生。 BC3H1细胞的谱系 将以MHOX和β-微管蛋白疾病的测定为特征 中胚层和神经标记。如果适当的话， 将测试在BC3H1肌原性能力上增加的MHOX表达。结果 这项工作将有助于理解所需因素的平衡 成功的肌发生，因此将有助于理解 异常发育或差异化状态的丧失问题。