生理和病理状态中心肌蛋白troponin基因的调控

The proposal is a part of our studies on the regulation of myofibril protein expression in the heart. The troponin I (TnI) gene family is used as a model to study the regulation of myofibril gene expression during the maturation of cardiac muscle tissues. Fetal, or slow skeletal TnI (ssTnI) is expressed in the embryonic and fetal heart, and adult, or cardiac TnI (cTnI) expression is up-regulated after birth to replace ssTnI and predominates throughout adulthood in most mammals including humans. The mechanisms of the postnatal fetal-to-adult TnI isoform switch and ssTnI gene inactivation during heart development are still not clear. .In our previous studies, we have demonstrated that up-regulated cTnI itself is not a signal molecule that causes the down-regulation of ssTnI in the heart and ssTnI expression time-course in the heart can be partially influenced by thyroid hormone. We believe that the ssTnI gene regulatory region and the nuclear co-factors in cardiac myocytes should be critical for the regulation of ssTnI gene. We have successfully cloned mouse ssTnI gene regulatory region that includes the gene promoter and a 5' end intragenic region containing the first two non-coding exons, two introns and part of exon 3. To study mouse TnI gene has unique advantage since mouse is a more popular tool in biomedical research using transgenic models. Preliminary studies on this gene regulatory region have shown that it contains a potential promoter activity. In this proposal, we will characterize the cloned mouse ssTnI gene regulatory region to test the hypothesis that decreased enhancers and their binding activity or increased inhibitors in cardiac myocytes result in the repression of ssTnI gene expression during heart development. Three specific aims have been developed to address this hypothesis. Aim 1 will analyze the promoting activity of non-coding exons and introns on mouse ssTnI gene regulatory region. Aim 2 will determine the role of potential trans-factors (especially Yin Yang 1) on ssTnI expression in cardiac myocytes. Aim 3 will explore the mechanisms underlying the inactivation of fetal gene expression during heart development. .It is clinically important to elucidate the mechanisms underlying TnI gene regulation since TnI loss causes diastolic dysfunction and sudden cardiac death. The re-expression of fetal TnI, in case it is needed, may help the heart to maintain a normal function.

肌钙蛋白（TnI）基因调控研究已成为心脏发育过程中蛋白表达调节研究的典范。目前已证实，哺乳动物胚胎型TnI（ssTnI）主要在心脏胚胎期表达，而成年型心脏TnI（cTnI）表达上调最终取代ssTnI，但胚胎型向成年型TnI表达转换及ssTnI表达失活的机制现尚不清楚。前期研究中发现cTnI不是导致ssTnI失活的因素,ssTnI基因表达调节区与核因子的相互作用决定了ssTnI表达水平。我们已成功克隆了ssTnI基因表达调节区，此区有潜在的启动活性。因此，我们将对该表达调节区域进行深入研究，通过降低增强因子表达或提高抑制子活性影响ssTnI表达，阐明该调节区中基因5'端非编码区的启动活性，观察各反式因子对顺式元件的识别与结合，深入探讨小鼠心脏发育过程中ssTnI基因失活的机制。研究TnI基因调节具有重要临床意义，重新开放失活的ssTnI基因将有助于弥补心脏TnI不足造成的心脏功能异常和疾病。

本项目是对TnI基因的启动子区域进行了活性检测, 测定TnI启动子活性区的表观遗传修饰如甲基化, 乙酰化对该基因表达的影响和调控。并把在基因调控上的成果直接应用到野生型和转基因小鼠，进行整体动物实验，观察干预和不干预时乙酰化，甲基化对in vivo动物心脏TnI表达的影响。通过研究，我们阐明了表观遗传修饰在TnI基因表达中的作用，我们的研究结果证明在正常生理或在疾病发生的病理期可以通过表观遗传修饰（甲基化或乙酰化）来调控心脏中某些心肌蛋白基因的表达从而来纠正某些基因表达不正常而导致的病理现象和疾病。我们的研究成果发表了15篇SCI文章，在整个项目中，培养了六名硕士研究生，一名博士研究生。在美国实验室资助培训了三位中，青年学者，用请进来走出去的办法加强了国内外科研交流和协作。