Mechanisms in transcriptional regulation during cardiac hypertrophy

心脏肥大过程中的转录调控机制

• 批准号: 8561869
• 负责人: Maha Abdellatif
• 金额: $ 37.84万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-26 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8561869
• 关键词: Actins; Attention; Basal transcription factor genes; CDK9 Protein Kinase; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cells; Collagen; DNA; Data; Development; Disease model; Down-Regulation; Drug Design; Essential Genes; Extracellular Matrix; Functional disorder; Future; Gene Expression; Gene Expression Regulation; General Transcription Factors; Genes; Genetic Transcription; Grant; Heart; Heart Hypertrophy; Heart failure; Housekeeping; Housekeeping Gene; Hypertrophy; Immune response; Knowledge; MAPK1 gene; Messenger RNA; MicroRNAs; Molecular; Mus; Muscle Cells; Nature; Oligonucleotides; Organogenesis; Pathogenesis; Pathology; Polymerase; Proteins; RNA Polymerase II; Regulation; Role; Sequence Analysis; Spliced Genes; Technology; Therapeutic; Time; Transcription Factor TFIIB; Transcriptional Regulation; Up-Regulation; Work; chromatin immunoprecipitation; deep sequencing; genome-wide; locked nucleic acid; overexpression; pressure; promoter; public health relevance; skeletal; tool

DESCRIPTION (provided by applicant): Cardiac hypertrophy is characterized by a generalized increase in gene expression that is commensurate with the increase in myocyte size and mass, on which is superimposed more robust changes in the expression of specialized genes. While transcriptional regulation of some of those genes has been validated, we do not have comprehensive, genome-wide, knowledge of which genes are regulated by transcription vs. those that may be independently regulated by posttranscriptional mechanisms involving microRNA. Additionally, we do not know the mode of transcriptional regulation - de novo RNA polymerase II (pol II) recruitment vs. the release of paused pol II - or the regulators involved. One of the earliest changes observed after applying pressure overload on a mouse heart, is the downregulation of miR-1, which precedes any other miRNA changes, the increase in cardiac mass, or contractile dysfunction. This suggested that miR-1 might be a cause rather than an effect of the underlying pathogenesis. Our preliminary data show that miR-1 targets two major components of basic transcription, general transcription factor 2B (TFIIB) and cyclin-dependent kinase 9 (Cdk9), which are the key regulators of pol II recruitment and elongation, respectively. Inhibiting miR-1 with locked nucleic acid- modified anti-miR oligo in the heart is sufficient for inducing upregulation of these targets, and conversely overexpression of miR-1 suppresses their expression. More significantly, chromatin immunoprecipitation-deep sequencing analysis (ChIP-Seq) reveals that supplementing cardiac myocytes with miR-1 suppresses de novo pol II recruitment on a subset of genes, while inducing pausing on another, in concordance with its suppression of TFIIB and Cdk9, respectively. The same subsets of genes are inversely regulated during cardiac hypertrophy as miR-1 is downregulated. In general, the regulation by de novo pol II recruitment and that by the release of promoter-paused pol II seem to be mutually exclusive. The former mainly regulates ~6% of genes mainly including those with specialized functions (e.g. contractile, extracellular matrix, immune response...etc), while the latter involve ~ 25% of expressed genes mainly including housekeeping/essential genes (e.g. protein and mRNA turnover genes, basal transcription factor, splicing genes...etc). These results led to the hypotheses for this grant. i- Downregulation of miR-1 is required for upregulation of TFIIB and Cdk9 during cardiac hypertrophy and, accordingly, the associated changes in gene expression. ii- Selective inhibition of TFIIB in the heart during cardiac hypertrophy will inhibit de novo recruitment of pol II to the promoters of a subset of genes (~6%) including those involved in the development of cardiomyopathy (e.g. ANF, BNP, alpha skeletal actin, collagen, etc.) This will not inhibit the increase in cardiac mass but will ameliorate contractile dysfunction during hypertrophy. iii- Selective inhibition of Cdk9 in the heart during cardiac hypertrophy will inhibit promoter clearance of paused pol II on all essential/housekeeping genes (~25%) in the heart (e.g. Vdac1, pinin, TFIIB, Cdk9, MAPK1, etc). This will inhibit the increase in cardiac mass and result in precipitous cardiac failure. The specific aims are 1) Identify the mechanisms involved in the regulation of TFIIB and Cdk9, and basic gene transcription, during cardiac hypertrophy. 2) Determine the role of TFIIB in gene transcription and the development of cardiac hypertrophy. 3) Determine the role of Cdk9 in gene transcription and the development of cardiac hypertrophy.

描述（由申请方提供）：心脏肥大的特征在于基因表达的普遍增加，其与肌细胞大小和质量的增加相称，其上叠加了特化基因表达的更稳健变化。虽然其中一些基因的转录调控已经得到验证，但我们没有全面的，全基因组的知识，哪些基因是由转录调控的，而哪些基因可能是由涉及microRNA的转录后机制独立调控的。此外，我们不知道转录调控的模式-从头RNA聚合酶II（pol II）招募与暂停pol II的释放-或涉及的监管机构。对小鼠心脏施加压力超负荷后观察到的最早变化之一是miR-1的下调，其先于任何其他miRNA变化、心脏质量增加或收缩功能障碍。这表明miR-1可能是潜在发病机制的原因而不是影响。我们的初步数据显示，miR-1靶向基础转录的两个主要组分，一般转录因子2B（TFIIB）和细胞周期蛋白依赖性激酶9（Cdk 9），它们分别是pol II募集和延伸的关键调节因子。在心脏中用锁核酸修饰的抗miR oligo抑制miR-1足以诱导这些靶标的上调，相反，miR-1的过表达抑制它们的表达。更重要的是，染色质免疫沉淀-深度测序分析（ChIP-Seq）揭示，用miR-1补充心肌细胞抑制了一个基因子集上的从头pol II募集，同时诱导另一个基因子集上的暂停，分别与其对TFIIB和Cdk 9的抑制一致。当miR-1下调时，相同的基因子集在心脏肥大期间反向调节。一般来说，重新招募pol II和释放启动子暂停pol II的调节似乎是相互排斥的。前者主要调节约6%的基因，主要包括那些具有特殊功能的基因（例如收缩，细胞外基质，免疫反应等）。等），而后者涉及约25%的表达基因，主要包括管家/必需基因（例如蛋白质和mRNA周转基因、基础转录因子、剪接基因...等等）。这些结果导致了这项赠款的假设。i-在心脏肥大期间，miR-1的下调是TFIIB和Cdk 9上调所必需的，因此，基因表达的相关变化。ii-在心脏肥大期间选择性抑制心脏中的TFIIB将抑制pol II重新募集到一个基因子集（~6%）的启动子，包括参与心肌病发展的那些（例如ANF、BNP、α骨骼肌动蛋白、胶原蛋白等）。这不会抑制心脏质量的增加，但会改善肥大期间的收缩功能障碍。iii-在心脏肥大期间选择性抑制心脏中的Cdk 9将抑制 心脏中所有必需/管家基因（例如Vdac 1、pinin、TFIIB、Cdk 9、MAPK 1等）上暂停的pol II的启动子清除率（~25%）。这将抑制心脏质量的增加，并导致突发性心力衰竭。具体目标是：（1）确定所涉机制， 心肌肥大过程中TFIIB和Cdk 9的调节以及基础基因转录。2)确定TFIIB在基因转录和心肌肥大发展中的作用。3)确定Cdk 9在基因转录和心肌肥厚发展中的作用。