Mechanisms in transcriptional regulation during cardiac hypertrophy

心脏肥大过程中的转录调控机制

• 批准号: 8725735
• 负责人: Maha Abdellatif
• 金额: $ 38.96万
• 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/8725735
• 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（由申请人提供）：心脏肥大的特征是基因表达的普遍增加，这与肌细胞大小和质量的增加相一致，在其上叠加了专用基因表达的更大变化。尽管这些基因中的某些基因的转录调节已得到验证，但我们没有全面的，全基因组的知识，对哪些基因受转录的调节，而与可能受到涉及microRNA的文字后机制进行独立调节的知识。此外，我们不知道转录调控的方式-DE NOVO RNA聚合酶II（POL II）招募与暂停POL II或所涉及的调节器的释放。在小鼠心脏上施加压力超负荷后观察到的最早变化之一是miR-1的下调，它在其他miRNA变化，心脏质量增加或收缩功能障碍之前。这表明miR-1可能是基本发病机理的原因，而不是其作用。我们的初步数据表明，miR-1靶向基本转录因子2B（TFIIB）和细胞周期蛋白依赖性激酶9（CDK9）的两个主要组成部分，它们分别是POL II募集和伸长率的关键调节剂。用锁定的核酸修饰的抗MIR寡寡核能抑制miR-1足以诱导这些靶标的上调，相反，miR-1的过表达抑制了它们的表达。更重要的是，染色质免疫沉淀 - 深度测序分析（CHIP-SEQ）表明，用miR-1补充心肌细胞在基因子集中抑制了从头pol II的募集，同时诱导了另一个基因的暂停，并与TFIIB和CDK9的抑制作用一致。随着miR-1的下调，相同的基因子集在心脏肥大期间受到呈负调节。通常，从头pol II招募的法规，并且通过释放启动者pol II的规定似乎是相互排斥的。前者主要调节约6％的基因，其中包括具有专业功能的基因（例如收缩，细胞外基质，免疫反应等），而后者涉及大约25％的表达基因，主要是家政/基本基因（例如蛋白质和mRNA失误基因，基本转录基因，基本转录因子，剪接基因等）。这些结果导致了这笔赠款的假设。 MiR-1的I-在心脏肥大过程中上调TFIIB和CDK9需要miR-1的下调，因此，基因表达的相关变化。 II-心脏肥大期间对TFIIB对心脏中TFIIB的选择性抑制作用将抑制从头招募POL II为基因子集的启动子（约6％）（包括涉及心肌病的发展的启动子）肥大。心脏肥大期间心脏中CDK9对CDK9的选择性抑制作用将抑制 在心脏中所有必需/管家基因（例如VDAC1，Pinin，tfiib，tfiib，cdk9，mapk1等）上暂停的POL II的启动子清除率。这将抑制心脏质量的增加，并导致心脏衰竭。具体目的是1）确定所涉及的机制 心脏肥大期间TFIIB和CDK9的调节以及基本基因转录。 2）确定TFIIB在基因转录和心脏肥大的发展中的作用。 3）确定CDK9在基因转录和心脏肥大的发展中的作用。