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Mechanisms in transcriptional regulation during cardiac hypertrophy

心脏肥大过程中的转录调控机制

基本信息

批准号：
9064832
负责人：
Maha Abdellatif
金额：
$ 39.75万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-26 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9064832
关键词：
Actins Attention 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 Genes Genetic Transcription Grant Health 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 skeletal tool transcription factor

项目摘要

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(申请人提供)：心肌肥厚的特征是基因表达的普遍增加，这与心肌细胞大小和质量的增加相称，在此基础上叠加了特定基因表达的更强劲的变化。虽然其中一些基因的转录调控已经得到验证，但我们还没有全面的、全基因组范围的知识，即哪些基因受转录调控，哪些基因可能由涉及microRNA的转录后机制独立调控。此外，我们不知道转录调控的模式-从头RNA聚合酶II(PolII)的招募与暂停的PolII的释放-或涉及的调节器。在对小鼠心脏施加压力超负荷后，观察到的最早的变化之一是miR-1的下调，这在任何其他miRNA变化、心脏质量增加或收缩功能障碍之前。这表明miR-1可能是潜在发病机制的原因而不是结果。我们的初步数据表明，miR-1靶向基本转录的两个主要成分，通用转录因子2B(TFIIB)和细胞周期蛋白依赖性蛋白激酶9(CDK9)，这两个蛋白分别是PolII招募和延伸的关键调节因子。用锁定的核酸修饰的抗miR-1寡核苷酸在心脏中抑制miR-1足以诱导这些靶点的上调，反过来，miR-1的过表达则抑制它们的表达。更重要的是，染色质免疫沉淀深度测序分析(ChIP-Seq)显示，补充心肌细胞miR-1抑制了一组基因上的从头PII募集，同时诱导了另一组基因的暂停，这与其分别对TFIIB和CDK9的抑制一致。当miR-1下调时，相同的基因亚群在心肌肥厚过程中被反向调控。总体而言，通过重新招募POLII和释放启动子暂停的POLII的调控似乎是相互排斥的。前者主要调控~6%的基因，主要包括具有特殊功能的基因(如收缩、细胞外基质、免疫反应等)，后者涉及~25%的表达基因，主要包括管家/必需基因(如蛋白质和mRNA转换基因、基础转录因子、剪接基因等)。这些结果导致了对这笔赠款的假设。在心肌肥厚过程中，miR-1的i-下调是TFIIB和CDK9上调所必需的，从而导致相关基因表达的变化。II-在心肌肥厚期间选择性抑制TFIIB将抑制PolII从头招募到一组基因的启动子(~6%)，包括那些参与心肌病发展的基因(如ANF、BNP、α骨骼肌动蛋白、胶原等)。这不会抑制心脏质量的增加，但会改善肥厚时的收缩功能障碍。III-在心肌肥厚过程中选择性抑制心脏中的CDK9将抑制暂停的PolII对心脏中所有必需/管家基因(如Vdac1、Pinin、TFIIB、CDK9、MAPK1等)的启动子清除(~25%)。这将抑制心脏质量的增加，并导致急性心力衰竭。具体目标是1)确定涉及的机制心肌肥厚过程中TFIIB、CDK9及基础基因转录的调控。2)确定TFIIB在基因转录和心肌肥厚发生发展中的作用。3)确定CDK9在基因转录和心肌肥厚发生发展中的作用。