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.
The regulation of gene expression is fundamental to organogenesis and pathogenesis. A change in the mRNA level of a gene can be attributed to either transcriptional and/or posttranscriptional regulation. While posttranscriptional regulation has recently attracted much attention, especially with regards to microRNA, our knowledge is limited regarding the extent and mode of transcriptional regulation of the genes involved in cardiac hypertrophy. Also, we have no knowledge of the mechanisms involved in the generalized increase in cell mRNA and proteins that defines hypertrophy. This information is critical for the general understanding of the nature of a given pathology and for the future of drug design. The advent of deep sequencing technology has allowed us for the first time to investigate the nature of the transcriptional regulation during cardiac hypertrophy in a genome-wide fashion, using RNA polymerase II chromatin immunoprecipitation followed by deep sequencing of the attached DNA fragments. This is the first study of its kind in a disease model. The results have revealed unique aspects of transcriptional regulation in general and in cardiac hypertrophy in specific. This proposal will investigate the mechanisms underlying these findings and the trigger of transcriptional changes during cardiac hypertrophy. The work also seeks to inhibit different aspects of transcription, including genes that are involved in contractile dysfunction during cardiac hypertrophy, which we predict will be a potential therapeutic tool. We hope that this information will provide more detailed and in depth understanding of the molecular mechanisms underlying cardiac hypertrophy and failure that would advance drug design.
| Shin, Hyewon; He, Minzhen; Yang, Zhi et al. (2018) Transcriptional regulation mediated by H2A.Z via ANP32e-dependent inhibition of protein phosphatase 2A. Biochim Biophys Acta Gene Regul Mech 1861:481-496 |
| Sayed, Danish; Yang, Zhi; He, Minzhen et al. (2015) Acute targeting of general transcription factor IIB restricts cardiac hypertrophy via selective inhibition of gene transcription. Circ Heart Fail 8:138-48 |
| Pfleger, J; He, M; Abdellatif, M (2015) Mitochondrial complex II is a source of the reserve respiratory capacity that is regulated by metabolic sensors and promotes cell survival. Cell Death Dis 6:e1835 |
| Sayed, Danish; He, Minzhen; Yang, Zhi et al. (2013) Transcriptional regulation patterns revealed by high resolution chromatin immunoprecipitation during cardiac hypertrophy. J Biol Chem 288:2546-58 |
