The recognition of the hierarchies of genetic pathways coordinating heart development remains incomplete, in part because of the difficulties to identify the critical cis-acting sequences that are required for the regionalized expression of genes during cardiogenesis. Here we propose an integrative system to identify the critical components of the regulatory network of TBX20, a gene that plays important roles in heart development. To achieve this, we propose to utilize a combination of an in vivo zebrafish and mouse reporter assay to identify heart enhancers in the TBX20 locus. We will carry out a saturation scan for enhancers in the TBX20 locus, systematically testing sequences regardless of their pattern of evolutionary conservation, with the goal of identifying the critical enhancers that coordinate TBX20 expression during cardiogenesis. We will carry out a detailed characterization of the spatial and temporal domains of these heart enhancers during embryogenesis, leading to a broad understanding of the components of the TBX20 regulatory network. We will test the necessity of each of these enhancers for the proper expression of TBX20. Using engineered Bacterial Artificial Chromosomes (BACs), we will delete each enhancer that drives expression in multiple domains of the heart. This experimental design will allow us to infer the necessity of individual components of the regulatory network for the proper temporal, spatial and quantitative expression of TBX20. In summary, we will use zebrafish and mouse in vivo assays to characterize the cis-regulatory network that coordinates TBX20 expression during cardiogenesis, and evaluate how the individual components of the network orchestrate the dynamic and complex pattern of TBX20 expression.
Congenital heart diseases (CHD) may result from mutations in protein-coding exons of genes that encode for that build the individual structures of the developing. But mutations in regulatory elements of these genes, which determine where, when and how much protein will be made from a gene may also underlie the genetic base of several forms of CHD. We lack systematic strategies to identify the DNA elements that control gene expression, and assays that evaluate how multiple regulatory sequences interact to orchestrate the expression of genes throughout cardiac development. Using Tbx20 as a paradigm, we propose a system to comprehensively identify regulatory elements of genes involved in heart development and dissect the molecular bases of the coordinated expression of this gene in the developing heart. Our results will further the understanding of heart development and allow us to investigate the roles of mutations in gene regulatory sequences in CHD.
| Sakabe, Noboru Jo; Savic, Daniel; Nobrega, Marcelo A (2012) Transcriptional enhancers in development and disease. Genome Biol 13:238 |
| Arnolds, David E; Liu, Fang; Fahrenbach, John P et al. (2012) TBX5 drives Scn5a expression to regulate cardiac conduction system function. J Clin Invest 122:2509-18 |
| Smemo, Scott; Campos, Luciene C; Moskowitz, Ivan P et al. (2012) Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease. Hum Mol Genet 21:3255-63 |
| Distler, Margaret G; Plant, Leigh D; Sokoloff, Greta et al. (2012) Glyoxalase 1 increases anxiety by reducing GABAA receptor agonist methylglyoxal. J Clin Invest 122:2306-15 |
| Sakabe, Noboru J; Aneas, Ivy; Shen, Tao et al. (2012) Dual transcriptional activator and repressor roles of TBX20 regulate adult cardiac structure and function. Hum Mol Genet 21:2194-204 |
| Shen, Tao; Aneas, Ivy; Sakabe, Noboru et al. (2011) Tbx20 regulates a genetic program essential to adult mouse cardiomyocyte function. J Clin Invest 121:4640-54 |
| Taher, Leila; McGaughey, David M; Maragh, Samantha et al. (2011) Genome-wide identification of conserved regulatory function in diverged sequences. Genome Res 21:1139-49 |
| Arnolds, David E; Chu, Alison; McNally, Elizabeth M et al. (2011) The emerging genetic landscape underlying cardiac conduction system function. Birth Defects Res A Clin Mol Teratol 91:578-85 |
| Savic, Daniel; Distler, Margaret G; Sokoloff, Greta et al. (2011) Modulation ofTcf7l2 expression alters behavior in mice. PLoS One 6:e26897 |
| Narlikar, Leelavati; Sakabe, Noboru J; Blanski, Alexander A et al. (2010) Genome-wide discovery of human heart enhancers. Genome Res 20:381-92 |
Showing the most recent 10 out of 14 publications
