Transcriptional regulation is essential for mediating proper gene expression during cardiovascular development. Over the last decade, multiple transcription factors and chromatin regulators have been implicated in the development of congenital heart disease (CHD) highlighting the importance of understanding how gene expression is regulated during early heart development. As CHD results from changes that occur early in development, determining the genetic and epigenetic mechanisms that coordinate cell fate decisions, tissue morphogenesis and cell lineage commitment during early cardiac development will be instrumental in elucidating the causes of CHD. Essential for mammalian development, the histone variant H2AZ has been shown to mediate patterning of chromatin states necessary for the proper execution of developmental programs in embryonic stem cells, however little is known about how H2AZ coordinates gene expression patterns during lineage commitment in the cardiovascular system. In light of the prominent role H2AZ plays in setting up chromatin states during embryogenesis, I hypothesize that H2AZ is an essential mediator of gene expression during early cardiac differentiation. By using a defined in vitro cardiac differentiation system, I propose to determine how the genome-wide patterns and requirement of H2AZ change during early cardiac development. To accomplish this objective, in specific aim 1, I will perform chromatin immunoprecipitation followed by deep sequencing at three stages of cardiac differentiation to determine the localization of H2AZ during mammalian heart development. In tandem I will also determine the localization of a select set of histone modifications known to correlate with distinct chromatin states.
In specific aim 2, I will evaluate changes in gene expression and histone modifications after depletion of H2AZ through inducible RNA interference at the cardiac progenitor and cardiac myocyte stages of heart development. Defining the genome-wide occupancy and requirement of H2AZ through cardiac differentiation will be critical to determine how this variant contributes to changing epigenetic landscapes essential for proper gene expression during cardiac differentiation and may help to identify unknown genes essential for heart development.
CHD is the primary cause of infant morbidity worldwide and represents a significant public health concern. As CHD results from problems that occur during embryonic development, this research aims to understand how chromatin structure regulates cardiac specific genes during embryogenesis to correctly pattern the developing heart. Additionally, this research will provide valuable information that will aid in the development of stem cell based therapies for cardiac related diseases.
|Lodato, Michael A; Ng, Christopher W; Wamstad, Joseph A et al. (2013) SOX2 co-occupies distal enhancer elements with distinct POU factors in ESCs and NPCs to specify cell state. PLoS Genet 9:e1003288|