Genetics, Cell Biology, and Development (GCD)
Molecular, Cellular, Developmental Biology and Genetics (MCDB&G), Biomedical Informatics and Computational Biology (BICB) (Kuang Lab)
My project focuses on RNA transcription and the role of proximal-promoter pausing (PPP) in stem cell differentiation. RNA Polymerase II (Pol II) pauses before transcription at as many as 40-70% of genes, but the mechanisms of PPP and the dynamics of pausing in stem cell differentiation are poorly understood. The first aim of my project is to describe pausing dynamics in multiple, disease-relevant cell types. I will perform ChIP-seq in differentiated and undifferentiated cells using antibodies specific to Pol II and key pausing proteins. We anticipate that comparing ChIP-seq data of undifferentiated and differentiated cells will reveal Pol II and other pausing antibody associations that are unique to different cell types. Pause factor knock-down and RNA-seq data will compliment this approach to reveal the genes most regulated by pausing to uncover where pause release is required for differentiation. The second aim of my project is to uncover mechanisms of pause release using a FLAG-tagged construct of the active kinase responsible for Pol II phosphorylation and release. Immunoprecipitation of this construct in both undifferentiated and differentiated cells will reveal proteins that recruit this kinase during differentiation. Overexpression of such a protein may prove a useful tool in overcoming the rate-limiting step of transcription and increasing the rate of differentiation. Overall, my project seeks to address the complex process of PPP and release from pausing into productive elongation. Understanding these mechanisms is the first step to achieving the goal of as faster and more efficient method of stem cell differentiation.