Mechanisms of Nuclear Body Formation in the Early Embryo

SUMMARY: Coordinated gene regulation is especially critical in the developing embryo where the zygotic genome initiates transcription for the first time. A conserved strategy to facilitate coordinated gene regulation is formation of conserved structures called nuclear bodies, sub-nuclear regions at which specific factors accumulate to perform regulatory and transcriptional functions. The histone locus body (HLB) is a conserved nuclear body that regulates the histone genes, and I discovered that the zinc finger protein, CLAMP (Chromatin Linked Adaptor for MSL Proteins), is essential to HLB regulation in Drosophila melanogaster. My research now aims to address the key question: How does CLAMP act in a context-specific manner to initiate nuclear body formation during the critical developmental stage of zygotic genome activation? In Aim 1 of this project I will define the role of CLAMP in chromatin accessibility and factor recruitment during zygotic genome activation. I will perform ATAC-seq and transcription factor ChIP-seq from sexed embryo pools in the presence and absence of CLAMP, around the time point of zygotic genome activation. I will receive mentorship from my mentor, Dr. Erica Larschan at Brown University. These experiments will address how chromatin state affects nuclear body formation and how the composition of chromatin changes at ZGA when nuclear bodies assume their unique properties. In Aim 2 I will use genetic tools to determine the developmental timing requirements of CLAMP at the HLB and the specific qualities of cis-elements that allow HLB formation, under mentorship from my co-mentor Dr. Robert Duronio at the University of North Carolina-Chapel Hill. This aim will compare the mechanism(s) of HLB initiation and maintenance throughout development. In Aim 3 I will perform Proteomics of Isolated Chromatin Segments in early embryos in the presence and absence of CLAMP, followed by mass spectrometry analysis. I will identify unknown HLB components and their reliance on CLAMP recruitment, under mentorship from my co-mentor Dr. Paul Schedl at Princeton University. This aim will illuminate the mechanism of early embryonic nuclear body formation, including the ordered recruitment of known and novel factors over developmental time. I will perform the proposed experiments while also gaining experience and critical background in developmental biology and proteomics. I will also develop my teaching and mentoring skills through hands on experience and leadership/laboratory management courses. My proposed research will generate significant new experimental directions, and my proposed career development will support me, as I initiate my independent laboratory.