Determinants of Context Specific Transcription Factor Function

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Project Summary: The developing embryo relies on gene expression to be orchestrated on strict temporal and quantitative levels. Without proper coordination, the zygote suffers severe consequences such as growth issues and even embryo termination. Early embryonic gene regulation is carried out by maternally deposited transcription factors which are able to find their cognate cis sequences and activate the zygotic genome with tremendous efficacy despite the crowded environment of the nucleus. Once correctly targeted, transcription factors can perform context- specific functions across the genome even while sharing similar cis element sequence structure. I aim to define the relationship between transcription factors and their cis sequences by asking what are the contributions of DNA sequence and genomic context to transcription factor function in the early embryo? I will use the excellent genetic model system Drosophila melanogaster to investigate context-specific functions of the maternally deposited transcription factor Chromatin-Linked Adapter for MSL Proteins (CLAMP). CLAMP targets GA-rich cis elements in the embryonic genome, but performs several distinct functions depending on genomic context. For example, CLAMP primes the male X-chromosome for dosage compensation, regulates promoters genome-wide, and promotes formation of the conserved histone locus body (HLB), which regulates developmental and cell cycle expression of the replication-dependent histone genes. CLAMP targeting occurs in the embryo and when CLAMP is depleted, embryos terminate before the zygotic genome is activated. Although CLAMP targets similar cis elements in all three contexts, it recruits very different locus-specific transcription factors. I hypothesize that CLAMP inputs both DNA sequence information and genomic context to perform its context-specific functions. In Aim 1, I will engineer fly lines carrying a transgenic histone locus in which I have replaced the natural cis elements with CLAMP-recruiting GA-rich elements from different genomic origins. I will assess how these cis elements impact early embryo HLB formation, CLAMP binding, and histone gene transcription to determine how similar cis elements from different origins impact CLAMP function. In Aim 2, I will perform (A) in vitro gel-shift assays and (B) in vivo experiments, in which I vary the length of the CLAMP-recruiting cis elements and determine binding affinity and transcriptional output. In this aim, I will determine the contribution of cis element affinity to transcription factor function. Overall, the proposed aims will define how cis element DNA sequence and genomic context influence context-specific transcription factor function in the early embryo.