This proposal is a revision of renewal application for 1R01GM080372-01 "Function and regulation of the ETS transcriptional repressor TEL1/Yan". Developmental programs are driven by transcription factors that act upon cis-regulatory enhancer elements to coordinate precise patterns of gene expression. Two members of the conserved ETS family of transcription factors, a repressor Yan and an activator Pointed, known as TEL1 and ETS1/ETS2 in humans, act as downstream effectors of the receptor tyrosine kinase/Ras/MAPK signaling pathway to orchestrate the balance between proliferation and differentiation in a variety of cell types. In addition to the ETS DNA binding motif, Yan/TEL1 and Pointed/ETS1/2 share a second conserved domain, the sterile alpha motif (SAM), which mediates homo- and hetero-typic protein-protein interactions. SAM-mediated interactions modulate network output during normal cell fate transitions and provide an oncogenic driving force in the functional fusions produced by chromosomal translocations targeting the Tel1 locus. The goals of this proposal are to elucidate the mechanisms of Yan/TEL1-mediated repression and to understand the contribution of homotypic SAM- mediated interactions to these mechanisms. Because the signaling molecules and networks we are studying have conserved functions in mammals, and because their dysregulation contributes to oncogenic transformation in a number of leukemia and solid tumors, the discoveries resulting from these investigations will improve our understanding of mechanisms underlying human development and disease.
Aim 1 will investigate the molecular mechanisms by which the repressor Yan is recruited to chromatin to regulate gene expression. We will define how Yan-Pointed competition manifests at the level of chromatin occupancy and regulation of target gene expression. We will test the provocative idea that in addition to its canonical role as a sequence-specific DNA binding repressor, Yan also acts as a chromatin associated factor.
Aim 2 will explore the hypothesis that Yan's complex chromatin occupancy profile reflects a novel mechanism for buffering gene expression against genetic and environmental noise. Using state of the art in vivo molecular genetics technologies, we will determine the individual contribution of Yan-bound regions to regulating the expression of its target genes under both optimal conditions and in the face of genetic or environmental variation. Using 3C and ChIP-qPCR, we will investigate the influence of three-dimensional chromatin interactions on Yan occupancy and regulation of gene expression.
Aim 3 will investigate the molecular mechanisms of active Yan-mediated repression. Using an integrative combination of molecular genetics, bioinformatics and biochemical approaches, we will identify the components of Yan transcriptional complexes that determine the differential repressor activity of Yan monomers versus polymers and explore how the extent of polymerization impacts repressive activity in vivo.
The combination of unsurpassed genetic tractability and the extensive evolutionary conservation of developmental signaling networks make Drosophila an ideal model system in which to elucidate the molecular mechanisms that ensure accurate and robust cell fate transitions during development. Our approach is to investigate how specific cell fates in the developing embryo and eye are specified through the coordinate action of two antagonistic transcription factors that respond to and modulate signaling downstream of the receptor tyrosine kinase signaling pathway. Because the signaling molecules and networks we are studying have conserved functions in mammals, and because their dysregulation contributes to oncogenic transformation in a number of leukemia and solid tumors, the discoveries resulting from our investigations will improve understanding of human development and disease.
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