Monday, February 6, 2023
2/6/2023
PROJECT SUMMARY Cellular differentiation is driven by changes in gene expression. To transition from one type of cell to another, cells must simultaneously activate genes that promote differentiation while repressing genes that antagonize faithful progression. How gene repression is achieved amidst widespread transcriptional activation is not fully understood. A recently described mode of gene repression, which relies on integrated transcriptional and translational interference, exemplifies the coordination of transcriptional activation and gene repression. In this form of gene regulation, a long undecoded transcript isoform (LUTI) is transcribed from a gene-distal promoter, leading to repressive co-transcriptional chromatin modifications over the gene-proximal promoter4, 6, 34. In contrast to the efficiently translated, canonical transcript derived from the proximal promoter, the LUTI contains upstream open reading frames in its 5’ extended sequence which prevent translation of the protein coding sequence4, 5, 34. First observed in budding yeast gametogenesis - the differentiation program required to produce sex cells - and later during the yeast unfolded protein response and in human cells12, 36, LUTI-based regulation allows cells to toggle between a repressive and coding transcript throughout differentiation. Furthermore, LUTI-dependent interference over the proximal promoter is tunable and reversible, permitting dynamic developmental gene expression. While the chromatin modifications induced by LUTI transcription have been described, trans-acting factors required for LUTI-dependent nucleosome positioning changes have yet to be uncovered. This proposal seeks to uncover these factors using forward genetics in yeast and to characterize their role in developmental gene regulation. Preliminary work from these approaches implicates the Swi/Snf chromatin remodeling complex in LUTI-based transcriptional interference. Experiments proposed in aim 1 will investigate the role of the Swi/Snf complex in LUTI- based regulation during yeast gametogenesis, first by assaying regulation of the model LUTI gene NDC80 and then on a global scale. Results from aim 2 will reveal whether the LUTI transcriptional interference mechanism is conserved from yeast to humans. Aim 2 also seeks to uncover a functional role for LUTI-based gene repression during differentiation of human embryonic stem cells to the endoderm fate. Together, these experiments will illuminate the molecular requirements for LUTI-based gene repression and functional significance of LUTI transcription in development.
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