Saturday, April 27, 2024
4/27/2024
PROJECT SUMMARY The overall goal of this proposal is to systematically characterize the cellular functions of transcription factor isoforms. Transcription factors (TFs) are master regulators of gene expression and as such play key roles in a variety of biological processes, including cell growth and differentiation. The human genome is estimated to harbor ~1600 TF genes; however, most of these are expressed as a series of protein isoforms arising from mRNAs with alternative starts, ends, or splicing. Though a handful of alternative TF isoforms are known to play functionally important (and distinct) roles in the cell, the overwhelming majority— thousands of proteins—remain entirely uncharacterized. Moreover, splicing aberrations are a hallmark of cancer, and mis-expression of TF isoforms can contribute to tumorigenesis. Thus, decoding the roles of TF isoforms is key to a systems-level understanding of gene regulatory networks (GRNs) in development and disease. In my previous work, I found widespread changes in DNA binding, protein-protein interactions, and transcriptional activation in exogenous assays across a collection of >700 TF isoforms. These results underscore the need to perturb and characterize TF isoforms in their endogenous cellular context to truly understand their roles in GRNs. Functional genomics approaches such as high throughput perturbation screens have revolutionized our understanding of gene functions. I aim to apply and extend these functional genomics approaches to study isoforms, which have remained elusive due to technical limitations. I propose to use a combination of state-of-the-art genomic technologies to decode the functions of TF isoforms in breast cancer cells. I will leverage long read RNA-sequencing to perform rigorous isoform-aware analyses (Aim 1) and isoform-specific high throughput experiments (Aims 2 and 3). In Aim 1, I will re-analyze existing CRISPR/Cas9 knock-out databases from the Cancer Dependency Map Consortium to identify candidate isoform-specific phenotypes in breast cancer. In Aim 2, I will establish a platform for robust and specific knock-down of individual isoforms in mammalian cells using RNA-targeting CRISPR/Cas systems. In Aim 3, I will use tunable libraries of TF ORFs to quantitatively tune isoform expression across a range of physiological levels. The proposed work includes technology development in the mentored K99 phase, which I will then leverage during the independent R00 phase to probe the mechanisms of TF isoform function in breast cancer via single-cell screening approaches. Successful execution of these aims will begin to decode the “dark matter” of alternative TF isoforms, laying a foundation for future studies of alternative isoforms more broadly. By combining new training in high- throughput screening approaches during the K99 phase with my existing expertise in gene regulation, TF biology, and bioinformatics, this transition to independence award will position me well to start my own research group that uses interdisciplinary genomic methods to probe GRNs in development and disease.
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