Maia-Silva, Diogo (March 2023) MECHANISMS OF GENE TRANSCRIPTION BY TP63 AND THE MEDIATOR COMPLEX IN SQUAMOUS PANCREATIC CANCER. PhD thesis, Cold Spring Harbor Laboratory.
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Abstract
The transcription factor p63 is a major lineage-specific growth dependency in adenosquamous pancreatic carcinoma, a highly aggressive variant of pancreatic cancer. When expressed, p63 drives cancer cell survival, invasion, and marked transcriptomic and histopathological changes that are characteristic of squamous cell carcinomas. While the pro-tumorigenic role of p63 in adenosquamous carcinomas has been previously established, the mechanisms underlying the powerful transcriptional activating function of this transcription factor remain largely unknown. Here, we developed and employed functional genomic, genetic and biochemical approaches to define the core molecular determinants of p63 function in adenosquamous carcinomas. Using intracellular FACS-based genome-wide CRISPR screens, we uncover MED12, a component of the CDK8 kinase module of the Mediator complex, as a critical cofactor of p63. Genomic characterization of p63 and MED12 showed that they coregulate squamous gene expression by co-occupying similar loci on chromatin, and that p63 controls the recruitment of MED12 to its target loci. In vitro reconstitution and other biochemical assays demonstrate a direct interaction between p63 and MED12, thereby establishing MED12 as a direct, novel coactivator of p63. Finally, we reveal that p63 imposes a selective hypersensitivity to MED12 loss, which is particularly deleterious for adenosquamous and squamous cell carcinomas compared to other cancer lineages. Our findings on MED12 highlight the importance of understanding the functional determinants of the Mediator complex in vivo. To comprehensively address this question, we generated and screened CRISPR exon tiling libraries of all Mediator genes to define their critical protein segments and domains. Coupling this extensive genetic characterization to detailed, publicly available structural information, we propose a three-dimensional structural-functional model of the human Mediator. Our studies unveil MED12 as a novel p63-instructed vulnerability of adenosquamous and squamous carcinomas, and define important mechanisms of transcriptional activation by p63 and the Mediator complex.
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