UC Berkeley

The cellular control of gene expression is a fundamental process in biological life, and one that is still incompletely understood. DNA is packaged into chromatin inside human cells which is bound by sequence-specific transcription factors and their accompanying chromatin coactivator complexes. These molecular assemblies modify chromatin and regulate mRNA production from nearby and distal genes but how they accomplish these diverse functions is still unclear. In the first part of my thesis, I focus on one particularly elusive chromatin coactivator complex, SAGA, and, through collaboration with the Nogales lab, I characterize its molecular and atomic features using cryo-electron microscopy. The resulting structure reveals many interesting physical features that would have been unpredictable using previous models of SAGA from yeast, including large architectural rearrangements and elucidation of the structural incorporation of non-conserved subunits such as the splicing module. Furthermore, we are able to demonstrate the biological utility of a human-specific structure of SAGA by identifying patient mutations which likely interrupt non-conserved interfaces, making testable predictions of their disease etiologies. The high-resolution characterization of hSAGA paves the way for detailed future mechanistic studies which may interrogate SAGA’s many complex roles in human gene expression.

My work then zooms out to identify and characterize regulators of gene expression in an understudied system within human developmental biology, that of placental development. Studies into the molecular mechanisms of placental development have the potential for tremendous impact on human health where many deadly pregnancy diseases currently have no cures or prevention. Defects in placental stem cell (trophoblast) differentiation have been described in deadly pregnancy diseases such as preeclampsia where patient placentas show impaired cytotrophoblast cell fusion and decreased expression of endogenous retroviral fusogens such as syncytin-2, that correlate with disease severity. Here, I demonstrate new tools to identify and characterize molecular drivers of differentiation in the cell-cell fusion of human placental trophoblasts. I demonstrate the optimization of cellular and molecular tools in the Forskolin-induced BeWo human cell model of placental syncytium formation with the goal of building an imaging-based arrayed CRISPR knockout screen to perform an unbiased search for genes required for placental cell differentiation and fusion. These experiments demonstrate the optimization of high efficiency gene knockouts in BeWo cells and cell fusion assays allowing for high-confidence readout of cell fusion perturbations at scale. Taking a candidate approach, I then investigate a conserved transcription factor, TFEB, and its roles in human placental cell fusion. I reveal that genetic deletion of TFEB and its paralogue TFE3 in BeWo cells results in reduced expression of syncytin-1 and syncytin-2 and functionally impaired cell-cell fusion. Finally, I use confocal and single molecule imaging to elucidate TFEB-chromatin interactions during differentiation, prompting the creation of new models of TFEB activity during trophoblast differentiation. These studies illustrate the ability to identify new regulators of human placental cell fusion using optimized tools in BeWo cells and to deeply characterize the molecular behavior of such regulators at the single molecule level. Such interdisciplinary work demonstrates the vision to use tools which span biological scales to uncover basic mechanisms of gene expression and lay the groundwork for identifying and characterizing novel therapeutic targets which could save lives during pregnancy.