CRISPR-mediated Chemical Genetics Define Transcription Factor Gene Networks and Mechanisms of Control

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ABSTRACT Sequence-specific transcription factors are critical mediators of cellular adaptation in response to both extracellular and intracellular cues. These rapidly changing transcriptional programs facilitate everything from cell fate decisions to stress responses, and the disruption of transcription factor function and/or expression is associated with disease states including developmental disorders, neurologic disorders, and cancer. While it is clear that the appropriate regulation of gene expression is critical for normal cellular function, historically, our ability to understand how sequence-specific transcription factors rapidly and specifically alter transcriptional programs has been limited by a toolbox of very slow genetic and knockdown strategies that take days to weeks before transcription factor activity can be assayed. Therefore, while direct transcriptional effects occur within minutes to hours, these models take days to establish resulting in the detection of secondary and/or compensatory transcriptional changes that often mask the direct/immediate effects of transcription factor disruption. In order to overcome these technical limitations, we use CRISPR-mediated genome editing to introduce degron tags into endogenous transcription factor loci. This chemical-genetic approach results in rapid transcription factor degradation (minutes to hours) following PROTAC treatment, and effectively collapses the timeframe for assaying transcriptional changes, chromatin states, and genome-wide transcription factor occupancy from days to hours. We complement these studies with proteomics-based approaches to identify associated complexes and cooperating transcription factors, and are beginning to incorporate single cell and single molecule imaging approaches to interrogate heterogeneity within transcriptional responses. Combined, these approaches are allowing us to address fundamental questions in the transcription field including how transcription factors are influenced by and exert influence over the chromatin landscape, how multiple sequence- specific transcription factors cooperate to regulate gene expression, and how enhancer activity influences promoter activation.