Systematic Characterization Of Transcriptional Regulators By Site-specific Protein Recruitment

Student: Nader Alerasool

Supervisor: Dr. Mikko Taipale

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Meeting ID: 942 7705 8176 

Passcode: 954673 

Abstract

Many transcriptional regulatory proteins and chromatin-associated factors have been identified and their DNA binding sites elucidated. The functional role of most, however, remains unclear. Furthermore, many regulators tend to be multifunctional as their roles can vary depending on the genomic context where they bind. Here I present a novel pooled approach for systematically testing the transcriptional activity of thousands of proteins. By utilizing dCas9, I have developed an ORFeome-wide tethering screen and recruited ~14,000 human proteins to the basal promoter of a reporter gene integrated into the human genome. In a pooled screen, I identified over 200 proteins capable of robustly activating the reporter. Known activators, such as mediator components, were highly enriched in the screen. Interestingly, many highly homologous transcription factors had strikingly different activation profiles, suggesting they form functionally distinct groups despite similar DNA binding specificities. Moreover, a number of novel transcriptional activators were identified. Characterization of these factors with affinity purification coupled to mass spectrometry indicated they activate transcription by interacting with distinct activator complexes. Utilizing the same platform to recruit protein fragments of known activators, I was able to identify multiple new transactivation domains in a single experiment. Finally, I profiled several KRAB domains for their ability to repress both a reporter and endogenous targets. This led me to identify a potent repressor that outperformed other CRISPRi platforms described to date.