RNA Regulation of Transcription Factor Activity

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Project Summary/Abstract Nimble and responsive transcriptional control is central to all processes of life, and hence this process is regulated at a number of levels. During the last grant period, we discovered a robust, and unexpected, RNA- binding activity inherent in representative proteins from two major classes of transcriptional factors (TFs): the pluripotency factor Sox2 of the high mobility group box (HMGB) family and the glucocorticoid nuclear hormone receptor (GR). Their RNA-binding activity was found to be mediated by their DNA-binding domains and directly compete with DNA binding to their respective promoter or enhancer sequences. Furthermore, we found that this activity was structure-specific rather than sequence specific in vitro. Both TFs strongly disfavored binding ssRNA. Instead, the pluripotency factor Sox2 bound dsRNA regions while GR bound exclusively to RNA hairpin structures. These observations raise critical questions regarding the role of TF RNA-binding activity in transcriptional regulation and provide the direct motivation for the research program described here. Our corroborating discovery of RNA association of Sox2 in mouse embryonic stem cells confirms that direct RNA binding occurs in vivo. The next steps are to further define and understand the specificity for the in vivo RNA targets and determine the impact TF RNA interactions has on the transcriptional program. This includes refining our understanding of what drives this RNA binding and determine how pervasive the activity is in other TFs. We have developed an integrated and strategic research program to achieve these goals. Aim 1 capitalizes on our observation that Sox2 directly interacts with RNAs in cells and develops this critical line of inquiry in GR to develop a comprehensive RNA interactome including eRNAs. We will investigate whether transcription factors use their RNA-binding activity to provide an alternate chromatin association strategy that impacts localization and gene regulation. Finally, we take advantage of our biochemical insights to design and validate DNA- and RNA-binding separation-of-function mutants, which will allow us to directly measure the impact of loss of RNA binding on the transcriptome. In Aim 2, we turn to understanding the molecular nature of RNA association to Sox2 and GR, first through the use of eCLIP strategies to identify consensus binding motifs and second through high resolution structure determination of TF/RNA complexes. In Aim 3 of this program, we will establish the generality of these observations to other important transcription factors, both within and beyond the HMGB and nuclear hormone receptor families, as represented by Sox2 and GR, respectively. Together, this proposal describes a comprehensive program seeking to determine the extent and role of RNA binding in modulating the transcriptional program through interaction with classic transcription factors. The impact of this program is high because understanding the roles of genome-wide pervasive transcription and how this activity influences diverse cellular processes remains a major unanswered question in biology.