Functional and mechanistic studies of XPC DNA-repair complex as transcriptional coactivator in embryonic stem cells

Abstract

The embryonic stem cell (ESC) state is transcriptionally controlled by OCT4, SOX2, and NANOG with cofactors, chromatin regulators, noncoding RNAs, and other effectors of signaling pathways. Uncovering components of these regulatory circuits and their interplay provides the knowledge base to deploy ESCs and induced pluripotent stem cells. We recently identified the DNA-repair complex xeroderma pigmentosum C (XPC)-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4/SOX2 transcriptional activation. Here we investigate the role of SCC genome-wide in murine ESCs by mapping regions bound by RAD23B and analyzing transcriptional profiles of SCC-depleted ESCs. We establish OCT4 and SOX2 as the primary transcription factors recruiting SCC to regulatory regions of pluripotency genes and identify the XPC subunit as essential for interaction with the two proteins. The present study reveals new mechanistic and functional aspects of SCC transcriptional activity, and thus underscores the diversified functions of this regulatory complex.transcription | pluripotency | protein-protein interactions | ChIP-seq | RNA-seq

Architecture of the human XPC DNA repair and stem cell coactivator complex

“…Finally, CETN2 may also contribute to this overall flexibility, because it can adopt different conformations depending on its metal-binding state (38); however, the resolution of the XPC-RAD23B subcomplex was not markedly improved, suggesting that this contribution to complex flexibility is minor, as would be expected for its relatively small mass contribution to the complex. The recently described requirement of RNA for the XPC complex to interact with its transcription partner SOX2 (25) invokes the idea of low-complexity domains or regions, perhaps interspersed throughout XPC, linking their inherent flexibility to a critical aspect of the XPC complex's function. The possibility that the mammalian-specific insertion within the TGD domain (residues 331-517; Fig.…”

“…F., unpublished). Similarly, although the N and C termini of XPC are critical for recruitment and stimulation of TFIIH at sites of damage for global nucleotide excision repair (15,16,44,45), the removal of the N-and C-terminal TFIIH-binding domains of XPC (residues 1-195 and 814-940, respectively) only impacts repair but not transcriptional activity (4,25).…”

“…Reflecting the ever-expanding repertoire of reported XPC roles is the number of known physical and functional interactors of the XPC complex, e.g., TFIIH (15,16), OGG1 (23), TDG (3), SOX2 (4,25) (Fig. 4), and OCT4 (4, 25), among others (46).…”

“…1A). Our recent work describing the involvement of RNA in mediating the XPC-SOX2 interaction adds an additional and potentially intriguing dimension to future structural studies in this regard (25). Additionally, it would be interesting to explore whether structural changes are imposed on the XPC complex upon binding to its partner proteins; further biochemical and structural work to assemble such larger protein assemblies is required.…”

“…4F). Using information on XPC's interaction domains with partner proteins (14,15,25,27,36), sequence homology between yeast Rad4 and human XPC (Fig. S5), as well as the docking of the Rad4/Rad23 crystal structure, we were able to generate a model indicating the predicted locations of the interaction domains on the XPC complex (Fig.…”