RNA polymerase III (Pol III) subunit RPC7α, which is encoded by POLR3G in humans, has been linked to both tumor growth and metastasis. Accordantly, high POLR3G expression is a negative prognostic factor in multiple cancer subtypes. To date, the mechanisms underlying POLR3G upregulation have remained poorly defined. We performed a large-scale genomic survey of mRNA and chromatin signatures to predict drivers of POLR3G expression in cancer. Our survey uncovers positive determinants of POLR3G expression, including a gene-internal super-enhancer bound with multiple transcription factors (TFs) that promote POLR3G expression, as well as negative determinants that include gene-internal DNA methylation, retinoic-acid induced differentiation, and MXD4-mediated disruption of POLR3G expression. We show that novel TFs identified in our survey, including ZNF131 and ZNF207, functionally enhance POLR3G expression, whereas MXD4 likely obstructs MYC-driven expression of POLR3G and other growth-related genes. Integration of chromatin architecture and gene regulatory signatures identifies additional factors, including histone demethylase KDM5B, as likely influencers of POLR3G gene activity. Taken together, our findings support a model in which POLR3G expression is determined with multiple factors and dynamic regulatory programs, expanding our understanding of the circuitry underlying POLR3G upregulation and downstream consequences in cancer.

RNA polymerase III (Pol III) produces noncoding RNAs involved in diverse cellular activities, including translation (tRNA, 5S rRNA, 7SL RNA), RNA processing (U6 snRNA, RPPH1, RMRP), and transcription regulation (7SK snRNA). In this way, Pol III activity must be broadly coupled with cellular demands for protein accumulation and growth, increasing in response to nutrient availability and decreasing during differentiation and exit from proliferation. However, the currently established mechanisms of Pol III regulation remain relatively limited, due in part to the few Pol III-centered protein-protein interaction (PPI) studies performed to date. To address this gap, we first investigated PPIs shared by multiple Pol III subunits to under-stand the macromolecular interactome of Pol III, with special attention directed at potential regulatory candidates. Our proteomic survey uncovers interactions between Pol III and the NuRD (Nucleosome Remodeling and Deacetylase) complex. Taken further, we show that NuRD localizes to active Pol III-transcribed genes and that its recruitment is Pol III-dependent but nonrandom, with peak occupancy and regulatory hallmarks converging on tRNA gene clusters associated with notably high expression levels. Inhibiting NuRD-associated histone deacetylase function reduces Pol III transcription at these sites, suggesting NuRD restricts Pol III and thereby modulates the global dynamic range of Pol III-derived RNA species. These findings are congruent with the transcriptionally repressive nature of NuRD and bring-to-light a new regulatory mechanism that may couple signaling events and changes in metabolic needs with the dynamic availability of specific tRNA pools.