Hormone Receptor Regulation of RNA Polymerase III

Project summary Steroid receptors are a subset of nuclear receptor (NR) transcription factors that are found only in vertebrates and regulate essential functions such as organismal development and reproduction, while also impacting aging, tumorigenesis, and cancer progression. Small lipophilic hormones bind to steroid receptors for estrogens (ER- alpha and ER-beta), progesterone (PR), glucocorticoids (GR), and androgens (AR) and transmit their signal through gene regulation. Since these small molecules can be synthetically modified and manufactured, a variety of pharmaceutical drugs provide critical medicines for diseases including metabolic disorders, reproduction, and cancer treatment. NR mechanism of action has been mostly studied at RNA polymerase II (Pol II) transcribed genes including protein-coding mRNAs and small/long non-coding RNAs. Multiple NRs act in complexes on DNA to activate or repress Pol II transcription. However, NR controlled cell transcriptomes are often not always tightly correlated with the proteome due to post-transcriptional regulation that is not completely understood. We have uncovered a second layer of coordinated NR activity through regulation of RNA Polymerase III (Pol III) transcribed genes. Pol III transcribes small RNAs essential for translation of mRNAs into protein including tRNAs and 5S rRNA and is a major node for controlling cell growth, stem cells, aging, and cancer. Negative regulation of Pol III is commonly through the conserved repressor Maf1. Very little is known concerning how NRs regulate Pol III in more complex mammalian cells and organisms. We discovered using genome-wide analyses of PR chromatin binding in ER+PR+ breast cancer cell lines and tumors that PR localizes at multiple tRNA genes. PR associates with the Pol III complex and decreases tRNA levels and protein synthesis. Progesterone recruits PR and retinoic acid receptor alpha (RARα) to tRNA genes near a conserved DNA sequence resembling an NR binding half site. Our hypothesis is that PR regulates Pol III transcription of tRNA genes through crosstalk with RARα and recruitment of Maf1 resulting in decreased levels of target tRNA genes and selective translation. Aim 1 will determine how PR associates at tRNA genes, the role of RARα and other steroid receptors, and the NR binding half site. Aim 2 will determine the role of Maf1 in PR modulation of Pol III transcription. Aim 3 will define hormone-induced changes in the tRNA pool and the impact on translational efficiency. Regulation of Pol III is vastly understudied compared to Pol II and crucial for normal and oncogenic cell phenotypes. Results of this study will define a novel mechanism of NR action at Pol III genes that will help explain i) an additional layer of hormone regulation that contributes to selective mRNA translation, and ii) how NRs converge on multiple cell polymerases to impact cell growth, differentiation, stemness, and tumor progression.