Browsing by Author "Greenleaf, Arno L"

Since RNA Polymerase II (RNAPII) transcribes much of the genome, it is well situated to encounter and initiate a response to various types of DNA damage. However, to date very little is known about any response of RNAPII to DNA damage outside of Transcription Coupled Nucleotide Excision Repair (TC-NER). A link between DNA damage response mechanisms and the C-terminal domain of RNAPII (CTD) is suggested by an overlap between proteins that bind the CTD and genes required for resistance to DNA damaging agents. In this thesis, I show that proper deployment of CTD associated proteins is required to respond to DNA damaging agents. Furthermore, I show that a CTD associated protein (Set2) is required for response to DNA damage, but its catalytic activity is not. Finally, I show that the recombinational ability of strains lacking the CTD kinase, Ctk1, is deficient. Based on these lines of evidence, I propose a novel CTD Associated DNA Damage Response (CAR) system of proteins that is required for proper response to DNA damaging agents.

Eukaryotic RNA polymerase II (RNAPII) not only synthesizes mRNA, but also coordinates transcription-related processes through the post-translational modification of its unique C-terminal repeat domain (CTD). The CTD is an RNAPII specific extension of the enzyme's largest subunit and consists of multiple repeating heptads with the consensus sequence Y₁S₂P₃T₄S₅P₆S₇. In Saccharomyces cerevisiae (Sc), RNAPII committed to productive elongation is phosphorylated at the S₂ positions of the CTD, primarily by CTDK-I (composed of the CDK-like Ctk1, the cyclin-like Ctk2, and Ctk3) the principal elongation-phase CTD kinase in Sc. Although responsible for the bulk of S₂ phosphorylation in vivo, Ctk1 coexists with the essential kinase Bur1 which also contributes to S₂ phosphorylation during elongation. In higher eukaryotes there appears to be only one CTD S₂ kinase: P-TEFb, which had been suggested to reconstitute the activity of both of the Sc S₂ CTD kinases. Based on comparative genomics, we hypothesized that the previously-unstudied Drosophila CDK12 (dCDK12) and little-studied human CDK12 and CDK13 (hCDK12 and hCDK13) proteins are CTD elongation-phase kinases, the metazoan orthologs of yeast Ctk1. Using fluorescence microscopy we show that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNAPII, but is distinct from that of P-TEFb. Chromatin immunoprecipitation experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes in a pattern reminiscent of a S₂ CTD kinase. Appropriately, we show that dCDK12, hCDK12, and hCDK13 purified from nuclear extracts manifest CTD kinase activity in vitro and associate with CyclinK, implicating it as the cyclin subunit of the kinase. Most importantly we demonstrate that RNAi knockdown of dCDK12 in Drosophila cell culture and hCDK12 in human cell lines alters the phosphorylation state of the CTD. In an effort to further characterize the transcriptional roles of human CDK12/CyclinK we overexpress, purify to near homogeneity, and characterize, full-length hCDK12/CyclinK. Additionally, we also identify hCDK12 associated proteins via mass spectrometry, revealing interactions with multiple RNA processing factors, and attempt to engineer an analog sensitive CDK12 human cell line. Overall, these results demonstrate that CDK12 is a major elongation-associated CTD kinase, the ortholog of yCtk1. Our findings clarify the relationships between two yeast CDKs, Ctk1 and Bur1, and their metazoan homologues and draw attention to major metazoan CTD kinase activities that have gone unrecognized and unstudied until now. Furthermore, the results suggest that hCDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors.

The unique C-terminal domain (CTD) of RNA polymerase II (RNAPII), composed of tandem heptad repeats of the consensus sequence YSPTSPS, is subject to differential phosphorylation throughout the transcription cycle. Several RNA processing factors have been shown to bind the appropriately phosphorylated CTD, and this facilitates their localization to nascent pre-mRNA during transcription. In Saccharomyces cerevisiae, the mRNA export protein Yra1 (ALY/REF in metazoa) has been shown to cotranscriptionally associate with mRNA and is thought to deliver it to the nuclear pore complex for export to the cytoplasm. Based on a previous proteomics screen, I hypothesized that Yra1 is a bona fide phosphoCTD associated protein (PCAP) and that this interaction is responsible for the pattern of Yra1 cotranscriptional association observed in vivo. Using in vitro binding assays, I show that Yra1 directly binds the hyperphosphorylated form of the CTD characteristic of elongating RNAPII. Using truncations of Yra1, I determined that its phosphoCTD-interacting domain (PCID) resides in the segment comprising amino acids 18-184, which, interestingly, also contains the RNA Recognition Motif (RRM) (residues 77-184). Using UV crosslinking, I found that the RRM alone can bind RNA, although a larger protein segment, extending to the C-terminus (aa 77-226), displays stronger RNA binding activity. Even though the RRM is implicated in both RNA and CTD binding, certain RRM point mutations separate these two functions: thus, mutations that produce defects in RNA binding do not affect CTD binding. Both functions are important in vivo, in that RNA binding-defective or CTD binding-defective versions of Yra1 engender growth and mRNA export defects. I also report the construction and characterization of a useful new temperature sensitive YRA1 allele (R107AF126A). Finally, using chromatin immunoprecipitation, I demonstrate that removing the N-terminal 76 amino acids of Yra1 (all of the PCID up to the RRM) results in a 10-fold decrease in Yra1 recruitment to genes during elongation. These results indicate that the PCTD is likely involved directly in cotranscriptional recruitment of Yra1 to active genes.

The estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor (NR) with no natural ligand identified. Recent studies report that ERRα expression and activity correlate with poor prognosis in breast cancer. It is also suggested that ERRα is involved in tumor growth and progression, thus this receptor may be a therapeutic target in the treatment of breast cancer. However, the specific role of ERRα in breast cancer is not fully understood. Similar to other nuclear receptors, ERR has been suggested to regulate target gene transcription through both classical (direct DNA binding) and non-canonical (tethering mechanisms) to effect various aspects of tumor pathogenesis, such as angiogenesis, regulation of hypoxic response, tumor growth, and migration. Thus, the objective of this dissertation research is to explore the roles of ERRα in breast cancer by (a) identifying novel ERRα target genes important for tumor pathogenesis, (b) characterizing the molecular mechanism of non-canonical actions of ERRα-mediated gene transcription, and (c) examining the structure basis of ERRα antagonism for future pharmaceutical exploitation. First, we identified an ERRα target gene, ECM1, which is relevant to breast cancer angiogenesis. The role of ECM1 in angiogenesis was confirmed by endothelial tube formation assay. We further showed that knocking down ECM1 has a dramatic inhibitory effect on tumor xenograft growth. This result, for the first time, directly demonstrates the role of ECM1 in tumor environment and further sheds light on the significance of ERR&alpha-regulated genes in tumors angiogenesis. Next, we explored the molecular mechanism of ERRα non-canonical pathways using transcriptional reporter assay and ERRα DNA-binding domain (DBD) mutants. We discovered that the expression of carbonic anhydrase 9 (CA9), a target gene of one of the ERRα tethering partner hypoxia inducible factor-1 (HIF-1), does not require direct binding of ERRα to DNA but its DBD is indispensible. These results reflect on the importance of ERRα DBD even in the non-canonical signaling of ERRα, which brings challenges to dissecting ERRα canonical/non-canonical pathways in the future. Finally, to determine the molecular mechanisms underlying ERRα antagonism, we probed the conformations of ERRα upon antagonist treatments. M13 phage display was used to screen for ERRα-interacting peptides. We identified peptides that interact with ERRα in the activation function 2 (AF2) domain, some of which are able to distinguish the binding of different classes of ERRα antagonists. Cumulatively, these studies have explored the biological functions of ERRα and the molecular basis ERRα-mediated signaling pathways.