REGULATION OF EUKARYOTIC TRANSCRIPTIONAL ELONGATION AND ASSOCIATED DNA REPAIR

Author

Rwik Sen, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2016

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Bhaumik, Sukesh

Abstract

Transcriptional elongation is a crucial step in eukaryotic gene regulation whose mis-regulation leads to cellular pathologies. This makes it quite imperative to aim for a better understanding of the processes regulating transcriptional elongation. An important process promoting the association of RNA Polymerase II (RNAPII) with the coding region of the active gene and hence transcriptional elongation is the monoubiquitination of histone H2B at lysine 123. A complex of an E2 conjugase, Rad6p, and an E3 ligase, Bre1p, is essential for this process. Consistent with the role of histone H2B monoubiquitination in promoting the association of RNAPII with the active gene, this process was found to be impaired in the absence of Rad6p or point mutation of lysine 123 to arginine (H2B-K123R). Intriguingly, the association of RNAPII with the coding region of the active gene was not impaired in the absence of Bre1p, even though Bre1p is essential for histone H2B monoubiquitination. However, deletion of Bre1p’s RING domain that is essential for histone H2B monoubiquitination led to an impaired RNAPII association with the active gene. This observation indicates a role of the non-RING domain of Bre1p in repressing the association of RNAPII with the active gene, resulting in no net decrease in RNAPII occupancy in the absence of Bre1p. Taken together, my results implicated both the stimulatory and repressive roles of the histone H2B ubiquitin ligase Bre1p in regulation of RNAPII association with the coding regions of active genes and hence transcriptional elongation. Interestingly, my work also revealed that for efficient transcriptional elongation by histone H2B monoubiquitination, its optimum level needs to be maintained by a proper balance between Rad6p-Bre1p-mediated ubiquitination and de-ubiquitination (DUB) by the DUB module of SAGA. It was found that Sus1p, a subunit of the DUB module, promotes transcriptional elongation, DNA repair and replication via regulation of histone H2B DUB. In addition to Rad6p- Bre1p and the DUB module, global level of histone H2B monoubiquitination is also critically regulated by Cdk9, a kinase essential for phosphorylation of the serine 2 residue in the C-terminal domain (CTD) of RNAPII, which promotes transcriptional elongation. Apart from serine phosphorylation, proline residues at RNAPII-CTD undergo isomerization by proline isomerases, which also regulate transcription. One of the proline isomerases, Rrd1p, has been previously implicated in transcription in response to rapamycin treatment. Based on this fact and Rrd1p’s known interaction with RNAPII-CTD, we predicted that Rrd1p might regulate transcription independently of rapamycin treatment. In agreement with this hypothesis, our work revealed Rrd1p’s role in facilitating transcription of both rapamycin responsive and non-responsive genes in the absence of rapamycin treatment. Consistently, the absence of Rrd1p led to an impaired nucleosomal disassembly at the active gene, which correlates with the role of Rrd1p in promoting transcription. This is because maintenance of proper nucleosomal dynamics is essential for efficient transcription. It is known that transcriptional elongation is facilitated by the regulation of nucleosomal dynamics via the histone chaperone, FACT. Efficient chromatin reassembly in the wake of elongating RNAPII contributing to the fidelity of transcription is promoted by FACT. Being evolutionarily conserved among eukaryotes, FACT is also known to regulate DNA replication and repair, apart from transcription. Intriguingly, FACT has been found to be upregulated in cancers while its downregulation leads to tumor cell death. However, the mechanism which fine-tunes FACT for normal cellular functions remained unknown. My studies revealed a novel mechanism of regulation of FACT by the ubiquitin-proteasome system in yeast. San1p, an E3 ligase involved in nuclear protein quality control, was found to associate with the active gene and regulate transcriptional elongation through its E3 ligase activity- mediated turnover of Spt16p component of FACT. This regulation was found to maintain optimum level of Spt16p/FACT to engage with the active gene for proper transcriptional elongation, DNA repair and replication. In spite of playing such crucial roles in gene regulation, it was not known how FACT is targeted to the active gene. We discovered that a direct physical interaction between FACT and Cet1p, the mRNA capping enzyme, targets FACT to the active gene independently of Cet1p’s mRNA capping activity. Such targeting of FACT to the active gene leads to the release of promoter proximally paused-RNAPII into transcriptional elongation. However, the progress of RNAPII along the active gene during transcriptional elongation is frequently impeded by various kinds of damages along the underlying template DNA. Even though some of these lesions are co-transcriptionally repaired, it was not known whether the repair of extremely toxic DNA double-strand breaks (DSBs) was coupled to transcription. My results showed that DSBs at the transcriptionally active state of a gene are repaired faster than at the inactive state but such repair was not mediated by a co-transcriptional recruitment of DSB repair factors. This observation is in contrast to other DNA repair pathways such as nucleotide excision repair (NER) where repair factors are co-transcriptionally recruited to the lesion containing DNA. In this regard, we found that an NER factor, Rad14p, co-transcriptionally associates with the active gene in the absence of DNA damage to promote transcription, which unraveled a new role of Rad14p in transcription in addition its established role in NER. In summary, my results provide significant novel insights into the regulation of transcriptional elongation and associated processes leading to better understanding of eukaryotic gene expression.