NOVEL GENE REGULATORY MECHANISMS WITH IMPLICATIONS IN CANCER

Eukaryotic gene expression to proteins is a complex process that begins with transcription which is regulated by numerous regulatory factors and signals. Alterations in these regulatory factors that modulate gene expression are linked with a multitude of cellular pathologies including cancers. Thus, it is important to delineate these transcriptional regulatory mechanisms of gene expression. Therefore, a large number of studies have been aimed at understanding the mechanism of transcription at the level of initiation, elongation, and termination. In line with this, my dissertation work is focused towards elucidating novel regulatory mechanisms of transcription initiation and elongation. Our results illuminate genetically how TOR (target of rapamycin) signaling pathway regulates transcription initiation and hence, transcription, in response to nutrients. The process of transcription initiation at the promoter is followed by RNA polymerase II (Pol II) pausing at the promoter-proximal site for mRNA capping/quality control. Such promoter-proximal pausing of Pol II (paused Pol II) plays an important role in regulating transcription elongation. Our results unveil how paused Pol II is released to engage into productive elongation for mRNA synthesis. We show that the capping enzyme, Cet1, targets a transcription factor known as FACT (facilitates chromatin transcription) which subsequently recruits a transcription elongation factor, Paf1C (RNA polymerase II- associated factor 1 [Paf1] complex), to release the paused Pol II for productive transcription elongation for mRNA synthesis. During such transcription elongation, histones need to be evicted in front of Pol II and reassembled in the wake of Pol II, and this dynamic histone disassembly and reassembly are coordinated by a number of histone chaperones. The aforementioned transcription factor, FACT, is one such histone chaperone that plays a key role in histone reassembly during transcription elongation. Importantly, we find a new regulation of FACT, by the ubiquitin-proteasome system (UPS), and hence, histone dynamics at the coding sequence and transcription. Specifically, the Spt16 component of FACT is ubiquitinated by the E3 ubiquitin ligase San1, and subsequently degraded by the 26S proteasome in yeast. Such proteasomal regulation of Spt16 subunit of FACT regulates transcription, and impairment of this UPS regulation alters transcription, leading to cellular pathologies. Indeed, SPT16 has been found to be associated with a lot of cancers, and our results show that this proteasomal degradation of SPT16 is impaired in cancer cells. Further, upregulation of SPT16 is associated with alterations in transcription of genes linked to cancer. Subsequent to its synthesis, mRNA needs to be exported to cytoplasm for translation to proteins. Importantly, transcription elongation has been found to be coupled to mRNA export, and like elongation, mRNA export is also controlled by UPS. Our findings demonstrate the role of active transcription in the proteasomal degradation of a key mRNA export factor, Sub2, mediated via Mdm30 (an F-box protein), thus, enhancing our understanding of the UPS regulation of mRNA export. Taken together, my dissertation work elucidates novel regulatory mechanisms of gene expression in response to nutrients and UPS, with implications in cellular pathologies including cancers