Date of Award
Doctor of Philosophy
Molecular Biology, Microbiology and Biochemistry
Gene expression is central to normal cellular functions and health. Thus, misregulation/alteration of gene expression is associated with cellular pathologies and diseases. In eukaryotes, protein coding gene expression starts in the nucleus at the level of transcription that generates mRNA, which undergoes co-transcriptional processing such as 5’-end capping, 3’-end poly-adenylation, splicing of introns, and subsequent packaging for export through nuclear pores to the cytoplasm for translation to protein. Thus, transcription is an important step of eukaryotic gene expression, which is initiated by the activator protein bound at the specific upstream activating sequence of the gene. Activator targets co-activator that facilitates the assembly of general transcription factors (GTFs) and RNA polymerase II to form the pre-initiation complex (PIC) at the promoter for transcription initiation. Thus, co-activators are important regulators of gene activation. One such co-activator is SAGA (Spt-Ada-Gcn5-Acetyltransferase), a large complex consisting of 20 different proteins. SAGA has multiple structural and functional modules such as TAF (TBP-associated factor), SPT (Suppressor of Ty), HAT (Histone acetyltransferase) and DUB (De-ubiquitinase). During transcription initiation or gene activation, SAGA contacts with activator via its Tra1 subunit, and recruits TBP (TATA-box binding protein) to the promoter via its Spt3 and Spt8 subunits. Subsequently, TBP nucleates the PIC formation to initiate transcription. The PIC formation or transcription requires free DNA template. However, eukaryotic genome is highly packed in the form of chromatin that is an array of nucleosomes, each of which consists of two histone H2A-H2B dimers and one histone H3-H4 tetramer forming an octamer wrapped by about 146 bp (base pair) DNA double helix. Such chromatin/nucleosomal structure of eukaryotic genome occludes the transcription factors to bind the cognate DNA sequence. Therefore, PIC formation and transcription initiation are controlled by chromatin and the factors regulating chromatin. Importantly, in addition to stimulating the PIC formation, the co-activator SAGA regulates chromatin (and hence PIC formation and transcription) via its two distinct enzymatic activities catalyzing histone H3 acetylation and histone H2B de-ubiquitylation. These two enzymatic activities of SAGA are associated with gene expression and cellular functions, alteration of which is associated with cancer and other diseases. The enzymatic activity of the DUB module (that contains Ubp8, Sgf11, Sgf73/ataxin-7 and Sus1) of SAGA is controlled by its Sgf73/ataxin-7 subunit that connects the DUB module with the rest of SAGA, and hence maintains SAGA’s overall structural integrity and DUB activity. Consistently, the loss of Sgf73/ataxin-7 impairs SAGA’s overall structural integrity, PIC formation and transcription. Thus, functional alteration/misregulation of Sgf73/ataxin-7 is likely to develop cellular pathologies. Indeed, alteration of Sgf73/ataxin-7 abundance or its malfunction/mutation is associated with various diseases such as cancer, early childhood blindness, neurodegenerative disorders, attention-deficit/hyperactivity disorder and ocular coloboma. Therefore, it is important to know the factors/mechanisms that change the cellular level of Sgf73/ataxin-7 towards understanding the disease pathogenesis and therapeutic interventions. However, the regulation of Sgf73/ataxin-7 is poorly understood. In view of this, we have carried out experiments to understand the regulation of Sgf73/ataxin-7 in yeast and human cells. Our results in yeast reveal that Sgf73/ataxin-7 undergoes ubiquitylation and 26S proteasomal degradation, thus unveiling a novel ubiquitin-proteasome system (UPS) regulation of Sgf73/ataxin-7. Alteration of such regulation changes cellular Sgf73/ataxin-7 abundance that affects the PIC formation and transcription. Thus, UPS fine-tunes Sgf73/ataxin-7 in orchestrating the PIC formation and transcription. Therefore, our results reveal a novel UPS regulation of Sgf73/ataxin-7 with physiological relevance in gene expression in yeast. Likewise, we find in human cells that ataxin-7 undergoes ubiquitylation and proteasomal degradation. Alteration of such regulation changes ataxin-7’s abundance that is associated with transcription aberration and cellular pathologies, thus implicating altered UPS regulation of ataxin-7 in diseases. Therefore, our results unveil a novel UPS regulation of Sgf73/ataxin-7 in yeast and human cells for normal cellular health, and implicate the alteration of such regulation in diseases. Further, increased or decreased level of Sgf73 impairs the assembly of DUB with the rest of SAGA, thus altering histone H2B ubiquitylation that is associated with transcription elongation. Histone H2B ubiquitylation regulates transcription elongation in collaboration with an evolutionarily conserved chromatin remodeling factor, FACT (Facilitates chromatin transcription), via direct interaction. FACT is a heterodimer of Spt16 and Pob3 in yeast or SSRP1 in humans. Spt16 contains four conserved domains. One such domain is CTD (C-terminal domain) that is negatively charged and intrinsically disordered, and binds to histone H2A-H2B dimer, thus impairing histone-DNA interaction and hence chromatin assembly/disassembly. However, there has not been any study so far to analyze the role of this intrinsically disordered region (IDR) of Spt16 in regulation of chromatin dynamics and transcription in living cells. To address this, we have depleted the CTD/IDR of Spt16, and then analyzed chromatin assembly, disassembly and transcription. Intriguingly, our results reveal that the IDR of Spt16 facilitates chromatin disassembly at the promoter as well as PIC formation and transcription, even though this domain is not required for cellular viability. Thus, our results demonstrate the roles of an IDR of Spt16 in regulating chromatin and transcription, hence advancing our understanding of the FACT regulation of chromatin and gene expression. While FACT is known to regulate transcription and chromatin, it is intriguingly found to be upregulated in cancers, especially aggressive and poorly differentiated tumors. However, how FACT is upregulated in aggressive tumor cells remained unknown until our recent studies that demonstrated ubiquitylation and proteasomal degradation of the Spt16 subunit of FACT in yeast, mouse and human cells in controlling transcription of the genes including the ones associated with cancer. In yeast, Spt16 is ubiquitylated by San1 (an E3 ubiquitin ligase that is involved in nuclear protein quality control) and degraded by the 26S proteasome. However, the role of San1 in genome-wide transcription remained unknown until our recent studies. Such knowledge would provide novel function of a nuclear quality control protein in global gene expression. To address this, we analyzed the role of San1 in the PIC formation and RNA polymerase II elongation genome-wide. Intriguingly, our results demonstrate distinct roles of San1 in regulation of the PIC formation and elongating RNA polymerase II (and hence transcription) genome-wide, thus revealing novel gene regulation by a nuclear protein quality control factor, San1. To determine the role of San1 in regulating transcription via FACT, we carried out a set of experiments including TAP-MS (Tandem affinity purification – mass spectrometry). Our analysis revealed that FACT interacts with a large number of proteins involved in regulation of chromatin, transcription, DNA repair and replication, and several of these interactions are altered in the absence of San1. The factors with altered interactions in the absence of San1 include transcription and chromatin regulatory factors. Thus, our results provide insights as to how San1 regulates transcription via regulation of FACT and its interaction with other proteins, hence advancing our understanding of FACT’s interactions, functions and regulation. Collectively, our results reveal novel regulations of the key chromatin and transcription regulatory factors, SAGA and FACT, in controlling gene expression with mechanistic insights and implications in understanding disease pathogenesis (with impact on future targeted therapeutic development).
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