Throughout the past several decades it has become increasingly apparent that a large proportion of the human genome is transcribed, while only a small fraction (~2%) codes for protein. Among the non-coding transcriptome is a class of RNA termed long noncoding RNAs. Although recent estimates have suggested close to 20,000 potentially functional long noncoding RNAs within the human genome, the functions and mechanisms of these transcripts remain largely unexplored. In the studies presented here, we demonstrate novel functions and molecular mechanisms for the long noncoding RNA, LINC00662, in 2 different cellular processes: DNA damage repair and cancer cell migration. We found that in response to UV irradiation, LINC00662 is up-regulated through a splicing mechanism dependent upon hnRNP C binding to an Alu sequence within LINC00662 exon 3. Surprisingly, we also found that this UV-induced splicing mechanism is likely regulated by N6-methyladenosine (m6A) modification. We provide evidence that LINC00662 enhances non-homologous end joining through interaction with Ku70 and Ku80, leading to more efficient DNA damage repair. We also found that loss of LINC00662 alters cell morphology and migratory potential for cancer cells, likely through regulation of genes involved in extracellular matrix organization and eukaryotic translation elongation. Finally, we show that LINC00662 is often overexpressed in several cancers, and that altered LINC00662 expression is correlated with clinical outcomes for select cancers. Together, these studies further our understanding of the diverse regulatory roles for long noncoding RNAs in complex cellular processes, and implicate LINC00662 as a functionally significant long noncoding RNA for human health and disease.

Tumor suppressor PTEN functions differently in the cytoplasm and nucleus. Posttranslational modifications play important roles in regulating activities, functions and subcellular localizations of PTEN. Previously, our laboratory discovered that nuclear PTEN promotes acetylation of tumor suppressor p53. In the present study, we focus on contribution of nuclear PTEN to PML-IV-mediated cellular senescence and to high levels of free fatty acid (FFA)-induced oxidative stress.

PML-IV is a tumor suppressor and the major regulator of cellular senescence. We show that the C-terminal domain of PML-IV is required for induction of cellular senescence. Nuclear PTEN regulates cellular senescence via interaction with the C-terminus of PML-IV, which is essential for recruiting PTEN into PML-Nuclear Bodies (PML-NBs), enhancing p53 acetylation and inducing cellular senescence.

We also show that high FFA induces nuclear export of PTEN and down-regulates p53 acetylation and protein levels, which leads to inhibition of p53 downstream target GPx-1 and accumulation of ROS in endothelial cells. Furthermore, mTOR/S6K signaling induces phosphorylation of PTEN at Ser380, which decreases PTEN monoubiquitination, promotes PTEN nuclear export and leads to p53/GPx-1 inhibition. This study suggests that alterations of the two posttranslational modifications of PTEN caused by mTOR/S6K are responsible for oxidative stress induced by high FFA.

In summary, we show the regulation of cellular senescence by nuclear PTEN and provide a new mechanism of tumor suppression function of PTEN. We also demonstrate a novel pathway by which high levels of FFA induce oxidative stress through PTEN nuclear export and thus provide novel insight into the role of nuclear PTEN in metabolism.

The heterotrimeric PP2A holoenzyme consists of a scaffolding A, a catalytic C, and a variable regulatory B subunit. Previous study has reported that B56gamma subunit mediates the tumor suppressive function of PP2A. B56gamma specific PP2A inhibits cell transformation by dephosphorylating and activating p53. In addition, B56gamma specific PP2A possesses the p53 independent tumor suppressive function although its mechanism is unclear. In the present study, we demonstrate the mechanism of B56gamma tumor suppressive function inactivation through characterizing B56gamma mutations identified in human cancer.

First, we demonstrate that the A383G and F395C mutations disrupt B56gamma from interacting and activating p53. The mutants are unable to dephosphorylate p53 and thus lack the p53 dependent tumor suppressive function. Moreover, we identify the region adjacent to these mutations as the p53 binding domain. This finding implies that the bridging interaction between B56gamma and p53 is required for p53 dephosphorylation by PP2A.

Second, we show that the C39R mutation within the HEAT repeat 1 disrupts B56gamma binding with the AC core and thus abolishes the B56gamma tumor suppressive function completely. We further demonstrate that the intact HEAT repeat 1 is required for B56gamma to bind the AC core, providing structural insights into the B56gamma specific PP2A holoenzyme assembly.

Further characterization of additional B56gamma mutations reveals two classes of mutations with different mechanisms of tumor suppressive function inactivation. Mutations in the first class disrupt the interaction between B56gamma and the AC core and thus fail to regulate all substrates and completely lose the tumor suppressive function. Mutations in the second class prevent specific substrates from binding B56gamma and partially reduce the tumor suppressive function. Although it remains to be investigated how frequently these mutations occur, our results underline the importance of B56gamma tumor suppressive function in human cancer.

Finally, we identified the S220N mutation that specifically disrupts the p53 independent tumor suppressive function of B56gamma, suggesting that it may disrupt the binding of unknown substrates. Identification of these proteins would contribute to the further understanding of the tumor suppressor role of B56gamma in cells.

Transcription of protein coding genes and some non-coding RNAs relies on RNA polymerase II (pol II). Positioning the polymerase at the correct location at the start of a gene is aided by a series of general transcription factors (GTFs): TFIIA, B, D, E, F, and H. Together with pol II, these factors form the preinitiation complex. TFIID is first to recognize and bind to a gene promoter and helps facilitate recruitment of the other components. TAF1 is the largest subunit of TFIID and aside from its role as a GTF, it functions to regulate gene specific transcription. TAF1 has intrinsic kinase activity and has been shown to phosphorylate other members of the general transcription machinery as well as the tumor suppressor p53. Previous studies have shown that phosphorylation of p53 by TAF1 leads to dissociation from the p21 promoter and termination of transcription. Those studies also found that TAF1 kinase responds to fluctuations in cellular ATP, having a KmATP of 1.9mM. In the early stages following DNA damage, cells undergo cellular ATP depletion which was found to effect TAF1-mediated phosphorylation of p53 giving rise to a regulatory mechanism of p21 transcription. The studies presented here investigate the possibility that TAF1 regulates other sequence specific transcription factors, like p53, through phosphorylation. The work described in this dissertation includes the purification of many proteins for in vitro characterization of two novel TAF1 kinase targets, E2F1 and FOXM1. Furthermore, this research investigates the effect that TAF1-mediated phosphorylation has on E2F1 and FOXM1 promoter occupancy and transcription of target genes. My studies provide new insights into how TAF1 regulates gene-specific transcription through phosphorylation, broadening our understanding of this complex GTF.

Altered cellular metabolism is considered a crucial hallmark of cancer, and has been an area of accelerated research. Interestingly, metabolic alterations in cancer can lead to changes in metabolite concentration, and these may play a role in tumorigenesis through functions that are not overtly metabolic. Tumor suppressor p53 plays a prominent role in cancer and much of human biology. Many functions have been attributed to p53, including roles in DNA repair and recombination through association with proteins involved in genome stability and chromatin modification. However, its broadest cellular effect is through its function as a transcription factor (TF). p53 is regulated through multiple post-translational modifications (PTMs) in response to a variety of cellular stresses including DNA damage. One important PTM is phosphorylation on the Thr55 residue, which is mediated by TBP Associated Factor 1 (TAF1) on the p21 promoter and leads to p53 promoter dissociation and termination of p21 transcription. TAF1 is the largest subunit of transcription factor TFIID and a cell cycle regulatory protein important for G1 phase progression. Interestingly, TAF1 phosphorylates p53 in a cellular ATP level-dependent manner. Because high glucose (HG) conditions lead to increased cellular ATP levels, we tested its effect on p53 function and found that global p53 binding to target promoters is reduced under HG conditions even in the presence of DNA damage. Functional analysis of cell growth inhibition as well as cell apoptosis suggests HG is a potent inhibitor of these p53-dependent cellular responses. Further, we examined the overall HG effect on the DNA damage-dependent gene expression changes and found that HG alters the DNA damage transcriptome. Importantly, HG affected the expression of genes that have already been shown to play a role in cancer. These studies suggest that cancer cells use the dynamic nature of cell metabolism to promote transformation via metabolite concentration. Altogether, this work establishes a molecular mechanism by which hyperglycemia may indirectly contribute to malignant transformation, providing an explanation for the association between diabetes and cancer.

Cancer is the second leading cause of global mortality. Accumulating evidence strongly suggests that aberrant alterations in signaling cascades can lead to uncontrolled cell growth, driving the progression of this disease. While the promotion of cancer cell growth by hyperglycemia has been extensively studied, the comprehensive mechanism underlying this relationship remains incompletely elucidated. In the current study, we present findings that high glucose levels (HG) indeed bolster DNA synthesis and cell cycle progression, fostering tumor cell growth, as revealed by genome-wide analyses. Moreover, our investigation identified E2F family as the central transcription factor responsible for the adaptive response induced by HG. The use of the pan-E2F inhibitor HLM006474 and shRNA-mediated E2F1 depletion, effectively counteracted HG-triggered DNA synthesis and cell growth. Furthermore, we establish that HG amplifies Rb1 phosphorylation, directly contributing to the activation of E2F1. Inhibition of Rb1 phosphorylation using the CDK2/4/6 inhibitor compound PF-3600 substantially mitigates the E2F1-mediated transcriptional activation of downstream DNA replication genes. Notably, among the E2F1 target genes induced by HG, RRM2 plays an essential role in the nucleotide synthesis by generating essential dNTPs required for DNA replication. Our investigation reveals that HG promotes intracellular dNTP levels in an E2F1-RRM2-dependent manner, closely associated with increased DNA synthesis and subsequent cancer cell growth. The RNR inhibitor Triapine effectively impedes RRM2-mediated elevation of intracellular dATP and dGTP, and DNA synthesis. To gain more insights into the metabolic alterations occurring in HG-treated cancer cells, we carried out the untargeted metabolomic profiling. This analysis not only reaffirms the activation of glycolysis in HG-treated cells, as evidenced by the elevated levels of lactic acid, but also illustrates noteworthy enhancements in the levels of sucrose and fructose in the polyol pathway. These findings provide another facet of how cancer cells utilize excess glucose to fuel their proliferation. Collectively, our findings decipher the oncogenic signaling and metabolic connections between hyperglycemia and cancer cell proliferation, mediated through the Rb1/E2F and polyol pathways. This study provides a detailed molecular mechanism that sheds light on how hyperglycemia directs tumor cells to enhanced cell cycle progression and cell proliferation.

Gene regulation is a complex mechanism and our knowledge of molecular pathways remains limited. In particular, current research examining transcriptional regulation emphasizes events that precede the initiation of transcription. However, correct regulation also requires turning off transcription when it’s no longer needed. Because DNA-bound activators interact with and recruit components of the basal transcription apparatus to the promoter, these basal factors could, in theory, mark the activators. Presumably, through post-translational modification after transcription, this process could lead to transcription termination. To determine this mechanism, previous studies have elucidated the relationship between the TATA-Binding Protein Associated Factor 1 (TAF1) and p53 in response to Ultra Violet (UV) DNA damage. The p53 tumor suppressor is a transcription factor that plays a critical role in guarding cell genomes against DNA damage. Among the genes induced by p53 is p21, which induces cell cycle arrest. TAF1 phosphorylates p53 at Thr55 on the p21 promoter, leading to p53 dissociation from the promoter and transcription termination. TAF1 is the largest subunit of general transcription factor TFIID and possesses intrinsic protein kinase activity. Because TAF1 is a component of general transcription machinery bound to many promoters, the following research was designed to elucidate how TAF1 contributes to transcriptional regulation via it’s DNA binding activities and kinase activities. This dissertation aims to reveal TAF1’s genome-wide response to UV induced DNA damage. My studies included an in-depth look into TAF1’s recruitment to the promoter via core promoter elements, novel TAF1 kinase targets, and the regulation of gene expression upon DNA damage via TAF1. These studies will provide important insights into how TAF1 impacts genome-wide transcription regulation and explores how to regulate transcription termination. Since the regulation of gene expression is a vital process for the integrity of organisms, this discovery provides an in-depth understanding of the cellular development of a wide range of species.

In response to cellular stresses, the transcription factor p53, a tumor suppressor, controls cell cycle arrest, DNA repair, or senescence by modulating the expression of target genes. Phosphorylation of p53 at threonine-residue 55 (p53-Thr55) by the TBP-associated factor 1 (TAF1), a subunit of the general transcription factor TFIID, suppresses the interaction of p53 with target genes and promotes nuclear export of p53, resulting in degradation of p53 and G1 cell cycle progression. TAF1-mediated p53-Thr55 phosphorylation in response to UV damage or high glucose (HG) treatment terminates the transcription of p21, an important regulator of G1 progression.

In Chapter 2, as a first step to identify additional p53 target genes regulated by TAF1-mediated p53-Thr55 phosphorylation through genome-wide transcription and protein-DNA interaction assays, I recapitulated the HG-induced, TAF1-, and p53-dependent repression of p21 and confirmed the conditions under which maximal repression of p21 can be observed. Furthermore, my preliminary results suggest that HG can alter p53 protein levels in response to UV damage and provide a model for how HG increases the cancer risk of diabetics.

In Chapter 3, I collected lists of p53- and TAF1- target genes, and HG-regulated genes via bioinformatics. I identified a set of genes commonly activated in response to different stress conditions: 12 p53-activated and 11 p53-repressed TAF1 target genes differentially expressed by HG, suggesting that these genes are regulated via TAF1-mediated p53-Thr55 phosphorylation. Furthermore, I describe a number of transcription factors representing putative targets for regulation via TAF-mediated phosphorylation.

In Chapter 4, I describe tools to further investigate the relationship between TAF1 and p53. A 2x3'BS p21-LUC construct shall allow discernment of the role of different p53 DNA binding sites in the differential recruitment of TAF1 by DNA bound p53. Two TAF1 kinase mutant expression constructs shall facilitate the future elucidation of the TAF1 kinase domain responsible for p53-Thr55 phosphorylation in vivo. Finally, my preliminary results suggest that the p53 target gene NOXA is regulated via TAF1-mediated p53-Thr55 phosphorylation in response to UV, indicating that TAF1-mediated phosphorylation of p53-Thr55 may be involved in regulating the expression of a subset of p53 target genes.

The relationship between metabolism and cancer is formed through molecular mechanisms which allow for the growth of all cells in our body. Unfortunately, cancer cells rely on similar growth pathways found in normal healthy cells but reprogram them to adopt a cellular growth advantage. Recent research has shown that there is a higher occurrence of cancer, such as breast, liver, and colon cancer, in patients with diabetes. Diabetes is characterized by altered metabolism; to understand how these alterations relate to increased cancer incidences, our lab investigated the molecular effects of a high glucose treatment in cancer cells, as this mimics the hyperglycemic conditions in diabetic patients. Previously our laboratory demonstrated that TAF1, a general transcription factor with intrinsic kinase activity, phosphorylates p53, a tumor suppressor and transcription factor known to protect the cell from DNA damage and maintain genomic integrity. Phosphorylation of p53 at Thr55 results in dissociation of p53 and TAF1 from promoters leading to transcription termination. Importantly, this regulation event contributes to cell cycle progression from G1 to S phase through dissociation from the p21 promoter. In addition, the lab has shown that TAF1 kinase activity is governed by cellular ATP concentrations, and may function as an ATP sensor in cells. Because TAF1 kinase is cellular ATP dependent, HG may increase tumorigenesis in vivo through Thr55-phosphorylation and inhibition of p53. This dissertation aims to provide additional evidence for the role TAF1 kinase and p53 regulation plays in a diabetic mouse model. My studies include creation of a T55A phosphorylation mutant through CRISPR/Cas9 genome editing, and the effects this mutation has on p53 regulation both in cell culture and in vivo. Further, I determine that our working model can be replicated in vivo, suggesting the importance of TAF1 kinase regulation and p53 inactivation in tumorigenesis. Additionally, I discover a new model for the role hyperglycemia plays in cell migration. Because the molecular mechanisms behind the link between diabetes and increased cancer risk are largely unknown, the discoveries shown here provide evidence that hyperglycemia can lead to cancer progression through multiple pathways.

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