The phaseolin (phas) promoter drives the copious production of transcripts encoding the protein phaseolin during seed embryogenesis but is silent in vegetative tissues when a nucleosome is positioned over its three phased TATA boxes. Transition from the inactive state in transgenic Arabidopsis leaves was accomplished by ectopic expression of the transcription factor PvALF (Phaseolus vulgaris ABI3-like factor), and application of abscisic acid (ABA). PvALF belongs to a family of seed-specific transcriptional activators that includes the maize viviparious1 (VP1) and the Arabidopsis abscisic acid-insensitive3 (ABI3) proteins. The major goal of the study is to gain insight to the regulation of seed-specific gene expression in three different aspects. First, since ABI3 (homolog of PvALF) is involved in ABA-mediated expression of several seed-specific protein genes in Arabidopsis, understanding its transcriptional regulation will provide insight to the mechanism by which PvALF expression is controlled. To achieve this, ABI3 promoter deletion analysis using either $-glucuronidase (gus) or green fluorescent protein (gfp) reporter gene fusions have identified various regulatory regions within the ABI3 promoter including two upstream activating sequences and a minimal seed specific expression region. In addition, a 405 bp 5' UTR was shown to play a negative role in ABI3 expression, possibly through post-transcriptional mechanisms. Second, placement of PvALF expression under control of an estradiol-inducible promoter permitted chronological ChIP analysis of changes in histone modifications, notably increased acetylation of H3-K9, as phas chromatin is remodeled (potentiated). A different array of changes (trimethylation of H3-K4) is associated with ABA-mediated activation. In contrast, H3-K14 acetylation decreased upon phas potentiation and increased on activation. Whereas decreases in histone H3 and H4 levels were detected during PvALF-mediated remodeling, slight increases occurred following ABA-mediated activation, suggesting the restoration of histone-phas interactions or the redeposition of histones in the phas chromatin. The observed histone modifications thus provide insight to the factors involved in euchromatinization and activation of a plant gene. Finally, ectopically expressed ABI5 and PvALF renders the activation of phas ABA-independent, suggesting ABI5 acts downstream of ABA during phas activation.

Histone Deacetylase (HDAC) plays a vital role in cellular processes, for example gene expression, cell growth, and apoptosis. Finding drug candidates to inhibit the over activity of HDACs in cancer is a growing area of interest. Inhibitors, thus far, have three important motifs to be studied: the zinc binding group, a hydrophobic linker, and a cap group. By altering these groups on the inhibitor, not only can activity be increased but also selectivity within the classes of HDACs. We present the design of two novel sets of molecules that contain either a 1,2,3-triazole or 1,2,4-triazole. The 1,2,3-triazoles were synthesized using “click chemistry” with a novel pyridyl triazine catalyst. The 1,2,4-triazoles were synthesized utilizing substitution chemistry. This set of molecules was designed after suberoylanilide hydroxamic acid (SAHA) but replaced the hydroxamate with the triazole as the zinc binding group. The activity of these inhibitors against HDAC 1, HDAC 6, and SIRT 1 were tested using the Biomol Fluor de Lys in vitro kits. Though none of the synthesized compounds were strong activators or inhibitors of any of the classes of HDACs, trends were observed that could lead to the design of more potent inhibitors.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 118-130.

Histone deacetylase (HDAC) inhibition has recently emerged as a novel therapy for cancer treatment. However, currently approved histone deacetylase inhibitors (HDACi) are pan-inhibitors thus inhibiting all 11 zinc dependent HDAC isoforms including those not involved in tumorigenesis. These inhibitors are also associated with various side effects including a potentially fatal cardiotoxicity. To address these issues, isoform selective HDACi were designed and synthesized. The use of 3-hydroxy-pyridin-2-thione (3HPT) as zinc chelation group resulted in small molecules devoid of HDAC1 inhibition but active against HDAC6 and/or 8. Selected 3HPT containing HDACi displayed anticancer activity against various cancer cell lines including DU145, LNCaP and Jurkat. Surprisingly, the lead-compounds were very potent against Jurkat Jγ cells which are resistant to SAHA-induced apoptosis. HDACi were also targeted to cancer cells using folic or pteroic acids as targeting groups. Incorporation of the folic acid into the HDACi pharmacophoric model resulted in inhibitors selective for HDAC6, whereas pteroic-based HDACi inhibited both HDAC1 and 6. Only the pteroic-based inhibitors displayed anticancer activities against folate receptor overexpressing tumors such KB and HeLa. Furthermore, cell-based studies established the inhibition of HDAC1 as the basis for the anticancer activities of the pteroic-based HDACi.

Spt6 is a conserved eukaryotic transcription factor, known to interact with both nucleosomes and RNA polymerase II (RNAPII) to control transcription. We have initiated study of Spt6 in S. pombe in order to identify both novel and conserved roles in regulation of transcription and chromatin. We first constructed and analyzed spt6 mutants by several approaches. As Spt6 is known to be required for histone H3K36 methylation in both Saccharomyces cerevisiae and human cells, we examined the global levels of several histone modifications; we found that in S. pombe, Spt6 is required for both H3K4 and H3K36 trimethylation. We examined the chromatin state at two highly expressed genes, \(act1^+\) and \(pma1^+\), and found that there is a defect in recruitment of the methyltransferases responsible for those marks, Set1 and Set2, respectively. We also observed loss of nucleosomes, as well as a decrease in histone H2B monoubiquitylation. These results suggest that Spt6 plays an important role in chromatin regulation during transcription. We also conducted transcriptional analysis of an spt6 mutant by both microarray and high-throughput sequencing (RNA-seq) and discovered that Spt6 plays a critical role in maintaining the integrity of transcription genome-wide. We found that Spt6 is required to repress antisense transcription, with nearly 70% of genes having antisense transcripts increased by at least two-fold in an spt6 mutant. We also found that transcription of most long terminal repeats (LTRs) is derepressed. Finally, we found that a major class of transcripts elevated in the spt6 mutant is derived from heterochromatin, which is normally silenced. To study the heterochromatic silencing defect in greater detail, we analyzed the chromatin state of the pericentric repeats and found a decrease in H3K9 trimethylation, elevated levels of H3K14 acetylation, reduced recruitment of several known silencing factors and a loss of siRNA production. We also see a very modest increase in RNAPII recruitment. Based on this combination of phenotypes, Spt6 is likely to contribute to both transcriptional and post-transcriptional silencing mechanisms. Taken together, we have found that Spt6 plays several important roles to control transcription in both euchromatin and heterochromatin in S. pombe.

Chromatin structure is crucial to regulate access to the genome for processes such as a transcription, recombination, DNA repair, and DNA replication. Spt6, a key factor involved in regulating chromatin structure, is conserved throughout eukaryotes. Spt6 has been shown to function in many aspects of gene expression, including nucleosome assembly, transcription initiation and elongation, and mRNA processing and export. In addition, Spt6 has several conserved domains; however, little is known about their functions. I have performed a structure-function analysis of Spt6 using three separate approaches. First, I employed a random insertion mutagenesis that has identified sixty-seven mutants. While these mutants did not provide information regarding known domains, some have phenotypes that may prove useful for future study. Second, in a collaborative project with Romier lab, I studied the functional roles of the Spt6 SH2 domains. I have shown that deletion of the region of Spt6 encoding the SH2 domains causes severe mutant phenotypes without affecting Spt6 protein levels, demonstrating the importance of the SH2 domains of Spt6. Third, in an additional collaboration with the Romier lab, I showed that mutations that alter the region of Spt6 that interacts with the conserved transcription factor Spn1 impair Spt6 functions in vivo. Overall, this multi-pronged structure-function analysis of Spt6 has provided new insights into the tandem SH2 domains of Spt6, the Spt6-Spn1 interaction, and the uses and limitations of insertion mutagenesis. In addition, I have attempted to explore a possible role for Spt6 in transcription-associated mutagenesis. After employing several types of in vivo assays, I conclude that a possible role for Spt6 in transcription-associated mutagenesis is uncertain, as the results (with respect to a role for Spt6) reproducibly vary depending on the assay used. Thus, understanding this aspect of Spt6 biology awaits better assays and understanding of transcription-associated mutagenesis. Overall, the work in this dissertation will serve to further elucidate the mechanisms of Spt6 in chromatin regulation, transcription, and DNA damage repair.

Humans are exposed to arsenicals through many routes with the most common being drinking water. Exposure to arsenic has been associated with an increased incidence of skin, lung, liver, prostate, and bladder cancer. Although the relationship between arsenic exposure and carcinogenesis is well documented, the mechanisms by which arsenic participates in tumorigenesis are not fully elucidated. We evaluated the potential epigenetic component of arsenical action by assessing the histone acetylation and DNA methylation state of 13,000 human gene promoters in a cell line model of arsenical-mediated malignant transformation. We show changes in histone H3 acetylation and DNA methylation occur during arsenical-induced malignant transformation, each of which is linked to the expression state of the associated gene. These epigenetic changes occurred non-randomly and targeted common promoters whether the selection was performed with arsenite [As(III)] or with the As(III) metabolite monomethylarsonous acid [MMA(III)]. The epigenetic alterations of these promoters and associated malignant phenotypes were stable after the removal of the transforming arsenical. One of the affected regions was the promoter of WNT5A. This gene is transcriptionally activated during arsenical induced malignant transformation and its promoter region exhibited alterations in each of the four histone modifications examined which were linked to its transcriptional activation. Experimental reduction of WNT5A transcript levels resulted in abrogated anchorage independent growth, suggesting a participative role for the epigenetic remodeling of this promoter region in arsenical-induced malignant transformation. Taken together, these data suggest that arsenicals may participate in tumorigenesis by stably altering the DNA methylation and histone modifications associated with targeted genes, uncovering a likely set of participative genes and representing a mechanism to potentially explain the latency associated with arsenic-induced malignancy.

Long terminal repeat (LTR) retrotransposons account for approximately 10% of mouse and 8% of human genomes and may play a role in modifying gene expression. Many species harbor retrotransposon families encompassing both autonomous and non-autonomous members. Specifically, the mouse Early Transposon (ETn) family members lack all retroviral genes but are transcriptionally and retrotranspositionally active, causing over 20 known insertional germline mutations. ETns owe their retrotransposition potential to proteins encoded by structurally intact MusD retrotransposons with whom they share LTRs. ETn elements are transcribed at a much higher level than MusD retrotransposons in embryos and undifferentiated cells, suggesting their evasion of host restriction mechanisms. However, mechanisms responsible for the replicative success of non-autonomous retrotransposon subfamilies over their coding-competent relatives are poorly understood. In the first stage of my research, I analyzed regulatory sequences in an ETn LTR responsible for its high promoter activity in the undifferentiated cell line P19. I found that three GC-boxes that may function as Sp1/Sp3 binding sites act synergistically and are indispensable for undifferentiated cell-specific promoter activity of the LTR. Sp1 binding partners may be responsible for the restricted ETn expression. Moreover, I have shown that unlike many retroviruses, ETn elements possess multiple transcription initiation sites and that they have amplified via intracellular retrotransposition in the P19 teratocarcinoma cell line. In the next step of my research, I performed analysis of epigenetic mechanisms as a means of ERV suppression. Specifically, I showed that in embryonic stem cells, autonomous MusD retrotransposons are epigenetically suppressed to a greater degree than non-autonomous ETn retrotransposons, illustrated by a higher level of DNA methylation and a lower level of active histone modifications. I hypothesize that MusD elements may be silenced by DNA methylation and repressive chromatin spreading into the LTR from the CpG-rich internal retroviral sequence absent in ETn elements. I propose that internal structure largely devoid of high CG content enables ETn elements to evade host-imposed transcriptional repression, contributing to their high mutagenic activity in the mouse germline.

The budding yeast, Saccharomyces cerevisiae, serves as an excellent model for identifying fundamental mechanisms of DNA repair. A Local Coherence Detection (LCD) algorithm that uses biclustering to assign genes to multiple functional sub-groups was applied on the chromosome E-MAP containing genetic interactions among genes involved in nuclear processes. Using this method, we found that Asf1 and Rtt109, genes that are together required for histone H3K56 acetylation, cluster together with Ctf4, Ctf18, Ctf8 and Dcc1, genes important for efficient sister chromatid cohesion. It is known that H3K56 acetylation is required for post-repair chromatin reassembly at sites of DNA double-strand breaks (DSBs). The cohesion genes were previously implicated in the repair of some DNA DSBs, but the nature of their involvement has not been reported. The experimental data in my thesis work suggest that Ctf4, Ctf8, Ctf18 and Dcc1 function in the post-repair chromatin reassembly pathway.

No description available.