Colorectal cancer (CRC) is one of the most prevalent cancers worldwide and is recognized as the second leading cause of cancer-related deaths in North America. CRC follows a hierarchal tumor organization, the root being a small population of self-renewing and highly tumorigenic colon cancer stem cells (CSC). There is an epigenetic signature that exists within these colon CSCs contributing to their maintenance and dynamic plasticity. A key hallmark of this colon CSC epigenetic signature is the Histone-3 Lysine-9 di-methylation (H3K9me2) histone mark which is overrepresented in many types of cancer. Pharmacological modulation of this epigenetic signature (i.e through H3K9me2 modulation) serves as a novel way to selectively target and eliminate human CSCs while sparing normal progenitor cells. Direct inhibitors of key methyltransferases such as G9a, have been identified to have high specificity, however none of these inhibitors have shown success during early stages of clinical trials, leaving us to question the clinical relevance. My research has shown that the overexpression of histone methyltransferase G9a in colon cancer serves as a risk factor for patients and is associated with shorter-relapse free survival. G9a activity has been shown to be essential for the maintenance of embryonic-like transcriptional signature which promotes self-renewal, tumorigenicity and an undifferentiated state. This work provides insights into the role of G9a as a driver of a cancer stem cell phenotype. To combat the toxicity and issues associated with targeting the catalytic activity of G9a, I utilized a phenotypic screening pipeline consisting of thousands of clinically-approved compounds, and identified CSC-bioactive epigenetic inhibitors showing promise in CSC-like models. RNA-seq profiling, dose response treatments and molecular techniques were used to confirm the selectivity of these candidates to colon-CSC like cells with minimal impact on normal progenitors. The lead candidate compound, vanoxerine (VXN) restricts organoid-initiating capacity of patient derived colon CSCs in serial 3D organoid formation assays that I developed throughout my research project. Using two murine syngeneic models resembling microsatellite instability and stability in CRC, I found this compound to be successful in decreasing primary tumor volumes compared to vehicle control mice, through epigenetic reprogramming and infiltration of immune cells. Drug treated tumors that were harvested, dissociated and re-injected into secondary mice showed diminished tumor initiating capacity compared to vehicle controls. Furthermore, the target of vanoxerine, SLC6A3, was investigated and the expression pattern characterized revealed a new potential biomarker for colon cancer stem cells. This SLC6A3-G9a axis discovered for the first time in colon cancer and serves as a novel and important pathway to block H3K9me2 deposition in CRC, rewiring the CSC epigenome and suppressing neoplastic self-renewal and tumor-initiating functions. Together, the identified repurposed compound selectively targets and modulates the epigenetic signature of CSCs which diminishes the tumor initiating function of these cells. This hints at an interesting interaction between CRC stemness and tumor immunology, a promising future therapeutic avenue.

Herein we report the structure-activity and structure-physicochemical property relationships of a series of class I selective ortho-aminoanilides targeting the “foot-pocket” in HDAC1&2. To balance the structural benefits and the physicochemical disadvantages of these substances, we started with a set of HDACi related to tacedinaline (CI-994) and evaluated their solubility, lipophilicity (log D7.4) and inhibition of selected HDAC isoforms. Subsequently, we selected the most promising “capless” HDACi and transferred its ZBG to our previously published scaffold featuring a peptoid-based cap group. The resulting hit compound 10c (LSH-A54) showed favorable physicochemical properties and is a potent, selective HDAC1/2 inhibitor. The following evaluation of its slow binding properties revealed that LSH-A54 binds tightly to HDAC1 in an induced-fit mechanism. The potent HDAC1/2 inhibitory properties were reflected by attenuated cell migration in a modified wound healing assay and reduced cell viability in a clonogenic survival assay in selected breast cancer cell lines.

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Artificial insemination (AI) using cryopreserved sperm has an important positive impact on cattle production. Fertility is the most critical trait controlling livestock production; however, molecular, cellular, and physiological determinants of bull fertility and sperm freezability are not well understood. Better understanding of molecular, cellular, and physiological underpinnings of bull fertility may increase the success rate of AI. The objective of this study was to test the hypothesis that expression dynamics of sperm nuclear proteins, post-translational modifications (PTM) of sperm Histone 4 (H4), and seminal plasma metabolome are associated with bull fertility and sperm freezability (P = 0.043). Flow cytometry experiments were conducted to quantify H4 and acetylated histone 4 (H4ac) in sperm from high and low fertility Holstein bulls. The analysis of flow cytometry experiments clarified that retained levels of H4ac in bull sperm are associated with bull fertility. In addition, gas chromatography-mass spectrometry (GC-MS) was applied to ascertain the amino acid concentration of seminal plasma from bull semen with various freezability. A total of 21 amino acids and isomers were identified, and phenylalanine was positively associated with sperm post-thaw viability (r = 0.57, P-value = 0.043). Lastly, a quantitative western blotting experiment was utilized to ascertain relative quantification of sperm nuclear proteins including protamine 1 (PRM1), protamine 2 (PRM2), Histone 3 (H3), and H4. Also, sperm functional parameters including acrosome reaction, DNA fragmentation index, PAWP expression were analyzed using flow cytometry. In addition, immunocytochemistry experiments were applied to analyze sperm chromatin decondensation ability. The analyses of western blotting experiments revealed that the relative abundance of PRM2 in poor freezability sperm (PF) was greater than those in good freezability sperm (GF) (P = 0.0259). The relative abundance of retained H3 was greater in PF bulls than in GF bulls (1.02 ± 0.005 and 0.969 ± 0.021, respectively; P = 0.0272). There was a positive correlation between the abundance of retained H4 and sperm decondensation state (r = 0.71, P = 0.05). These results are important because they can help advance fundamental andrology and the assisted reproductive technologies both for cattle and other mammals, including humans and endangered species.

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DNA methylation regulates gene expression through a complex network of protein/protein and protein/DNA interactions in chromatin. The maintenance methylase, DNA methyltransferase 1 (DNMT1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications in somatic cells. The mechanistic details that explain how DNMT1 catalytic action is directed in a chromatin setting are not well understood. We hypothesize that post translational modifications and a variety of protein-protein interactions processes are key regulatory elements that set the methylation of CpG elements essential for normal growth behavior in somatic cells. These fundamental processes can be disrupted by DNA damage leading to inappropriate gene silencing and loss of growth control in somatic cells. First, we show that DNMT1 is post-translationally modified by sumoylation and we have mapped these sumoylation sites by defined mutations. Sumoylated DNMT1 is catalytically active on genomic DNA in vivo and substantially increases the enzymatic activity of DNMT1 both in vitro and in chromatin. These data establish that sumoylation modulates the endogenous activity of a prominent epigenetic maintenance pathway in cells. Second, we investigated novel mechanisms whereby somatic cells can erase then reset DNA methylation events in somatic cells. In this study, the relationship between DNA damage and gene silencing was explored. To this end, we generated a HeLa cell line containing a specialized GFP reporter cassette (DRGFP) containing two mutated GFP genes and a unique ISceI restriction endonuclease site. These cells do not express GFP. A unique double strand break is then delivered by transfecting in the gene for I-SceI. About 4% of the cells produced a functional GFP by gene conversion and homologous recombination (HR); however roughly half iv of the GFP recombinants expressed the gene poorly and this was attributed to gene silencing. Silencing of the GFP expressing cell clones was due to DNA methylation and could be reversed using a drug that inhibits global methylation (5-aza-2'-deoxycytidine). Approximately half of the repaired genes were heavily methylated, and half were hypomethylated. That is, a key intermediate methylation state after HR repair is hemimethylated DNA, defined as methylation limited to one strand. Evidence is given that DNMT1 is acting as a de novo methylase at the HR repair patches in cells. Moreover, the DNA damage inducible protein, GADD45, interacts specifically with the catalytic domain of DNMT1 and GADD45 binds with extremely high affinity to hemimethylated DNA sites. Thus, GADD45 is a key regulatory element in silencing of HR repaired DNA segments and appears to inhibit the activity of DNMT1. Consistent with these results, we found that GADD45 increased the expression of recombinant GFP following HR repair, further suggesting its role in orchestrating strand specific DNA methylation by DNMT1. Since these experiments were performed in live cells, there is strong physiological relevance. We propose that DS DNA damage and the resulting HR process involves precise, strand selected DNA methylation mediated by the prominent methylase enzyme, DNMT1. Moreover, DS DNA break repair through HR and gene conversion, may potentially erase and reset DNA methylation patterns and therefore alter the expression of repaired genes. The overall process is tightly regulated by the DNA damage inducible protein GADD45, which may coordinate strand specific methylation by recruiting DNMT1 to HR repair templates. The ability of GADD45 to modulate DNMT1 catalytic activity may explain its role as a passive mediator of demethylation that has been reported by other groups. The overall process of silencing post DNA repair is a strong evolutionary force that may predispose cells to malignant transformation

Chromatin is decorated by a large array of biochemical modifications made to DNA and histone proteins. These modifications—and the broader organizational structure of chromatin—provide an important additional layer of information that is superimposed upon genome sequence and thus are widely referred to as the epigenome. The epigenome helps control which genes are expressed in a given context to produce the gene expression patterns that underlie the many different cellular phenotypes that arise during an organism’s development, and determine how these gene expression patterns are subsequently maintained for the life of an organism. The epigenetically heritable states are maintained and transmitted by self-propagating epigenetic mechanisms that persist in the absence of an initial stimulus. These epigenetic programs are generally thought to be controlled by core regulatory networks involving molecular writers and readers of chromatin marks. Guided by these principles, in this dissertation, we establish an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system consists of synthetic factors that can write and read m6A, and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we use our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits are able to program epigenetic functions de novo, conceptually validating “read-write” architectures. This dissertation outlines a synthetic framework for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells. / 2021-08-30T00:00:00Z

Transcription factor LSF is an oncogene in Hepatocellular Carcinoma (HCC). HCC is the sixth most common cancer worldwide and the second highest cause of cancer-related death globally. LSF is overexpressed in human HCC cell lines, compared to normal hepatocytes, and expression levels show significant correlation with the stage and grades of the disease. Current treatments for HCC are insufficient, especially given the frequency of late stage diagnoses. Therefore, it is necessary to understand the molecular mechanism of HCC disease to aid in targeted and effective treatments. Most investigations of the regulation of LSF activity have focused on its post-translational modifications in response to cellular proliferation and signal transduction. Chromatin modifications and epigenetic mechanisms of LSF-mediated gene regulation had not been investigated. Given that alterations of epigenetic writers or readers have been demonstrated in a large fraction of HCC patient samples, I examined the connection between LSF and epigenetic regulators. In particular, LSF is shown to interact with DNA methyltransferase 1 (DNMT1) and Ubiquitin like with PHD and Ring Finger Domains (UHRF1), with consequences for global DNA methylation and transcription patterns. Additionally, I identified unexpected, pairwise associations between LSF, histone methyltransferase SET8, and tubulin, both in vitro and in vivo. The interactions were identified by proteomics analyses, co-localization, co-immunoprecipitation, and direct protein-protein interaction studies in vitro. Strikingly, both LSF and SET8 associate with microtubules, leading to the discovery that SET8 methylates α-tubulin at several novel, specific lysines. This suggests parallels between regulation of chromatin by the histone code and regulation of microtubule function by the tubulin code. Surprisingly, LSF enhances tubulin methylation by SET8 in vitro and FQI1, a specific LSF small molecule inhibitor, reduces tubulin methylation. Furthermore, LSF promotes, and FQI1 inhibits, tubulin polymerization in vitro. Taken together, these findings suggest that SET8 is a microtubule-associated methyltransferase that LSF recruits to microtubules to enhance tubulin modification. The results indicate that both LSF and SET8 have cellular implications beyond their roles in gene transcription and histone methylation. Finally, this discovery of the dual functions for LSF and SET8 set up the possibility for connections between epigenetic and cytoskeleton modifications in cancer. / 2021-12-24T00:00:00Z