Cereal endosperm represents half of all human food calories and serves as the primary feedstock for livestock. The regulatory mechanism of cereal endosperm development is largely unknown. Polycomb complex has been shown to play a key role in the regulation of endosperm development in Arabidopsis, but its role in cereal endosperm development remains obscure. In addition, the enzyme activities of all the plant polycomb complexes have not been demonstrated in vitro. Here we purified the rice OsFIE2-polycomb complex using tandem affinity purification and demonstrated its specific H3 methyltransferase activity. We found that the OsFIE2 gene product was responsible for H3K27me3 production specifically in vivo and the gene expression was not regulated by imprinting. Genetic studies showed that a severe reduction of OsFIE2 expression led to completely endospermree seeds and a moderated reduction of OsFIE2 expression resulted in smaller seeds and loss of seed dormancy. Genome wide ChIP-seq analyses found that a large number of endosperm specific regulatory genes and storage nutrient metabolic pathway genes were directly regulated by H3K27me3 modification in the rice endosperm. Our results suggest that OsFIE2-polycomb complex positively regulates rice endosperm development and grain filling via a mechanism different from that in Arabidopsis. In this dissertation, lysine acetylation, an important posttranslational modification in rice was also studied. Lysine acetylation is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases. Recent advances in high-throughput proteomics have greatly contributed to the global analysis of lysine acetylation and a large number of proteins of diverse biological function in mammalian and bacterial cells have been shown to be acetylated. However, the extent of lysine acetylation in non-histone proteins remains largely unknown in plants, especially in cereal crops. Here we report a large scale study of lysine acetylation in rice. We identified 112 lysine acetylated sites on 80 proteins with diverse biological functions. Immunoblot studies further validated the presence of a large number of acetylated nonhistone proteins. Overall, our results suggest that lysine acetylation may constitute an important regulatory mechanism for a large number of proteins including both histones and nonhistone proteins.

University of Arizona Student Showcase 2012 President’s Award, University of Arizona Student Showcase 2012 1st Place Biological Sciences, AACR Special Meeting Pancreatic Cancer: Progress and Challenges Scholar-in-Training Award / Guidance molecules from the Netrin, Slit, Ephrin, and Semaphorin gene families were originally described as cues for the directional guidance of axons in the developing nervous system. More recently, members of these families have been found to have critical roles in epithelial development, angiogenesis and cancer. The SLIT2/ROBO1 signaling axis has properties of a potential tumor suppressor pathway via the inhibition of epithelial cell growth, directional migration, ductal morphogenesis, and is epigenetically silenced in lung, colon and breast cancers. We proposed that changes in SLIT2 and ROBO1 expression in pancreatic ductal adenocarcinoma may mediate ductal expansion following the conversion of PanIN precursor lesions to invasive carcinoma. The SLIT2 receptor, ROBO1, is a member of the immunoglobulin (Ig) superfamily and is required for lung and mammary development in mammals. ROBO1 has an alternative splice variant, DUTT1, and these two variants have different initial exons and initiating codons which may suggest the two proteins have distinct functions. In our studies, we found that all pancreatic cancer cell lines and primary pancreatic cancer specimens express only the DUTT1 isoform. We also determined that as pancreatic cancer cell lines become KRAS-independent, ROBO1 expression increases. Furthermore, using immunohistochemistry (IHC), we found that ROBO1 protein expression in primary pancreatic cancer tissue specimens is localized to the ductal compartment with no stromal staining seen. In normal pancreas, ROBO1 expression is weak while its ligand SLIT2 is strongly expressed in both the acinar and ductal compartments in vitro and in vivo. Mammals encode three SLIT genes (SLIT1-3). The secreted SLIT2 protein is not diffusible, but has a cleavage site within its EGF-like repeats creating two fragments which allow it to act either as a short or long range guidance cue with each fragment appearing to have its own cell-association characteristics. The 5’ promoter of SLIT2 has been shown to be methylated resulting in gene silencing in early stages of several epithelial cancers suggesting a possible tumor suppressor role. miR-218-1 is an intronic microRNA found between exons 15 and 16 of the SLIT2 gene and targets a complimentary sequence in the ROBO1 3’ untranslated region (UTR) indicative of a potential regulation of receptor availability in the presence of ligand. In our studies, we determined that the KRAS-dependent pancreatic cancer cell lines express SLIT2 and ROBO1 in a cell autonomous manner. The KRAS-independent cell lines, however, have silenced SLIT2 and miR-218-1 expression. Using IHC we found that high levels of SLIT2 are seen in normal pancreas localized to the acinar and ductal compartments. Reduced SLIT2 expression is seen in primary pancreatic cancer tissue specimens. We confirmed that loss of SLIT2 mRNA in KRAS-independent lines was due to DNA hypermethylation shown by methylation specific PCR and Sequenom analysis. Chromatin immunoprecipitation analysis shows that silencing histone marks are found in the 5’ promoter of the SLIT2 gene in KRAS-independent lines. Treatment with demethylating agents reactivate SLIT2 and miR-218-1 expression suggesting that epigenetic mechanisms controlling the SLIT2 promoter also regulate miR-218-1 expression. Overall, our data establishes that the SLIT2/ROBO1 signaling axis is a dynamic pathway in pancreatic cancer that can act in the tumor expansion and progression along intrapancreatic neurons that express the SLIT2 ligand.

Ovarian cancer is the leading cause of death among gynecologic malignancies. The risk of developing ovarian cancer in a woman’s lifetime is 1 to 2 in 100. This high rate of development and death from the tumorigenesis is a result of its asymptomatic manifestation. Ovarian cancer is usually found in its advanced stage; therefore the survival rate is lower than for other types of cancers. The most common type of ovarian cancer, serous epithelial ovarian cancer, arises from the surface epithelium of the ovary and less frequent from the fallopian tubes or uterus. The treatment of surgery is limited by the fact that most ovarian cancers are detected after they have metastasized. Chemotherapy is often difficult because of the lack of sufficient target specific drugs. Typically, platinum in combination with other drugs is provided as the standard treatment. These combinations exhibit higher toxicity, are often not target specific, and frequently despite treatment, the tumor relapses. Current studies suggest epigenetics plays a significant role in carcinogenesis by the silencing of tumor suppressor genes (TSG). Histone modifications and the methylation of specific cytosine phosphate guanosine (CpG) residues in the upstream region of genes silence the TSG. Many clinical trials are in progress to develop combination therapies utilizing histone deacetylase inhibitors (HDACi), and DNA methyl transferase I (DNMTI) inhibitors, in combination with other cytotoxic agents. HDACi are known to be effective against different types of leukemia’s, such as Cutaneous T-cell Lymphoma; however, they are not as effective against solid tumors when used as a single agent. Our laboratory was one of the first to demonstrate that HDACi, in addition to its known property to increase histone acetylation, additionally decrease CpG island methylation in the upstream region of TSG. This demethylation causes re-expression of TSG. Our laboratory hypothesizes that re-expression of TSG sensitize cancer cells to other cytotoxic drugs. In an effort to develop improved therapy for ovarian cancer, we employed a combination therapy, which includes epigenetic drugs, HDACi, in combination with calpain protease inhibitor, calpeptin. Calpain is a ubiquitous protease usually activated in cardiovascular diseases and cancer cells. The present study discerns that combination of HDACi and calpeptin produce more than additive growth inhibition of diverse ovarian cancer cells. HDACi re-expressed TSG. Additionally, the observed growth inhibition of ovarian cancer cells was caused by cell-cycle arrest, induction of apoptosis, followed by autophagy. The phosphorylation of growth promoting signaling protein, Mitogen Activated Protein Kinase 1 (ERK), was inhibited. In addition, the inhibitors also partially inhibited phosphorylation of anti-apoptotic protein V-ask Murine Thyomoma Viral Oncogene Homolog 1 (Akt). Collectively, the outcome of this study suggests that epigenetic drugs (HDACi) sensitize the diverse ovarian cancer cell lines by re-expression of TSG, followed by cell death, when treated in combination with calpain inhibitor, calpeptin.

abstract / Psychiatry / Doctoral / Doctor of Philosophy

Epigenetics encompasses the cellular mechanisms that affect gene expression without changing an organism’s actual DNA sequence. Currently, there are three main methods of cellular epigenetic gene regulation: histone modifications, DNA modifications, and non-coding RNAs. Histone and DNA modifications are commonly referred to as ‘tags’ and alter the physical and biochemical environment around the residue or base. Tags are recognized or removed by various protein/enzyme ‘readers’ or ‘erasers’ that result in various changes in DNA transcription. One of the most studied epigenetic modifications, methylation at the 5’-position of cytosine, is successively oxidized by the Ten-eleven translocase (TET1, TET2, and TET3) family of enzymes and results in demethylation. TET2 is found in a complex with histone modifying proteins that link demethylation with histone modifications and eventually changes in gene expression. One such protein, O-linked N-acetylglucosamine transferase (OGT), is an enzyme that transfers N-acetylglucosamine (GlcNAc) moieties onto –OH groups of serine and threonine residues. Due to the co-localization of TET2 and OGT on chromatin, we sought to investigate the possibility of GlcNAcylated 5-hydroxymethylcytosine (5-GlcNAc-hmC). / 2018-06-16T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / The emerging field of epigenetics is expanding our understanding of how biological diversity is generated in the face of genetic limitations. One epigenetic mechanism in particular, DNA methylation, has demonstrated a dynamic range during neural development. Here, we provide evidence that DNA methylation occurs as a cell unique program aiding in the regulation of neurodevelopmental gene expression. DNA methylation has demonstrated sensitivity to external inputs ranging from stress to chemical exposure and dietary factors. To explore DNA methylation as a means of communicating early-life stress to the brain, we utilized a mouse model of fetal alcohol spectrum disorders (FASD). FASD presents a range of neurodevelopmental deficits and is a leading cause of neurodevelopmental disabilities in the United States. Predicated on the knowledge of alcohol's teratogenic role in brain development, we describe that the normal pattern of cortical DNA methylation and epigenetic correlates is similarly impacted by prenatal alcohol exposure. Due to the biochemical interaction of alcohol metabolism and the pathways regulating DNA methylation synthesis, we further investigated whether dietary manipulation could normalize the cortical DNA methylation program and aid in the protection of FASD characteristics. We found that the alcohol sensitive DNA methylation landscape is dually capable of registering dietary intervention, demonstrating normalization of disease-related patterns in the cortex and improved neurodevelopmental gene expression and morphology. Finally, we investigated the DNA methylation landscape in a crucial corticodevelopmental gene to more accurately define the breadth and scope of the environmental impacts at the nucleotide level. We found that alcohol and dietary supplementation are selective for regions associated with transcriptional control. Collectively, the evidence supports that DNA methylation plays a regulatory role in development and that its sensitivity to external inputs is dynamic and detectable at the smallest genomic level. Importantly, DNA methylation landscapes are adaptable and thus bear diagnostic and therapeutic potential.

No description available.

The aims of the work presented in this thesis were: to investigate the role of methylation of 14-3-3σ (a key regulator of p53-mediated G2/M arrest and of translational control during mitosis) in colorectal cancer using colorectal cancer cell lines and fresh colorectal tumours; to investigate any relationship between 14-3-3σ methylation status and gene expression; to determine whether aberrant methylation is associated with cell cycle defects and other factors known to contribute to colorectal carcinogenesis. PCR bisulphite sequencing showed that 78% (7/9) of colorectal cancer cell lines were unmethylated in the 14-3-3σ upstream promoter region (UPR). The unmethylated cell lines expressed high levels of 14-3-3σ, while methylated cell lines expressed negligible levels of 14-3-3σ protein or mRNA. Methylated colorectal cancer cell lines were treated with 5-aza-2’-deoxycytidine and demethylation was confirmed by MSP analysis. However, demethylation did not induce 14-3-3σ re-expression in the methylated cell lines, suggesting that CpG methylation may not be the only mechanism of transcriptional control. In contrast to colorectal cancer cell lines, 90% (89/99) of fresh colorectal tumours were methylated at CpG dinucleotides within the 14-3-3σ UPR. Bisulphite sequencing analysis of individual clones from 14-3-3σ methylated tumours (n =3) demonstrated that the clones displayed methylated CpG levels of approximately 41%. In agreement with previous PCR bisulphite sequencing analysis, there were a low percentage of methylated CpG dinucleotides (~ 15%) in clones from the 14-3-3σ unmethylated tumours (n =3). Unmethylated tumours expressed significantly higher levels of 14-3-3σ in comparison to methylated tumours (p =0.03), indicating that 14-3-3σ methylation may be associated with expression. PCR bisulphite sequencing analysis of matched normal mucosa tissues indicated that the 14-3-3σ UPR was methylated in all samples. Preliminary studies therefore suggest that there is tumour-specific loss of 14-3-3σ methylation in colorectal tumours within the 14-3-3σ UPR and CpG island. There were no apparent clinico-pathological correlations with 14-3-3σ methylation status. Whilst 14-3-3σ methylation was associated with expression in fresh colorectal tumours, there was no significant difference in expression levels between unmethylated colorectal tumours and matched methylated normal tissue. Bisulphite sequencing analysis of individual clones from normal tissues (from patients free of cancer) revealed that the 14-3-3σ UPR and CpG island was methylated at the majority of CpG sites analysed in colonic tissue (422/495, ~85.2%) and approximately half (795/1557, 51.1%) of CpG sites in skin samples (n =3). Furthermore, higher levels of 14-3-3σ protein were observed in skin tissue samples compared to normal colonic tissue, suggesting that 14-3-3σ CpG island methylation may be associated with tissue-specific expression. Experiments to assess the relationship between 14-3-3σ methylation and general methylation defects, suggest that methylation differences in 14-3-3σ were not simply a consequence of more general methylation phenomena well described in colorectal cancer. Nearest Neighbor analysis showed no evidence of generalised hypomethylation. Furthermore, MethyLight analysis of the CpG Island Methylator Phenotype (CIMP) showed no relationship between 14-3-3σ methylation status and CIMP; since, 1/5 (20%) tumours methylated at 14-3-3σ UPR and 1/5 (20%) tumours unmethylated at 14-3-3σ UPR were CIMP positive. In vitro functional assays showed that overexpression of 14-3-3σ in SW480 cells (14-3-3σ methylated) delayed the apoptotic response to UV-C, compared to control SW480 cells. This suggests that 14-3-3σ may protect colorectal cancer cells from apoptosis. MTT assays showed that overexpression of 14-3-3σ in SW480 cells resulted in a trend of increasing proliferation with a significant increase on day 4, compared to controls SW480 cells (p <0.01). Furthermore, FACS-sorted SW480 cells overexpressing 14-3-3σ, showed a significant shift to S-phase from G1 compared to control SW480 cells (p <0.01). Western blot analysis and immunohistochemistry revealed no relationship between p53 status and methylated 14-3-3σ in fresh tumours, while there was no relationship between published p53 status for colorectal cancer cell lines and 14-3-3σ methylation status defined experimentally. I have presented data which shows that methylation status of 14-3-3σ varies between colorectal cancer tissue, colorectal cancer cell lines and normal colonic tissue. Overexpression of 14-3-3σ appears to contribute to colorectal cancer carcinogenesis, raising the hypothesis that 14-3-3σ expression and function may at least in part be dependent on CpG methylation.

Thesis advisor: Hugh P. Cam / Chromatin is the site of numerous structural features that contribute to the regulation of the genome. Although numerous posttranslational modifications to the histone proteins that make up chromatin have been identified, it remains unclear whether and to what extent these modifications might regulate transposons and other repetitive sequences. One such modification is methylation of histone H3 lysine 4 (H3K4me), which is catalyzed by Set1 and its associated complex Set1C/COMPASS. Although H3K4me is associated with actively transcribed regions in euchromatin, an emerging body of evidence suggests that Set1-mediated transcriptional control is often repressive. This thesis work describes expanded functions for Set1C/COMPASS as a regulatory module with roles throughout the genome. We identify novel locus-dependent repressive functions for Set1 at repetitive genomic regions. Interestingly, Set1 has multiple repressive modes that are dependent and independent of H3K4me. Additionally, we show that Set1 controls the nuclear organization of Tf2 retrotransposons by antagonizing H3K4 acetylation. We describe how the roles of Set1 in the nuclear organization and transcriptional repression of Tf2 cooperate to restrict Tf2 transposition. Finally, we identify an H3K4-dependent role in countering the reduced dosage of histone H3 genes to help maintain genome stability and silencing of Tf2s and pericentromeric heterochromatin. Our study considerably expands the regulatory repertoire of an important histone modifier and highlights the multifaceted function by a highly conserved chromatin-modifying complex with diverse roles in genome control. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Epigenetic regulation is a mechanism by which heritable changes in gene expression are controlled by chromatin status rather than primary DNA sequence. Changes in chromatin structure affect accessibility of DNA elements to the transcriptional machinery and thus affect transcription activity of the gene. A key event in this process is reversible modification of core histones, which is catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDs, HDAs, or HDACs). In general, histone deacetylation is related to transcriptional gene silencing, whereas acetylation is associated with gene activation.To study the role of histone deacetylase in plant gene regulation and development, we generated constitutive antisense histone deacetylase 1 (CASH) transgenic plants. AtHD1 is a homolog of RPD3 protein, a global transcriptional regulator in yeast. Expression of the antisense AtHD1 caused dramatic reduction in endogenous AtHD1 transcription, resulting in accumulation of acetylated histones. Down-regulation of histone deacetylation caused a variety of growth and developmental abnormalities and ectopic expression of tissue-specific genes. However, changes in genomic DNA methylation were not detected in repetitive DNA sequences in the transgenic plants.We also identified a T-DNA insertion line in exon 2 of AtHD1 gene (athd1-t1), resulting in a null allele at the locus. The complete inhibition of the AtHD1 expression induced growth and developmental defects similar to those of CASH transgenic plants. The phenotypic abnormalities were heritable across the generations in the mutants. When the athd1-t1/athd1-t1 plants were crossed to wild-type plants, the mutant phenotype was corrected in the F1 hybrids, which correlated with the AtHD1 expression and reduction of histone H4 Lys12 acetylation. Microarray analysis was applied to determine genome-wide changes in transcriptional profiles in the athd1-t1 mutant. Approximately 6.7% (1,753) of the genes were differentially expressed in leaves between the wild-type (Ws) and the athd1-t1 mutant, whereas 4.8% (1,263) of the genes were up- or down-regulated in flower buds of the mutant. These affected genes were randomly distributed across five chromosomes of Arabidopsis and represented a wide range of biological functions. Chromatin immunoprecipitation assays indicated that the activation for a subset of genes was directly associated with changes in acetylation profiles.