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Pediatric medulloblastoma (MB) is a cerebellar brain tumor namely characterized by its origination in early development, as early as embryogenesis. MB is thought to originate from the highly heterogeneous granular neuron precursor (GNP) cell population that resides within the rhombic lip of the dorsal hindbrain region, and is particularly susceptible to the effects of the oncogenic Sonic Hedgehog (SHH) signaling pathway. Patched 1 (Ptch1), typically a transmembrane SHH pathway tumor suppressor gene, is mutated in 20% of MB cases, otherwise known as SHH-group MBs. This mutation in MB presents as a loss of heterozygosity (LOH), where the wild type allele of Ptch 1 is deleted. Ptch 1 receptor silencing activates downstream target genes such as proto-oncogene Smoothened (Smo) and allows for the initiation of tumorigenesis. However, the molecular basis for Ptch1 LOH in MB remains elusive. We have discovered a cancer-testis antigen, Protamine 1 (Prm 1), that is present in the Ptch 1 locus in SHH-group MB tumors. By utilization of the RNAscope technique, we confirm mRNA expression of Prm 1 in cerebellar tumor tissue, predominantly from tumor cells, but not in stromal cells. These studies reveal that tumor cells highjack the promoter of Ptch 1 to express Prm 1, promoting tumor progression. These findings establish the mechanism for Ptch 1 LOH in SHH-group MB, and provide the rationale to define the cell of origin for SHH group MB based on Prm 1 expression. / Biomedical Sciences

Cutaneous malignant melanoma is the deadliest form of skin cancer with a rising incidence rate. Epidemiological studies show exposure to ultraviolet radiation (UVR) cause 80% of melanomas. However, the underlying molecular mechanisms by which UVR promotes melanomagenesis are unclear. The mutagenic properties of UVR are incontrovertible; however, well-studied driver mutations of melanomagenesis (BRAF V600E and NRAS Q61L/R) do not bear UVR signature mutations and so the role UVR mutations play in the early initiation of melanoma remains controversial. This highlights the gap in knowledge of the initial critical molecular mechanisms of UVR-induced melanomas and warrant investigating non-mutational mechanisms as causal factors of UVR-induced melanomagenesis. Aberrant DNA methylation is a signature of melanoma and regulates expression of important tumor suppressors. While epigenetic dysregulation is an important aspect of melanoma etiology, it has never been investigated in the context of UVR. We hypothesize that these initial UVR-induced DNA methylation changes may sensitize a field of melanocytes to acquiring subsequent complementary spontaneous and/or UVR-induced genetic mutations and render them susceptible to melanomagenesis. My preliminary data demonstrate that UVR can modulate DNA methylation in melanocytes and suggests a pigment dependent mechanism. UVR-induced DNA methylation changes in highly pigmented melanocytes primarily in intergenic regions as areas of active transcription were protected from 5’mC changes. Additionally, UVR induced long-term transcriptional changes in both dark and light pigmented melanocytes suggesting multiple epigenetic mechanisms being altered. Evaluation of the protein regulation of the enzymes involved in writing or erasing 5’mC point towards a dysregulation in TET2. Further work is needed to determine if changes in TET2 could contribute to the observed methylation changes. To determine if these methylation changes had any significance to melanoma development, they were compared to the skin cutaneous melanoma cohort in the TCGA database which found a modest correlation in UVR-induced methylation changes and those found in melanoma patients. Interestingly, 5’mC at UVR-sensitive sites was prognostic of patient survival. A highly pigmented human melanoma cell line was UV-irradiated to see if DNA methylation can also be affected in transformed cells; however, no changes were observed. This suggests UV-induced methylation contributes to early changes in melanoma development and/or other relevant physiological changes within the melanocytes. Altogether, these data identify a novel non-mutation mechanism by which UVR may contribute of melanomagenesis. / Cancer Biology & Genetics

It is well known that chromatin components are key players in establishing and maintaining spatial and temporal gene expression in plants, however, little is known about the epigenetic regulation on cell wall degradation and regeneration. This study aimed to 1) investigate the global proteome and phosphoproteome of rice chromatin, and 2) characterize changes in chromatin components and chromatin structure associated with cell wall degradation and regeneration, and 3) characterize the differentially regulated proteins and eventually explore the mechanism. In this dissertation, we examine proteins copurified with chromatin using both 2-DE gel and shotgun approaches from rice (Oryza sativa) suspension cells. Nine hundred seventy-two distinct protein spots were resolved on 2-DE gels and 509 proteins were identified by MALDI-MS/MS following gel excision, these correspond to 269 unique proteins. When the chromatin copurified proteins are examined using shotgun proteomics, a large number of histone variants in addition to the four common core histones were identified. Furthermore, putative phosphoproteins copurified with chromatin were examined using Pro-Q Diamond phosphoprotein stain and followed by MALDI-MS/MS. Our studies provided new insights into the chromatin composition in plants. To study the epigenetic regulation of the cell wall degradation and regeneration, we examined cellular responses to the enzymatic removal of the cell wall in rice suspension cells using proteomic approaches. We found that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Microscopy examination and chromatin decondensation assay further showed that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses revealed substantial histone modification changes, particularly H3K18AC and H3K23AC, are associated with the degradation and regeneration of the cell wall. Labelree comparative proteome analyses further revealed that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, particularly cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.

Cancer is the second leading cause of death in females. According to the American Cancer Society, there are 327,660 new cases in breast and gynecological cancers estimated in 2014, placing emphasis on the need for cancer prevention and new cancer treatment strategies. One important approach to cancer prevention involves phytochemicals, biologically active compounds derived from plants. A variety of studies on the impact of dietary compounds found in cruciferous vegetables, green tea, and spices like curry and black pepper have revealed epigenetic changes in female cancers. Thus, an important emerging topic comprises epigenetic changes due to the modulation of noncoding RNA levels. Since it has been shown that non-coding RNAs such as microRNAs and long non-coding RNAs are aberrantly expressed in cancer, and furthermore are linked to distinct cancer phenotypes, understanding the effects of dietary compounds and supplements on the epigenetic modulator non-coding RNA is of great interest. This article reviews the current findings on nutrition-induced changes in breast and gynecological cancers at the non-coding RNA level.

Suppression of genes involved in DNA repair, tumor suppression and detoxification through epigenetic modifications has been implicated in the etiology of cancer. As such analysis of promoter methylation patterns in genes frequently down regulated in breast cancer in non-cancerous subjects may serve as an indicator of breast cancer risk. CpG-island hypermethylation of single genes has been detected in cells isolated from nipple aspirate and ductal lavage, yet both isolation methods yield insufficient cells to complete an extensive analysis on any one donor sample. As an alternative we have turned to magnetic separation of human mammary epithelial cells from breast milk. Initial studies with these cells, which are detailed in chapter one, show that a breast milk sample provides sufficient epithelial cells to isolate high quality RNA for gene expression analyses or genomic DNA for methylation analysis of multiple genes. Using quantitative RT-PCR of RNA collected from these samples we detected differences in the mRNA levels for six genes known to be down regulated in breast cancers: BRCA1, p16, CDH1, TMS-1, GSTPi, and SFRP1. Additionally using methyl-specific PCR (MSP) we assayed for a small panel of genes frequently methylated in cancer and found them to be unmethylated in the few breast milk samples examined. However, given the small number of CpG sites which can be assayed by the MSP technique it is not surprising that methylation was not detected in disease-free subjects. With methods for collecting breast milk samples and processing them for genetic material established we turned to a more comprehensive study of DNA methylation in larger population of donors which is detailed in chapter two. Utilizing a highly sensitive and highly quantitative methylation analysis technique known as Pyrosequencing we examined age-related methylation patterns for RASSF1A, TMS-1, CDH1, SFRP1, GSTPi, and CRBP1 in genomic DNA purified from exfoliated epithelial cells magnetically enriched from breast milk (n=111) and whether the protective effects conveyed by early pregnancy could be partly due to decreases in DNA hypermethylation. Although firm answers about early pregnancy were inconclusive based on our sample pool, this body of work lays down a solid foundation for future studies.