Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation.

The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries.

Cell Fate Specification and the Regulation of RNA-dependent DNA Methylation in the Arabidopsis Root Meristem

The Arabidopsis root apical meristem (RAM) is a complex tissue capable of generating all the cell types that ultimately make up the root. The work presented in this thesis takes advantage of the versatility of high-throughput sequencing to address two independent questions about the root meristem. Although a lot of information is known regarding the cell fate decisions that occur at the RAM, cortex specification and differentiation remain poorly understood. In the first part of this thesis, I used an ethylmethanesulfonate (EMS) mutagenized marker line to perform a forward genetics screen. The goal of this screen was to identify novel genes involved in the specification and differentiation of the cortex tissue. Mapping analysis from the results obtained in this screen revealed a new allele of BRASSINOSTEROID4 with abnormal marker expression in the cortex tissue. Although this allele proved to be non-cortex specific, this project highlights new technology that allows mapping of EMS-generated mutations without the need to map-cross or back-cross. In the second part of this thesis, using fluorescence activated cell sorting (FACS) coupled with high throughput sequencing, my collaborators and I generated single-base resolution whole genome DNA methylomes, mRNA transcriptomes, and smallRNA transcriptomes for six different populations of cell types in the Arabidopsis root meristem. We were able to discover that the columella is hypermethylated in the CHH context within transposable elements. This hypermethylation is accompanied by upregulation of the RNA-dependent DNA methylation pathway (RdDM), including higher levels of 24-nt silencing RNAs (siRNAs). In summary, our studies demonstrate the versatility of high-throughput sequencing as a method for identifying single mutations or to perform complex comparative genomic analyses.

Modulating unimolecular charge transfer by exciting bridge vibrations.

Ultrafast UV-vibrational spectroscopy was used to investigate how vibrational excitation of the bridge changes photoinduced electron transfer between donor (dimethylaniline) and acceptor (anthracene) moieties bridged by a guanosine-cytidine base pair (GC). The charge-separated (CS) state yield is found to be lowered by high-frequency bridge mode excitation. The effect is linked to a dynamic modulation of the donor-acceptor coupling interaction by weakening of H-bonding and/or by disruption of the bridging base-pair planarity.

A longitudinal study of epigenetic variation in twins.

DNA methylation is a key epigenetic mechanism involved in the developmental regulation of gene expression. Alterations in DNA methylation are established contributors to inter-individual phenotypic variation and have been associated with disease susceptibility. The degree to which changes in loci-specific DNA methylation are under the influence of heritable and environmental factors is largely unknown. In this study, we quantitatively measured DNA methylation across the promoter regions of the dopamine receptor 4 gene (DRD4), the serotonin transporter gene (SLC6A4/SERT) and the X-linked monoamine oxidase A gene (MAOA) using DNA sampled at both ages 5 and 10 years in 46 MZ twin-pairs and 45 DZ twin-pairs (total n=182). Our data suggest that DNA methylation differences are apparent already in early childhood, even between genetically identical individuals, and that individual differences in methylation are not stable over time. Our longitudinal-developmental study suggests that environmental influences are important factors accounting for interindividual DNA methylation differences, and that these influences differ across the genome. The observation of dynamic changes in DNA methylation over time highlights the importance of longitudinal research designs for epigenetic research.

Histone modifications within the human X centromere region.

Human centromeres are multi-megabase regions of highly ordered arrays of alpha satellite DNA that are separated from chromosome arms by unordered alpha satellite monomers and other repetitive elements. Complexities in assembling such large repetitive regions have limited detailed studies of centromeric chromatin organization. However, a genomic map of the human X centromere has provided new opportunities to explore genomic architecture of a complex locus. We used ChIP to examine the distribution of modified histones within centromere regions of multiple X chromosomes. Methylation of H3 at lysine 4 coincided with DXZ1 higher order alpha satellite, the site of CENP-A localization. Heterochromatic histone modifications were distributed across the 400-500 kb pericentromeric regions. The large arrays of alpha satellite and gamma satellite DNA were enriched for both euchromatic and heterochromatic modifications, implying that some pericentromeric repeats have multiple chromatin characteristics. Partial truncation of the X centromere resulted in reduction in the size of the CENP-A/Cenp-A domain and increased heterochromatic modifications in the flanking pericentromere. Although the deletion removed approximately 1/3 of centromeric DNA, the ratio of CENP-A to alpha satellite array size was maintained in the same proportion, suggesting that a limited, but defined linear region of the centromeric DNA is necessary for kinetochore assembly. Our results indicate that the human X centromere contains multiple types of chromatin, is organized similarly to smaller eukaryotic centromeres, and responds to structural changes by expanding or contracting domains.

Coordinated activation of candidate proto-oncogenes and cancer testes antigens via promoter demethylation in head and neck cancer and lung cancer.

BACKGROUND: Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported. We used an integrative method to analyze expression in primary head and neck squamous cell carcinoma (HNSCC) and pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC. METHODOLOGY/PRINCIPAL FINDINGS: We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS. Aberrant BORIS expression correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC, induced coordinated proto-oncogene specific promoter demethylation and expression in non-tumorigenic cells, and transformed NIH3T3 cells. CONCLUSIONS/SIGNIFICANCE: Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs in aerodigestive cancers, and BORIS is implicated in the coordinated promoter demethylation and reactivation of epigenetically silenced genes in human cancers.

Chromatin accessibility mapping identifies mediators of basal transcription and retinoid-induced repression of OTX2 in medulloblastoma.

Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes.

Inactivation of the von Hippel-Lindau tumor suppressor leads to selective expression of a human endogenous retrovirus in kidney cancer.

A human endogenous retrovirus type E (HERV-E) was recently found to be selectively expressed in most renal cell carcinomas (RCCs). Importantly, antigens derived from this provirus are immunogenic, stimulating cytotoxic T cells that kill RCC cells in vitro and in vivo. Here, we show HERV-E expression is restricted to the clear cell subtype of RCC (ccRCC) characterized by an inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene with subsequent stabilization of hypoxia-inducible transcription factors (HIFs)-1α and -2α. HERV-E expression in ccRCC linearly correlated with HIF-2α levels and could be silenced in tumor cells by either transfection of normal VHL or small interfering RNA inhibition of HIF-2α. Using chromatin immunoprecipitation, we demonstrated that HIF-2α can serve as transcriptional factor for HERV-E by binding with HIF response element (HRE) localized in the proviral 5' long terminal repeat (LTR). Remarkably, the LTR was found to be hypomethylated only in HERV-E-expressing ccRCC while other tumors and normal tissues possessed a hypermethylated LTR preventing proviral expression. Taken altogether, these findings provide the first evidence that inactivation of a tumor suppressor gene can result in aberrant proviral expression in a human tumor and give insights needed for translational research aimed at boosting human immunity against antigenic components of this HERV-E.

Utility of a molecular prescreening program in advanced colorectal cancer for enrollment on biomarker-selected clinical trials.

BACKGROUND: Incorporation of multiple enrichment biomarkers into prospective clinical trials is an active area of investigation, but the factors that determine clinical trial enrollment following a molecular prescreening program have not been assessed. PATIENTS AND METHODS: Patients with 5-fluorouracil-refractory metastatic colorectal cancer at the MD Anderson Cancer Center were offered screening in the Assessment of Targeted Therapies Against Colorectal Cancer (ATTACC) program to identify eligibility for companion phase I or II clinical trials with a therapy targeted to an aberration detected in the patient, based on testing by immunohistochemistry, targeted gene sequencing panels, and CpG island methylation phenotype assays. RESULTS: Between August 2010 and December 2013, 484 patients were enrolled, 458 (95%) had a biomarker result, and 157 (32%) were enrolled on a clinical trial (92 on biomarker-selected and 65 on nonbiomarker selected). Of the 458 patients with a biomarker result, enrollment on biomarker-selected clinical trials was ninefold higher for predefined ATTACC-companion clinical trials as opposed to nonpredefined biomarker-selected clinical trials, 17.9% versus 2%, P < 0.001. Factors that correlated positively with trial enrollment in multivariate analysis were higher performance status, older age, lack of standard of care therapy, established patient at MD Anderson, and the presence of an eligible biomarker for an ATTACC-companion study. Early molecular screening did result in a higher rate of patients with remaining standard of care therapy enrolling on ATTACC-companion clinical trials, 45.1%, in contrast to nonpredefined clinical trials, 22.7%; odds ratio 3.1, P = 0.002. CONCLUSIONS: Though early molecular prescreening for predefined clinical trials resulted in an increase rate of trial enrollment of nonrefractory patients, the majority of patients enrolled on clinical trials were refractory to standard of care therapy. Within molecular prescreening programs, tailoring screening for preidentified and open clinical trials, temporally linking screening to treatment and optimizing both patient and physician engagement are efforts likely to improve enrollment on biomarker-selected clinical trials. CLINICAL TRIALS NUMBER: The study NCT number is NCT01196130.