Dissertations / Theses on the topic 'Epigenomics and epigenetics'

Barley (Hordeum vulgare) is an economically important crop species with a large diploid genome. Around a half of the barley genome and a fifth of the genes are constrained within a low-recombining pericentromeric (LR-PC) region. I explored the LR-PC gene component with a genomic investigation of gene expression, diversity and evolution. Chromatin environments were also explored in the LR and high recombining (HR) regions by surveying the genic and genomic distributions of nine histone modifications. Firstly, regions of HR and LR were identified and compared for gene evolution, expression and diversity. LR regions of the barley genome were found to be restrictive for gene evolution and diversity, but not gene expression. I employed a bioinformatics approach to identify ancient gene pairs in barley to determine the long-term effects of residency in those regions upon gene evolution. Gene pair loss in LR regions was found to be elevated relative to the HR regions. Applying the same method to rice and Brachypodium distachyon revealed the same situation, suggesting a universal process in the grasses for loss of gene pairs in LR regions. The chromosomal distributions of transposable elements (TEs) were also explored and examined for correlations with recombination rate. Secondly, I developed a chromatin immunoprecipitation followed by Next Generation Sequencing (ChIP-seq) protocol for the investigation of histone modifications in barley seedlings. A protocol was optimised for the fixation, extraction and sonication of barley chromatin. The protocol was applied using antibodies against 13 different histone modifications. Following DNA library construction and Illumina sequencing, a bioinformatics pipeline was devised to analyse the sequence data. NGS reads were mapped to a custom assembly of the barley cultivar Morex reference genome sequence before peak calling. Genomic and genic locations were determined for the covalently modified histones. Four modifications were discarded from further study on the basis of low peak numbers or unexpected chromosomal locations. The remaining nine modifications were classified into four groups based on chromosomal distributions. Groupings were closely mirrored by peak sharing relationships between the modifications except histone H3 lysine-27 tri-methylation (H3K27me3). In addition, chromatin states representing local chromatin environments were defined in the barley genome using the peak sharing data. Mapping the states onto the genome revealed a striking chromatin structure of the gene-rich chromosome arms. A telomere-proximal region bearing high levels of H3K27me3-containing states was found adjacent to an interior gene-rich region characterised by active chromatin states lacking H3K27me3. The LTR retroelement-rich interior was found to be associated with repressive chromatin states. The histone modification status of TE classes were also probed revealing unexpected differences relating to the genomic and genic distributions of these elements. Finally, a genome browser was created to host the information publicly.

Obesity and type 2 diabetes (T2D) are major risk factors for cardiovascular and other diseases and are currently undergoing an increase in global prevalence. The work presented in my thesis addresses the role epigenetics, specifically DNA methylation, plays in the susceptibility to obesity and T2D and deals with methodological issues in the analysis of DNA methylation data. I first combined epigenome-wide DNA methylation data across 38 adipose tissue samples with corresponding SNP and mRNA data for the same subjects. At 5% false discovery rate (FDR), methylation of 149 regions associated with at least one cis-SNP. When 19 of the 149 regions were tested for association in an additional 181 independent samples, five regions replicated. These results indicate a genetic influence on DNA methylation in adipose tissue. I then analysed 90 epigenome-wide methylation samples taken from 15 South Asian controls and 30 T2D cases participating in the LOLIPOP study at two time points ∼7 years apart. I found global differences at both follow-up and baseline between the normal glucose tolerant and T2D groups, as well as strong differences with aging. I further used the main EpiMigrant data from 2,687 individuals, with 36 samples measured in duplicate to assess approaches to quality control, data normalisation and batch correction through control probe adjustment. A null hypothesis for epigenome-wide association studies (EWAS) by permutation testing and I investigated the effects of correlation between individual methylation markers. Using the developed methods, I carried out an EWAS of body mass index (BMI) with subsequent meta-analysis amongst 10,261 individuals of European and South Asian ancestry. DNA methylation markers at 187 genetic loci were associated with BMI. Mendelian randomisation experiments suggested that association of DNA methylation with BMI is the consequence of BMI. Lastly, I tested haplotypes of 85 SNPs currently known to be associated with T2D and 118 SNPs associated with obesity traits for an enrichment of CpG creating or abrogating SNPs and found that 9 T2D and 23 obesity SNPs showed a significant difference in CpG count between the SNP alleles as established by permutation testing. Amongst these is FTO, a locus which has been previously been shown to have a haplotype-specific methylation effect. My work provides novel insights into the role of DNA methylation in metabolic diseases. The methods that I developed to robustly detect association are flexible and scalable and will further be useful for larger, future EWAS.

Les cellules placentaires portent un génome différent du génome maternel, puisque 50% de leurs gènes proviennent du génome paternel. Cependant, comme les cellules cancéreuses après la transformation néoplasique, elles réussissent à envahir les tissus de leur hôte, échapper à son système immunitaire et induire une angiogenèse afin d’établir la grossesse. Les cellules cancéreuses et placentaires arborent aussi une différence majeure : alors que de tels mécanismes typiques des cancers sont incontrôlés dans les cellules cancéreuses, ils sont spatialement et temporairement contrôlés dans les cellules placentaires saines. Ainsi, le recherche sur le « concept cancer/placenta » – l’utilisation du placenta pour mieux comprendre le cancer – peut aboutir à l’identification de biomarqueurs et d’approches thérapeutiques innovantes en oncologie, tout comme en gynécologie-obstétrique. Par exemple, les efforts de recherche portant sur l’expression des gènes CGB, codant pour la sous-unité ß de l’hormone chorionique gonadotrope humaine, dans les cellules cancéreuses et placentaires a mené au développement d’un biomarqueur largement utilisé pour la prise en charge de multiples cancers. Il est aussi intéressant de noter que ce même biomarqueur est aussi utilisé pour le dépistage d’aneuploïdies fœtales. De même, le clonage d’INSL4, codant pour le précurseur du peptide placentaire précoce ressemblant à l’insuline (pro-EPIL), dans des cellulaires placentaires précoces, a mené au développement d’un biomarqueur faisant actuellement l’objet d’études cliniques. Avec l’émergence de l’épigénétique, des études de la méthylation de l’ADN, la caractéristique épigénétique la mieux comprise, ont montré que les loci de gènes CGB et INSL4 sont hypométhylés dans les cellules cancéreuses et placentaires ; ce qui pourrait refléter l’hypométhylation globale caractéristique de ces deux types cellulaires. Par conséquent, le projet doctoral présenté dans cette thèse a exploré les modifications des paysages épigénétiques des cellules placentaires au cours de la grossesse et des cellules cancéreuses au cours de la transformation néoplasique. Ce projet a contribué initialement au développement d’un test d’immunoanalyse qui détecte l’hCGß de type II, spécialement codée par un sous-groupe de gènes CGB et détectée dans le sérum de patients atteints de cancers non-placentaires et de trisomie 21 fœtale. Ce test d’immunoanalyse, avec un test similaire développé pour la détection de pro-EPIL, a aussi été utilisé pour des études de preuve de concept précoces quant à l’effet de la méthylation de l’ADN sur l’expression de l’hCGß de type II et de pro-EPIL dans des surnageants de culture cellulaire. En fin de compte, ce projet a mené à la première comparaison directe et pan-génomique de la méthylation de l’ADN dans des cellules cancéreuses au cours de la transformation néoplasique et dans des cellulaires placentaires au cours de la grossesse. Cette étude a porté sur des données, disponibles publiquement, générées à partir de biopsies de 13 types de tumeurs, de villosités choriales (tissus placentaires) et d’autres tissus sains. Elle a également porté sur des données originales générées par nos soins à partir d’échantillons placentaires uniques : des cellules cytotrophoblastiques isolées de villosités choriales ex vivo. Toutes les données inclus dans cette étude ont été générées sur une plateforme de puces à ADN pour la mesure de la méthylation au niveau de 485 512 sites CpG pour chaque échantillon. En combinant, des logiciels innovants reposant sur la puissance d’algorithmes de lissage statistique et sur un solide rationnel biologique, cette étude a ainsi contribué à l’identification de motifs d’hypométhylation à l’échelle du mégabase distinguant les cellules placentaires du début de la grossesse de celles de la fin de la grossesse tout comme ils distinguent les cellules cancéreuses des cellules normales. (...)
Placental cells carry a genome different from the maternal genome, as 50% of it originate from the paternal genome. However, like cancer cells after neoplastic transformation, they successfully invade their host tissues, escape its immune system and induce angiogenesis in order to establish the pregnancy. Cancer and placental cells also display a major discrepancy: while such hallmarks of cancer mechanisms are uncontrolled in cancer cells, they are spatially and temporally controlled in healthy placental cells. Thus, research on the “cancer/placenta concept” – the use of the placenta to better understand cancer – can lead to innovative biomarkers and therapeutic approaches in oncology as well as in gynecology and obstetrics. For example, research efforts on the expression of the CGB genes, encoding for the human chorionic gonadotropin beta subunit (hCGß), in cancer and placental cells have led to the development of a biomarker widely used for the management of various cancers. Interestingly, this same biomarker is also used for the screening of fetal aneuploidies. Likewise, the cloning of INSL4, encoding for the precursor of the early placenta insulin-like peptide (pro-EPIL) in early pregnancy placental cells, has led to the development of a biomarker currently investigated in the clinical setting. Following the rise of epigenetic, studies on DNA methylation, the most well understood epigenetic mark, showed that the loci of CGB genes and INSL4 are hypomethylated in cancer and placental cells, which may reflect a global hypomethylation also characteristic of these cells. Therefore, the doctoral project presented in this dissertation had explored modifications in the epigenetic landscape of placental cells throughout pregnancy and cancer cells throughout neoplastic transformation. This project initially contributed to the development of an immunoassay detecting type II hCGß, specifically encoded by a subset of CGB genes and detected in the serum of patients with non-placental cancers and fetal Down Syndrome. This immunoassay, along with another one directed to pro-EPIL, was also used for an early proof of concept study regarding the effect of DNA methylation on the expression of type II hCGß and pro-EPIL in cell culture supernatants. Ultimately, this project led to the first direct genome-wide comparison of DNA methylation in cancer cells throughout neoplastic transformation and in placental cells throughout pregnancy. It included publically available data generated from biopsies of 13 types of tumors, chorionic villi (placental tissues) and other normal tissues. It also included original data generated from unique placental samples: villous cytotrophoblastic cells isolated ex vivo from chorionic villi. All datasets were generated on a microarray platform measuring DNA methylation at 485,512 CpG sites in each sample. Combining innovative software that leverages the power of statistical smoothing algorithms and a strong biological rationale, this study thus contributed to the identification of megabase-scale patterns of hypomethylation distinguishing early pregnancy from late pregnancy placenta cells as they distinguish normal from cancers cells. Strikingly, the affected genomic regions encompassed genes related to hallmarks of cancer mechanisms such as epithelial-mesenchymal transition (EMT), innate and acquired immune response, and hypoxia. Taken together, these results suggest the hypothesis that patterns of DNA methylation might contribute to “cancer/placenta epigenetic switches” allowing placental implantation and neoplastic transformation when turned “on”, while preventing the placenta to degenerate into an aggressive tumor when turned “off”

DNA methylation is a crucial epigenetic modification involved in numerous biological processes. However, despite its functional importance, the evolutionary history of this modification and the mechanisms diving such changes are poorly understood. The aim of this thesis is to provide a better understanding of DNA methylation in the context of human recent evolution. We identified and described hundreds of regions presenting a human-specific DNA methylation pattern compared to great apes. We also analyzed for the first time the relationship between DNA methylation changes and sequence evolution at both nucleotide and protein level. In summary, this research reveals new insights into the evolutionary properties of DNA methylation and the interpretation of inter-species non-coding variation
La metilación del ADN es una modificación epigenética implicada en numerosos procesos biológicos. Sin embargo, a pesar de su relevancia funcional, se sabe muy poco sobre su historia evolutiva y los mecanismos que generan estos cambios. El objetivo de esta tesis es proporcionar una mejor compresión de la metilación del ADN en el contexto de la evolución humana reciente. Hemos identificado y descrito cientos de regiones que presentan un patrón de metilación especifico de humanos. Así mismo, hemos analizado por primera vez la relación entre los cambios en metilación y la evolución de la secuencia tanto a nivel nucleotídico como proteico. En resumen, esta investigación revela nuevos conocimientos sobre las propiedades evolutivas de la metilación del ADN y la interpretación de la variación no codificante entre especies.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2016.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 203-225).
One of the fundamental aims of biology is to determine what lies at the root of differences across individuals, species, diseases, and cell types. Furthermore, the sequencing of genomes has revolutionized the ways in which scientists can investigate biological processes and disease pathways; new genome-wide, high-throughput experiments require computer scientists with a biological understanding to analyze and interpret the data to improve our understanding about life science. This provides us with a key opportunity to use computational techniques for new biological discoveries. While genetic variation plays an important role in influence phenotype, sequence alone cannot account for all differences: for example, different types of cells in an individual have varying function and attributes, but identical genetic makeup. This highlights the importance of studying epigenetic changes, which are dynamic chemical changes to and around the DNA. While the DNA of every cell in an individual is the same, the epigenetic context for that DNA varies from cell to cell. In this way, these epigenetic differences play a crucial role in gene regulation, with epigenetic changes both causing and recording regulatory mechanisms. In this thesis, we combine the power of computational, statistical, and data science approaches with the new wave of epigenetic data at a genome-wide level in a number of ways. First, in chapter 2, we demonstrate the importance of computational analysis at an epigenomic level by identifying an epigenomic signature of the olfactory receptor gene family that gives insight into the mechanism behind monogenic gene regulation. Next, in chapter 3, we explain our development of ChromDiff, a novel statistical and information theoretic computational methodology to identify chromatin state differences in groups of samples. In our methodology, we use correction for external covariates to isolate the relevant signal, and as a result, we find that our method outperforms existing computational methods, with further validation through randomized simulations. In chapter 4, we apply our methodology to characteristics including sex, developmental age, and tissue type, we unveil relevant chromatin states and genes that distinguish the groups of epigenomes, with further validation of our results through differential expression analysis and gene set enrichment. In chapter 5, we show the power of integrative analysis through the combination of DNA methylation data with chromatin state profiles, cell types, sample groups, experimental technologies, and histone mark data to reveal insightful epigenetic patterns and relationships. Finally, in chapter 6, we identify "hidden" or "unknown" covariates in epigenomic data by using agnostic principal component analysis on our samples to discover similarities between our known covariates and the identified components. In summation, our research highlights the importance of both algorithm development and method application for epigenomic questions, reaffirming the importance of interdisciplinary research that brings together cutting-edge techniques in computer science with appropriate biological hypotheses and data. While questions and analysis must be carefully paired in an informed manner to produce meaningful, interpretable, and believable results in computational biology, our work here provides a sampling of the vast potential for scientific discovery at the intersection of the fields of computer science and biology.
by Angela Yen.
Ph. D.

Epigenetic dysfunction is a known contributor in carcinogenesis, and is emerging as a mechanism involved in toxicant-induced malignant transformation for environmental carcinogens such as arsenicals. In addition to aberrant DNA methylation of single genes, another manifestation of epigenetic dysfunction in cancer is agglomerative DNA methylation, which can participate in long-range epigenetic silencing that targets many neighboring genes and has been shown to occur in several types of clinical cancers. Using in vitro model systems of toxicant-induced malignant transformation, we found hundreds of aberrant DNA methylation events that emerge during malignant transformation, some of which occur in an agglomerative fashion. In an arsenite-transformed prostate epithelial cell line, the protocadherin (PCDH), HOXC and HOXD gene family clusters are targeted for agglomerative DNA methylation. Aberrant DNA methylation in general occurred more often within H3K27me3 stem cell domains. We found a striking association between enrichment of H3K9me3 stem cell domains and toxicant-induced agglomerative DNA methylation. Global gene expression profiling of the arsenite-transformed prostate epithelial cells showed that gene expression changes and DNA methylation changes were negatively correlated, but less than 10% of the hypermethylated genes were down-regulated. These studies confirm that a majority of the DNA hypermethylation events occur at transcriptionally repressed, H3K27me3 marked genes. In contrast to aberrant DNA methylation targeting H3K27me3 pre-marked silent genes, we found that actively expressed ZNF genes marked with H3K9me3 on their 3' ends, are preferred targets of DNA methylation linked gene silencing. H3K9me3 mediated gene silencing of ZNF genes was widespread, occurring at individual ZNF genes on multiple chromosomes and across ZNF gene family clusters. At ZNF gene promoters, H3K9me3 and DNA hypermethylation replaced H3K4me3, resulting in a widespread down-regulation of ZNF gene expression which accounted for 8% of all the down-regulated genes in the arsenical-transformed cells. In summary, these studies associate arsenical exposure with agglomerative DNA methylation of gene family clusters and widespread silencing of ZNF genes by DNA hypermethylation-linked H3K9me3 spreading, further implicating epigenetic dysfunction as a driver of arsenical-induced carcinogenesis.

Multiple computational algorithms were developed for analyzing ChIP-seq datasets of histone modifications. For basic ChIP-seq data processing, the problems of ambiguous short sequence read mapping and broad peak calling of diffuse ChIP-seq signals were solved by novel statistical methods. Their performance was systematically evaluated compared with existing approaches. The potential utility of finding meaningful biological information was demonstrated by the applications on real datasets. For biological question driven data mining, several important topics were selected for algorithm developments, including hypothesis-driven insulator prediction, unbiased chromatin boundary element discovery and combinatorial histone modification signature inference. The integrative computational pipeline for insulator prediction not only produced a list of putative insulators but also recovered specific associated chromatin and functional features. Selected predictions have been experimentally validated. The unbiased chromatin boundary element prediction algorithm was feature-free and had the capability to discover novel types of boundary elements. The predictions found a set of chromatin features and provided the first report of tRNA-derived boundary elements in the human genome. The combinatorial chromatin signature algorithm employed chromatin profile alignments for unsupervised inferences of histone modification patterns. The signatures were associated with various regulatory elements and functional activities. Both the computational advantages and the biological discoveries were discussed.

Epigenetic modifications, such as DNA methylation and histone modifications, play important roles in gene expression and regulation, and are highly involved in cellular processes such as stem cell pluripotency/differentiation and tumorigenesis. Chromatin immunoprecipitation (ChIP) is the technique of choice for examining in vivo DNA-protein interactions and has been a great tool for studying epigenetic mechanisms. However, conventional ChIP assays require millions of cells for tests and are not practical for examination of samples from lab animals and patients. Automated microfluidic chips offer the advantage to handle small sample sizes and facilitate rapid reaction. They also eliminate cumbersome manual handling. In this report, I will talk about three different projects that utilized microfluidic immunoprecipitation followed by next genereation sequencing technologies to enable low input and high through epigenomics profiling. First, I examined RNA polymerase II transcriptional regulation with microfluidic chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) assays. Second, I probed the temporal dynamics in the DNA methylome during cancer development using a transgenic mouse model with microfluidic methylated DNA immunoprecipitation followed by next generation sequencing (MeDIP-seq) assays. Third, I explored negative enrichment of circulating tumor cells (CTCs) followed by microfluidic ChIP-seq technology for studying temporal dynamic histone modification (H3K4me3) of patient-derived tumor xenograft on an immunodeficient mouse model during the course of cancer metastasis. In the first study, I adapted microfluidic ChIP-seq devices to achieve ultrahigh sensitivity to study Pol2 transcriptional regulation from scarce cell samples. I dramatically increased the assay sensitivity to an unprecedented level (~50 K cells for pol2 ChIP-seq). Importantly, this is three orders of magnitude more sensitive than the prevailing pol2 ChIP-seq assays. I showed that MNase digestion provided better ChIP-seq signal than sonication, and two-steps fixation with MNase digestion provided the best ChIP-seq quality followed by one-step fixation with MNase digestion, and lastly, no fixation with MNase digestion. In the second study, I probed dynamic epigenomic changes during tumorigenesis using mice often require profiling epigenomes using a tiny quantity of tissue samples. Conventional epigenomic tests do not support such analysis due to the large amount of materials required by these assays. In this study, I developed an ultrasensitive microfluidics-based methylated DNA immunoprecipitation followed by next-generation sequencing (MeDIP-seq) technology for profiling methylomes using as little as 0.5 ng DNA (or ~100 cells) with 1.5 h on-chip process for immunoprecipitation. This technology enabled me to examine genome-wide DNA methylation in a C3(1)/SV40 T-antigen transgenic mouse model during different stages of mammary cancer development. Using this data, I identified differentially methylated regions and their associated genes in different periods of cancer development. Interestingly, the results showed that methylomic features are dynamic and change with tumor developmental stage. In the last study, I developed a negative enrichment of CTCs followed by ultrasensitive microfluidic ChIP-seq technology for profiling histone modification (H3K4Me3) of CTCs to resolve the technical challenges associated with CTC isolation and difficulties related with tools for profiling whole genome histone modification on tiny cell samples.
Ph. D.

Cancer progression is driven by cumulative changes that promote and maintain the malignant phenotype. Epigenetic alterations are central to malignant transformation and to the development of therapy resistance. Changes in DNA methylation, histone acetylation and methylation, noncoding RNA expression and higher-order chromatin structures are epigenetic features of cancer, which are independent of changes in the DNA sequence. Despite the knowledge that these epigenetic alterations disrupt essential pathways that protect cells from uncontrolled growth, how these modifications collectively coordinate cancer gene expression programs remains poorly understood. In this dissertation, I utilize molecular and informatic approaches to define and characterize the genome-wide epigenetic patterns of two important human cancer cell models. I further explore the dynamic alterations of chromatin structure and its interplay with gene regulation in response to therapeutic agents. In the first part of this dissertation, pancreatic ductal adenocarcinoma (PDAC) cell models were used to characterize genome-wide patterns of chromatin structure. The effects of histone acetyltransferase (HAT) inhibitors on chromatin structure patterns were investigated to understand how these potential therapeutics influence the epigenome and gene regulation. Accordingly, HAT inhibitors globally target histone modifications and also impacted specific gene pathways and regulatory domains such as super-enhancers. Overall, the results from this study uncover potential roles for specific epigenomic domains in PDAC cells and demonstrate epigenomic plasticity to HAT inhibitors. In the second part of this dissertation, I investigate the dynamic changes of chromatin structure in response to estrogen signaling over a time-course using Estrogen Receptor (ER) positive breast cancer cell models. Accordingly, I generated genome-wide chromatin contact maps, ER, CTCF and regulatory histone modification profiles and compared and integrated these profiles to determine the temporal patterns of regulatory chromatin compartments. The results reveal that the majority of alterations occur in regions that correspond to active chromatin states, and that dynamic chromatin is linked to genes associated with specific cancer growth and metabolic signaling pathways. To distinguish ER-regulated processes in tamoxifen-sensitive and in tamoxifen-resistant (TAMR) cell models, we determined the corresponding chromatin and gene expression profiles using ER-positive TAMR cancer cell derivatives. Comparison of the patterns revealed characteristic features of estrogen responsiveness and show a global reprogramming of chromatin structure in breast cancer cells with acquired tamoxifen resistance. Taken together, this dissertation reveals novel insight into dynamic epigenomic alterations that occur with extrinsic stimuli and provides insight into mechanisms underlying the therapeutic responses in cancer cells.

Mestrado em Biotecnologia Molecular
Sabe-se hoje que o genoma humano, para além da sua sequencia nucleotídica, revela várias alterações químicas no DNA, nomeadamente metilações das citosinas. Estas modificações estabelecem padrões específicos que podem ser transmitidos de uma geração para a seguinte e exercem controlo sobre os genes que são expressos a cada momento nas células, tecidos ou orgãos. Esta tese teve como objectivos: explorar as principais bases de dados que contêm dados epigenómicos relevantes; obter ficheiros fastq de bibliotecas bisulfite-seq aplicando métodos de data mining a dados reais de bases de dados públicas de sequenciação de segunda geração; alinhar e mapear estes ficheiros usando software adequado (Methy-Pipe); fazer uma análise comparative por forma obter características associadas ao envelhecimento saudável de indivíduos e á evolução do epigenoma ao longo da vida; finalmente é esperado que, após atingidos os objectivos anteriores, se perceba o contributo do epienoma no envelhecimento saudável das populações .
It is already known today, that the human genome, in addition to its nucleotide sequence, shows multiple chemical modifications at the DNA level, namely cytosine methylations. These modifications changes establish specific patterns that can be transmitted from generation to generation and exercise control over the genes that are expressed at every moment in the life of the cells / tissues / organs. This thesis aimed to: understand the contribution of the epigenome to a healthy lifestyle; to explore the main databases containing relevant epigenomic data; to obtain fastq files of bisulfite-seq libraries by applying data mining methods to real data from next generation public databases; to align and map these files using adequate software (Methy Pipe); to do a comparative analysis in order to identify features associable to a healthy aging of individuals and the evolution of the epigenome in humans throughout life.In doing so, it is expected that this work will contribute to the understanding of the contribution of the epigenome to a healthy lifestyle.

We developed a computational framework implemented as an R package for generation, visualization and functional and differential analysis of epigenome maps. Methods are provided for integrating and comparing data from different conditions or biological backgrounds, accounting and adjusting for systematic biases in order to provide an efficient and statistically robust base for differential analysis. We also provide methods for general data assessment and quality control, such as functions to study chromatin domain conservation between epigenomic backgrounds, to detect gross technical outliers and to help in the selection of candidate marks for de-novo epigenome mapping.
Hem desenvolupat una metodologia computational implementada en forma de paquet pel llenguatge R per la generació i visualització de mapes epigenòmics, així com per dur a terme el seu anàlisi funcional i diferencial. Proporcionem mètodes per la integració i comparació de dades provinents de diferents condicions, identificant i eliminant biaixos sistemàtics per obtenir una base amb robustesa estadística per l'anàlisi diferencial. També proporcionem funcions per dur a terme un control de qualitat de les dades, per estudiar la conservació i integritat dels dominis de cromatina, per detectar errors tècnics i per ajudar en la selecció de factors epigenètics candidats per la generació de mapes epigenòmics 'de-novo'.

Mestrado em Biomedicina Molecular
Epigenetics can be defined as changes in the genome that are inherited during cell division, however without direct modify the DNA sequence. These genomic changes are supported by three major epigenetic mechanisms: DNA methylation, histone modification and small RNAs. Different epigenetic marks function by regulating gene transcription, because when these processes are altered, this triggers various diseases such as cancer. Thus, one main objective was to study the epigenetics signals in the human genome, meaning, whether there is dependence observed between the context and the occurrence of epigenomic marking. For this purpose we used histone epigenomes available in the NIH Roadmap Epigenomics Mapping Consortium database that contains various types of cells and various types of tissues. The present study employed different statistical methodologies, namely, statistical tests, effect size measures, residue analysis and hierarchical classification. With this analysis, we compared genomic contexts of epigenetic marking among chromosomes and among epigenomes. Complementing the analysis with a control scenario, without marking and factoring the CG content. As a result of this study, it was possible to identify one dependency between the context and the occurrence of epigenetic marking and we were able to identify specific genomic contexts in histone modifications.
A epigenética pode ser definida pela ocorrência de modificações no genoma, que são herdadas durante a divisão celular, não havendo no entanto modificações directas na sequência do DNA. Estas modificações genómicas são apoiadas em três grandes mecanismos epigenéticos: metilação do DNA, modificação de histonas e pequenos RNAs. Estas diferentes marcas epigenéticas podem ter como função regular a transcrição genética, pois quando existe algum tipo de alteração nestes processos, pode desencadear-se em diversas patologias como o cancro. Assim, o objectivo principal deste trabalho é estudar os sinais epigenéticos no genoma humano, ou seja, observar se existe dependência entre o contexto e a ocorrência da marcação epigenómica. Para esse efeito foram utilizados os epigenomas das histonas disponíveis na base de dados do NIH Roadmap Epigenomics Mapping Consortium que contêm vários tipos de células e vários tipos de tecidos. No presente estudo são empregues diferentes metodologias estatísticas, nomeadamente testes estatísticos, medidas do tamanho do efeito, análise de resíduos e classificação hierárquica. Com esta análise, comparam-se contextos genómicos da marcação epigenética entre cromossomas e entre epigenomas. Complementando a análise com um cenário de controlo, sem marcação e factorizando pelo teor de CG. Foi possível identificar uma dependência entre o contexto e a ocorrência de marcação epigenética, sendo possível identificar contextos genómicos específicos para as modificações das histonas.

Craniofacial development is a tightly regulated event that requires expression of many genes at a precise space-temporal specificity. Interference in the regulation of such genes and their pathways is known to lead to abnormal phenotypes affecting the face and cranium. In this manner, regulation of these pathways is further complicated by interaction between genetic and environmental factors such that disturbance to either may result in craniofacial malformation, as orofacial clefts. Despite several at-risk loci have been identified, they do not completely explain the high heritability observed for the orofacial clefts and many questions remain open. For example, concerning the orofacial clefts transcriptome, the gene pathways which may be dysregulated and the affected cellular processes are still poorly understood. Further, if there is gene expression dysregulation in orofacial clefts, the causes leading to that need to be elucidated, such as the investigation of epigenetic factors. Also, since the multifactorial contribution makes environment relevant to this malformation, epigenetic and epigenomic differences in orofacial clefts should clarified. At last, rare syndromic forms of orofacial clefts with still unknown molecular cause and mechanisms should be elucidated in order to better understand craniofacial development and their impact in non-syndromic forms. Therefore, the main objective of this study was to investigate the molecular mechanisms involved in the aetiology of orofacial clefts, which was focused in gene expression and epigenetic analysis in non-syndromic cleft lip and/or palate (NSCL/P) as well as genetic, gene expression, animal modelling and epigenetics in Richieri-Costa-Pereira Syndrome (RCPS), a rare autosomal recessive syndromic form of orofacial cleft. We found significant transcriptome differences in NSCL/P in comparison to controls, revealing the BRCA1-dependent DNA damage repair pathway as compromised in NSCL/P cells leading to DNA damage accumulation. Next, we studied the potential of DNA methylation in those cells and found a slight but significant increase of BRCA1 promoter DNA methylation in NSCL/P cells and a distinct DNA methylation distribution, point to a possible epigenetic contribution in this phenomenon. We also evaluated the contribution of DNA methylation in 8q24.21 region, one of the most replicated regions in NSCL/P Genome-wide association studies and found no significant differences in our sample. Attempting to investigate DNA methylation in NSCL/P in an epigenomic level, we analysed methylomes and found 578 methylation variable positions in NSCL/P, highly enriched in regulatory regions and in relevant gene pathways for craniofacial development as Epithelial-Mesenchymal Transition pathway. We also studied effect of DNA methylation in familial NSCL/P displaying incomplete penentrance and found a significant increase of CDH1 promoter hypermethylation in penetrant cases in comparison to non-penetrants. Finally, by the use of different sequencing strategies and identity-by-descent analysis we mapped the mutation region of RCPS to EIF4A3 5\'UTR/promoter and found a complex structure of expanded repeats in RCPS patients leading to EIF4A3 downregulation. We were also able to validate the phenotypes using an animal modelling strategy in zebrafish. Because those repeats are CG rich, we investigated whether they were submitted to DNA hypermethylation in RCPS patients as a cause for EIF4A3 hypomorphism, however we found no evidence of methylation increase in RCPS. In conclusion, we were able to associate dysregulated pathways to NSCL/P susceptibility and DNA methylation differences to both non-familial and familial NSCLP. Besides, we were able to identify the genetic cause of RCPS, which now can be molecularly diagnosed. Altogether, our results add to the understanding of craniofacial development and the aetiology of orofacial clefts
O desenvolvimento craniofacial é um evento finamente regulado que requer a expressão de muitos genes em uma precisão espaço-temporal específica. A interferência na regulação de tais genes e suas respectivas vias é sabidamente causadora de fenótipos que afetam a face e o crânio. Neste sentido, a regulação destas vias é decorrente da interação entre fatores genéticos e ambientais, de tal forma que a perturbação de quaisquer destes fatores pode resultar em malformações craniofaciais, como as fissuras orofaciais. Apesar dos muitos loci de risco já identificados, estes não explicam completamente a alta herdabilidade observadas nas fissuras orofaciais e muitas questões permanecem em aberto. Por exemplo, em relação ao transcriptoma em fissuras orofaciais, as vias genéticas que podem estar desreguladas, assim como processos celulares afetados em decorrência, são ainda pouco compreendidos. Além disso, se há desregulação na expressão de genes em fissuras orofaciais, as causas que levam a essas diferenças necessitam ser elucidadas, como, por exemplo, por meio da investigação de fatores epigenéticos. Também, uma vez que o componente multifatorial torna a influência do ambiente relevante para esta malformação, diferenças epigenéticas e epigenômicas nas fissuras orofaciais devem ser melhor compreendidas. Por fim, formas raras e sindrômicas de fissuras orofaciais sem elucidação de causa moleculares devem ser estudadas para que melhor se compreenda o desenvolvimento craniofacial e o impacto destes mecanismos moleculares em formas não-sindrômicas. Portanto, nosso objetivo principal neste estudo foi investigar os mecanismos moleculares envolvidos na etiologia das fissuras orofaciais, com o foco na análise de expressão gênica e epigenètica em fissuras de lábio-palatinas não-sindrômicas (FL/P NS) e também o estudo genético, de expressão gênica, modelagem animal e epigenética na Síndrome de Richieri-Costa-Pereira (RCPS), uma forma sindrômica e autossômica recessiva de fissura orofacial. Nós encontramos diferenças significantes no transcriptoma de FL/P NS em comparação com controles, que revelaram o comprometimento da via do BRCA1 no reparo ao dano de DNA e o acúmulo de dano de DNA em células FL/P NS. Em seguida, nós estudamos o potencial da metilação de DNA nestas células e encontramos um pequeno, porém significante, aumento de metilação de DNA no promotor do BRCA1 e uma distribuição diferente de metilação, apontando para uma possível contribuição epigenética na desregulação do gene. Nós também avaliamos a contribuição da metilação de DNA na região 8q24.21, uma das mais associadas às FL/P NS por meio de Genome-wide association studies, porém não encontramos diferenças significantes na nossa amostra. Com o intuito de investigar a metilação de DNA em FL/P NS em uma escala epigenômica, nós analisamos o perfil de metilomas e encontramos 578 sítios diferencialmente metilados nas FL/P NS, altamente enriquecidos em regiões regulatórias e em vias relevantes para o desenvolvimento craniofacial como a via de Transição Epitélio-Mesenquimal. Nós também estudamos o efeito da metilação de DNA em casos famílias de FL/P NS com penetrância incompleta e encontramos um aumento significativo de metilação do promotor do CDH1 nos casos penetrantes em comparação aos não-penetrantes. Por último, por meio de diferentes estratégias de sequenciamento e análise de segregação de haplótipos nós mapeamos a mutação de RCPS na região 5\'UTR/promotor do EIF4A3 e encontramos uma estrutura complexa de expansão de repetições nos pacientes RCPS, ocasionando a diminuição da expressão do EIF4A3. Nós também reproduzimos fenótipos comparáveis aos da RCPS por meio de modelo animal em zebrafish. Uma vez que tais repetições são ricas em CG, nós investigamos se estas poderiam ser submetidas à metilação de DNA em pacientes RCPS como uma causa para a redução dos transcritos do EIF4A3, porém não encontramos evidências de aumento de metilação em RCPS. Em conclusão, nós conseguimos associar vias gênicas desreguladas à susceptibilidade para as FL/P NS e diferenças de metilação de DNA tanto em casos familiais como não-familiais de FL/P NS. Além disso, identificamos a causa genética de RCPS, sendo que a síndrome pode ser agora diagnosticada molecularmente. Em conjunto, nossos resultados adicionam ao conhecimento do desenvolvimento craniofacial e na etiologia das fissuras orofaciais

El càncer ha estat tradicionalment considerat una malaltia fonamentalment genètica, però recentment s'està fent palès que la desregulació de mecanismes epigenètics contribueix en gran manera al desenvolupament tumoral. Al bell mig de la intersecció entre la genètica i l'epigenètica s'hi troben els factors reguladors de la cromatina (CRFs, en anglès), que són un focus important de recerca a causa de la seva potencial utilitat en teràpies contra el càncer. En aquesta tesi, determino l'estat transcriptòmic de cèl·lules normals i tumorals basant-me en informació epigenètica i regulatòria, i descric l'existència d'una sincronització global de l'expresió gènica en què la regulació controlada per Polycomb es manifesta com a un dels dos components principals. Presento una anàlisi sobre com la baixa expressió dels gens regulats per Polycomb contribueix a l'avenç del càncer de mama i a la transició entre epitel·li i mesènquima. A més, identifico aquesta baixa expressió com a factor valuós de pronòstic independent. Aprofitant les dades genòmiques de càncer que han estat posades a la disposició del públic recentment, també avaluo l'estat mutacional dels CRFs en molts tumors humans provinents de diferents teixits i línies cel·lulars de càncer. Els resultats indiquen que 39 CRFs són potencialment conductors del procès cancerígen en almenys un teixit, malgrat que molts d'ells es torben mutats en freqüències relativament baixes. Finalment, presento un recurs per a visualitzar i analitzar alteracions genòmiques entre línies cel·lulars de càncer en el context de la resistència a fàrmacs i de la informació sobre alteracions de
Cancer has traditionally been regarded as a genetic disease, but recently it is becoming apparent that the deregulation of epigenetic mechanisms greatly contributes to tumour development. At the crossing of genetics and epigenetics lie chromatin regulatory factors (CRFs), which are the focus of intense research due to their potential usefulness in anticancer therapy. In this thesis, I determine the transcriptomic state of normal and tumour cells based on epigenetic and regulatory information, and describe the existence of a global synchronisation of gene expression in which Polycomb regulation arises as one of the two main components. I present an analysis on how the under-expression of Polycomb regulated genes contributes to breast cancer progression and epithelial to mesenchymal transition. Furthermore, I identify this under-expression as a valuable independent prognostic factor. Taking advantage on the wealth of cancer genomics data made available recently, I also evaluate the mutational status of CRFs across many human tumours from different tissues and cancer cell lines, and find that 39 CRFs are potential cancer drivers in at least one tissue, even though most of them are mutated at relatively low frequencies. Finally, I present a resource to visualise and analyse genomic alterations across cancer cell lines in the context of drug sensitivity/resistance and the information on somatic tumour alterations.

L'épigénome est défini par l’ensemble de l’information chromatinienne autre que celle fournie par la séquence ADN. Au sein d'une même espèce et pour un type cellulaire donné, chaque individu présente des caractéristiques particulières de l'épigénome. Les épi-polymorphismes, définis comme étant les différences inter-individus de marques chromatiniennes, sont encore partiellement caractérisés et peuvent être liés aux phénotypes de chacun. La première partie de mon travail a été d'identifier et d'interpréter chez S.cerevisiae l'impact des épi-polymorphismes de modification des queues d'histones. Pour y parvenir, j'ai cartographié les épigénomes de cinq modifications différentes (3 acétylations et 2 méthylations) chez trois souches de levures issues de différents isolats naturels. Par une méthode de ChIP-seq et le développement d'un outil informatique, j'ai comparé les épigénomes de ces souches à l'échelle de nucléosomes individuels. L'étude des propriétés génomiques des épi-polymorphismes m'a alors permis de découvrir certaines caractéristiques encore inconnues et décrites dans ce manuscrit.Par ailleurs, j'ai voulu aborder le lien entre épi-polymorphismes et réponse transcriptionnelle à l'environnement. Pour cela, j'ai construit un jeu de souches mutantes dérivées de souches naturelles, où certains épi-polymorphismes ne peuvent plus être maintenus. J'ai analysé par RNA-seq les transcriptomes de certaines de ces souches avant et après un changement environnemental. Malheureusement, l'analyse des résultats a révélé que la qualité des données ne permettent pas d'établir le lien recherché mais les outils mis en place sont désormais disponibles.J'ai enfin étudié la dynamique d'évolution d'un épigénome en présence ou en l'absence de pression de sélection. Pour cela, j'ai suivi une modification d'histone (l'acétylation de la lysine 14 de l'histone H3) chez la levure pendant 1.000 générations dans deux conditions d'évolution expérimentale différentes : l'une sélective, l'autre neutre. J'ai mis en évidence des différences remarquables et inattendues entre ces deux régimes évolutifs. Des études mécanistiques détaillées restent à faire pour caractériser la nature et les propriétés de ces différences
Epigenome is defined as the entire chromatin information other than the DNA sequence. Within a given species and for a given cell type, each indivual has specific epigenomic characteristics. Epigenomic differences between individuals (refered to as 'epi-polymorphisms') remain poorly characterized, although cases were reported where they could be linked to phenotypic differences. In my thesis, I used the model organism S. cerevisiae to identify histone modification epi-polymorphisms and study their biological impact. I profiled the epigenome of five different histone modifications (3 acetylations and 2 methylations) in three natural yeast strains. By ChIP-seq methods and software developments, I compared these strains at single-nucleosome resolution and discovered novel characteristics of these epi-polymorphisms which are described in this manuscript.Furthermore, I constructed a research framework to investigate the link between epi-polimorphisms and response to environmental cues. For this, I built a set of mutant strains derived from natural strains but where some epi-polymorphisms can no longer be maintained. I analyzed by RNA-seq the transcriptomes of some of these mutant strains before and after an environmental shift. Unfortunately, the quality of this initial data produced was not sufficient to link epi-polymorphisms to differntial responses, but the strain resources remain available for further investigations. Finally, I studied the evolutionary dynamics of epi-polymorphisms in the presence or absence of selection pressure. To do so, I followed the evolution of H3K14ac for 1.000 generations under two conditions of yeast experimental evolution ( selective or neutral). Marked differences were observed between the two regimes, revealing unexpected consequences of the presence of selection. Further mechanistic studies will be needed to elucidate the full properties of these differences

Des changements de méthylation de l'ADN peuvent affecter l'expression des gènes et pour certains être transmis au travers des générations. De telles " épimutations " qui concernent des groupes de cytosines à proximité ou dans les gènes sont donc une source potentielle de variation phénotypique héritable en absence de changements de la séquence de l'ADN. Chez les plantes la méthylation de l'ADN est cependant principalement observée au niveau des séquences répétées. Il reste à déterminer dans quelle mesure les changements de méthylation au niveau de ce type de séquences peuvent être héritées et affecter les phénotypes. Afin de répondre à ces questions, plus de 500 épiRIL (epigenetic Recombinant Inbred Lines) quasi-isogéniques a été générée chez Arabidopsis thaliana. Cette population a été obtenue par le croisement d'un parent sauvage et d'un parent mutant pour le gène DDM1 présentant une très forte réduction du taux de méthylation de l'ADN. Après un rétrocroisement de la F1 avec une plante sauvage, les individus sauvages pour le gène DDM1 ont été sélectionnés et propagées sur 6 générations par autofécondation. Nous avons montré par l'analyse du méthylome de plus de 100 épiRIL que l'hypométhylation induite par ddm1 présente selon les séquences affectées différents degrés de transmission au travers des générations. La réversion de l'hypométhylation concerne des régions associées à une abondance élevée en sRNA de 24 nt. Nous avons utilisé l'hypométhylation stablement transmise dans les épiRIL induite par ddm1 afin de détecter des QTL (Quantitative Trait Loci) affectant le temps de floraison et la longueur de la racine primaire, deux caractères pour lesquels les variations observées dans les épiRIL présentent une héritabilité importante. En dernier lieu, nous avons recherché par différentes approches les variations causales de ces QTL.

Mammalian development is orchestrated by global transcriptional changes, which drive cellular differentiation, giving rise to diverse cell types. The mechanisms that mediate the temporal control of early differentiation can be studied using embryonic stem cell (ESCs) and embryonal carcinoma cells (ECCs) as model systems. In these stem cells, differentiation signals induce transcriptional repression of genes that maintain pluripotency (PpG) and activation of genes required for lineage specification. Expression of PpGs is controlled by these genes’ proximal and distal regulatory elements, promoters and enhancers, respectively. Previously published work from our laboratory showed that during differentiation of ESCs, the repression of PpGs is accompanied by enhancer silencing mediated by the Lsd1/Mi2-NuRD-Dnmt3a complex. The enzymes in this complex catalyze histone H3K27Ac deacetylation and H3K4me1/2 demethylation followed by a gain of DNA methylation mediated by the DNA methyltransferase, Dnmt3a. The absence of these chromatin changes at PpG enhancers during ESC differentiation leads to their incomplete repression. In cancer, abnormal expression of PpG is commonly observed. Our studies show that in differentiating F9 embryonal carcinoma cells (F9 ECCs), PpG maintain substantial expression concomitant with an absence of Lsd1-mediated H3K4me1 demethylation at their respective enhancers. The continued presence of H3K4me1 blocks the downstream activity of Dnmt3a, leading to the absence of DNA methylation at these sites. The absence of Lsd1 activity at PpG enhancers establishes a “primed” chromatin state distinguished by the absence of DNA methylation and the presence of H3K4me1. We further established that the activity of Lsd1 in these cells was inhibited by Oct3/4, which was partially repressed post-differentiation. Our data reveal that sustained expression of the pioneer pluripotency factor Oct3/4 disrupts the enhancer silencing mechanism. This generates an aberrant “primed” enhancer state, which is susceptible to activation and supports tumorigenicity.

As differentiation proceeds and multiple layers of cells are produced in the early embryo, the inner cells are depleted of O₂, which triggers endothelial cell differentiation. These cells form vascular structures that allow transport of O₂ and nutrients to cells. Using ESC differentiation to endothelial cells as a model system, studies covered in this thesis work elucidated a mechanism by which the transcription factor Vascular endothelial zinc finger 1 (Vezf1) regulates endothelial differentiation and formation of vascular structures. Our data show that Vezf1-deficient ESCs fail to upregulate the expression of pro-angiogenic genes in response to endothelial differentiation induction. This defect was shown to be the result of the elevated expression of the stemness factor Cbp/p300-interacting transactivator 2 (Cited2) at the onset of differentiation. The improper expression of Cited2 sequesters histone acetyltransferase p300 from depositing active histone modifications at the regulatory elements of angiogenesis-specific genes that, in turn, impedes their activation.

Besides the discovery of epigenetic mechanisms that regulate gene expression during differentiation, our studies also include development of a sensitive method to identify activities of a specific DNA methyltransferase at genomic regions. In mammals, DNA methylation occurs at the C5 position of cytosine bases. The addition of this chemical modification is catalyzed by a family of enzymes called DNA methyltransferases (Dnmts). Current methodologies, which determine the distribution of Dnmts or DNA methylation levels in genomes, show the combined activity of multiple Dnmts at their target sites. To determine the activity of a particular Dnmt in response to an external stimulus, we developed a method, Transition State Covalent Crosslinking DNA Immunoprecipitation (TSCC-DIP), which traps catalytically active Dnmts at their transition state with the DNA substrate. Our goal is to produce a strategy that would enable the determination of the direct genomic targets of specific Dnmts, creating a valuable tool for studying the dynamic changes in DNA methylation in any biological process.

In eukaryotic systems, the genetic material of the cell –DNA– is packed into a protein-dense structure called chromatin. Chromatin structure is critical for preservation of the genetic material as well as coordination of vital processes such as DNA replication, transcription and DNA damage repair. The fundamental repeating unit of chromatin is nucleosome which is composed of an octamer of small alkaline proteins called histones and the DNA wrapped around this octamer. The nucleosomes are then packed into higher-order structures leading to formation of 3D chromatin architecture. The chromatin is a dynamic structure; the spacing between nucleosomes, or the folding of the larger chromatin segments is subjected to alterations during embryonic development, tissue specifications or simply during any event that require gene expression changes. Failure in proper regulation of chromatin structure has been associated with embryonic defects and disease such as cancer.

This work has focused on a class of ATP-dependent chromatin remodeling complexes known as switch/sucrose-non-fermentable (SWI/SNF) or BRG-associated factors (BAF) complex. This family of complexes act on chromatin and alter its physical structure by mobilizing histones or nucleosome particles through the activity of its ATPase –BRG1 or BRM, enabling more accessible DNA for the other factors such as transcription factors to localize and recruit transcription machinery. In particular, we discovered and biochemically defined a novel version of this family of chromatin complexes that we named as GLTSCR1/1L-BAF (GBAF). GLTSCR1 and GLTSCR1L are two uncharacterized paralogous proteins that have been identified as BRG1-interacting proteins. Biochemically surveying the essence of this interaction, we realized that these proteins incorporates into a previously unknown SWI/SNF family complex that lacks well-characterized SWI/SNF subunits such as ARID1/2, BAF170, BAF47; instead, uniquely comprise GLTSCR1/1L and bromodomain-containing protein BRD9. Focusing on the GLTSCR1 subunit, we observed that its absence is well-tolerated by many different cell types except slight growth retardation by prostate cancer cells. Expanding the cohort of prostate cancer cells, we realized that not the paralogous subunits GLTSCR1 or GLTSCR1L but unique and non-redundant subunit BRD9 is the major GBAF-dependence in prostate cancer cells. We observed that especially the androgen-receptor positive cell lines have severe growth defects upon BRD9 knockdown or inhibition. In vivo, we showed that xenografts with BRD9 knockdown prostate cancer cells (LNCaP) have smaller tumor size. We demonstrated that BRD9 inhibition can block the expression of androgen-receptor targets. Similarly, BRD9 knockdown and treatment with antiandrogen drug (enzalutamide) has overlapping transcriptional effects. Mechanistically, we showed that BRD9 interacts with AR and it colocalizes with AR in subset of AR -binding sites. Surprisingly, we realized that BRD9 depletion has similar transcriptional and phenotypic effects as BET protein inhibitors. BET protein family contains 4 bromodomain containing proteins (BRD2, BRD3, BRD4, BRDT). These proteins were previously shown to be critical for AR-dependent gene expression. We detected interaction between BRD9 and BRD2/4. We demonstrated that BRD4 and BRD9 had shared binding sites on genome, a fraction of which are co-bound by AR. At particular target sites we showed that BRD9 localization is dependent on BET proteins, but not the other way around. Taking together, we provided some evidences that GBAF targeting through BRD9 can be a novel therapeutic approach for prostate cancer. Growing body of reports suggested that current therapy options targeting the androgen receptor is failing due to acquired resistance. Therefore, targeting the AR pathways via its coregulators such as BET proteins or SWI/SNF complexes can serve as potent alternative approaches. Further research is needed to elucidate the roles of GBAF and BET proteins in androgen receptor independent prostate cancer cells, which are still responsive to GBAF or BET manipulations although to a lesser extent.

The transcriptional repressor Polycomb Repressive Complex 1 (PRC1) is critical for stem cell maintenance and proper differentiation and as such is involved in the development and progression of cancer. Canonical PRC1, composed of PCGF, PHC, RING and CBX, binds histone H3 lysine 27 trimethylation (H3K27me3) allowing for ubiquitination, chromatin compaction and subsequently transcriptional silencing. In mammals, each subunit has multiple paralogs creating functional and compositional diversity. The greatest diversity is contributed by the CBX targeting subunit with five mutually exclusive paralogs (CBX2/4/6/7/8). The CBX paralogs contain an N-terminal chromodomain for methyllysine binding. There has been interest in the CBX paralogs due to their misregulation in various cancers and the “druggability” of the chromodomain histone interaction. However, the unique biochemical and transcriptional functions of the paralogs are unclear. Expression changes during lineage specification and the context-dependent misregulation of CBX paralogs in cancers suggest the paralogs have paralog-specific functions. However, little has been done to define differences in paralog-mediated chromatin binding and regulation. This work utilizes a variety of approaches to tease apart the biological and biochemical functions of the CBX paralogs in chromatin binding and oncogenesis. In this dissertation, we identify a combinatorial therapeutic strategy using a CBX chromodomain inhibitor to enhance chemotherapeutic response. Further, this work demonstrates a role for CBX8 and its chromodomain in glioblastoma oncogenesis suggesting it may serve as a therapeutic target. Finally, we identify a binding mechanism for the CBX8 chromodomain in which DNA and H3K27me3 binding contribute to full chromatin association.

DNA organization is an intricate and dynamic process. The approximately two meters of DNA in a single cell is wrapped around small proteins called histones. Histones can be compacted into dense coils or loosely distributed along DNA, allowing for cells to control gene expression. This combination of DNA and histones forms chromatin. This work has focused on understanding the role of Polybromo1 (PBRM1), which is a member of a chromatin remodeling complex. PBRM1 is mutated in 3% of all human cancers and is mutated in 40% of renal clear cell carcinomas (ccRCC), the most common type of kidney cancer. Through my work characterizing PBRM1 as a tumor suppressor, we have found PBRM1 acts as a stress sensor. PBRM1 is a member of the Polybromo1 BRG1 associating factors (PBAF) complex which is a subtype of the larger BAF family of chromatin remodelers. Although BAF is essential for cell viability, knockdown of PBRM1 shows minor phenotypic changes in many cell types under standard cell culturing conditions. However, when cells without PBRM1 experience external stress, the reactive oxygen species levels in the cells are elevated and remain high compared to cells with wild type PBRM1. Depending on the cellular environment of the cell, increase in ROS can be growth promoting or growth inhibiting. PBRM1 is a structurally unique protein, containing two bromo-adjacent homologs, a high mobility group and six tandem bromodomains. Due to the multiple reader domains, it is likely PBRM1 acts to target the complex. Taking advantage of a RCCC cell line not expressing PBRM1, we re-expressed full length PBRM1 containing an asparagine to alanine mutation in each bromodomain, disrupting the acetyl-lysine binding. We have found that the bromodomains are cooperative and are facilitating binding of PBAF to chromatin. We found defects in PBRM1’s ability to suppress growth, bind to chromatin, and regulate gene expression when any of the bromodomains were mutated besides the third bromodomain. These results correlated with patient data. Using acetylated histone peptides, we have identified potential combinations of marks that PBRM1 prefers over single marks. Further work needs to be done to characterize how these histone modifications are altered under stress and they contribute to the role of PBRM1 in stress response.

Lung cancer is the third most prevalent cancer in the world; however it is the leading cause of cancer related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for ~85% of the lung cancer cases. The current strategies to treat NSCLC patients with frequent causal genetic mutations is through targeted therapeutics. Approximately 10-35% of NSCLC patient tumors have activated mutations in the Epidermal Growth Factor Receptor (EGFR) resulting in uncontrolled cellular proliferation. The standard-of care for such patients is EGFR-Tyrosine Kinase Inhibitors (EGFR-TKIs), a class of targeted therapeutics that specifically inhibit EGFR activity. One such EGFR-TKI used in this study is erlotinib. Following erlotinib treatment, tumors rapidly regress at first; however, over 50% of patients develop erlotinib resistance within a year post treatment. Development of resistance remains to be the major challenge in treatment of NSCLC using EGFR-TKIs such as erlotinib.

In approximately 60% of cases, acquired erlotinib resistance in patients is attributed to a secondary mutation in EGFR, whereas in about 20% of cases, activation of alternative signaling pathways is the reported mechanism. For the remaining 15-20% of cases the mechanism of resistance remains unknown. Therefore, it can be speculated that the common methods used to identify genetic mutations in tumors post erlotinib treatment, such as histologic analysis and genetic screening may fail to identify alterations in epigenetic mediators of erlotinib resistance, also including microRNAs (miRNAs). MiRNAs are short non-coding RNAs that post-transcriptionally negatively regulate their target transcripts. Hence, in this study two comprehensive screens were simultaneously conducted in erlotinib sensitive cells: 1) a genome-wide knock-out screen, conducted with the hypothesis that loss of function of certain genes drive erlotinib resistance, 2) a miRNA overexpression screen, conducted with the hypothesis that certain miRNAs drive the development of erlotinib resistance when overexpressed. The overreaching goal of the study was to identify novel drivers of erlotinib resistance such as microRNAs or other epigenetic factors in NSCLC.

The findings of this study led to the identification of a tumor suppressive protein and an epigenetic regulator, SUV420H2 (KMT5C) that has never been reported to be involved in erlotinib resistance. On the other hand, the miRNA overexpression screen identified five miRNAs that contribute to erlotinib resistance that were extensively analyzed using multiple bioinformatic tools. It was predicted that the miRNAs mediate erlotinib resistance via multiple pathways, owing to the ability of each miRNA to target multiple transcripts via partial complementarity. Importantly, a correlation between the two screens was identified clearly supporting the use of two simultaneous screens as a reliable technique to determine highly significant miRNA-target interactions. Overall, the findings from this study suggest that epigenetic factors, such as histone modifiers and miRNAs function as critical mediators of erlotinib resistance, possibly belonging to the 15-20% of NSCLC cases with unidentified mechanisms involved in erlotinib resistance.

DNA methylation is an epigenetic modification that is nearly ubiquitous. Eukaryotic DNA methylation contributes to the regulation of gene expression and maintaining genome integrity. In mammals, DNA methylation occurs primarily on the C5 carbon of cytosine in a CpG dinucleotide context and is catalyzed by the DNA methyltransferases, DNMT1, DNMT3A and DNMT3B. While dnmt3a and dnmt3b genes are highly homologous, the enzymes have distinct functions. Some previous reports suggested differences in the enzymatic behavior of DNMT3A and 3B, which could affect their biological roles. The goal of my thesis work was to characterize kinetics mechanisms of DNMT3A and 3B, and to identify the similarities and differences in their catalytic properties that contribute to their distinct biological functions. Given the sequence similarity between the enzymes, we asked whether DNMT3B was kinetically similar to DNMT3A. In a series of experiments designed to distinguish between various kinetics mechanisms, we reported that unlike DNMT3A, DNMT3B methylated tandem CpG on DNA in a processive manner. We also reported that the disruption of the R-D interface, critical for the cooperativity of DNMT3A, had no effect on DNMT3B activity, supporting the non-cooperative mechanism of this enzyme.

DNMT3A is frequently mutated in numerous cancers. Acute Myeloid Leukemia (AML) is a malignancy of hematopoietic stem cells in which numerous patients exhibit a high frequency of the heterozygous somatic mutation Arg882His in DNMT3A. Through thorough consensus motif building, we discovered a strong similarity in CpG flanking sequence preference between DNMT3A Arg882His variant and DNMT3B enzyme. Moreover, we found that the variant enzyme has the same kinetics mechanism as DNMT3B, indicating a gain-of-function effect caused by the mutation. This change is significant because the variant enzyme can aberrantly methylate DNMT3B targets in AML cells and effect global gene expression. In particular, given that DNMT3B has been shown to have oncogenic properties, this suggests that the Arg882His variant can acquire similar oncogenic properties and drive AML development.

Taken together, my thesis work provides novel insights into the relationship between the biochemical properties and the biological functions of DNMT3A and 3B.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree in Master of Science (Medicine) in the Division of Human Genetics
Epigenetic mechanisms regulate gene expression, a particularly important activity during foetal development. DNA methylation contained within promoter and regulatory intergenic regions influence gene activity. In utero alcohol exposure as a result of maternal consumption during pregnancy has been associated with disruption of foetal DNA methylation and gene expression, leading to neurological dysfunction, growth retardation and facial anomalies. While similar phenotypes in offspring have been associated with chronic preconception paternal alcohol exposure, the mechanisms underlying these effects remain largely unexplored. This study aimed to: (1) validate significant changes in sperm DNA methylation in a list of ten candidate genes in male mice chronically exposed for ten weeks to ethanol (n=10) compared to a calorie-equivalent sucrose solution (n=10); (2) validate significant changes in gene expression in candidate genes in the brain, liver and placenta of E16.5 embryos sired by ethanol (n=24) compared to sucrose (n=24) treated male mice; (3) quantify DNA methylation changes in candidate genes in the three embryonic tissues. (4) Lastly, previously generated microarray data were reanalysed using bioinformatics tools to generate a top ranked candidate differentially expressed gene list that was used to identify and analyse biological functions or pathways significantly over represented among these genes using PANTHER and DAVID. This study was unable to provide validation for most of the significant differences observed in the sperm DNA methylome in the original study, most likely because of the low sperm DNA concentration. Significant methylation differences were however observed at individual CpG sites in three candidate genes (Igf1r, Odc1, Depdc1b) in specific tissues of embryos sired by ethanol-exposed males relative to embryos sired by sucrose-treated males. There was concordance in the direction of altered gene expression between the cases and controls using the microarray and real-time PCR approaches for two genes in the brain (Grm7 and Zfp317), three genes in the liver (Igf1r, Vwf and Depdc1b) and one gene in the placenta vii (Vwf). However, none of the candidate genes selected for validation showed statistically significant changes. This may be a result of the modest fold changes observed in the microarray experiment that as shown in many cases, often do not replicate. The remainder of the genes showed no changes in expression in the test embryos relative to the control. The functional enrichment analysis revealed biological processes that were over represented in the brain and liver indicating that they may be more vulnerable to the effects of alcohol, compared to the placenta. Overall, the study could not provide a statistically significant correlation between methylation changes in the sperm that were inherited by the offspring which subsequently dysregulated gene expression in the embryo. However, as trends toward significance and significant DNA methylation changes were observed in the embryonic tissues, this study supports the idea that preconception paternal alcohol exposure can induce epigenetic alterations in a locus and organ specific manner within offspring.
MT2016

Epigenetic studies hold the promise of addressing some of the fundamental questions of human biology including development, cell differentiation, and the aetiological mechanisms of complex disease. Over the last years, several new large scale high throughput technologies have been developed to allow genome wide profiling of epigenetic signals such as DNA methylation and histone modifications. Two of such technologies were developed in our laboratory enabling a genome wide microarray based profiling of DNA methylation signatures and a high throughput method for the site specific interrogation of the density of methylated cytosine. Using these techniques, we identified a DNA methylation difference in the 3’UTR of the DLX1 gene with potentially functional implications to discordance in risk taking behavior in a single pair of MZ twins. We modeled a power analysis on the effect size of the detected difference and determined that approximately 6~25 discordant twin pairs will be adequate to yield 80% power across the entire 12 K CpG island microarray platform using our epigenomic microarray profiling technique. We performed a DNA methylome analysis of MZ twins in white blood cells (WBC), buccal epithelial cells, and gut (rectum) biopsies (N=57 pairs in total) using 12K CpG island microarrays providing the basis for the first annotation of epigenetic metastability of ~6,000 unique genomic regions in MZ twins. We performed a classical twin study on DNA methylation differences in WBC and buccal epithelial cells from 39 pairs of MZ twins to 40 pairs of DZ twins. DZ co-twins exhibited significantly higher epigenetic difference compared to the MZ co-twins in buccal cells (p=1.2x10-294). While such higher epigenetic discordance in DZ twins can result from DNA sequence differences, our in silico SNP analyses and comparison of methylomes in inbred vs. outbred mice favour the hypothesis that this is due to epigenomic differences in the zygotes. This study suggests that molecular mechanisms of heritability may not be limited to DNA sequence differences.

Prostate cancer is one of the most frequently diagnosed cancers and failure to manage localized disease contributes to the majority of deaths. Radiation therapy (RT) is a common treatment for localized prostate cancer and uses ionizing radiation (IR) to damage DNA. Although RT is potentially curative, tumors often recur and progress to terminal disease. The cellular response to RT is multidimensional. For example, cells respond to a single dose of IR by activating the DNA damage response (DDR) to repair the DNA. Targeting proteins involved in the DDR is an effective clinical strategy to sensitize cancer cells to RT. However, multiple radiation treatments, as in fractionated ionizing radiation (FIR), can promote neuroendocrine differentiation (NED). FIR-induced NED is an emerging resistance mechanism to RT and tumors that undergo NED are highly aggressive and remain incurable.

Currently, the only clinical approach that improves RT for prostate cancer treatment is androgen deprivation therapy (ADT). ADT blocks androgen receptor (AR) signaling which inhibits the repair of DNA damage. In 2017, my lab reported that targeting Protein arginine methyltransferase 5 (PRMT5) blocks AR protein expression. Therefore, targeting PRMT5 may also sensitize prostate cancer cells to RT via a novel mechanism of action.

This dissertation focuses on the role of PRMT5 in the cellular response to IR and the goal of my work is to validate PRMT5 as a therapeutic target to enhance RT for prostate cancer treatment. I demonstrate that PRMT5 has several roles in the cellular response to IR. Upon a single dose of IR, PRMT5 cooperates with pICln to function as a master epigenetic activator of DDR genes and efficiently repair IR-induced DNA damage. There is an assumption in the field that the methyltransferase activity and epigenetic function of PRMT5 is dependent on the cofactor MEP50. I demonstrate that PRMT5 can function independently of MEP50 and identify pICln as a novel epigenetic cofactor of PRMT5. During FIR, PRMT5, along with both cofactors MEP50 and pICln, are essential for initiation of NED, maintenance of NED, and cell survival. Targeting PRMT5 also sensitizes prostate cancer xenograft tumors in mice to RT, significantly reduces and delays tumor recurrence, and prolongs overall survival. Incredibly, while 100% of control mice died due to tumor burden, targeting PRMT5 effectively cured ~85% of mice from their xenograft tumor. Overall, this work provides strong evidence for PRMT5 as a therapeutic target and suggests that targeting PRMT5 during RT should be assessed clinically.

The emergence of high-throughput methodologies after the conclusion of the Human Genome Project has brought genomic and epigenomic wide studies to the forefront of current research of biological and biomedical knowledge. Currently, the focus in genetic mutations as primary cause of certain disorders is not so relevant as before, since it was demonstrated that epigenetic mechanisms are involved in cellular programming and gene regulation providing adaptive variants of a given gene to a changing environment with an association to cellular differentiation. The research in the DNA methylation field has already revealed essential facts as the existence of methylation in CpG islands and alternative contexts that influence gene expression in tissue-specific manner. The influence of lifestyle choices in aging processes has also been related to methylome variations. And, in the case of cancer, the cooperation of epigenetic and genetic information is essential to understand the progress of cancer development as well as the silencing of key regulatory genes. An overall hypomethylation in cancer genome leads to oncogene activation whereas hypermethylation in specific regions is associated with silencing of tumour suppressor genes. For that reason, the research for new therapeutic approaches to cancer and aging is a current issue of the scientific community that work in the epigenomic field. In order to contribute to the study of mammalian epigenomes during lifespans, this research focused on the usage of public databases datasets to further investigation about DNA methylation across aged individuals in order to extract tissue-specific markers related with healthy aging. The validation of results was made through the usage of samples, form healthy individuals with good or bad cognitive performances, available in iBiMED. In both situations the genes ELOVL2 (cg16867657) and FHL2 (cg06639320) were identified as good markers of age
O aparecimento de metodologias de sequenciação de elevado rendimento após a conclusão do Projeto do Genoma Humano foi um avanço fundamental para a pesquisa biológica e biomédica na área da genómica. Embora as mutações genéticas tenham sido durante décadas o foco principal na causa de certas desordens, atualmente demonstrou-se que os mecanismos epigenéticos estão envolvidos na programação celular e na regulação genética, providenciando variações adaptativas do mesmo gene a um determinado ambiente e possuindo ainda uma associação direta com a diferenciação celular. O desenvolvimento científico no campo da metilação de DNA revela atualmente factos essenciais na biologia molecular, como a existência de metilação nas ilhas CpG e em contextos alternativos que influenciam a expressão genética nos diferentes tecidos humanos. Para além disso, a influência dos estilos de vida no processo de envelhecimento já demonstrou estar relacionada com o estado do epigenoma, nomeadamente com as variações no metiloma humano. No caso do cancro, a cooperação dos fatores genéticos e epigenéticos é essencial para a compreensão do desenvolvimento desta patologia no organismo humano nomeadamente através do silenciamento de genes reguladores essenciais. Uma hipometilação global no genoma do cancro conduz geralmente a uma ativação de oncogenes enquanto que hipermetilações localizadas estão associadas com o silenciamento de genes supressores de tumores. Por estes motivos, o desenvolvimento de novas terapias para o cancro ou o envelhecimento torna-se um tópico de interesse pela comunidade científica da área da epigenómica. Com o objetivo de desenvolver estes temas e melhorar a determinação de variações globais no epigenoma humano, esta investigação desenvolveu-se com base na utilização de dados de bases de dados públicas de indivíduos saudaveis de forma a extrair marcadores de metilação diferenciada em variados tecidos ao longo do envelhecimento saudável. O projeto foi validado através da utilização de amostras saúdaveis e de indivíduos com boas ou más performances cognitivas disponíveis no iBiMED. Em ambas as situações os genes ELOVL2 (cg16867657) e FHL2 (cg06639320) foram identificados como bons marcadores da idade dos indivíduos
Mestrado em Biotecnologia

Many imprinted genes are necessary for normal human development. Approximately 70 imprinted genes have been identified in humans. I developed a novel approach to identify candidate imprinted genes in humans using the premise that imprinted genes are often associated with nearby parent-of-origin-specific DNA differentially methylated regions (DMRs). I identified parent-of-origin-specific DMRs using sodium bisulfite-based DNA (CpG) methylation profiling of uniparental tissues, mature cystic ovarian teratoma (MCT) and androgenetic complete hydatidiform mole (AnCHM), and biparental tissues, blood and placenta. In support of this approach, the CpG methylation profiling led to the identification of parent-of-origin-specific differentially methylated CpG sites (DMCpGs) in known parent-of-origin-specific DMRs. I found new DMRs for known imprinted genes NAP1L5 and ZNF597. Most importantly, I discovered many new DMCpGs, which were associated with nearby genes, i.e., candidate imprinted genes. Allelic expression analyses of one candidate imprinted gene, AXL, suggested polymorphic imprinting of AXL in human blood.