Orientador: Regina Maria Barretto Cicareli / Banca: Raquel Mantuaneli Scarel Caminaga / Banca: Maurício Bacci Junior / Resumo: Alterações súbitas ou prolongadas no meio ambiente podem ter influências deletérias na composição do código da vida, o (DNA). A epigenética é a visão moderna da nossa interação com o meio em que vivemos. De acordo com a Organização Mundial de Saúde, "morte súbita" é aquela que acontece até 24 horas desde o início da sintomatologia. A morte súbita de causa cardíaca, definida como uma morte natural inesperada em pessoas sem antecedentes cardiovasculares pré-conhecidos (com carácter fatal) revela-se, em todos os estudos efetuados até o momento, como a principal causa de morte na atividade física, podendo acometer tanto recém-nascidos como adultos. Diversos genes foram identificados em associação a SQTL (síndrome do QT longo - doenças genéticas que causam arritmias cardíacas potencialmente fatais), onde 90% dos casos estão relacionados a mutações no gene responsável pelo canal de sódio SCN5A (locus LQT3) e no gene MTR. A metilação do DNA é uma alteração epigenética que atua na regulação da expressão gênica, e pode estar relacionada com a morte súbita de origem cardíaca. Na metilação do DNA ocorre a adição de um radical metil (CH3) no carbono 5 de citosina geralmente seguida por guanina (dinucleotídeo CpG), catalisada por enzimas DNA metiltransferases (DNMTs). Metodologias relativamente simples permitem o conhecimento da existência de metilação no DNA. O tratamento do DNA genômico com bissulfito de sódio converte as citosinas (C) não metiladas em uracilas (U), mas não afeta as citosinas (C) metiladas, procedendo a seguir à amplificação por PCR específica para metilação e sequenciamento dos produtos selecionados a partir do DNA tratado com bissulfito. Este estudo teve como objetivos investigar diferenças nos padrões de metilação que pudessem estar associados ao desenvolvimento... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Prolonged or sudden changes in the environment may have deleterious influences on the content of the code of life, the (DNA). Epigenetics is the modern view of our interaction with the environment in which we live. According to the World Health Organization, "sudden death" is that which happens within 24 hours from the onset of symptoms. Sudden death from cardiac causes, defined as an unexpected natural death in people without cardiovascular history foreknown (with fatal character) shows up in all studies performed to date, as the leading cause of death in physical activity can affect both newborns and adults. Several genes have been identified in association with LQTS (long QT syndrome - genetic diseases that cause potentially fatal cardiac arrhythmias), where 90% of cases are related to mutations in the gene responsible for the sodium channel SCN5A (LQT3 locus) and the MTR gene. DNA methylation is an epigenetic modification that acts in the regulation of gene expression, and may be related to sudden cardiac death. DNA methylation occurs on the addition of a methyl group (CH3) carbon-5 of cytosine generally followed by guanine (CpG dinucleotide), enzyme catalyzed DNA methyltransferases (DNMTs). Methodologies allow relatively simple knowledge of the existence of DNA methylation. The treatment of genomic DNA with sodium bisulfite converts cytosine (C) at uracilas unmethylated (U), but does not affect cytosines (C) methylated by making Following amplification by methylation specific PCR and sequencing of the products selected from DNA treated with bisulfite. This study aimed to investigate differences in methylation patterns that could be associated with the development and / or recurrence of cardiovascular diseases. Therefore we assessed the pattern of methylation in the promoter region of genes... (Complete abstract click electronic access below) / Mestre

The Polycomb group (Pc-G) and trithorax group (trx-G) genes play crucial roles in development by regulating expression of homeotic and other genes that control cell fate. Both groups catalyse modifications in chromatin, including histone methylation, leading to epigenetic changes in gene activity. The trx-G antagonises the function of Pc-G genes by activating Pc-G target genes, and consequently trx-G mutants suppress Pc-G mutants. The trx-G genes are relatively poorly characterised in plants. We identified a novel trx-G candidate SUPRESSOR OF POLYCOMB 12 (SOP12) by a genetic screen for suppressors of mutants for the Arabidopsis Pc-G gene CURLY LEAF (CLF). Thus sop12 mutations have no discernible phenotype in wild type backgrounds but partially suppress the leaf curling and early flowering phenotypes of clf mutants. Molecular cloning shows that SOP12 encodes a Harbinger transposase nuclease-like protein which is conserved in green plants, although key residues required for the catalytic activity of the nuclease domain are not conserved. In sop12 clf double mutants, many CLF target genes are down-regulated relative to clf mutant, which suggests SOP12 is a general activator of Pc-G target genes instead of a target of CLF or a late flowering suppressor. The CLF gene encodes an H3K27me3 histone methyltransferase, however chromatin immunoprecipitation (ChIP) analysis indicates that SOP12 does not antagonise Pc-G by removing H3K27me3 methylation, which is consistent with the fact that sop12 suppresses mutants for another Pc-G gene, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), which is not involved in H3K27me3 deposition. . Rather, genetic analysis shows that sop12 enhances the phenotype of mutants of EARLY FLOWERING IN SHORT DAYS (EFS), a trx-G gene involved in deposition of H3K36me3, and of ULTRAPETALA 1 (ULT1), a plant specific trx-G gene. The enhancement indicates SOP12 may act together with ULT or EFS proteins, or at least regulate the same targets in synergistic ways. For example, SOP12 activates AP3 expression, a role which overlaps with EFS. Yeast two hybrid screening and imunoprecipitation followed by Mass spectrometry were performed to identify numerous potential SOP12 interacting proteins but await further validation. One protein (SUP1) identified through yeast two hybrid screens was independently identified by another group as a Pc-G suppressor, suggesting that SOP12 and SUP1 may act in a common complex to regulate Pc-G targets. Collectively, my data suggests that SOP12 represents a domestic transposase that has acquired a role as a novel, plant specific trx-G members.

The N-terminal tails of histones are subject to many chemical modifications that are involved in a variety of biological functions. Histone methylation is a major epigenetic modification found in both single and multicellular organisms and is directly involved in the regulation of gene expression. Methylation of lysine 9 of histone 3 (H3K9) has been shown to have diverse functions depending on the number of methyl groups added; H3K9me1 marks the active promoters, while H3K9me2 and H3K9me3 are present within inactive gene promoters and pericentric heterochromatin. G9a, also known as euchromatic histone-lysine N-methyltransferase 2 (Ehmt2), is a histone methylase that catalyses addition of mono- and dimethyl groups to H3K9 in euchromatic regions of the genome to silence genes. Therefore, it is a vital component of the gene expression regulation machinery. In mouse embryonic stem (ES) cells, G9a forms a stable heterodimer with the G9a-like protein (GLP or Ehmt1), which is further stabilised by the C2H2-type zinc finger protein, widely interspaced zinc finger protein (WIZ). These three proteins form the core G9a complex, which is essential for mouse development. Lack of any G9a complex member leads to embryonic lethality at E9.5 with severe growth defects. The ankyrin repeat domain of G9a/GLP can bind to H3K9me1/2 with high affinity in vitro (Collins et al. 2008). This enables the self-recruitment of the G9a complex to sites with H3K9me1/2 and maintenance of the mark. However, the initial recruitment of the G9a complex to sites lacking H3K9me1/2 mark during differentiation is poorly understood. Neither G9a nor GLP has a DNA/RNA binding domain, so recruitment of the G9a complex to specific sites must be mediated by other binding partners of the G9a complex. Using mass spectrometry, I was able to identify a number of zinc finger proteins as binding partners of G9a. Among these, WIZ was identified in stoichiometric amounts to G9a and GLP, and is a potential DNA binding protein similar to other C2H2-type zinc fingers. The aim of this study was to determine the role of WIZ in the recruitment of the G9a complex to specific sites. I showed that knockdown of WIZ had no significant effect on the chromatin binding of G9a in undifferentiated mouse ES cells, which indicates WIZ is dispensable in the maintenance of H3K9me2. However, I observed a 30% decrease in the G9a levels upon WIZ knockdown, which shows that WIZ might have a role in stabilising G9a. Using recombinant WIZ zinc finger pairs, I was able to show that the 3rd and 4th zinc finger of WIZ bind DNA in vitro. Furthermore, using the systematic evolution of ligands exponential enrichment (SELEX) approach I demonstrated that the zinc fingers of WIZ preferentially bind to G-rich double-stranded DNA sequences. Binding site analysis with synthetic DNA indicated that WIZ ZF3-4 require two binding sites that are a certain distance apart from each other for efficient binding. In addition, ZF3-4 binds ssDNA with higher affinity than dsDNA, and binding to ssDNA is sequence-independent. This study shows for the first time that mouse WIZ zinc finger pairs can bind DNA and RNA in vitro. Therefore, sequence-specific recruitment of G9a might be mediated by WIZ during differentiation. Furthermore, DNA binding preference of WIZ might suggest that WIZ-mediated recruitment of G9a to establish H3K9me2 could occur at the R-loops where G-rich DNA forms a hybrid with newly transcribed RNA or at the G-rich repetitive sequences.

In 2106, there were 171,550 new cases of blood cancers and over one million people in the United States living with one of these disorders. Bone marrow transplants have good outcomes, but these procedures require a donor who is a perfect match, and thus many patients are unable to receive treatment. It is important to find patient-derived treatments, such as molecules which stimulate hematopoietic stem cell (HSC) formation without the need for a donor. Therefore, a study was initiated to use human-induced pluripotent stem cell (hiPSC) technology to make a patient-derived, personalized HSC. Epigenetic regulators are divided into readers, writers, and erasers, and each of these classes has shown some effect on HSC formation. Writers add functional groups to deoxyribonucleic acid (DNA) and histone proteins, whereas erasers remove them. Readers are groups on transcription factors which interpret these changes and increase or decrease the recruitment of transcriptional machinery accordingly. In this study, a screen of 12 different candidate molecules with distinct epigenetic targets in casper zebrafish was conducted at 36 hours postfertilization (hpf) to reveal increases or decreases in definitive HSC development. The two writer molecules, C646 (histone acetyltransferase, or HAT, inhibitor) and OICR9249 (WDR5 inhibitor), and the two eraser molecules, Ex-527 (Sirt1 inhibitor) and JIB-04 (bromodomain inhibitor), showed varying degrees of increasing HSC formation. Of these molecules, C646 created the most significant increase and was further tested in the zebrafish at 48 and 72 hpf and in a murine model using ex vivo technique and a colony-forming unit (CFU) assay. In contrast to these results, the two eraser molecules, entinostat (class I histone deacetylase, or HDAC, inhibitor) and vorinostat (general HDAC inhibitor), were found to decrease HSC formation in zebrafish. The overall findings of this study provide insight into specific epigenetic regulators in HSC development and identify particular epigenetic markers that could regulate HSC formation from endothelial cells. This discovery will be a stepping stone in utilizing patient-derived hemogenic endothelial cells as a novel source of bone marrow-independent HSCs to treat patients with leukemia, lymphoma, and bone marrow cancers. / 2019-07-11T00:00:00Z

No description available.

Preterm birth (PTB) is a global public health problem that has significant adverse effects on neonatal mortality and morbidity. Progress in understanding the pathological mechanisms underlying PTB has been greatly hampered by the complex and polygenic nature of the disease. As a result, a multifaceted approach may hold promise for identifying true causal factors. The main objective of this thesis is to identify genes that play a role in the etiology of PTB using experimental data derived from different molecular levels (genome, transcriptome, and epigenome). To achieve this goal, we performed association studies using a candidate gene approach to identify genetic factors contributing to PTB. Our analysis of genetic variants in three OXT pathway genes (oxytocin (OXT), oxytocin receptor (OXTR), and leucyl/cystinyl aminopeptidase (LNPEP)) revealed several common polymorphisms in LNPEP that show significant association with prematurity. Large-scale sequence analysis of the OXTR gene identified several novel rare coding variants that might be of etiologic importance. Our results suggest that these variants, in aggregate, appear to make some contribution to susceptibility to PTB. We also examined the gene expression profiles in the human placenta to identify, at the transcriptomic level, candidate genes for PTB. Using splicing-sensitive microarray and deep sequencing technologies, we identified transcriptome signatures that differ between term (with and without labor) and preterm placental tissues and between placental and other human tissues. The transcriptome data were analyzed not only at the gene-level, but also at the exon-level, enabling the detection of alternative splicing events. The exon-level analysis revealed more frequent disruption of alternative splicing in preterm than term placental tissues, indicating that alternative splicing may represent one possible mechanism contributing to PTB. Our study at the epigenomic level was pursued through investigation of placental DNA methylation profiles. We, using a genome-wide approach, detected a panel of genes showing labor- and gestational age-associated methylation differences. Selected genes were validated using bisulfite sequencing and methylation-specific PCR. SLC30A3, a validated differentially methylated gene between term labor and preterm labor amnion tissues, for instance, may potentially play a role in the pathogenesis of PTB. Taken together, this thesis work provides a valuable source of novel candidate genes for PTB, and future research using integrative systems biology approaches may shed light on the molecular mechanisms underlying this complex, heterogeneous disease.

Doctor of Philosophy (PhD) / The field of epigenetics emerged primarily from studies in Drosophila, and is now being studied intensively by mammalian biologists. In order to increase our knowledge of epigenetic gene control in the mouse, I have studied modifiers of epigenetic gene silencing. My main method of investigation involved the characterisation of mutants from a sensitised ENU mutagenesis screen performed previously in our laboratory. The screen was carried out in an FVB/NJ strain carrying a variegating GFP transgene expressed in erythrocytes. To date we have recovered 12 dominant (D) and seven recessive (R) mutant mouse lines from this screen that display altered transgene expression. We have named these Mommes (Modifiers of murine metastable epialleles). I investigated the phenotype and attempted to identify the underlying causative mutation of two of these Momme mutants. MommeD6 is a semi-dominant, homozygous lethal mutation that acts as a suppressor of variegation with respect to the GFP transgene. This mutation has a large effect on the level of expression of the transgene in expressing cells, but little effect on the percentage of cells expressing the transgene. MommeD6 is linked to a 2.5 Mbp interval on chromosome 14. MommeD9 is a semi-dominant, homozygous lethal mutation that acts as an enhancer of variegation with respect to the GFP transgene. Mutants have a tendency to become obese as they age, show abnormal haematology profiles, and females develop infertility. MommeD9 is linked to a 17.4 Mbp region on chromosome 7. I produced and studied a strain carrying the same GFP transgene but in a new strain background, C57BL/6J. This strain provided an opportunity to look for strain-specific modifiers of expression of the GFP transgene. Several regions were mapped to chromosomal locations. Further work will be needed to identify the genes involved. This mouse will be useful in future mutagenesis screens of this type.

The dynamic changes in chromatin play a significant role in lineage commitment and differentiation. These epigenetic modifications control gene expression through recruitment of transcription factors. While the active mark H3K4me3 is present around the transcription start site on the gene, the function of the H3K36me3 mark is unknown. A number of H3K36-specific histone methyltransferases (HMTs) have been identified, however the focus of this study is the HMT Hypb. To elucidate the role of H3K36me3 in mediating expression of developmentally-regulated loci, native chromatin immunoprecipitation (N-ChIP) was performed at a subset of genes. Upon differentiation, we observe that H3K36me3 becomes enriched at the 3’ end of several muscle-specific genes. To further investigate the role of H3K36me3 in myogenesis, a lentiviral-mediated knockdown of the H3K36 HMT Hypb was performed in muscle myoblasts using shRNA. Upon Hypb knockdown, we were surprised to observe enhanced myogenesis. N-ChIP was also performed on differentiated Hypb knockdown cell lines in order to look at H3K36me3 enrichment on genes involved in muscle differentiation. N-ChIP data show a drop in H3K36me3 enrichment levels on myogenin and Ckm genes. The possible occupancy of Hypb on the coding regions of muscle-specific genes was experimentally observed by cross-linked chromatin immunoprecipitation (X-ChIP) on differentiated C2C12 cells and subsequently confirmed by X-ChIP on knockdown lines where the occupancy was lost. A model is proposed that links the observed phenotype with H3K36me3.

Eukaryotic organisms utilize post-translational modifications of highly conserved histone proteins to control gene expression programs. Methylation of lysine 4 on histone H3 (H3K4me) in particular, is thought to be associated with actively transcribed DNA. Paradoxically, recent evidence has suggested that H3K4me has a repressive function as well. Pho23, a member of the highly conserved ING family of tumour suppressor proteins, binds H3K4me and is a component of the gene repressive complex, Rpd3L. My genetic analysis suggests that Pho23 controls transcriptional repression via H3K4me and that Pho23 is itself regulated by the sequence-specific DNA-binding protein Ume6. Moreover, this Ume6-regulated function appears to be governed by Ume6 phosphorylation by Mck1, an evolutionarily conserved kinase. Finally, while Ume6/Pho23 are known to function together with the histone deacteylase Rpd3, my findings suggest the existence of an Rpd3-independent function for Pho23.

This study examined DNA methylation patterns at promoters of endothelial cell (EC)-enriched genes during differentiation of mouse ES cells towards the EC. We have previously shown that eNOS, CD31, VE-cadherin and vWF, which have an EC-enriched pattern of gene expression are differentially methylated between EC and vascular smooth muscle cells. Given that differential promoter DNA methylation is functionally important we asked when these distinct patterns are established. Using the hanging drop method to differentiate ES cells, followed by FACS, we isolated early (EB-day4 VEGFR2-positive) and late (EB-day7 CD31-positive) endothelial progenitor cells. Though current paradigms suggest that lineage-restricted genes are methylated in ES cells, we show heterogeneous promoter DNA methylation. We show DNA demethylation at the CD31 promoter in EB-day 7 CD31-positive cells. In contrast, the eNOS promoter is still heavily methylated in EB-day 7 CD31 positive cells compared with murine EC where there is no DNA methylation.