<p><p>Eukaryotic genomes have extensive flexibility and plasticity to modify transcription and replication programs, yielding a myriad of differentiated cell types and survival mechanisms to adverse environmental conditions. As these genomic processes require precise localization of DNA-binding factors, their dynamic temporal and spatial distributions provide dramatically different interpretations of a static genome sequence. DNA-binding factors must compete with nucleosomes, the basic subunit of chromatin, for access to the underlying DNA sequence. Even though the spatial preferences of these proteins are partially explained by DNA sequence alone, the complete genome occupancy profile has remained elusive, and we currently have a limited understanding of how DNA-binding protein configurations directly impact transcription and replication function.</p></p><p><p>Profiling the entire chromatin environment has typically required multiple experiments to capture both DNA-binding factors and nucleosomes. Here, we have extended the traditional micrococcal nuclease (MNase) digestion assay to simultaneously resolve both nucleosomes and smaller DNA-binding footprints in <i>Saccharomyces cerevisiae</i>. Visualization of protected DNA fragments revealed a nucleotide-resolution view of the chromatin architecture at individual genomic loci. We show that different MNase digestion times can capture nucleosomes partially unwrapped or complexed with chromatin remodelers. Stereotypical DNA-binding footprints are evident across all promoters, even at low-transcribed and silent genes. By aggregating the chromatin profiles across transcription-factor--binding sites, we precisely resolve protein footprints, yielding <i>in vivo</i> insights into protein-DNA interactions. Together, our MNase method, in one experiment, provides an unprecedented assessment of the entire chromatin structure genome-wide.</p></p><p><p>We utilized this approach to interrogate how the replication program is regulated by the chromatin environment surrounding DNA replication initiation sites. Pre-replicative complex (pre-RC) formation commences with recruitment of the origin recognition complex (ORC) to specific locations in the genome, termed replication origins. Although successful pre-RC assembly primes each site for S-phase initiation by loading the Mcm2-7 helicase, replication origins have substantially different activation times and efficiencies. We posited that replication origin function is substantially impacted by the local chromatin environment. Here, we resolved a high-resolution ORC-dependent footprint at 269 replication origins genome-wide. Even though ORC in <i>S. cerevisiae</i> remains bound at replication origins throughout the cell cycle, we detected a subset of inefficient origins that did not yield a footprint until G1, suggesting a more transient ORC interaction prior to pre-RC assembly. Nucleosome movement accommodated the pre-RC-induced expansion of the ORC-dependent footprint in G1, leading to increased activation efficiency. Mcm2-7 loading is preferentially directed to one side of each replication origin, in close proximity to the origin-flanking nucleosome. Our data demonstrates that pre-RC components are assembled into multiple configurations <i>in vivo</i>.</p></p><p><p>We anticipate that extending chromatin occupancy profiling to many different cell types will reveal further insights into genome regulation.</p></p> / Dissertation

Made available in DSpace on 2015-04-09T12:28:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-06-30Bitstream added on 2015-04-09T12:48:26Z : No. of bitstreams: 1 000815808.pdf: 368367 bytes, checksum: 730f4d89fb923e167df061884d63fd71 (MD5) / O objetivo deste estudo foi avaliar o empacotamento da cromatina e morfometria da cabeça de espermatozoides criopreservados de touros Bos taurus indicus da raça Nelore de diferentes idades e a influência do grau de compactação da cromatina na produção in vitro de embriões (PIV). Foram utilizados 40 animais mantidos em centro de coleta e processamento de sêmen (CCPS) e distribuídos em três grupos: Grupo Jovens (entre 1,8 e 2 anos), Grupo Adultos (entre 3,5 e 7 anos) e Grupo Senis (entre 8 e 14,3 anos). Os ejaculados foram congelados de acordo com os padrões pré-estabelecidos do CCPS. Utilizou-se a coloração de azul de toluidina, que permite a avaliação simultânea da cromatina e morfometria da cabeça espermática, a cromomicina A3 (CMA3) para analisar a protaminação espermática e a PIV para o desenvolvimento embrionário. O azul de toluidina foi avaliado pela microscopia óptica e a CMA3 pelo citômetro de fluxo. Na PIV foram realizadas quatro repetições por grupo, com 25 a 30 oócitos em cada repetição. A análise seminal revelou que espermatozoides de touros jovens obtiveram maiores valores de área (A), perímetro (P) e largura (L) quando comparados a adultos e senis (Jovens: A=1848,5±119,79, P=10,23±0,29, L=1,95±0,1; Adultos: A=1672,9±104,46, P=9,86±0,33, L=1,81±0,06; Senis: A=1723,1±124,41, P=9,97±0,33, L=1,83±0,09; P<0,0001) e apresentaram maior deficiência de protaminação quando analisado pela CMA3 (Jovens: 1,57±0,76; Adultos: 1,09±0,63, Senis: 0,90±0,59; P<0,05). Da mesma forma, variáveis de tamanho (A, P, L) e protaminação espermática, avaliado pela CMA3, obtiveram correlação negativa com a idade e positiva com a elipsidade (P<0,05). Espermatozoides com anormalidades na cromatina obtiveram maior área quando comparado àqueles sem alteração de cromatina (P<0,0001). Não houve diferença significativa na PIV quando sêmen com maior e menor parcela de alteração de cromatina foi ... / The aim of this study was to evaluate chromatin condensation and sperm head morphometry of cryopreserved semen samples from Bos taurus indicus bulls of different ages and the influence of the degree of chromatin condensation on in vitro embryo production (IVP). A total of 40 animals kept in an Artificial Insemination Centre were divided into three groups: Young Group (1.8 to 2 years), Adult Group (3.5 to 7 years) and Senile Group (8 to 14.3 years). The ejaculates were frozen in accordance with established standards of the Artificial Insemination Centre. The thawed semen samples were evaluated with toluidine blue, which analyzes the chromatin condensation and sperm head morphometry simultaneosly, chromomicin A3 (CMA3) to analyze the protamination and in vitro embryo production to analyze the embryonic development. The toluidine blue was evaluated by optical microscopy and the CMA3 by flow cytometry. Semen analyses revealed that spermatozoa of young bulls had higher values of area (A), perimeter (P) and width (W) when compared to adults and senile (Young: A = 1848.5 ± 119.79, P = 10. 23 ± 0.29, W = 1.95 ± 0.1; Adults: A = 1672.9 ± 104.46, P = 9.86 ± 0.33, W = 1.81 ± 0.06; Senile: A = 1723.1 ± 124.41, P = 9.97 ± 0.33, W = 1.83 ± 0.09; P<0.0001) and had higher protamination deficiency when analyzed with CMA3 (Young: 1.57 ± 0.76, Adults: 1.09 ± 0.63, Senile: 0.90 ± 0.59, P<0.05). Variable size (A, P, W) and sperm protamination evaluated by CMA3 showed negative correlation with age and a positive with ellipticity (P<0.05). Spermatozoa with abnormal chromatin obtained larger area when compared to those with normal chromatin (P<0.0001). There was no significant difference in IVP using semen with higher and lower altered chromatin condensation, evidencing that the proportion of spermatozoa with abnormal chromatin (4 to 16.15%) did not affect the embryonic development. Our results indicate that sperm head of young bulls are ...

In eukaryotic cells, transcription factors and polymerases must access DNA in the context of nucleosomes and chromatin. The accessibility of DNA sequences to such trans-acting factors is an important feature of gene regulatory elements, including promoters. In vertebrates, the majority of gene promoters coincide with CpG islands (CGIs), which remain free from DNA methylation and exhibit elevated CpG densities. This hypomethylated and CpG-rich state at CGI promoters is associated not only with transcriptional activity, but also with high levels of chromatin accessibility. However, the causes and consequences of such chromatin accessibility remain unclear. To address this, I have profiled chromatin accessibility in mouse embryonic stem cells (ESCs). In addition to confirming that CGI accessibility is independent of transcriptional activity, I was able to demonstrate that the loss of DNA methylation in ESCs resulted in increased chromatin accessibility at a subset of CpG-rich repetitive elements, suggesting that non-methylated CpG-rich sequences may, at least partially, facilitate open chromatin states. This was supported by preliminary work targeting bacterial CpG-rich sequences into the mouse genome, where they were sufficient to establish novel regions of chromatin accessibility. To examine potential mechanisms by which hypomethylated DNA could serve to promote chromatin accessibility, I profiled chromatin accessibility in mouse ESCs lacking various chromatin-modifying proteins which are normally enriched at CGIs, with the histone demethylases KDM2A/B linked to maintaining open chromatin at CGIs. As an alternative approach to understanding the causes of chromatin accessibility in mouse ESCs, I examined the mechanism by which the pioneer transcription factor OCT4 is able to access previously inaccessible chromatin, and reveal that it requires the chromatin remodeller BRG1 to remodel chromatin and facilitate transcription factor binding at distal regulatory elements. Ultimately, this work provides an insight into some of the molecular determinants of chromatin accessibility in mouse ESCs, although many of the consequences of such chromatin states remain unclear.

abstract: In eukaryotes, DNA is packed in a highly condensed and hierarchically organized structure called chromatin, in which DNA tightly wraps around the histone octamer consisting of one histone 3-histone 4 (H3-H4) tetramer and two histone 2A- histone 2B (H2A-H2B) dimers with 147 base pairs in an almost two left handed turns. Almost all DNA dependent cellular processes, such as DNA duplication, transcription, DNA repair and recombination, take place in the chromatin form. Based on the critical importance of appropriate chromatin condensation, this thesis focused on the folding behavior of the nucleosome array reconstituted using different templates with various controllable factors such as histone tail modification, linker DNA length, and DNA binding proteins. Firstly, the folding behaviors of wild type (WT) and nucleosome arrays reconstituted with acetylation on the histone H4 at lysine 16 (H4K16 (Ac)) were studied. In contrast to the sedimentation result, atomic force microscopy (AFM) measurements revealed no apparent difference in the compact nucleosome arrays between WT and H4K16 (Ac) and WT. Instead, an optimal loading of nucleosome along the template was found necessary for the Mg2+ induced nucleosome array compaction. This finding leads to the further study on the role of linker DNA in the nucleosome compaction. A method of constructing DNA templates with varied linker DNA lengths was developed, and uniformly and randomly spaced nucleosome arrays with average linker DNA lengths of 30 bp and 60 bp were constructed. After comprehensive analyses of the nucleosome arrays' structure in mica surface, the lengths of the linker DNA were found playing an important role in controlling the structural geometries of nucleosome arrays in both their extended and compact forms. In addition, higher concentration of the DNA binding domain of the telomere repeat factor 2 (TRF2) was found to stimulate the compaction of the telomeric nucleosome array. Finally, AFM was successfully applied to investigate the nucleosome positioning behaviors on the Mouse Mammary Tumor Virus (MMTV) promoter region, and two highly positioned region corresponded to nucleosome A and B were identified by this method. / Dissertation/Thesis / Ph.D. Chemistry 2010

Chromatin remodelling proteins support a variety of cellular functions and utilise the energy from ATP hydrolysis to either reposition or evict nucleosomes. One such protein, Lymphoid specific helicase (LSH), regulates DNA methylation in mammalian cells cooperatively with DNA Methyltransferase 3B (DNMT3B) through binding of the N-terminal domain of LSH. The correct functioning of LSH is essential for heterochromatin formation, with a knockout of LSH causing perinatal lethality or severe developmental abnormalities. There is little biochemical data and no structural data on LSH. Therefore, we aim to determine the structural characteristics and regulatory mechanism of LSH in vitro. LSH was expressed in an optimised insect cell system which increased protein yield 25-fold with greater than 95% purity. LSH is monomeric with increased thermal stability upon ATP or ADP binding. Full length LSH could not be crystallised therefore a core ATPase region of LSH missing the N-terminal domain was identified through limited proteolysis. This also provided evidence the N-terminal domain of LSH is disordered, which was proven through biophysical characterisation of LSH1-176. Expression of the LSH ATPase region was weak and the protein was unstable; suggesting the N-terminal domain of LSH is required for LSH stability. Therefore, complementary structural methods were used to study LSH. Crosslinking mass-spectrometry revealed the N and C termini are in close proximity, suggesting flexible linking regions, which was supported by limited proteolysis experiments. Negative staining Electron Microscopy defined LSH as a tri-lobal and elongated structure which could harbour the ATPase region in the two spherical lobes. 3D modelling of SAXS data obtained of LSH was in agreement with EM data. To understand molecular mechanisms of LSH, functional studies investigating LSH:DNA and LSH:DNMT3B interactions were performed. LSH had a KD for dsDNA of 0.4 μM in solution. LSH does not bind ssDNA nor does it have a greater affinity for methylated dsDNA. LSH was found to bind the dsDNA overhangs of nucleosomes but not to core nucleosomes, suggesting LSH solely interacts with DNA in chromatin and not histones. A stable complex of LSH:DNMT3B could not be achieved in vitro, however, other components for complex formation may have been missing. This study has improved our understanding of LSH structure, biophysical properties and its biochemical interaction with DNA and nucleosomes. This study has laid the foundations for the structural investigations of a LSH:nucleosome and potentially a LSH:DNMT3B complex in vitro to gain a greater understanding of how functional domains of LSH regulates its enzymatic function.

In eukaryotes, DNA is wrapped around a group of proteins termed histones that are required to precisely control gene expression during development. The amino acids of both the globular domains and unstructured tails of these histones can be modified by chemical moieties, such as methylation, acetylation and ubiquitination. The ‘histone code’ hypothesis proposes that specific combinations of these and other histone modifications contain transcriptional information, which guides the cell machinery to activate or repress gene expression in individual cell types. Chromatin immunoprecipitation (ChIP) experiments using undifferentiated stem cell populations have identified the genomic co-localisation of histone modifications reported to have opposing effects on transcription, which is known as bivalency. The human α-globin promoter, a well-established model for the study of transcriptional regulation, is bivalent in embryonic stem (ES) cells and this bivalency is resolved once the ES cells terminally differentiate (i.e. only activating or repressing marks remain). In a humanised mouse model, the deletion of a bone fide enhancer within the human α-globin locus results in heterogeneous expression patterns in primary erythroid cells. Notably, this correlates with an unresolved bivalent state at this promoter in terminally differentiated cells. Using this mouse model it is not feasible to ascertain whether the transcriptional heterogeneity observed in the cells lacking an α-globin enhancer is reflective of epigenetic heterogeneity (i.e. a mixed population of cells) rather than co-localisation of bivalent histone modifications within the same cells. Furthermore, the functional contribution of bivalency to development has yet to be described. To address these difficulties, I aimed to generate a fluorescent reporter system for human α-globin to facilitate the separation of transcriptionally heterogeneous erythroid cells. This model will provide material for ChIP studies on transcriptionally active and inactive populations to determine whether the epigenetic bivalency is reflective of a mixed cell population or true bivalency. In addition, I aimed to produce epigenetic editing tools to target bivalent promoters, which in combination with in vitro differentiation assays would provide an interesting framework to test the function of bivalency during development. In this study, I extensively tested gene-editing strategies for generating a fluorescent reporter knock-in in humanised mouse ES cells. I validated the suitability of humanised mouse ES cell lines for gene targeting studies and optimised a robust in vitro differentiation protocol for studying erythropoiesis. I utilised both recombineering and CRISPR/Cas9 gene editing tools in tandem with PiggyBac transposon technology, to knock-in the reporter gene. I made significant steps in gene targeting and successfully inserted the reporter downstream of the α-globin gene. I also generated a cloning system to express site-specific DNA-binding domains (TALEs) fused to epigenetic regulators with the aim to resolve bivalent histone modifications in vitro. From preliminary tests using these fusion proteins targeting Nrp1, a bivalent promoter in mES cells, I observed mild but significant changes in gene expression although histone modifications were unchanged. The various tools generated and tested in this study provide a solid foundation for future development of genetic and epigenetic editing at the human α-globin and other bivalent loci.

Deformed epidermal autoregulatory factor 1 (DEAF1) is a transcription factor that binds to (T/C)TCG(G/T) half-sites and has been shown to be involved in human diseases of cancer, diabetes, depression and intellectual disorders. We used chromatin immunoprecipitation assays to assess endogenous levels of DEAF1 and RNA polymerase II occupancy on the promoter and 5'UTR of the DEAF1 gene. In exponentially growing HEK293 cells, low levels of DEAF1 bind to sequences between -718 and +232, with +1 marking the start of translation. Within 0.5 hr of treating the cells with 500 µM H2O2, DEAF1 occupancy is increased between 7-18 fold at B (-718/-569), -577/-444, C (-432/-299), D (-205/-112) and E(-97/17). There were no statistically significant changes in either RNA polymerase II phospho-serine 5 (RNA PolII pS5) or RNA polymerase II phospho-serine 2 (RNA PolII pS2) binding with H2O2 treatment compared to control. With media change, there is an increase in RNA PolII pS2 and pS5 occupancy at both a distal site -1462/-1326 and in the coding region at 133/232, while no significant change in DEAF1 occupancy was detected. DEAF1 occupancy at the DEAF1 promoter and 5'UTR are inversely correlated with RNA polymerase II occupancy, however, there were no measurable differences in DEAF1 RNA levels at 0.5 hr and 1 hr time points. In summary, these data indicate that there is increased occupancy of DEAF1 at its own promoter following stress, which inversely affects occupancy of RNA polymerase at proximal promoter and 5'UTR sites of the DEAF1 gene.

Les complexes histones méthyltransférases SET1, hautement conservés de la levure aux mammifères, sont ciblés aux régions promotrices par la protéine CFP1/CXXC, résultant en l’implémentation de la méthylation de la lysine 4 de l’histone H3 (H3K4me), modification post-traductionnelle influençant l’expression des gènes selon le contexte chromatinien. La présence de plusieurs complexes SET1 distincts dans différents systèmes modèles eucaryotes a compliqué l’étude de leurs fonctions dans un contexte développemental. Caenorhabditis elegans contient une seule protéine homologue de SET1, SET-2, et d’uniques homologues des autres sous-unités du complexe, RBBP5, ASH2, WDR5, DPY30 et CFP1. Cependant, la composition biochimique du complexe n’a pas été décrite. En couplant des expériences de co-immunoprécipitation avec des analyses de spectrométrie de masse, j’ai identifié le complexe SET-2/SET1 dans les embryons de C. elegans. D’autre part, j’ai montré que le complexe SET-2/SET1 co-immunoprécipite aussi un autre complexe conservé modifiant la chromatine et j’ai mis en évidence les interactions mises en jeu entre ces deux complexes. Mon analyse génétique a démontré que les mutants de perte de fonction des sous-unités des deux complexes partagent des phénotypes communs, en cohérence avec des fonctions développementales communes. Le laboratoire a également entrepris des expériences de transcriptomique et d’immunoprécipitation de la chromatine montrant un nouveau rôle de CFP-1 dans le recrutement de ce complexe au niveau de sites spécifiques de la chromatine. / The highly conserved SET1 family complexes are targeted by CFP1/CXXC protein to promoter regions through multivalent interactions to implement methylation of histone H3 Ly4 (H3K4me), a modification that correlates with gene expression depending on the chromatin context. The presence of distinct SET1 complexes in multiple eukaryotic model systems has hampered studies aimed at identifying the complete array of functions of SET1/MLL regulatory networks in a developmental context. Caenorhabditis elegans contains one SET1 protein, SET-2, one MLL-like protein, SET-16, and single homologs of RBBP5, ASH2, WDR5, DPY30 and CFP1. The biochemical composition of the complex however, has not been described. Through the use of co-immunoprecipitation coupled to mass spectrometry-based proteomics, I identified the SET-2/SET1 complex in C. elegans embryos. Most importantly, I showed that the SET-2/SET1 complex also co-immunoprecipitates another conserved chromatin-modifying complex and I highlighted the interactions involved between these two complexes. My genetic analysis revealed that loss of function mutants of the two complex subunits share common phenotypes, consistent with common developmental functions. The laboratory has also undertaken transcriptomic and chromatin immunoprecipitation experiments showing that CFP-1 has a role in the binding of this complex at specific chromatin regions.

DNA methylation is an epigenetic mark that is deposited throughout the genome of mammals and plays an important role in the maintenance of transcriptionally repressive states across cell divisions. There are two major mechanisms by which DNA methylation has been proposed to act: one involves the recognition of the mark by protein complexes containing histone deacetylases (HDACs) that can remodel the local chromatin. Alternatively, methylation has been suggested to directly affect the interaction between transcription factors and their cognate binding sequence. The aim of this research was to determine the contributions of these two mechanisms in cells. The importance of HDAC activity in mediating DNA methylation-dependent transcriptional repression was assessed by comparing the genes and retrotransposons that are upregulated in response to DNA methylation loss or the disruption of HDAC activity. To this purpose, we performed whole-genome transcriptional analysis in wild type and DNA methylation-deficient mouse embryonic stem cells (DNMT.TKO mESCs) in the presence and absence of the HDAC inhibitor trichostatin A. Our data suggests that there are few genes whose repression is solely dependent on the recruitment of HDACs by DNA methylation in mESCs. Rather it appears that DNA methylation and HDAC-mediated silencing represent two independent layers of repression that converge at certain transcriptional elements. To investigate the contribution of DNA methylation on the genome-wide occupancy of transcription factors, we compared the global chromatin accessibility landscape and the binding profile of candidate transcription factors in the absence or presence of DNA methylation. We found that loss of DNA methylation associates with localised gains in accessibility, some of which can be linked to the novel binding of transcription factors such as GABPA, MAX, NRF1 and YY1. Altogether, our results present new insights into the interplay between DNA methylation and histone deacetylation and their impact on the localisation of transcription factors from different families.

abstract: The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial proteins expressed in human cell lines, yet they exhibit an organizing principle: that genes and proteins may be treated as modular units that can be moved from their native organism to a novel one. However, protein behavior is always unpredictable; drop-in functionality is not guaranteed. My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli. 1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin. 2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2017