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Computational Approaches for the Analysis of Chromosome Conformation Capture Data and Their Application to Study Long-Range Gene Regulation: A DissertationLajoie, Bryan R. 10 February 2016 (has links)
Over the last decade, development and application of a set of molecular genomic approaches based on the chromosome conformation capture method (3C), combined with increasingly powerful imaging approaches have enabled high resolution and genome-wide analysis of the spatial organization of chromosomes. The aim of this thesis is two-fold; 1), to provide guidelines for analyzing and interpreting data obtained from genome-wide 3C methods such as Hi-C and 3C-seq and 2), to leverage the 3C technology to solve genome function, structure, assembly, development and dosage problems across a broad range of organisms and disease models.
First, through the introduction of cWorld, a toolkit for manipulating genome structure data, I accelerate the pace at which *C experiments can be performed, analyzed and biological insights inferred. Next I discuss a set of practical guidelines one should consider while planning an experiment to study the structure of the genome, a simple workflow for data processing unique to *C data and a set of considerations one should be aware of while attempting to gain insights from the data.
Next, I apply these guidelines and leverage the cWorld toolkit in the context of two dosage compensation systems. The first is a worm condensin mutant which shows a reduction in dosage compensation in the hermaphrodite X chromosomes. The second is an allele-specific study consisting of genome wide Hi-C, RNA-Seq and ATAC-Seq which can measure the state of the active (Xa) and inactive (Xi) X chromosome. Finally I turn to studying specific gene – enhancer looping interactions across a panel of ENCODE cell-lines.
These studies, when taken together, further our understanding of how genome structure relates to genome function.
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Identification of a Hybrid Lethal Gene on the X Chromosome of Caenorhabditis briggsaeDougherty, John Kelly January 2019 (has links)
No description available.
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Étiologie du biais de l'inactivation du chromosome X (ICX) dans les cellules sanguines des femmes vieillissantes : sélection hémizygote et acquisition de mutations somatiquesAyachi, Sami 04 1900 (has links)
Les cellules souches hématopoïétiques (CSH) assurent une production constante des cellules
sanguines tout au long de la vie, mais sont vulnérables à l’acquisition de mutations pouvant
mener à une transformation maligne. Les mutations qui confèrent un avantage de croissance
entraîneront une prolifération clonale. L’étude de la clonalité est centrale à la compréhension
de ces phénomènes. Historiquement, l’analyse de la clonalité a été possible grâce au principe
de l’inactivation du chromosome X (ICX) chez les femmes qui entraîne la création de deux
populations cellulaires, celle avec le X-paternel actif et celle avec le X-maternel actif. Une
déviation (biais) de la proportion théorique de 1 :1 entre ces deux populations peut supposer
une dominance clonale.
Nous avons démontré un biais significatif de l’ICX chez les femmes avec l’âge. Ce
phénomène peut être expliqué par plusieurs causes dont la sélection hémizygote (un des deux
X possède des allèles plus forts que l’autre) et l’acquisition de mutations dans une CSH.
Nous posons l’hypothèse que ces deux phénomènes coexistent et peuvent être distingués par
une approche génomique.
Nous avons recruté une cohorte de 2996 femmes canadiennes-françaises âgées entre 37 et
101 ans composée de 2172 individus issus de 321 familles et de 824 individus non
apparentés. Deux tissus biologiques ont été recueillis : le sang périphérique (PMN,
monocytes, lymphocytes T, lymphocytes B) et des cellules buccales. Le ratio de l’ICX a été
déterminé par la méthode HUMARA, l’analyse de gènes associés à l’hématopoïèse clonale
(19 gènes) a été faite par la méthode de séquençage NGS, et la cohorte a été génotypée à
700 625 loci polymorphiques de l’ADN (SNP). Des analyses bioinformatiques ont été
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appliquées pour étudier la contribution génétique au biais de l’ICX. Nous démontrons que :
(i) le biais de l’ICX est plus prévalent dans les cellules sanguines par rapport aux cellules
épithéliales et maximal dans les cellules myéloïdes; (ii) le biais augmente avec l’âge
seulement dans les cellules sanguines et que cette influence est plus marquée pour les
neutrophiles; (iii) la concordance du biais est très importante pour les différents types
cellulaires sanguins, suggérant un mécanisme opérant au niveau de la CSH ; (iv) il y a une
composante héréditaire liée au biais de l’ICX; (v) la présence de mutations acquises (TET2,
DNMT3A, etc.) explique seulement une partie du biais ; (vi) à l’aide d’analyses par liaison
génétique la présence d’une région sur le chromosome X à Xq21 (LOD score 4.9) qui est
associée au biais des lymphocytes T et une autre sur le chromosome 1 à 1q21 (LOD score
6) qui est associée au biais des neutrophiles.
Nous avons départagé la contribution liée à l’acquisition de mutations somatiques et identifié
pour la première fois des régions liées à une prédisposition génétique. Nos travaux se
poursuivront d’une part par l’analyse de gènes candidats dans les régions identifiées, et
d’autre part nous tenterons d’identifier les cibles génétiques qui confèrent un potentiel de
transformation maligne en utilisant une approche basée sur l’analyse du méthylome, de
l’hydroxyméthylome et du transcriptome que nous venons de valider.
Notre étude démontre la complexité de l’adaptation de l’hématopoïèse au vieillissement et
ouvre des portes sur l’identification de facteurs prédisposant aux cancers hématologiques. / Hematopoietic stem cells (HSC) ensure a constant lifelong production of blood cells, but are
vulnerable to acquisition of mutations, which may lead to malignant transformation.
Mutations that confer a growth advantage will lead to clonal derivation of cells. The study
of clonality is central to the understanding of hematopoiesis adaptation to aging. Historically,
the first clonality assays were based on the principle of X-chromosome inactivation (XCI)
in women. Women are mosaics with half the cells with the paternal X active and the other
half with the maternal one. A skewing from the theoretical 1:1 ratio between these two
populations of cells could infer clonal derivation of cells.
More than 20 years ago, our team demonstrated, through analysis of (XCI) in women, that
skewing increases with age. This intriguing phenomenon can be explained by several
etiology including hemizygous selection (one of the 2 Xs has stronger alleles) or the
acquisition of mutations giving a growth advantage. The first etiology is genetically
predetermined and the second, acquired in somatic cells of bone marrow. We hypothesize
that these two phenomena coexist and can be distinguished with a genomic approach.
To test our hypothesis, we investigated skewing in a cohort of 2996 French-Canadian women
aged 37 to 101 comprised of 2172 related individuals from 321 families and 824 unrelated
individuals. We analyzed XCI ratios at the HUMARA locus in epithelial cells, neutrophils,
T-cells, monocytes, B-lymphocytes. We genotyped the cohort for clonal hematopoiesis and
looked for germline heritable components by genome wide association studies and linkage
analyses. We document that skewing was more prevalent in blood cells than in epithelial
cells, and maximal in myeloid cells. Skewing increases with age only in blood cells. Intra-
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individual correlation of skewing blood cell types was strongly correlated, suggesting
selection influences operating at the HSC. Sibship analyses demonstrated heritability which
was strongest when parental origin of skewing was taken into account. Clonal hematopoiesis
accounted only for a small proportion of the skewing trait but its importance increased in the
very old. Linkage analysis identified a region at Xq21 for skewing occurring in T-cells (LOD
score 4.9) suggesting a hemizygous cell selection influence. We also identified a region at
1q21 for skewing in neutrophils (LOD score 6) suggesting a gene-gene interaction with Xlinked
genes.
We have demonstrated that age-associated skewing is a complex trait caused in part by
acquired mutations and genetic predisposition variants. We will pursue our investigation
using a candidate gene approach in the two identified regions and will try to identify genetic
targets of oncogenic potential by a method based on analysis of the methylome,
hydroxymethylome and transcriptome that was have validated in this cohort.
This thesis demonstrates the complexity of the adaptation mechanisms of hematopoiesis to
aging and set the stage to identification of factors predisposing to hematological cancers.
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Mapping Hybrid Lethal Genes on the X Chromosome of C. BriggsaeBittorf, Blaine E. 08 June 2018 (has links)
No description available.
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Molekulárně genetické a biochemické studie vybraných dědičných metabolických onemocnění, vývoj a aplikace nových metod. / Molecular genetic and biochemical studies of selected inherited metabolic disorders, development and applications of new methodsMušálková, Dita January 2016 (has links)
Inherited metabolic disorders (IMD) form a diverse group of several hundred different diseases with a relatively high cumulative incidence (stated up to 1:600). They are associated with accumulation of the substrates and lack of the products in specific metabolic pathways, which is caused by deficiency of the enzyme or its activator, or dysfunction of the transport protein. However, the underlying cause is at the DNA level. The grounds for different phenotype manifestation in patients with the same genotype are often not known. During my work at the Institute of Inherited Metabolic Disorders, I designed several new methods for the research of IMD and applied them in the patients and their families. I created procedures for the isolation of lysosomal membranes that are used for the research of lysosomal storage disorders and general properties of lysosomes. Next, I introduced several novel assays for determination of the X-inactivation ratio, which led to a significant increase of informative women. Nowadays, we use these methods in heterozygous women with X-linked diseases in order to study the influence of X-inactivation on the manifestation of the diseases. The cases of a girl with mucopolysaccharidosis type II, a girl with OTC deficiency and a family with the mutation in HPRT1 gene are described...
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Exploring the structural and functional dynamics of the X-inactivation centre locus during development / Exploration de la dynamique fonctionnelle de l’architecture du locus Xic lors du développement / Investigação da dinâmica funcional e estrutural do locus Xic durante o desenvolvimento embrionário de ratinhoGalupa, Rafael 19 September 2017 (has links)
La régulation de l’expression génique chez les mammifères dépend de l’organisation tridimensionnelle des chromosomes, en particulier à l’échelle des communications entre les séquences régulatrices et leurs promoteurs cibles. Ainsi, les chromosomes sont organisés en une nouvelle architecture consistant en domaines d’interactions topologiques (TADs, acronyme anglais). Mon projet de thèse avait pour but de caractériser les mécanismes moléculaires impliqués dans cette architecture et leurs importances au cours du développement embryonnaire, pour un locus bien particulier, le Xic (acronyme anglais pour X-inactivation centre). Le Xic contient les éléments régulateurs nécessaires pour initier l’inactivation du chromosome X (ICX), un phénomène épigénétique spécifique du développement des mammifères femelles, rendant l’un des deux chromosomes X inactif du point de vue transcriptionnelle. L’ICX permet d’égaliser l’expression des gènes liés au X entre les sexes chez les mammifères. Le Xic est organisé au moins en deux TADs mais une partie du locus reste encore non identifiée. Je présente ici une analyse fonctionnelle approfondie des différents éléments régulateurs au sein du Xic, comprenant des enhancers, des gènes d’ARNs non codants et des éléments structurels. Après avoir créé une série d’allèles mutés chez la souris et les cellules souches embryonnaires murines, j’ai caractérisé l’impact de ces réarrangements génomiques sur le paysage structurel et transcriptionnel du Xic. J’ai identifié des nouveaux acteurs dans la régulation de ce locus, en particulier des séquences régulatrices conservées chez les mammifères placentaires et des éléments structurels importants pour la formation d’une frontière entre les deux TADs du Xic, importante pour leur séparation et régulation. Je décris aussi la découverte de communication entre ces TADs, ce qui constitue un mécanisme inédit de régulation génique pendant le développement. Ce travail contribue à un nouveau niveau de compréhension des lois qui régissent l’organisation des TADs dans le contexte de la régulation génique chez les mammifères. / Mammalian gene regulatory landscapes rely on the folding of chromosomes in the recently discovered topologically associating domains (TADs), which ensure appropriate communication between cis-regulatory elements and their target promoters. The aim of my PhD project was to characterise the molecular mechanisms that govern this novel architecture and its functional importance in the context of a critical and developmentally regulated locus, the X-inactivation centre (Xic). The Xic contains the necessary elements to trigger X-chromosome inactivation, an epigenetic phenomenon that occurs during the development of female mammals to transcriptionally silence one of the X-chromosomes and equalise X-linked gene expression between sexes. The Xic is partitioned into at least two TADs, but its full extent is unknown. Here, I present a comprehensive functional analysis of different cis-regulatory elements within the Xic, including enhancer-like regions, long noncoding RNA loci and structural elements. Upon generating a series of mutant alleles in mice and murine embryonic stem cells, I characterised the impact of these genomic rearrangements in the structural and transcriptional landscape of the Xic and identified novel players in the regulation of this locus, including cis-acting elements conserved across placental mammals and structural elements critical for the insulation between the Xic TADs. I also found evidence for communication across TADs at this locus, which provides new insights into how regulatory landscapes can work during development. This study also extends our understanding of the rules governing the organisation of TADs and their chromatin loops in the context of mammalian gene regulation. / Nos mamíferos, a regulação da expressão genética depende da organização tridimensional dos cromosomas, em particular ao nível da comunicação regulatória entre promotores e enhancers. A esta escala, descobriu-se recentemente que os cromossomas estão organizados em domínios de interações topológicas (conhecidos como TADs, no acrónimo inglês) que se pensa providenciarem uma base estrutural para as paisagens de regulação transcricional dos genes. O meu projecto de tese teve como objectivo caracterizar os mecanismos moleculares responsáveis por esta arquitectura e a sua importância funcional no contexto de um locus crítico para o desenvolvimento embrionário, o centro de inactivação do cromossoma X (Xic, acrónimo inglês). O Xic contém os elementos genéticos necessários e suficientes para iniciar a inactivação do cromossoma X, um fenómeno epigenético que ocorre durante o desenvolvimento das fêmeas de mamíferos para silenciar um dos cromosomas X e igualar a expressão dos genes do X entre indivíduos XX e XY. O Xic está organizado em pelo menos dois TADs, mas o seu intervalo genético completo permanece desconhecido. Apresento nesta tese uma análise funcional e detalhada de diferentes sequências reguladoras presentes no Xic, incluindo regiões do tipo enhancer, genes de ARNs não codificantes e elementos estruturais. Após a criação de diversos alelos mutantes (deleções, inserções, inversões) em ratinho e em células estaminais embrionárias, através das recentes técnicas de engenharia genética, TALENs e CRISPR/Cas9, caracterizei o impacto destes rearranjos genéticos na paisagem topológica e transcricional do Xic, o que permitiu a identificação de novos actores moleculares na regulação deste locus. Em particular, descobrimos sequências de regulação transcricional altamente conservadas em mamíferos placentários e elementos estruturais importantes para a formação da fronteira entre os dois TADs do Xic. Descrevo também evidência de que há comunicação entre os dois TADs neste locus, o que compromete os modelos actuais do modus operandis dos TADs, e por isso contribui para um novo nível de compreensão dos mecanismos que regulam a expressão genética durante o desenvolvimento.
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Molekulárně genetické a biochemické studie vybraných dědičných metabolických onemocnění, vývoj a aplikace nových metod. / Molecular genetic and biochemical studies of selected inherited metabolic disorders, development and applications of new methodsMušálková, Dita January 2016 (has links)
Inherited metabolic disorders (IMD) form a diverse group of several hundred different diseases with a relatively high cumulative incidence (stated up to 1:600). They are associated with accumulation of the substrates and lack of the products in specific metabolic pathways, which is caused by deficiency of the enzyme or its activator, or dysfunction of the transport protein. However, the underlying cause is at the DNA level. The grounds for different phenotype manifestation in patients with the same genotype are often not known. During my work at the Institute of Inherited Metabolic Disorders, I designed several new methods for the research of IMD and applied them in the patients and their families. I created procedures for the isolation of lysosomal membranes that are used for the research of lysosomal storage disorders and general properties of lysosomes. Next, I introduced several novel assays for determination of the X-inactivation ratio, which led to a significant increase of informative women. Nowadays, we use these methods in heterozygous women with X-linked diseases in order to study the influence of X-inactivation on the manifestation of the diseases. The cases of a girl with mucopolysaccharidosis type II, a girl with OTC deficiency and a family with the mutation in HPRT1 gene are described...
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Experimental and theoretical analysis of X-chromosome inactivation as a paradigm for epigenetic memory and molecular decision-makingMutzel, Verena 19 October 2021 (has links)
X-Chromosom-Inaktivierung (XCI) ist der Mechanismus, den Säuger zur Dosiskompensierung zwischen weiblichen und männlichen Zellen verwenden. XCI wird ausgelöst durch die monoallelische Hochregulation der langen nicht-kodierenden RNA Xist von einem der zwei X-Chromosomen in weiblichen Zellen. Die Xist RNA vermittelt dann das Ausschalten der Gene auf diesem X-Chromosom. Das wirft einige interessante Fragen auf: Wie zählen Zellen ihre X-Chromosomen und stellen sicher, dass genau eines aktiv bleibt? Wie entscheiden sie, welches X-Chromosom aktiv bleibt und welches ausgeschaltet wird? Und wie erinnern sie sich an diese Entscheidung und behalten sie stabil bei durch alle weiteren Zellteilungen?
Mithilfe eines stochastischen Modells zeigen wir, dass diese XCI Regulation prinzipiell durch nur zwei Regulatoren erklärt werden kann: Ein global (in trans) agierender XCI Aktivator und ein lokal (in cis) agierender XCI Repressor. Dieses Netzwerk aus nur zwei Regulatoren kann die Xist Expressionsmuster in verschiedenen Säugerspezies reproduzieren, von der Maus bis zum Mensch. Es sagt außerdem voraus, dass Zellen in der Lage sind, biallelische zu monoallelischer Xist Expression zu korrigieren, eine Vorhersage, für die wir tatsächlich experimentelle Belege finden. Mit einem mechanistischen Modell zeigen wir, dass das cis-Gedächtnis über den Xist Expressionszustand durch Antisense-Transkription zustande kommen könnte. Auf dieser Hypothese aufbauend untersucht der zweite Teil der Arbeit das Potential von Antisense-Transkription, ein lokales Gedächtnis über den Expressionszustand eines Gens zu generieren, genauer. Diese Analyse sagt vorher, dass Antisense-Repression den Expressionszustand eines Lokus tatsächlich für einige Tage stabil erhalten kann. / X-chromosome inactivation (XCI) is the mechanism for dosage compensation between the sexes in mammals. It is initiated through monoallelic upregulation of the long non-coding RNA Xist from one X chromosome, which mediates almost complete transcriptional silencing of this X chromosome. XCI regulation raises intriguing and thus far unanswered questions: How do cells count their X chromosomes and ensure that exactly one stays active? How do they make a mutually exclusive choice for one inactive X chromosome, and how do they then stably maintain this choice throughout subsequent cell divisions?
Using stochastic modeling, we show that XCI onset only requires two regulators: A trans-acting Xist activator that ensures female specificity and a cis-acting Xist repressor that allows stable maintenance of alternative Xist expression states. This two-regulator network can recapitulate Xist expression patterns across different species and makes a novel prediction that is validated experimentally: Cells are able to revert biallelic Xist expression to monoallelic expression. With a mechanistic stochastic model we show that Xist's antisense transcript Tsix might be the cis-acting Xist repressor, uncovering the molecular mechanism behind the stabilization of the alternative Xist expression states. Building upon Tsix' possible functional role in stabilizing alternative Xist expression states on the active and inactive X chromosome, the second part of this thesis investigates the potential of antisense transcription to maintain a transient transcriptional memory. We find that mutual repression between a pair of antisense genes can allow the locus to remember the transcription state it has acquired due to a past signal for several days.
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