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Regulation of gene expression in macrophage immune responseAlasoo, Kaur January 2017 (has links)
Gene expression quantitative trait loci (eQTL) mapping studies can provide mechanistic insights into the functions of disease-associated variants. However, many eQTLs are cell type and context specific. This is particularly relevant for immune cells, whose cellular function and behaviour can be substantially altered by external cues. Furthermore, understanding mechanisms behind eQTLs is hindered by the difficulty of identifying causal variants. We differentiated macrophages from induced pluripotent stem cells from 86 unrelated, healthy individuals derived as part of the Human Induced Pluripotent Stem Cells Initiative. We generated RNA-seq data from these cells in four experimental conditions: naïve, interferon- gamma (IFNɣ) treatment (18h), Salmonella infection (5h), and IFNγ treatment followed by Salmonella infection. We also measured chromatin accessibility with ATAC-seq in 31-42 individuals in the same four conditions. We detected gene expression QTLs (eQTLs) for 4326 genes, over 900 of which were condition-specific. We also detected a similar number of transcript ratio QTLs (trQTLs) that influenced mRNA processing and alternative splicing. Macrophage eQTLs and trQTLs were enriched for variants associated with Alzheimer’s disease, multiple autoimmune disorders and lipid traits. We also detected chromatin accessibility QTLs (caQTLs) for 14,602 accessible regions, including hundreds of long-range interactions. Joint analysis of eQTLs with caQTLs allowed us to greatly reduce the set of credible causal variants, often pinpointing to a single most likely variant. We found that caQTLs were less condition- specific than eQTLs and ~50% of the stimulation-specific eQTLs manifested on the chromatin level already in the naive cells. These observations might help to explain the discrepancy between strong enrichment of diseases associations in regulatory elements but only modest overlap with current eQTL studies, suggesting that many regulatory elements are in a ‘primed’ state waiting for an appropriate environmental signal before regulating gene expression.
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Identificação e caracterização de proteinas que se associam 'in vitro' a fita telomerica rica em G de 'Leishmania (Leishmania) amazonensis' / Identification and characterization of proteins that associate in vitro with the Leishmania (Leishmania) amazonensis G-rich telomeric strandFernandez, Maribel Fernandez 15 October 2004 (has links)
Orientador: Maria Isabel Nogueira Cano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T16:01:48Z (GMT). No. of bitstreams: 1
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Previous issue date: 2004 / Resumo: Os terminais dos cromossomos de Leishmania (Leishmania) amazonensis contêm repetições teloméricas mantidas pela enzima telomerase da seqüência 5'-TTAGGG-3', a qual é conservada também em outros tripanosomatídeos e outros eucariotos. Utilizando-se frações protéicas purificadas a partir de extratos S100 e nuclear, foi possível se detectar atividade de telomerase e três complexos proteína-DNA que se associam in vitro com seqüências teloméricas ricas em G do parasita. A atividade de telomerase de L. (L.) amazonensis foi detectada por ensaio TRAP ("Telomeric Repeat Amplification Protocol") e apresenta características comuns às telomerases já descritas em outros tripanosomatídeos.
Os complexos proteína-DNA foram identificados por EMSA ("Electrophoretic Mobility
Shift Assays") e ensaios de "UV cross-linking" e denominados LaGT1-3 (Leishmania
amazonensis G-strand telomeric protein 1-3). Eles não são formados: i) com DNA telomérico na forma de dupla fita e com fita rica em C, ii) em experimentos de competição específica usando oligonucleotídeos de seqüências de DNA telomérico, ou iii) após o tratamento enzimático com proteinase K. LaGT1 foi o complexo mais específico, o qual é formado com todas as seqüências de DNA telomérico (Tel1-6, Tel30 e Tel36) e não se associa à seqüência telomérica de Tetrahymena. As proteínas que formam os três complexos associam-se com uma seqüência de RNA telomérico rica em G e um complexo similar a LaGT1 é formado com DNA telomérico na forma de dupla fita contendo uma projeção 3' simples fita terminal. A estimativa das constantes de dissociação (Kd) do complexo LaGT1 associado a seqüência telomérica na forma de DNA e dos complexos LaGT2 e LaGT3 associados a seqüências teloméricas na forma de DNA ou RNA,
demonstra que estes se encontram na ordem de nM, característica comum a proteínas que ligam DNA na forma de simples fita. Os componentes protéicos dos complexos LaGT2 e LaGT3 foram purificados por cromatografia de afinidade e identificados após renaturação, como bandas de ~35 kDa e ~52 kDa, respectivamente. O componente protéico de < 15 kDa de LaGT1 foi purificado em gel como um complexo irradiado com luz UV de ~18-20 kDa. Os componentes protéicos de LaGT2 e LaGT3 e o complexo irradiado LaGT1 foram digeridos em gel com tripsina e os peptídeos trípticos resultantes foram analisados por espectrometria de massa utilizando-se as técnicas: "Matrix-Assisted Laser Desorption/Ionization-Time of Flight" (MALDI-TOF) e "Liquid Chromatography Electrospray Ionization tandem Mass Spectrometry" (LC/ESIMS/MS), a análise dos espectros demonstrou que o componente protéico de ~35 kDa de LaGT2 é homólogo da proteína Rbp38 de Leishmania spp. enquanto, o componente protéico de ~52 kDa
de LaGT3 é similar à subunidade 1 da proteína de replicação A (Rpa-1) de Leishmania spp. O componente protéico de < 15 kDa de LaGT1 provavelmente uma proteína de L. (L.) amazonensis que ainda não foi identificada. A identificação das proteínas por espectrometria de massa, possibilitou a clonagem dos genes que codificam às mesmas. Neste trabalho apresentamos a caracterização parcial do gene que codifica o componente protéico do complexo LaGT3: subunidade 1 da proteína de replicação A de L. (L.) amazonensis (LaRpa-1). A análise da sua organização genômica demonstrou que o mesmo encontra-se provavelmente em baixo número de cópias ou em cópia única no genoma do parasita e que ele se localiza em uma banda cromossômica de ~0,8 Mb. Foi realizada também a análise conformacional parcial da interação entre a proteína nativa renaturada LaRpa-1 e o DNA telomérico rico em G de L. (L.) amazonensis (oligonucleotídeo Tel6) por espectroscopia de fluorescência mostrando-se um aumento da intensidade da fluorescência quando ocorre essa interação / Abstract: The chromosomal ends of Leishmania (Leishmania) amazonensis contain conserved 5'- TTAGGG-3' telomeric repeats, that are maintained by telomerase. Using Telomeric Repeat Amplification Protocol (TR AP) we detected telomerase activity in protein fractions purified from S100 and nuclear extracts. Protein complexes that associate in vitro with telomeric DNA sequences, LaGT1-3 (Leishmania amazonensis G-strand telomeric protein), were identified and characterized by electrophoretic mobility shift assays (EMSA) and UV cross-linking using protein fractions purified from S100 and nuclear extracts. The three complexes did not form i) with double strand DNA (dsDNA) and the C-rich telomeric strand, ii) in competition assays using specific telomeric DNA oligonucleotides, or iii) after pre-treatment with proteinase K. LaGT1 was the most specific, it is formed with all DNA telomeric sequences (Tel1-6, Tel30 and Tel36) and did not bind a Tetrahymena telomeric sequence. All three LaGTs associated with an RNA sequence cognate to the telomeric G-rich strand and a complex similar to LaGT1 is formed with a dsDNA bearing a 3' G-overhang tail. The dissociation constants (Kd) for LaGT1 complexed with telomeric DNA sequence, and for LaGT2-3 complexed with telomeric DNA and RNA sequences were in the nM range. The protein components of LaGT2 and LaGT3 were purified by affinity chromatography and identified, after renaturation, as ~35 kDa and ~52 kDa bands, respectively. The < 15 kDa protein component of LaGT1 was gel-purified as a UV crosslinked complex of ~18-20 kDa. LaGT2 and LaGT3 protein bands and the irradiated LaGT1 complex were digested by trypsin and the resulting peptides were analysed by mass
espectrometry techniques: Matrix-Assisted Laser Desorption/Ionization-Time of Flight
(MALDI-TOF) and by Liquid Chromatography Electrospray Ionization tandem Mass
Spectrometry (LC/ESI-MS/MS). The fingerprint analysis showed that the ~35 kDa protein
component of LaGT2 was homologous to the Leishmania spp. Rbp38 protein, whereas the ~52 kDa component of LaGT3 was similar to the putative subunit 1 of replication protein A of Leishmania spp. and the < 15 kDa protein component of LaGT1 was probably a novel
Leishmania protein. The gene encoding the protein-forming complexe LaGT3 (LaRpa-1) were cloning and parcially caracterized, the genomic organization analysis of LaRPA-1 gene showed that it is present probably in low copy number or a single copy and was mapped on a chromosome of ~0,8 Mb. The partial conformational analysis of the interaction between nativeLaRpa-1 and the telomeric G-rich DNA (Tel6) was performed using fluorescence spectroscopy, this analysis showed an increase of the fluorescence when the interaction occurs / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular
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Cardiac-enriched BAF chromatin-remodeling complex subunit Baf60c regulates gene expression programs essential for heart development and functionSun, Xin, Hota, Swetansu K., Zhou, Yu-Qing, Novak, Stefanie, Miguel-Perez, Dario, Christodoulou, Danos, Seidman, Christine E., Seidman, J. G., Gregorio, Carol C., Henkelman, R. Mark, Rossant, Janet, Bruneau, Benoit G. 15 January 2018 (has links)
How chromatin-remodeling complexes modulate gene networks to control organ-specific properties is not well understood. For example, Baf60c (Smarcd3) encodes a cardiac-enriched subunit of the SWI/SNF-like BAF chromatin complex, but its role in heart development is not fully understood. We found that constitutive loss of Baf60c leads to embryonic cardiac hypoplasia and pronounced cardiac dysfunction. Conditional deletion of Baf60c in cardiomyocytes resulted in postnatal dilated cardiomyopathy with impaired contractile function. Baf60c regulates a gene expression program that includes genes encoding contractile proteins, modulators of sarcomere function, and cardiac metabolic genes. Many of the genes deregulated in Baf60c null embryos are targets of the MEF2/SRF co-factor Myocardin (MYOCD). In a yeast two-hybrid screen, we identified MYOCD as a BAF60c interacting factor; we showed that BAF60c and MYOCD directly and functionally interact. We conclude that Baf60c is essential for coordinating a program of gene expression that regulates the fundamental functional properties of cardiomyocytes.
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Modifications de l'organisation de la chromatine liées à l’entrée en sénescence et son impact sur la réplication du génome / Impact of chromatin structure modification in senescence on the DNA replication programBesnard, Emilie 16 December 2010 (has links)
L'entrée en sénescence, considérée comme un arrêt irréversible du cycle, se caractérise par une modification de l'organisation de la chromatine formant de véritables foyers d' hétérochromatine spécifiques (SAHF) coordonnée à une modification d'expression génique et à un déclin progressif de la compétence à répliquer le génome. Ainsi, au cours de ma thèse, j'ai voulu comprendre en quoi ces changements d'organisation du génome pouvaient influer sur la distribution et l'activation des origines d e réplication lors de l'entrée en sénescence réplicative ou déclenchée de façon prématurée par l'inhibition d'un modulateur de chromatine, la protéine à activité Histone AcétylTransférase p300. Pour étudier ces régulations, j'ai utilisé le peignage moléculaire d'ADN réplicatif qui permet de suivre les fourches de réplication et d'évaluer la distribution moyenne des origines. De plus, à l'aide de la purification de brins naissants aux origines de réplication couplée à un séquençage haut débit, nous avons cartographié la position de ces origines sur l'ensemble du génome humain et étudier un ensemble de facteurs pouvant intervenir dans ce déterminisme. Grâce à cette étude, nous avons pu suivre finement les modifications d'activité des origines associées à l'entrée en sénescence. De plus, afin de mieux comprendre les mécanismes d'activation des origines de réplication, nous avons étudié en collaboration avec l'équipe du Dr Fisher, le rôle de Cdk1 et de Cdk2 dans l'activation des origines dans le modèle Xénope. / Senescence entry, considered as an irreversible cell cycle arrest, is characterized by modifications of chromatin organization forming specific heterochromatin foci (SAHF) coordinated to modification of gene expression and the progressive loss of capacity to replicate the genome. During my PhD, we investigated whether these changes in genome organization might induce modifications in the distribution and the activity of replication origins during replicative senescence entry and in prematurely induced senescence by inhibition of a chromatin modulator, the Histone AcetylTransferase p300. To study these regulations, we used the replicating DNA combing allowing to follow the progression of replication forks and to evaluate the mean distribution of origins. By using the nascent strand purification assay coupled to deep sequencing, we mapped the position of replication origins in the whole human genome and studied some factors which could be involve d with this determinism. Thanks to this study, we followed finely the modifications of activity of replication origins associated to senescence entry. Moreover, in order to better understand the mechanisms of activation of origins, we studied in collaboration with Dr Fisher's team, the role of Cdk1 and Cdk2, in the activity of replication origins in the Xenopus model.
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Long non-coding RNAs interact with PRC1 to impact Polycomb group protein recruitment and expression of Polycomb regulated genesRay, Mridula Kumari 04 February 2016 (has links)
Long non-coding RNAs (lncRNAs) are increasingly recognized as important regulators of genomic processes and cellular specification. Many lncRNAs regulate chromatin by functionally impacting the epigenetic state through direct interactions with chromatin-modifying proteins. We developed a protocol to enrich for chromatin-lncRNA interactions and used this technique to identify several candidate lncRNAs that interact with the Polycomb group (PcG) proteins. Our immunoprecipitation protocol uses a crosslinked chromatin fraction as the input and employs stringent washes and cross-validation techniques to dramatically decrease mRNA signal (as a metric of transient interactions or false positives), and increase the dynamic range of conventional RNA immunoprecipitation protocols. Applying this protocol to the PRC1 component Bmi1, we have identified 11 PcG-interacting lncRNA candidates whose expression impacts the transcription of many other chromatin factors and PcG targets. We focus on knockdown of one lncRNA candidate, CAT7, which increases expression of several homeobox-containing transcription factors as well as chromatin interacting proteins, including Trithorax group proteins, Jumanji-domain containing proteins, and PcG-like proteins in HeLa cells. Consistent with the observed increase in gene expression, knockdown of CAT7 decreases PcG binding (Suz12, H3K27me3 and Bmi1) at the promoter of the homeodomain protein Mnx1, located at the boundary of an adjacent gene desert. During early motor neuron differentiation from embryonic stem cells, knockdown of CAT7 is accompanied by changes in expression of master regulators of neuronal specification: increased upregulation Mnx1, upregulation of Isl1, and downregulation of Irx3, as well as changes in expression to several other PcG-regulated targets. Overall, this protocol is the first of its kind to efficiently identify de novo interactions between the PcG proteins and lncRNAs which impact PcG binding or PcG target gene expression.
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Structural and Biochemical Dissection of the KMT2 Core ComplexZhang, Pamela Peng January 2015 (has links)
Histone H3 lysine 4 (H3K4) methylation is an evolutionarily conserved mark commonly associated with transcription activation in eukaryotes. In mammals, this post-translational modification is deposited by the KMT2 family of H3K4 methyltransferases. Biochemical studies have shown that the enzymatic activity of the KMT2 enzymes is regulated by a core complex of four evolutionarily conserved proteins: WDR5, RbBP5, ASH2L and DPY30, collectively known as WRAD, which are all important for global H3K4 methylation. However, how these proteins interact and regulate the activity of the KMT2 enzymes is not well investigated. During my PhD, I have used structural and biochemical approaches to determine the interactions underlying formation of the core complex and regulation of KMT2 enzymatic activity. My research have shown that 1) WDR5 uses two peptide-binding clefts on opposite sides of its β-propeller domain to bridge the KMT2 enzymes to the regulatory subunit RbBP5, 2) the WDR5 peptidyl-arginine-binding cleft exhibits plasticity to accommodate the binding of all KMT2 enzymes and 3) RbBP5 S350 phosphorylation stimulates formation of the RbBP5-ASH2L complex and H3K4 methylation by the mammalian KMT2 enzymes. Collectively, these studies have provided the structural basis for understanding the important interactions governing KMT2 complex assembly and activity.
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SNF2H-Mediated Chromatin Remodelling and Its Regulation of the Pluripotent StateCook, David January 2016 (has links)
In embryonic stem cells (ESCs), the SWI/SNF, CHD, and INO80 families of ATP-dependent chromatin remodellers have been implicated in maintaining pluripotency-associated gene expression, however the involvement of ISWI family remodellers has yet to be defined. Here, we explore the importance of the mammalian ISWI homologue SNF2H (Smarca5) by deriving a conditional knockout mouse ESC line and observing the consequences of SNF2H depletion on the pluripotent state. Cre-mediated deletion of Snf2h disrupts hallmark characteristics of pluripotency, resulting in distinct morphological changes; reduced expression of the master transcription factors Oct4, Sox2, and Nanog; and reduced alkaline phosphatase activity.
To understand the mechanisms of SNF2H-mediated regulation, we mapped SNF2H-bound nucleosomes genome-wide. SNF2H is broadly distributed across the genome, but is preferentially enriched at active regulatory regions and transcription factor binding sites. These findings demonstrate the importance of SNF2H in ESCs and shed light on genome-wide mechanisms of transcriptional regulation.
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The Role of Effector Caspases in DNA Damage Response and Chromatin Remodeling During Myogenic DifferentiationAl-Khalaf, Mohammad January 2017 (has links)
Effector caspase activation is a critical regulatory step in apoptosis, as well as an essential inductive cut in numerous non-death related processes that occur within complex cell systems. Here we report two novel studies detailing mechanisms by which effector caspase activation advances muscle cell differentiation. In the first study, we demonstrate that caspase 3 triggered DNA damage leads to rapid formation of XRCC1 repair foci within differentiating myonuclei, which dissipates as the maturation program proceeds. Skeletal myoblast deletion of XRCC1 does not impact cell growth, yet leads to perinatal lethality, with sustained DNA damage and impaired myofiber development. These observations demonstrate that the temporal deployment of the XRCC1-related DNA repair mechanisms are effector caspase mediated, and essential for muscle cell differentiation. In the second study, we sought to investigate whether effector caspase enzymes altered chromatin structure to promote the early differentiation of muscle progenitor cells. Past research has shown that Matrix Attachment region proteins known as Special AT-rich binding proteins are expressed abundantly in stem and progenitor cells, showing rapid decrease in expression as the cell advances into its mature phenotype. Here we demonstrate that effector caspase-7 is responsible for cleavage of Satb2, rather than caspase 3. Satb2 degradation alters the expressed genetic profile leading to acceleration of the muscle differentiation program. Our cumulative work adds novel roles in which effector caspases are vital in the development of cells.
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The effect of chromatin structure on P element-induced male recombination in Drosophila melanogasterFitzpatrick, Kathleen Anne January 1985 (has links)
Dysgenic male recombination (MR) induced by the P strains T-007 and OKI rarely, if ever, occurs in the heterochromatin of chromosome two. One possible explanation is that the lack of heterochromatic exchange is due to the highly condensed chromatin in this region. Butyrate (a suspected modifier of chromatin structure) induced significant levels of heterochromatic MR in dysgenic hybrids derived from crosses involving two different P strains. This finding is consistent with the hypothesis that chromatin structure can influence the insertion and excision of P elements and hence MR. Analogous experiments were performed using third chromosome suppressor of variegation (Su(var)) mutations. Neither suppressor mutation induced any heterochromatic MR, suggesting that the mode of action of these Su(var) genes is different from, and more specific than, that of butyrate. One of the mutations (325) which is thought to influence meiotic recombination frequencies, causes some alterations in euchromatic MR in crosses involving the OKI strain. The other mutation, 318, affects neither meiotic nor dysgenic recombination. Su(var) 325 is the first known "factor" to influence meiotic and dysgenic recombination similarly. / Science, Faculty of / Zoology, Department of / Graduate
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Insights into the comparative biological roles of S. cerevisiae nucleoplasmin-like FKBPs Fpr3 and Fpr4Savic, Neda 07 January 2020 (has links)
The nucleoplasmin (NPM) family of acidic histone chaperones and the FK506-binding (FKBP) peptidyl proline isomerases are both linked to chromatin regulation. In vertebrates, NPM and FKBP domains are found on separate proteins. In fungi, NPM-like and FKBP domains are expressed as a single polypeptide in nucleoplasmin-like FKBP (NPL-FKBP) histone chaperones. Saccharomyces cerevisiae has two NPL-FKBPs: Fpr3 and Fpr4. These paralogs are 72% similar and are clearly derived from a common ancestral gene. This suggests that they may have redundant functions. Their retention over millions of years of evolution also implies that each must contribute non-redundantly to organism fitness. The redundant and separate biological functions of these chromatin regulators have not been studied. In this dissertation I take a systems biology approach to fill this knowledge gap.
First, I refine the powerful synthetic genetic array (SGA) method of annotating gene-gene interactions, making it amenable for the analyses of paralogous genes. Using these ‘paralog-SGA’ screens I define distinct genetic interactions unique to either Fpr3 or Fpr4, shared genetic interactions common to both paralogs, and masked genetic interactions which are direct evidence for processes where these enzymes are functionally redundant. I provide transcriptomic evidence that Fpr3 and Fpr4 cooperate to regulate genes involved in polyphosphate metabolism and ribosome biogenesis. I identify an important role for Fpr4 at the 5’ ends of protein coding genes and the non-transcribed spacers of ribosomal DNA. Finally, I show that yeast lacking Fpr4 exhibit a genome instability phenotype at rDNA, implying that this histone chaperone regulates chromatin structure and DNA access at this locus. Collectively, these data demonstrate that Fpr3 and Fpr4 operate separately, cooperatively and redundantly to regulate a variety of chromatin environments. This work is the first comprehensive and comparative study of NPL-FKBP chaperones and as such represents a significant contribution to our understanding of their biological functions. / Graduate
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