5,973 research outputs found
Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming
Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors
Impact of Chromatin Structures on DNA Processing for Genomic Analyses
Chromatin has an impact on recombination, repair, replication, and evolution of DNA. Here we report that chromatin structure also affects laboratory DNA manipulation in ways that distort the results of chromatin immunoprecipitation (ChIP) experiments. We initially discovered this effect at the Saccharomyces cerevisiae HMR locus, where we found that silenced chromatin was refractory to shearing, relative to euchromatin. Using input samples from ChIP-Seq studies, we detected a similar bias throughout the heterochromatic portions of the yeast genome. We also observed significant chromatin-related effects at telomeres, protein binding sites, and genes, reflected in the variation of input-Seq coverage. Experimental tests of candidate regions showed that chromatin influenced shearing at some loci, and that chromatin could also lead to enriched or depleted DNA levels in prepared samples, independently of shearing effects. Our results suggested that assays relying on immunoprecipitation of chromatin will be biased by intrinsic differences between regions packaged into different chromatin structures - biases which have been largely ignored to date. These results established the pervasiveness of this bias genome-wide, and suggested that this bias can be used to detect differences in chromatin structures across the genome
YAP integrates the regulatory Snail/HNF4α circuitry controlling epithelial/hepatocyte differentiation
Yes-associated protein (YAP) is a transcriptional co-factor involved in many cell processes, including development, proliferation, stemness, differentiation, and tumorigenesis. It has been described as a sensor of mechanical and biochemical stimuli that enables cells to integrate environmental signals. Although in the liver the correlation between extracellular matrix elasticity (greatly increased in the most of chronic hepatic diseases), differentiation/functional state of parenchymal cells and subcellular localization/activation of YAP has been previously reported, its role as regulator of the hepatocyte differentiation remains to be clarified. The aim of this study was to evaluate the role of YAP in the regulation of epithelial/hepatocyte differentiation and to clarify how a transducer of general stimuli can integrate tissue-specific molecular mechanisms determining specific cell outcomes. By means of YAP silencing and overexpression we demonstrated that YAP has a functional role in the repression of epithelial/hepatocyte differentiation by inversely modulating the expression of Snail (master regulator of the epithelial-to-mesenchymal transition and liver stemness) and HNF4α (master regulator of hepatocyte differentiation) at transcriptional level, through the direct occupancy of their promoters. Furthermore, we found that Snail, in turn, is able to positively control YAP expression influencing protein level and subcellular localization and that HNF4α stably represses YAP transcription in differentiated hepatocytes both in cell culture and in adult liver. Overall, our data indicate YAP as a new member of the HNF4/Snail epistatic molecular circuitry previously demonstrated to control liver cell state. In this model, the dynamic balance between three main transcriptional regulators, that are able to control reciprocally their expression/activity, is responsible for the induction/maintenance of different liver cell differentiation states and its modulation could be the aim of therapeutic protocols for several chronic liver diseases
CTCF-mediated transcriptional regulation through cell type-specific chromosome organization in the {\beta}-globin locus
The principles underlying the architectural landscape of chromatin beyond the
nucleosome level in living cells remains largely unknown despite its potential
to play a role in mammalian gene regulation. We investigated the 3-dimensional
folding of a 1 Mbp region of human chromosome 11 containing the {\beta}-globin
genes by integrating looping interactions of the insulator protein CTCF
determined comprehensively by chromosome conformation capture (3C) into a
polymer model of chromatin. We find that CTCF-mediated cell type specific
interactions in erythroid cells are organized to favor contacts known to occur
in vivo between the {\beta}-globin locus control region (LCR) and genes. In
these cells, the modeled {\beta}-globin domain folds into a globule with the
LCR and the active globin genes on the periphery. By contrast, in non-erythroid
cells, the globule is less compact with few but dominant CTCF interactions
driving the genes away from the LCR. This leads to a decrease in contact
frequencies that can exceed 1000-fold depending on the stiffness of the
chromatin and the exact positioning of the genes. Our findings show that an
ensemble of CTCF contacts functionally affects spatial distances between
control elements and target genes contributing to chromosomal organization
required for transcription.Comment: Full article, including Supp. Mat., is available at Nucleic Acids
Research, doi: 10.1093/nar/gks53
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Dissecting the regulatory strategies of NF-kB RelA target genes in the inflammatory response reveals differential transactivation logics
Nuclear factor ÎșB (NF-ÎșB) RelA is the potent transcriptional activator of inflammatory response genes. We stringently defined a list of direct RelA target genes by integrating physical (chromatin immunoprecipitation sequencing [ChIP-seq]) and functional (RNA sequencing [RNA-seq] in knockouts) datasets. We then dissected each geneâs regulatory strategy by testing RelA variants in a primary-cell genetic-complementation assay. All endogenous target genes require RelA to make DNA-base-specific contacts, and none are activatable by the DNA binding domain alone. However, endogenous target genes differ widely in how they employ the two transactivation domains. Through model-aided analysis of the dynamic time-course data, we reveal the gene-specific synergy and redundancy of TA1 and TA2. Given that post-translational modifications control TA1 activity and intrinsic affinity for coactivators determines TA2 activity, the differential TA logics suggests context-dependent versus context-independent control of endogenous RelA-target genes. Although some inflammatory initiators appear to require co-stimulatory TA1 activation, inflammatory resolvers are a part of the NF-ÎșB RelA core response
Nucleosomal organisation over the ovine ÎČ-lactoglobulin gene
The genetic material of all higher organisms from yeast to mammals is organised
in the cell nucleus as a nucleoprotein complex called chromatin. The fundamental
repeating unit of chromatin, which covers nearly the entire DNA, is the nucleosome.
Each one comprises eight highly conserved protein subunits that sequester
approximately 146bp of DNA. Nucleosomes facilitate the highly condensed packaging
of DNA, most obvious in metaphase chromosomes, and also permit non-histone protein
factors access to the DNA in order to facilitate DNA replication, transcription and repair.For temporally and spatially specific gene activation to occur, chromatin
remodeling factors, transcription factors and RNA polymerase and its associated factors
must act in concert with the underlying nucleosome environment to effect transcription.
In some instances, this has shown to be a complex relationship. Nucleosomes are stably
positioned over transcription factor binding sites in some genes. This can prevent access
and therefore repress gene activation. In other genes, a positioned nucleosome is
required to wrap up DNA between separate transcription factor binding sites. Bringing
the sites together allows the binding factors to act cooperatively in initiating
transcription. Therefore, nucleosomes that are positioned over a specific DNA sequence
can have an instrumental role in gene regulation.To date, there have only been limited studies on the nucleosomal organisation of
genes in their natural environment. The majority of these studies have concentrated on
short regions of positioned nucleosomes spanning either repetitive DNA or the promoter
regions of specific genes. However, nucleosome positioning over an entire gene domain
may have a significant impact on its regulation and compaction. I have mapped the
nucleosomal organisation over lOkb of a tissue specific, temporally regulated gene using
the enzymatic probe, micrococcal nuclease and the chemical probe, cuprous
phenanthroline. The ovine p-lactoglobulin (BLG) gene studied has a well characterised
developmental profile, a minimal transcriptional domain and has been used extensively
as an expression cassette in transgenic animals to drive heterologous gene transcriptionWhen the gene is inactive, in the liver, it displays a tightly defined array of
positioned nucleosomes that modulate between two specific phases over the gene
domain. A similar, less tightly defined array is present when the gene is active, in the
mammary gland, except over the promoter and actively transcribing regions. The same
arrays arc present over the BLG promoter region in transgenic mice in both active and
inactive states. A monomer extension reaction provides in vitro evidence of the
positioning signals that are determined by DNA sequence alone. These show an
interesting correlation with the in vivo results.A number of other milk protein genes have a similar pattern of key transcription
factor binding sites over their promoter regions. If the nucleosome positions were
conserved in these genes, with respect to these binding sites, it might suggest a role for
positioned nucleosomes in their regulation. A total of three genes, each in two different
organisms, have been analysed to test for a correlation
Live Cell Imaging Unveils Multiple Domain Requirements for In Vivo Dimerization of the Glucocorticoid Receptor
Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.Fil: Presman, Diego Martin. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de QuĂmica BiolĂłgica; ArgentinaFil: Ogara, Maria Florencia. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de QuĂmica BiolĂłgica; ArgentinaFil: Stortz, Martin Dario. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de QuĂmica BiolĂłgica; ArgentinaFil: Alvarez, Lautaro Damian. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Unidad de MicroanĂĄlisis y MĂ©todos FĂsicos en QuĂmica OrgĂĄnica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de MicroanĂĄlisis y MĂ©todos FĂsicos en QuĂmica OrgĂĄnica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de QuĂmica OrgĂĄnica; ArgentinaFil: Pooley, John R.. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados Unidos. University of Bristol; Reino UnidoFil: Schiltz, R. Louis. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: GrĂžntved, Lars. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: Johnson, Thomas A.. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: Mittelstadt, Paul R.. National Cancer Institute. Laboratory of Immune Cell Biology; Estados UnidosFil: Ashwell, Jonathan D.. National Cancer Institute. Laboratory of Immune Cell Biology; Estados UnidosFil: Ganesan, Sundar. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados Unidos. National Institute of Allergy and Infectious Diseases; Estados UnidosFil: Burton, Gerardo. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Unidad de MicroanĂĄlisis y MĂ©todos FĂsicos en QuĂmica OrgĂĄnica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de MicroanĂĄlisis y MĂ©todos FĂsicos en QuĂmica OrgĂĄnica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de QuĂmica OrgĂĄnica; ArgentinaFil: Levi, Valeria. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de QuĂmica BiolĂłgica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de QuĂmica BiolĂłgica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de QuĂmica BiolĂłgica; ArgentinaFil: Hager, Gordon L.. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: Pecci, Adali. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologĂa, BiologĂa Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de QuĂmica BiolĂłgica; Argentin
Developmental Chromatin Restriction of ProâGrowth Gene Networks Acts as an Epigenetic Barrier to Axon Regeneration in Cortical Neurons
Axon regeneration in the central nervous system is prevented in part by a developmental decline in the intrinsic regenerative ability of maturing neurons. This loss of axon growth ability likely reflects widespread changes in gene expression, but the mechanisms that drive this shift remain unclear. Chromatin accessibility has emerged as a key regulatory mechanism in other cellular contexts, raising the possibility that chromatin structure may contribute to the ageâdependent loss of regenerative potential. Here we establish an integrated bioinformatic pipeline that combines analysis of developmentally dynamic gene networks with transcription factor regulation and genomeâwide maps of chromatin accessibility. When applied to the developing cortex, this pipeline detected overall closure of chromatin in subânetworks of genes associated with axon growth. We next analyzed mature CNS neurons that were supplied with various proâregenerative transcription factors. Unlike prior results with SOX11 and KLF7, here we found that neither JUN nor an activated form of STAT3 promoted substantial corticospinal tract regeneration. Correspondingly, chromatin accessibility in JUN or STAT3 target genes was substantially lower than in predicted targets of SOX11 and KLF7. Finally, we used the pipeline to predict pioneer factors that could potentially relieve chromatin constraints at growthâassociated loci. Overall this integrated analysis substantiates the hypothesis that dynamic chromatin accessibility contributes to the developmental decline in axon growth ability and influences the efficacy of proâregenerative interventions in the adult, while also pointing toward selected pioneer factors as highâpriority candidates for future combinatorial experiments
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