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1.
PLoS Pathog ; 18(6): e1010555, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35666761

RESUMO

The reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure, the reservoir of activatable proviruses must be eliminated while permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. In latently HIV-1 infected cells, a zinc finger protein tethered to the HIV-1 promoter produced a fluorescent signal as a protein of interest came in its proximity, such as the viral transactivator Tat when recruited to the nascent RNA. Tat is essential for viral replication. In these cells we assessed the proviral activation and chromatin composition. By linking Tat recruitment to proviral activity, we dissected the mechanisms of HIV-1 latency reversal and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasted to the continuous production of viral proteins. As expected, promoter H3K4me3 led to substantial expression of the provirus following T cell stimulation. However, the activation-induced cell cycle arrest and death led to a surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this cellular model was used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determined the effect of modifying enhancer chromatin on HIV-1 latency reversal. Only proviruses resembling active enhancers, associated with H3K4me1 and H3K27ac and subsequentially recognized by BRD4, efficiently recruited Tat upon cell stimulation. Tat-independent HIV-1 latency reversal of unknown significance still occurred. We present a method for single cell assessment of the microenvironment of the latent HIV-1 proviruses, used here to reveal how T cell stimulation modulates the proviral activity and how the subsequent fate of the infected cell depends on the chromatin context.


Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Linfócitos T CD4-Positivos , Proteínas de Ciclo Celular/genética , Cromatina , HIV-1/genética , Humanos , Proteínas Nucleares/genética , Provírus/fisiologia , Linfócitos T , Fatores de Transcrição/genética , Latência Viral/genética
2.
Mol Cell ; 59(6): 984-97, 2015 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-26321255

RESUMO

Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity.


Assuntos
Cromatina/genética , Células-Tronco Embrionárias Humanas/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Imunoprecipitação da Cromatina , Ritmo Circadiano , Corpos Embrioides/enzimologia , Epistasia Genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Células HCT116 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Lâmina Nuclear/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Ligação Proteica , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
3.
PLoS Pathog ; 16(1): e1008264, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999790

RESUMO

Human immunodeficiency virus type 1 (HIV-1) infection is a chronic condition, where viral DNA integrates into the genome. Latently infected cells form a persistent, heterogeneous reservoir that at any time can reactivate the integrated HIV-1. Here we confirmed that latently infected cells from HIV-1 positive study participants exhibited active HIV-1 transcription but without production of mature spliced mRNAs. To elucidate the mechanisms behind this we employed primary HIV-1 latency models to study latency establishment and maintenance. We characterized proviral transcription and chromatin development in cultures of resting primary CD4+ T-cells for four months after ex vivo HIV-1 infection. As heterochromatin (marked with H3K9me3 or H3K27me3) gradually stabilized, the provirus became less accessible with reduced activation potential. In a subset of infected cells, active marks (e.g. H3K27ac) and elongating RNAPII remained detectable at the latent provirus, despite prolonged proviral silencing. In many aspects, latent HIV-1 resembled an active enhancer in a subset of resting cells. The enhancer chromatin actively promoted latency and the enhancer-specific CBP/P300-inhibitor GNE049 was identified as a new latency reversal agent. The division of the latent reservoir according to distinct chromatin compositions with different reactivation potential enforces the notion that even though a relatively large set of cells contains the HIV-1 provirus, only a discrete subset is readily able to reactivate the provirus and spread the infection.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Cromatina/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Provírus/fisiologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , HIV-1/genética , Humanos , Provírus/genética , Ativação Viral , Montagem de Vírus , Latência Viral
4.
Nucleic Acids Res ; 48(13): 7154-7168, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32496538

RESUMO

Mono-ubiquitylation of histone H2B (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. It remained unclear whether Cdk9 and H2Bub1 cooperate to regulate the expression of individual genes. Here, we show that Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of ∼10% of fission yeast genes, with each perturbation affecting largely distinct subsets; ablation of both pathways de-represses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both modifications are enriched, and directly proportional to each other, in coding regions, and decrease abruptly around the cleavage and polyadenylation signal (CPS). Cdk9-dependence of antisense suppression at specific genes correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Genetic interactions link Cdk9, H2Bub1 and the histone deacetylase Clr6-CII, while combined Cdk9 inhibition and H2Bub1 loss impair Clr6-CII recruitment to chromatin and lead to decreased occupancy and increased acetylation of histones within gene coding regions. These results uncover novel interactions between co-transcriptional histone modification pathways, which link regulation of RNAPII transcription elongation to suppression of aberrant initiation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Histonas/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Elongação da Transcrição Genética , Fosforilação , Fatores de Elongação da Transcrição/metabolismo , Ubiquitinação
5.
Mol Cell ; 46(5): 691-704, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22681890

RESUMO

To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the nonessential genome. Using these signatures, we generated a catalog of 297 functional modules, and we assigned function to 144 previously uncharacterized genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional crosstalk between biological processes, suggesting general design principles of genetic interactomes.


Assuntos
Epistasia Genética , Evolução Molecular , Genes Fúngicos , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Genoma Fúngico , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Especificidade da Espécie
6.
Genome Res ; 25(6): 872-83, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25778913

RESUMO

Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation-exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally, genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation.


Assuntos
Genoma Fúngico , Histonas/genética , Nucleossomos/genética , Schizosaccharomyces/genética , Imunoprecipitação da Cromatina , Replicação do DNA , Bases de Dados Genéticas , Estudos de Associação Genética , Histonas/metabolismo , Metilação , Análise em Microsséries , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/metabolismo
7.
EMBO Rep ; 16(12): 1673-87, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26518661

RESUMO

The maintenance of open and repressed chromatin states is crucial for the regulation of gene expression. To study the genes involved in maintaining chromatin states, we generated a random mutant library in Schizosaccharomyces pombe and monitored the silencing of reporter genes inserted into the euchromatic region adjacent to the heterochromatic mating type locus. We show that Leo1-Paf1 [a subcomplex of the RNA polymerase II-associated factor 1 complex (Paf1C)] is required to prevent the spreading of heterochromatin into euchromatin by mapping the heterochromatin mark H3K9me2 using high-resolution genomewide ChIP (ChIP-exo). Loss of Leo1-Paf1 increases heterochromatin stability at several facultative heterochromatin loci in an RNAi-independent manner. Instead, deletion of Leo1 decreases nucleosome turnover, leading to heterochromatin stabilization. Our data reveal that Leo1-Paf1 promotes chromatin state fluctuations by enhancing histone turnover.


Assuntos
Cromatina/genética , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Heterocromatina/metabolismo , Código das Histonas , Histonas/genética , Mutação , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Interferência de RNA
8.
EMBO J ; 31(23): 4388-403, 2012 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-23103765

RESUMO

Nucleosome positioning governs access to eukaryotic genomes. Many genes show a stereotypic organisation at their 5'end: a nucleosome free region just upstream of the transcription start site (TSS) followed by a regular nucleosomal array over the coding region. The determinants for this pattern are unclear, but nucleosome remodelers are likely critical. Here we study the role of remodelers in global nucleosome positioning in S. pombe and the corresponding changes in expression. We find a striking evolutionary shift in remodeler usage between budding and fission yeast. The S. pombe RSC complex does not seem to be involved in nucleosome positioning, despite its prominent role in S. cerevisiae. While S. pombe lacks ISWI-type remodelers, it has two CHD1-type ATPases, Hrp1 and Hrp3. We demonstrate nucleosome spacing activity for Hrp1 and Hrp3 in vitro, and that together they are essential for linking regular genic arrays to most TSSs in vivo. Impaired arrays in the absence of either or both remodelers may lead to increased cryptic antisense transcription, but overall gene expression levels are only mildly affected.


Assuntos
Adenosina Trifosfatases/fisiologia , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação Fúngica da Expressão Gênica , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , DNA Helicases/química , Proteínas de Ligação a DNA/química , Dactinomicina/farmacologia , Deleção de Genes , Histonas/química , Modelos Biológicos , Mutação , Oligonucleotídeos Antissenso/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Transcrição Gênica , Transcriptoma
9.
PLoS Genet ; 8(9): e1002985, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23028377

RESUMO

Specialized chromatin containing CENP-A nucleosomes instead of H3 nucleosomes is found at all centromeres. However, the mechanisms that specify the locations at which CENP-A chromatin is assembled remain elusive in organisms with regional, epigenetically regulated centromeres. It is known that normal centromeric DNA is transcribed in several systems including the fission yeast, Schizosaccharomyces pombe. Here, we show that factors which preserve stable histone H3 chromatin during transcription also play a role in preventing promiscuous CENP-A(Cnp1) deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-A(Cnp1) incorporation at non-centromeric sites. FACT has little or no effect on CENP-A(Cnp1) assembly at endogenous centromeres where CENP-A(Cnp1) is normally assembled. In contrast, Clr6 complex II (Clr6-CII; equivalent to Rpd3S) histone deacetylase function has a more subtle impact on the stability of transcribed H3 chromatin and acts to prevent the ectopic accumulation of CENP-A(Cnp1) at specific loci, including subtelomeric regions, where CENP-A(Cnp1) is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-A(Cnp1) chromatin on centromeric DNA, bypassing the normal requirement for heterochromatin. Thus, our analyses show that alterations in the process of chromatin assembly during transcription can destabilize H3 nucleosomes and thereby allow CENP-A(Cnp1) to assemble in its place. We propose that normal centromeres provide a specific chromatin context that limits reassembly of H3 chromatin during transcription and thereby promotes the establishment of CENP-A(Cnp1) chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification.


Assuntos
Aminopeptidases/genética , Centrômero/genética , Proteínas Cromossômicas não Histona/genética , Chaperonas Moleculares/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Transcrição Gênica , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA/genética , Epigênese Genética , Heterocromatina/genética , Histonas/genética , Cinetocoros , Nucleossomos/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
10.
PLoS Comput Biol ; 7(8): e1002163, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21901086

RESUMO

Epigenetic regulation consists of a multitude of different modifications that determine active and inactive states of chromatin. Conditions such as cell differentiation or exposure to environmental stress require concerted changes in gene expression. To interpret epigenomics data, a spectrum of different interconnected datasets is needed, ranging from the genome sequence and positions of histones, together with their modifications and variants, to the transcriptional output of genomic regions. Here we present a tool, Podbat (Positioning database and analysis tool), that incorporates data from various sources and allows detailed dissection of the entire range of chromatin modifications simultaneously. Podbat can be used to analyze, visualize, store and share epigenomics data. Among other functions, Podbat allows data-driven determination of genome regions of differential protein occupancy or RNA expression using Hidden Markov Models. Comparisons between datasets are facilitated to enable the study of the comprehensive chromatin modification system simultaneously, irrespective of data-generating technique. Any organism with a sequenced genome can be accommodated. We exemplify the power of Podbat by reanalyzing all to-date published genome-wide data for the histone variant H2A.Z in fission yeast together with other histone marks and also phenotypic response data from several sources. This meta-analysis led to the unexpected finding of H2A.Z incorporation in the coding regions of genes encoding proteins involved in the regulation of meiosis and genotoxic stress responses. This incorporation was partly independent of the H2A.Z-incorporating remodeller Swr1. We verified an Swr1-independent role for H2A.Z following genotoxic stress in vivo. Podbat is open source software freely downloadable from www.podbat.org, distributed under the GNU LGPL license. User manuals, test data and instructions are available at the website, as well as a repository for third party-developed plug-in modules. Podbat requires Java version 1.6 or higher.


Assuntos
Sistemas de Gerenciamento de Base de Dados , Epigênese Genética , Epigenômica/métodos , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Algoritmos , Ciclo Celular/genética , Montagem e Desmontagem da Cromatina , Dano ao DNA , Genoma Fúngico , Cadeias de Markov , Modelos Genéticos , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
11.
Front Immunol ; 13: 1067767, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36561752

RESUMO

The establishment of a latency reservoir is the major obstacle for a cure of HIV-1. The shock-and-kill strategy aims to reactivate HIV-1 replication in HIV -1 latently infected cells, exposing the HIV-1-infected cells to cytotoxic lymphocytes. However, none of the latency reversal agents (LRAs) tested so far have shown the desired effect in people living with HIV-1. We observed that NK cells stimulated with a pan-caspase inhibitor induced latency reversal in co-cultures with HIV-1 latently infected cells. Synergy in HIV-1 reactivation was observed with LRAs prostratin and JQ1. The supernatants of the pan-caspase inhibitor-treated NK cells activated the HIV-1 LTR promoter, indicating that a secreted factor by NK cells was responsible for the HIV-1 reactivation. Assessing changes in the secreted cytokine profile of pan-caspase inhibitor-treated NK cells revealed increased levels of the HIV-1 suppressor chemokines MIP1α (CCL3), MIP1ß (CCL4) and RANTES (CCL5). However, these cytokines individually or together did not induce LTR promoter activation, suggesting that CCL3-5 were not responsible for the observed HIV-1 reactivation. The cytokine profile did indicate that pan-caspase inhibitors induce NK cell activation. Altogether, our approach might be-in combination with other shock-and-kill strategies or LRAs-a strategy for reducing viral latency reservoirs and a step forward towards eradication of functionally active HIV-1 in infected individuals.


Assuntos
Inibidores de Caspase , Infecções por HIV , HIV-1 , Células Matadoras Naturais , Latência Viral , Humanos , Inibidores de Caspase/farmacologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/fisiologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Latência Viral/imunologia
12.
Commun Biol ; 5(1): 357, 2022 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-35418589

RESUMO

HIV-1 infection induces a chronic inflammatory environment not restored by suppressive antiretroviral therapy (ART). As of today, the effect of viral suppression and immune reconstitution in people living with HIV-1 (PLWH) has been well described but not completely understood. Herein, we show how PLWH who naturally control the virus (PLWHEC) have a reduced proportion of CD4+CCR6+ and CD8+CCR6+ cells compared to PLWH on suppressive ART (PLWHART) and HIV-1 negative controls (HC). Expression of CCR2 was reduced on both CD4+, CD8+ and classical monocytes in PLWHEC compared to PLWHART and HC. Longer suppressive therapy, measured in the same patients, decreased number of cells expressing CCR2 on all monocytic cell populations while expression on CD8+ T cells increased. Furthermore, the CD4+CCR6+/CCR6- cells exhibited a unique proteomic profile with a modulated energy metabolism in PLWHEC compared to PLWHART independent of CCR6 status. The CD4+CCR6+ cells also showed an enrichment in proteins involved in apoptosis and p53 signalling in PLWHEC compared to PLWHART, indicative of increased sensitivity towards cell death mechanisms. Collectively, this data shows how PLWHEC have a unique chemokine receptor profile that may aid in facilitating natural control of HIV-1 infection.


Assuntos
Infecções por HIV , HIV-1 , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Controladores de Elite , Infecções por HIV/tratamento farmacológico , Humanos , Proteômica , Receptores CCR6/metabolismo
13.
Front Cell Infect Microbiol ; 11: 668637, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34026665

RESUMO

The Human Immunodeficiency Virus type 1 (HIV-1) integrates in the host genome as a provirus resulting in a long-lived reservoir of infected CD4 cells. As a provirus, HIV-1 has several aspects in common with an oncogene. Both the HIV-1 provirus and oncogenes only cause disease when expressed. A successful cure of both cancer and HIV-1 includes elimination of all cells with potential to regenerate the disease. For over two decades, epigenetic drugs developed against cancer have been used in the HIV-1 field to modulate the state of the proviral chromatin. Cells with an intact HIV-1 provirus exist in three states of infection: productive, inducible latent, and non-inducible latent. Here focus is on HIV-1, transcription control and chromatin structure; how the inducible proviruses are maintained in a chromatin structure that allows reactivation of transcription; and how transcription switches between different stages to allow for an abundance of different transcripts from a single promoter. Recently it was shown that a functional cure of HIV can be achieved by encapsulating all intact HIV-1 proviruses in heterochromatin, giving hope that epigenetic interventions may be used to end the HIV-1 epidemic.


Assuntos
Infecções por HIV , HIV-1 , Neoplasias , Linfócitos T CD4-Positivos , Epigênese Genética , Humanos , Provírus
14.
Nat Commun ; 12(1): 4800, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417450

RESUMO

Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction.


Assuntos
Cromatina/metabolismo , Genes Essenciais , Histonas/metabolismo , Lisina/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Ciclo Celular/genética , Linhagem Celular , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Humanos , Espectroscopia de Ressonância Magnética , Metilação , Camundongos , Modelos Biológicos , Nucleossomos/metabolismo , Conformação Proteica
15.
PLoS Genet ; 3(11): e207, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18039032

RESUMO

The long-term health outcome of prenatal exposure to arsenic has been associated with increased mortality in human populations. In this study, the extent to which maternal arsenic exposure impacts gene expression in the newborn was addressed. We monitored gene expression profiles in a population of newborns whose mothers experienced varying levels of arsenic exposure during pregnancy. Through the application of machine learning-based two-class prediction algorithms, we identified expression signatures from babies born to arsenic-unexposed and -exposed mothers that were highly predictive of prenatal arsenic exposure in a subsequent test population. Furthermore, 11 transcripts were identified that captured the maximal predictive capacity to classify prenatal arsenic exposure. Network analysis of the arsenic-modulated transcripts identified the activation of extensive molecular networks that are indicative of stress, inflammation, metal exposure, and apoptosis in the newborn. Exposure to arsenic is an important health hazard both in the United States and around the world, and is associated with increased risk for several types of cancer and other chronic diseases. These studies clearly demonstrate the robust impact of a mother's arsenic consumption on fetal gene expression as evidenced by transcript levels in newborn cord blood.


Assuntos
Intoxicação por Arsênico/genética , Inflamação/genética , Exposição Materna , NF-kappa B/genética , Efeitos Tardios da Exposição Pré-Natal/genética , Transdução de Sinais , Adulto , Animais , Sítios de Ligação , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Marcadores Genéticos , Genoma Humano/genética , Humanos , Recém-Nascido , Camundongos , Gravidez , Especificidade da Espécie , Tailândia , Fatores de Transcrição/metabolismo , Transcrição Gênica
16.
Sci Rep ; 10(1): 6055, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269268

RESUMO

Heterochromatin regulation is critical for genomic stability. Different H3K9 methylation states have been discovered, with distinct roles in heterochromatin formation and silencing. However, how the transition from H3K9me2 to H3K9me3 is controlled is still unclear. Here, we investigate the role of the conserved bromodomain AAA-ATPase, Abo1, involved in maintaining global nucleosome organisation in fission yeast. We identified several key factors involved in heterochromatin silencing that interact genetically with Abo1: histone deacetylase Clr3, H3K9 methyltransferase Clr4, and HP1 homolog Swi6. Cells lacking Abo1 cultivated at 30 °C exhibit an imbalance of H3K9me2 and H3K9me3 in heterochromatin. In abo1∆ cells, the centromeric constitutive heterochromatin has increased H3K9me2 but decreased H3K9me3 levels compared to wild-type. In contrast, facultative heterochromatin regions exhibit reduced H3K9me2 and H3K9me3 levels in abo1∆. Genome-wide analysis showed that abo1∆ cells have silencing defects in both the centromeres and subtelomeres, but not in a subset of heterochromatin islands in our condition. Thus, our work uncovers a role of Abo1 in stabilising directly or indirectly Clr4 recruitment to allow the H3K9me2 to H3K9me3 transition in heterochromatin.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Metilação de DNA , Instabilidade Genômica , Heterocromatina , Histona-Lisina N-Metiltransferase/genética , Mutação/genética , Interferência de RNA , Proteínas de Schizosaccharomyces pombe/genética
17.
Epigenetics ; 15(6-7): 702-714, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31900031

RESUMO

The transcriptional program that dictates haematopoietic cell fate and differentiation requires an epigenetic regulatory and memory function, provided by a network of epigenetic factors that regulate DNA methylation, post-translational histone modifications and chromatin structure. Disturbed epigenetic regulation causes perturbations in the blood cell differentiation program that results in various types of haematopoietic disorders. Thus, accurate epigenetic regulation is essential for functional haematopoiesis. In this study, we used a CRISPR-Cas9 screening approach to identify new epigenetic regulators in myeloid differentiation. We designed a Chromatin-UMI CRISPR guide library targeting 1092 epigenetic regulators. Phorbol 12-myristate 13-acetate (PMA) treatment of the chronic myeloid leukaemia cell line K-562 was used as a megakaryocytic myeloid differentiation model. Both previously described developmental epigenetic regulators and novel factors were identified in our screen. In this study, we validated and characterized a role for the chromatin remodeller CHD2 in myeloid proliferation and megakaryocytic differentiation.


Assuntos
Proteínas de Ligação a DNA/genética , Mielopoese , Proliferação de Células , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Humanos , Células K562 , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
18.
Life Sci Alliance ; 2(5)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31575705

RESUMO

Here, we investigate the function of fission yeast Fun30/Smarcad1 family of SNF2 ATPase-dependent chromatin remodeling enzymes in DNA damage repair. There are three Fun30 homologues in fission yeast, Fft1, Fft2, and Fft3. We find that only Fft3 has a function in DNA repair and it is needed for single-strand annealing of an induced double-strand break. Furthermore, we use an inducible replication fork barrier system to show that Fft3 has two distinct roles at blocked DNA replication forks. First, Fft3 is needed for the resection of nascent strands, and second, it is required to restart the blocked forks. The latter function is independent of its ATPase activity.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Mutação , Domínios Proteicos , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética
19.
Elife ; 72018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29714165

RESUMO

Human immunodeficiency virus (HIV) infection is currently incurable, due to the persistence of latently infected cells. The 'shock and kill' approach to a cure proposes to eliminate this reservoir via transcriptional activation of latent proviruses, enabling direct or indirect killing of infected cells. Currently available latency-reversing agents (LRAs) have however proven ineffective. To understand why, we used a novel HIV reporter strain in primary CD4+ T cells and determined which latently infected cells are reactivatable by current candidate LRAs. Remarkably, none of these agents reactivated more than 5% of cells carrying a latent provirus. Sequencing analysis of reactivatable vs. non-reactivatable populations revealed that the integration sites were distinguishable in terms of chromatin functional states. Our findings challenge the feasibility of 'shock and kill', and suggest the need to explore other strategies to control the latent HIV reservoir.


Assuntos
Linfócitos T CD4-Positivos/virologia , Cromatina , Infecções por HIV/virologia , HIV-1/fisiologia , Latência Viral , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Células Cultivadas , Infecções por HIV/imunologia , Infecções por HIV/patologia , Humanos , Vírion , Ativação Viral , Integração Viral
20.
J Bone Miner Res ; 22(1): 19-28, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17032150

RESUMO

UNLABELLED: Sclerostin is an osteocyte-derived negative regulator of bone formation. It inhibits BMP-stimulated bone formation both in vitro and in vivo but has no direct effect on BMP signaling. Instead, sclerostin inhibits Wnt signaling that is required for BMP-stimulated osteoblastic differentiation. INTRODUCTION: Sclerostin is a member of the Dan family of glycoproteins of which many members have been reported to antagonize BMP activity. Sclerostin has been shown to inhibit BMP-stimulated bone formation, but its mechanism of action seems to be different from classical BMP antagonists. In this study, we investigated the mechanism by which sclerostin inhibits BMP-stimulated bone formation. MATERIALS AND METHODS: DNA electroporation of calf muscle of mice using expression plasmids for BMP and sclerostin was used to study the effect of sclerostin on BMP-induced bone formation in vivo. Transcriptional profiling using microarrays of osteoblastic cells treated with BMP in the absence or presence of sclerostin was used to find specific growth factor signaling pathways affected by sclerostin. The affected pathways were further studied using growth factor-specific reporter constructs. RESULTS: BMP-induced ectopic bone formation in calf muscle of mice was prevented by co-expression of sclerostin in vivo. Transcriptional profiling analysis of osteoblastic cultures indicated that sclerostin specifically affects BMP and Wnt signaling out of many other growth signaling pathways. Sclerostin, however, did not inhibit stimulation of direct BMP target genes. Furthermore, we did not obtain any evidence for sclerostin acting as a direct BMP antagonist using a BMP-specific reporter construct. In contrast, sclerostin shared many characteristics with the Wnt antagonist dickkopf-1 in antagonizing BMP-stimulated bone formation and BMP- and Wnt-induced Wnt reporter construct activation. CONCLUSIONS: Sclerostin inhibits BMP-stimulated bone formation but does not affect BMP signaling. Instead, it antagonizes Wnt signaling in osteoblastic cells. High bone mass in sclerosteosis and van Buchem disease may, therefore, result from increased Wnt signaling.


Assuntos
Desenvolvimento Ósseo/fisiologia , Proteínas Morfogenéticas Ósseas/fisiologia , Marcadores Genéticos/fisiologia , Proteínas Wnt/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Desenvolvimento Ósseo/efeitos dos fármacos , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/farmacologia , Células Cultivadas , Eletroporação , Regulação da Expressão Gênica , Genes Reporter , Glicoproteínas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Músculo Esquelético/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Transfecção
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