Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
iScience ; 27(3): 109152, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38384833

RESUMO

HIV-1 latency results from tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. Here, we characterize PCI domain-containing 2 (PCID2) protein, a subunit of the transcription and export complex 2 (TREX2) complex, to enforce transcriptional repression and post-transcriptional blocks to HIV-1 gene expression during latency. PCID2 bound the latent HIV-1 LTR (long terminal repeat) and repressed transcription initiation during latency. Depletion of PCID2 remodeled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and latency reversal. Immunoprecipitation coupled to mass spectrometry identified PCID2-interacting proteins to include negative viral RNA (vRNA) splicing regulators, and PCID2 depletion resulted in over-splicing of intron-containing vRNA in cell lines and primary cells obtained from PWH. MCM3AP and DSS1, two other RNA-binding TREX2 complex subunits, also inhibit transcription initiation and vRNA alternative splicing during latency. Thus, PCID2 is a novel HIV-1 latency-promoting factor, which in context of the TREX2 sub-complex PCID2-DSS1-MCM3AP blocks transcription and dysregulates vRNA processing.

2.
J Comp Neurol ; 531(12): 1229-1243, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37125418

RESUMO

In vertebrates, the embryonic olfactory epithelium contains progenitors that will give rise to distinct classes of neurons, including olfactory sensory neurons (OSNs; involved in odor detection), vomeronasal sensory neurons (VSNs; responsible for pheromone sensing), and gonadotropin-releasing hormone (GnRH) neurons that control the hypothalamic-pituitary-gonadal axis. Currently, these three neuronal lineages are usually believed to emerge from uniform pools of progenitors. Here, we found that the homeodomain transcription factor Dbx1 is expressed by neurogenic progenitors in the developing and adult mouse olfactory epithelium. We demonstrate that Dbx1 itself is dispensable for neuronal fate specification and global organization of the olfactory sensory system. Using lineage tracing, we characterize the contribution of Dbx1 lineages to OSN, VSN, and GnRH neuron populations and reveal an unexpected degree of diversity. Furthermore, we demonstrate that Dbx1-expressing progenitors remain neurogenic in the absence of the proneural gene Ascl1. Our work therefore points to the existence of distinct neurogenic programs in Dbx1-derived and other olfactory lineages.


Assuntos
Mucosa Olfatória , Neurônios Receptores Olfatórios , Camundongos , Animais , Neurônios Receptores Olfatórios/metabolismo , Fatores de Transcrição/genética , Regulação da Expressão Gênica , Hormônio Liberador de Gonadotropina/metabolismo , Proteínas de Homeodomínio/genética
3.
Nucleic Acids Res ; 50(10): 5577-5598, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35640596

RESUMO

A major pharmacological strategy toward HIV cure aims to reverse latency in infected cells as a first step leading to their elimination. While the unbiased identification of molecular targets physically associated with the latent HIV-1 provirus would be highly valuable to unravel the molecular determinants of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has been challenging. Here we use a dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to probe the differential protein composition of the latent and activated HIV-1 5'LTR. Catchet-MS identified known and novel latent 5'LTR-associated host factors. Among these, IKZF1 is a novel HIV-1 transcriptional repressor, required for Polycomb Repressive Complex 2 recruitment to the LTR. We find the clinically advanced thalidomide analogue iberdomide, and the FDA approved analogues lenalidomide and pomalidomide, to be novel LRAs. We demonstrate that, by targeting IKZF1 for degradation, these compounds reverse HIV-1 latency in CD4+ T-cells isolated from virally suppressed people living with HIV-1 and that they are able to synergize with other known LRAs.


Assuntos
Infecções por HIV , HIV-1 , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/metabolismo , HIV-1/genética , Humanos , Fator de Transcrição Ikaros/genética , Provírus/genética , Talidomida/metabolismo , Talidomida/farmacologia , Fatores de Transcrição/metabolismo , Ativação Viral , Latência Viral
4.
mBio ; 12(6): e0298021, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34872356

RESUMO

To identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latent HIV-1 infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome, and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. A select set of candidate genes was functionally validated using short hairpin RNA (shRNA)-mediated depletion in latent HIV-1 infected J-Lat A2 and 11.1 T cell lines. We confirmed ADK, CHD9, CMSS1, EVI2B, EXOSC8, FAM19A, GRIK5, IRF2BP2, NF1, and USP15 as novel host factors involved in the maintenance of HIV-1 latency. Chromatin immunoprecipitation assays indicated that CHD9, a chromodomain helicase DNA-binding protein, maintains HIV-1 latency via direct association with the HIV-1 5' long terminal repeat (LTR), and its depletion results in increased histone acetylation at the HIV-1 promoter, concomitant with HIV-1 latency reversal. FDA-approved inhibitors 5-iodotubercidin, trametinib, and topiramate, targeting ADK, NF1, and GRIK5, respectively, were characterized for their latency reversal potential. While 5-iodotubercidin exhibited significant cytotoxicity in both J-Lat and primary CD4+ T cells, trametinib reversed latency in J-Lat cells but not in latent HIV-1 infected primary CD4+ T cells. Importantly, topiramate reversed latency in cell line models, in latently infected primary CD4+ T cells, and crucially in CD4+ T cells from three people living with HIV-1 (PLWH) under suppressive antiretroviral therapy, without inducing T cell activation or significant toxicity. Thus, using an adaptation of a haploid forward genetic screen, we identified novel and druggable host factors contributing to HIV-1 latency. IMPORTANCE A reservoir of latent HIV-1 infected cells persists in the presence of combination antiretroviral therapy (cART), representing a major obstacle for viral eradication. Reactivation of the latent HIV-1 provirus is part of curative strategies which aim to promote clearance of the infected cells. Using a two-color haploid screen, we identified 69 candidate genes as latency-maintaining host factors and functionally validated a subset of 10 of those in additional T-cell-based cell line models of HIV-1 latency. We further demonstrated that CHD9 is associated with HIV-1's promoter, the 5' LTR, while this association is lost upon reactivation. Additionally, we characterized the latency reversal potential of FDA compounds targeting ADK, NF1, and GRIK5 and identify the GRIK5 inhibitor topiramate as a viable latency reversal agent with clinical potential.


Assuntos
Infecções por HIV/genética , HIV-1/fisiologia , Haploidia , Latência Viral , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , Receptores de Ácido Caínico/genética , Receptores de Ácido Caínico/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Ativação Viral
5.
Sci Adv ; 6(33): eaba6617, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32851167

RESUMO

A leading pharmacological strategy toward HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.


Assuntos
Gliotoxina , Infecções por HIV , HIV-1 , Gliotoxina/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Células HeLa , Humanos , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas , Ribonucleoproteínas Nucleares Pequenas/química , Fatores de Transcrição/metabolismo
6.
Curr Opin Virol ; 38: 37-53, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31323521

RESUMO

HIV cure is impeded by the persistence of a strenuous reservoir of latent but replication competent infected cells, which remain unsusceptible to c-ART and unrecognized by the immune system for elimination. Ongoing progress in understanding the molecular mechanisms that control HIV transcription and latency has led to the development of strategies to either permanently inactivate the latent HIV infected reservoir of cells or to stimulate the virus to emerge out of latency, coupled to either induction of death in the infected reactivated cell or its clearance by the immune system. This review focuses on the currently explored and non-exclusive pharmacological strategies and their molecular targets that 1. stimulate reversal of HIV latency in infected cells by targeting distinct steps in the HIV-1 gene expression cycle, 2. exploit mechanisms that promote cell death and apoptosis to render the infected cell harboring reactivated virus more susceptible to death and/or elimination by the immune system, and 3. permanently inactivate any remaining latently infected cells such that c-ART can be safely discontinued.


Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Latência Viral/efeitos dos fármacos , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Ilhas de CpG , Metilação de DNA , Desenvolvimento de Medicamentos , Regulação Viral da Expressão Gênica , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/imunologia , Repetição Terminal Longa de HIV , Humanos , Ativação Transcricional , Carga Viral , Ativação Viral/efeitos dos fármacos , Latência Viral/genética , Latência Viral/imunologia , Replicação Viral
7.
Int Rev Cell Mol Biol ; 335: 191-243, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29305013

RESUMO

In this review, we cover transcription regulation of human immunodeficiency virus type 1 (HIV-1) gene expression, focusing on the invaluable contributions, made by HIV research over the years, toward the field of transcription. In this context, the HIV promoter can be considered to be a well-studied model promoter, which although a viral promoter, is subject to the same cellular regulatory mechanisms that modulate the transcriptional control of endogenous host cellular genes. The molecular control of HIV-1 transcription has been well studied and considerable knowledge toward development of alternative strategies for therapies aimed at eradicating both active but also latent HIV-1 has been obtained. Additionally, HIV-1 studies have provided insight into fundamental aspects of transcriptional regulation including transcriptional stochasticity, RNA polymerase II pausing, chromatin regulation of transcription, the role of the +1 nucleosome, the use of an RNA enhancer element, i.e., TAR, the discovery, and essential function of P-TEFb, and the super elongation complex in transcription elongation. These findings have been important not only in deciphering the mechanisms used by HIV-1 to regulate its gene expression and to establish and maintain HIV latency for therapeutic advancement, but were at the same time seminal in pushing the transcription field forward.


Assuntos
HIV-1/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Cromatina/metabolismo , Metilação de DNA/genética , Humanos , Modelos Biológicos
8.
Skelet Muscle ; 7(1): 12, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28587678

RESUMO

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is in most cases caused by a contraction of the D4Z4 macrosatellite repeat on chromosome 4 (FSHD1) or by mutations in the SMCHD1 or DNMT3B gene (FSHD2). Both situations result in the incomplete epigenetic repression of the D4Z4-encoded retrogene DUX4 in somatic cells, leading to the aberrant expression of DUX4 in the skeletal muscle. In mice, Smchd1 regulates chromatin repression at different loci, having a role in CpG methylation establishment and/or maintenance. METHODS: To investigate the global effects of harboring heterozygous SMCHD1 mutations on DNA methylation in humans, we combined 450k methylation analysis on mononuclear monocytes from female heterozygous SMCHD1 mutation carriers and unaffected controls with reduced representation bisulfite sequencing (RRBS) on FSHD2 and control myoblast cell lines. Candidate loci were then evaluated for SMCHD1 binding using ChIP-qPCR and expression was evaluated using RT-qPCR. RESULTS: We identified a limited number of clustered autosomal loci with CpG hypomethylation in SMCHD1 mutation carriers: the protocadherin (PCDH) cluster on chromosome 5, the transfer RNA (tRNA) and 5S rRNA clusters on chromosome 1, the HOXB and HOXD clusters on chromosomes 17 and 2, respectively, and the D4Z4 repeats on chromosomes 4 and 10. Furthermore, minor increases in RNA expression were seen in FSHD2 myoblasts for some of the PCDHß cluster isoforms, tRNA isoforms, and a HOXB isoform in comparison to controls, in addition to the previously reported effects on DUX4 expression. SMCHD1 was bound at DNAseI hypersensitivity sites known to regulate the PCDHß cluster and at the chromosome 1 tRNA cluster, with decreased binding in SMCHD1 mutation carriers at the PCDHß cluster sites. CONCLUSIONS: Our study is the first to investigate the global methylation effects in humans resulting from heterozygous mutations in SMCHD1. Our results suggest that SMCHD1 acts as a repressor on a limited set of autosomal gene clusters, as an observed reduction in methylation associates with a loss of SMCHD1 binding and increased expression for some of the loci.


Assuntos
Proteínas Cromossômicas não Histona/genética , Metilação de DNA , Loci Gênicos , Distrofia Muscular Facioescapuloumeral/genética , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Ilhas de CpG , Feminino , Heterozigoto , Humanos , Família Multigênica , Distrofia Muscular Facioescapuloumeral/metabolismo , Mutação , Mioblastos/metabolismo , Ligação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...