RESUMO
Transcriptional silencing of latent HIV-1 proviruses entails complex and overlapping mechanisms that pose a major barrier to in vivo elimination of HIV-1. We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent-AZD5582, an activator of the non-canonical NFκB pathway-to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of ING3 reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR. However, the combination of ING3 knockout accompanied with the activation of the non-canonical NFκB pathway via AZD5582 resulted in a dramatic increase in initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , Histonas/metabolismo , Proteínas Nucleares/metabolismo , HIV-1/fisiologia , Fatores de Transcrição/metabolismo , Latência Viral/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Soropositividade para HIV/genética , Provírus/genética , Linfócitos T CD4-Positivos , Proteínas de Homeodomínio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
Certain large DNA viruses, including those in the Marseilleviridae family, encode histones. Here we show that fused histone pairs Hß-Hα and Hδ-Hγ from Marseillevirus are structurally analogous to the eukaryotic histone pairs H2B-H2A and H4-H3. These viral histones form 'forced' heterodimers, and a heterotetramer of four such heterodimers assembles DNA to form structures virtually identical to canonical eukaryotic nucleosomes.
Assuntos
Vírus de DNA , DNA , Nucleossomos/metabolismo , DNA/química , DNA/metabolismo , Vírus de DNA/genética , Vírus de DNA/metabolismo , Histonas/química , Histonas/metabolismo , Ligação Proteica , Elementos Estruturais de Proteínas , Estrutura Terciária de ProteínaRESUMO
Mouse models of intestinal tumors have advanced our understanding of the role of gene mutations in colorectal malignancy. However, the utility of these systems for studying the role of epigenetic alterations in intestinal neoplasms remains to be defined. Consequently, we assessed the role of aberrant DNA methylation in the azoxymethane (AOM) rodent model of colon cancer. AOM induced tumors display global DNA hypomethylation, which is similar to human colorectal cancer. We next assessed the methylation status of a panel of candidate genes previously shown to be aberrantly methylated in human cancer or in mouse models of malignant neoplasms. This analysis revealed different patterns of DNA methylation that were gene specific. Zik1 and Gja9 demonstrated cancer-specific aberrant DNA methylation, whereas, Cdkn2a/p16, Igfbp3, Mgmt, Id4, and Cxcr4 were methylated in both the AOM tumors and normal colon mucosa. No aberrant methylation of Dapk1 or Mlt1 was detected in the neoplasms, but normal colon mucosa samples displayed methylation of these genes. Finally, p19(Arf), Tslc1, Hltf, and Mlh1 were unmethylated in both the AOM tumors and normal colon mucosa. Thus, aberrant DNA methylation does occur in AOM tumors, although the frequency of aberrantly methylated genes appears to be less common than in human colorectal cancer. Additional studies are necessary to further characterize the patterns of aberrantly methylated genes in AOM tumors.
Assuntos
Neoplasias do Colo/genética , Metilação de DNA , Modelos Animais de Doenças , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Azoximetano , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular , Neoplasias do Colo/induzido quimicamente , Conexinas/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas Quinases Associadas com Morte Celular , Humanos , Imunoglobulinas/genética , Proteínas Inibidoras de Diferenciação/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Proteínas de Membrana/genética , Camundongos , Proteína 1 Homóloga a MutL , Proteínas Nucleares/genética , Receptores CXCR4/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteína delta-2 de Junções ComunicantesRESUMO
BACKGROUND: DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters. RESULTS: Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and vice versa, in most pairs of tissues examined. CONCLUSION: The varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.