RESUMEN
We interrogated at nucleotide resolution the spatiotemporal order of chromatin changes that occur immediately following a site-specific double-strand break (DSB) upstream of the PHO5 locus and its subsequent repair by nonhomologous end joining (NHEJ). We observed the immediate eviction of a nucleosome flanking the break and the repositioning of adjacent nucleosomes away from the break. These early chromatin events were independent of the end-processing Mre11-Rad50-Xrs2 (MRX) complex and preceded the MRX-dependent broad eviction of histones and DNA end-resectioning that extends up to â¼8 kb away from the break. We also examined the temporal dynamics of NHEJ-mediated repair in a G1-arrested population. Concomitant with DSB repair by NHEJ, we observed the redeposition and precise repositioning of nucleosomes at their originally occupied positions. This re-establishment of the prelesion chromatin landscape suggests that a DNA replication-independent mechanism exists to preserve epigenome organization following DSB repair.
Asunto(s)
Roturas del ADN de Doble Cadena , Nucleosomas , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Nucleosomas/genéticaRESUMEN
Though the sequence of the genome within each eukaryotic cell is essentially fixed, it exists within a complex and changing chromatin state. This state is determined, in part, by the dynamic binding of proteins to the DNA. These proteins-including histones, transcription factors (TFs), and polymerases-interact with one another, the genome, and other molecules to allow the chromatin to adopt one of exceedingly many possible configurations. Understanding how changing chromatin configurations associate with transcription remains a fundamental research problem. We sought to characterize at high spatiotemporal resolution the dynamic interplay between transcription and chromatin in response to cadmium stress. Whereas gene regulatory responses to environmental stress in yeast have been studied, how the chromatin state changes and how those changes connect to gene regulation remain unexplored. By combining MNase-seq and RNA-seq data, we found chromatin signatures of transcriptional activation and repression involving both nucleosomal and TF-sized DNA-binding factors. Using these signatures, we identified associations between chromatin dynamics and transcriptional regulation, not only for known cadmium response genes, but across the entire genome, including antisense transcripts. Those associations allowed us to develop generalizable models that predict dynamic transcriptional responses on the basis of dynamic chromatin signatures.
Asunto(s)
Cromatina , Nucleosomas , Cromatina/genética , ADN/genética , Histonas/metabolismo , Nucleosomas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We report the case of a previously healthy 21-year-old woman who presented 6 days post-partum in acute fulminant hepatic failure. A liver ultrasound demonstrated normal echogenicity without discrete nodules while an enhanced computed tomography (CT) demonstrated innumerable 1- to 3-mm hypodense nodules throughout the liver with greater involvement of the left lobe. Liver biopsy confirmed herpes simplex virus infection. We believe this disease has a characteristic appearance on CT and prompt recognition can expedite diagnosis and therapy.
Asunto(s)
Hepatitis Viral Humana/diagnóstico , Herpes Simple/diagnóstico , Fallo Hepático Agudo/diagnóstico , Biopsia , Femenino , Hepatitis Viral Humana/complicaciones , Herpes Simple/complicaciones , Humanos , Hígado/diagnóstico por imagen , Fallo Hepático Agudo/complicaciones , Fallo Hepático Agudo/virología , Periodo Posparto , Simplexvirus , Tomografía Computarizada por Rayos X , Resultado del Tratamiento , Ultrasonografía , Adulto JovenRESUMEN
Infection with human immunodeficiency virus type 1 (HIV-1) causes an inexorable depletion of CD4(+) T cells. The loss of these cells is particularly pronounced in the mucosal immune system during acute infection, and the data suggest that direct viral cytopathicity is a major factor. Cell cycle arrest caused by the HIV-1 accessory protein Vpr is strongly correlated with virus-induced cell death, and phosphorylation of Vpr serine 79 (S79) is required to activate G(2)/M cell cycle blockade. However, the kinase responsible for phosphorylating Vpr remains unknown. Our bioinformatic analyses revealed that S79 is part of a putative phosphorylation site recognized by protein kinase A (PKA). We show here that PKA interacts with Vpr and directly phosphorylates S79. Inhibition of PKA activity during HIV-1 infection abrogates Vpr cell cycle arrest. These findings provide new insight into the signaling event that activates Vpr cell cycle arrest, ultimately leading to the death of infected T cells.
Asunto(s)
Ciclo Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , VIH-1/patogenicidad , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Humanos , Linfocitos/virología , Fosforilación , Serina/metabolismoRESUMEN
Cell surface determinants, cytokines and antibodies secreted by hematopoietic cells are used to classify their lineage and function. Currently available techniques are unable to elucidate multiple secreted proteins while also assigning phenotypic surface-displayed markers to the individual living cells. Here, a soft lithographic method, microengraving, was adapted for the multiplexed interrogation of populations of individual human peripheral blood mononuclear cells for secreted cytokines (IFN-gamma and IL-6), antigen-specific antibodies, and lineage-specific surface-expressed markers. Application of the method to a clinical sample from a recent-onset Type 1 diabetic subject with a positive titer of anti-insulin antibodies showed that approximately 0.58% of circulating CD19(+) B cells secreted proinsulin-reactive antibodies of the IgG isotype and 2-3% of circulating cells secreted IL-6. These data demonstrate the utility of microengraving for interrogating multiple phenotypes of single human cells concurrently and for detecting rare populations of cells by their secreted products.