RESUMEN
Critical illness myopathy (CIM) is an acquired, devastating, multifactorial muscle-wasting disease with incomplete recovery. The impact on hospital costs and permanent loss of quality of life is enormous. Incomplete recovery might imply that the function of muscle stem cells (MuSC) is impaired. We tested whether epigenetic alterations could be in part responsible. We characterized human muscle stem cells (MuSC) isolated from early CIM and analyzed epigenetic alterations (CIM n = 15, controls n = 21) by RNA-Seq, immunofluorescence, analysis of DNA repair, and ATAC-Seq. CIM-MuSC were transplanted into immunodeficient NOG mice to assess their regenerative potential. CIM-MuSC exhibited significant growth deficits, reduced ability to differentiate into myotubes, and impaired DNA repair. The chromatin structure was damaged, as characterized by alterations in mRNA of histone 1, depletion or dislocation of core proteins of nucleosome remodeling and deacetylase complex, and loosening of multiple nucleosome-spanning sites. Functionally, CIM-MuSC had a defect in building new muscle fibers. Further, MuSC obtained from the electrically stimulated muscle of CIM patients was very similar to control MuSC, indicating the impact of muscle contraction in the onset of CIM. CIM not only affects working skeletal muscle but has a lasting and severe epigenetic impact on MuSC.
Asunto(s)
Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Enfermedades Musculares , Humanos , Animales , Ratones , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Enfermedad Crítica , Calidad de Vida , Enfermedades Musculares/metabolismo , Músculo Esquelético/metabolismo , Células MadreRESUMEN
RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.
Asunto(s)
Roturas del ADN de Doble Cadena , Replicación del ADN , Animales , ADN/metabolismo , Reparación del ADN , Ratones , Fosforilación , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Immunoglobulin (Ig) class switch recombination (CSR) is the process occurring in mature B cells that diversifies the effector component of antibody responses. CSR is initiated by the activity of the B cell-specific enzyme activation-induced cytidine deaminase (AID), which leads to the formation of programmed DNA double-strand breaks (DSBs) at the Ig heavy chain (Igh) locus. Mature B cells use a multilayered and complex regulatory framework to ensure that AID-induced DNA breaks are channeled into productive repair reactions leading to CSR, and to avoid aberrant repair events causing lymphomagenic chromosomal translocations. Here, we review the DNA repair pathways acting on AID-induced DSBs and their functional interplay, with a particular focus on the latest developments in their molecular composition and mechanistic regulation.
Asunto(s)
Roturas del ADN de Doble Cadena , Cambio de Clase de Inmunoglobulina , Linfocitos B , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Reparación del ADN , Cadenas Pesadas de Inmunoglobulina/genéticaRESUMEN
The establishment of protective humoral immunity is dependent on the ability of mature B cells to undergo antibody gene diversification while adjusting to the physiological stressors induced by activation with the antigen. Mature B cells diversify their antibody genes by class switch recombination (CSR) and somatic hypermutation (SHM), which are both dependent on efficient induction of activation-induced cytidine deaminase (AID). Here, we identified PDGFA-associated protein 1 (Pdap1) as an essential regulator of cellular homeostasis in mature B cells. Pdap1 deficiency leads to sustained expression of the integrated stress response (ISR) effector activating transcription factor 4 (Atf4) and induction of the ISR transcriptional program, increased cell death, and defective AID expression. As a consequence, loss of Pdap1 reduces germinal center B cell formation and impairs CSR and SHM. Thus, Pdap1 protects mature B cells against chronic ISR activation and ensures efficient antibody diversification by promoting their survival and optimal function.
Asunto(s)
Diversidad de Anticuerpos , Linfocitos B/metabolismo , Genes de Inmunoglobulinas/genética , Animales , Linfocitos B/inmunología , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Muerte Celular , Diferenciación Celular , Línea Celular , Femenino , Técnica del Anticuerpo Fluorescente , Edición Génica , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.10275.].
RESUMEN
Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector functions of antibodies. CSR occurs via the formation and non-homologous end joining (NHEJ) repair of programmed DNA double-strand breaks (DSBs) at the immunoglobulin heavy chain locus. The DNA repair factors 53BP1 and Rif1 promote NHEJ and CSR by protecting DSBs against resection. However, to what extent repression of DNA end resection contributes to CSR is unknown. Here, we show that B lymphocytes devoid of 53BP1-Rif1-dependent DSB end protection activity undergo robust CSR. Inactivation of specific sets of phospho-sites within 53BP1 N-terminal SQ/TQ motifs abrogates Rif1 recruitment and inhibition of resection but only mildly reduces CSR. Furthermore, mutations within 53BP1 oligomerization domain abolish CSR without substantially affecting DNA end processing. Thus, inhibition of DNA end resection does not correlate with CSR efficiency, indicating that regulation of DSB processing is not a key determinant step in CSR.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Cambio de Clase de Inmunoglobulina , Proteína 1 de Unión al Supresor Tumoral P53/fisiología , Animales , Linfocitos B/inmunología , Roturas del ADN de Doble Cadena , Femenino , Humanos , Masculino , Ratones , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector component of antibody responses. CSR is initiated by activation-induced cytidine deaminase (AID), which targets transcriptionally active immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3' Igh super-enhancer, 3' regulatory region (3'RR), is essential for acceptor region transcription, but how this function is regulated is unknown. Here, we identify the chromatin reader ZMYND8 as an essential regulator of the 3'RR. In B cells, ZMYND8 binds promoters and super-enhancers, including the Igh enhancers. ZMYND8 controls the 3'RR activity by modulating the enhancer transcriptional status. In its absence, there is increased 3'RR polymerase loading and decreased acceptor region transcription and CSR. In addition to CSR, ZMYND8 deficiency impairs somatic hypermutation (SHM) of Igh, which is also dependent on the 3'RR. Thus, ZMYND8 controls Igh diversification in mature B lymphocytes by regulating the activity of the 3' Igh super-enhancer.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Cambio de Clase de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/genética , Proteínas Supresoras de Tumor/genética , Animales , Linfocitos B , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , ADN/genética , Elementos de Facilitación Genéticos , Reordenamiento Génico , Humanos , Dominios MYND , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Hipermutación Somática de Inmunoglobulina/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.
Asunto(s)
Carcinogénesis/metabolismo , Carcinogénesis/patología , Caspasa 8/metabolismo , Daño del ADN , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Animales , Apoptosis , Carcinoma Hepatocelular/patología , Proliferación Celular , Senescencia Celular , Enfermedad Crónica , Cruzamientos Genéticos , Reparación del ADN , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Femenino , Inestabilidad Genómica , Hepatectomía , Hepatocitos/patología , Histonas/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/metabolismo , Hígado/patología , Regeneración Hepática , Masculino , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Fosforilación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factores de RiesgoRESUMEN
Inflammation is a potent inducer of tumorigenesis. Increased DNA damage or loss of genome integrity is thought to be one of the mechanisms linking inflammation and cancer development. It has been suggested that NF-κB-induced microRNA-146 (miR146a) may be a mediator of the inflammatory response. Based on our initial observation that miR146a overexpression strongly increases DNA damage, we investigated its potential role as a modulator of DNA repair. Here, we demonstrate that FANCM, a component in the Fanconi Anemia pathway, is a novel target of miR146a. miR146a suppressed FANCM expression by directly binding to the 3' untranslated region of the gene. miR146a-induced downregulation of FANCM was associated with inhibition of FANCD2 monoubiquitination, reduced DNA homologous recombination repair and checkpoint response, failed recovery from replication stress, and increased cellular sensitivity to cisplatin. These phenotypes were recapitulated when miR146a expression was induced by overexpressing the NF-κB subunit p65/RelA or Helicobacter pylori infection in a human gastric cell line; the phenotypes were effectively reversed with an anti-miR146a antagomir. These results suggest that undesired inflammation events caused by a pathogen or over-induction of miR146a can impair genome integrity via suppression of FANCM.