RESUMEN
Disruption of cell division cycle associated 7 (CDCA7) has been linked to aberrant DNA hypomethylation, but the impact of DNA methylation loss on transcription has not been investigated. Here, we show that CDCA7 is critical for maintaining global DNA methylation levels across multiple tissues in vivo. A pathogenic Cdca7 missense variant leads to the formation of large, aberrantly hypomethylated domains overlapping with the B genomic compartment but without affecting the deposition of H3K9 trimethylation (H3K9me3). CDCA7-associated aberrant DNA hypomethylation translated to localized, tissue-specific transcriptional dysregulation that affected large gene clusters. In the brain, we identify CDCA7 as a transcriptional repressor and epigenetic regulator of clustered protocadherin isoform choice. Increased protocadherin isoform expression frequency is accompanied by DNA methylation loss, gain of H3K4 trimethylation (H3K4me3), and increased binding of the transcriptional regulator CCCTC-binding factor (CTCF). Overall, our in vivo work identifies a key role for CDCA7 in safeguarding tissue-specific expression of gene clusters via the DNA methylation pathway.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Nucleares , ADN , Metilación de ADN , Isoformas de Proteínas/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Ratones , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
BACKGROUND: Microrchidia proteins (MORCs) are involved in epigenetic gene silencing in a variety of eukaryotic organisms. Deletion of MORCs result in several developmental abnormalities and their dysregulation has been implicated in developmental disease and multiple cancers. Specifically, mammalian MORC3 mutations are associated with immune system defects and human cancers such as bladder, uterine, stomach, lung, and diffuse large B cell lymphomas. While previous studies have shown that MORC3 binds to H3K4me3 in vitro and overlaps with H3K4me3 ChIP-seq peaks in mouse embryonic stem cells, the mechanism by which MORC3 regulates gene expression is unknown. RESULTS: In this study, we identified that mutation in Morc3 results in a suppressor of variegation phenotype in a Modifiers of murine metastable epialleles Dominant (MommeD) screen. We also find that MORC3 functions as an epigenetic silencer of transposable elements (TEs) in mouse embryonic stem cells (mESCs). Loss of Morc3 results in upregulation of TEs, specifically those belonging to the LTR class of retrotransposons also referred to as endogenous retroviruses (ERVs). Using ChIP-seq we found that MORC3, in addition to its known localization at H3K4me3 sites, also binds to ERVs, suggesting a direct role in regulating their expression. Previous studies have shown that these ERVs are marked by the repressive histone mark H3K9me3 which plays a key role in their silencing. However, we found that levels of H3K9me3 showed only minor losses in Morc3 mutant mES cells. Instead, we found that loss of Morc3 resulted in increased chromatin accessibility at ERVs as measured by ATAC-seq. CONCLUSIONS: Our results reveal MORC3 as a novel regulator of ERV silencing in mouse embryonic stem cells. The relatively minor changes of H3K9me3 in the Morc3 mutant suggests that MORC3 acts mainly downstream of, or in a parallel pathway with, the TRIM28/SETDB1 complex that deposits H3K9me3 at these loci. The increased chromatin accessibility of ERVs in the Morc3 mutant suggests that MORC3 may act at the level of chromatin compaction to effect TE silencing.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Elementos Transponibles de ADN , Proteínas de Unión al ADN , Retrovirus Endógenos , Células Madre Embrionarias de Ratones , Animales , Cromatina , Proteínas de Unión al ADN/metabolismo , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Silenciador del Gen , Ratones , Células Madre Embrionarias de Ratones/metabolismoRESUMEN
Increasing use of next-generation sequencing technologies in clinical diagnostics allows large-scale discovery of genetic variants, but also results in frequent identification of variants of unknown significance (VUSs). Their classification into disease-causing and neutral variants is often hampered by the absence of robust functional tests. Here, we demonstrate that a luciferase reporter assay, in combination with ChIP-qPCR, reliably separates pathogenic ZBTB24 missense variants in the context of immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome from natural variants in healthy individuals and patients of other diseases. Application of our assay to two published ZBTB24 missense VUSs indicates that they are likely not to cause ICF2 syndrome. Furthermore, we show that rare gnomAD ZBTB24 missense variants in key residues of the C2H2-ZF domain lead to a loss of function phenotype that resembles ICF2, suggesting that these individuals are carriers of ICF syndrome. In summary, we have developed a robust functional test to validate missense variants in ZBTB24.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina/métodos , Luciferasas/metabolismo , Mutación Missense , Proteínas Represoras/genética , Animales , Células Cultivadas , Cara/anomalías , Predisposición Genética a la Enfermedad , Humanos , Luciferasas/genética , Ratones , Modelos Biológicos , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Fenotipo , Enfermedades de Inmunodeficiencia Primaria/genética , Dominios Proteicos , Proteínas Represoras/químicaRESUMEN
Gut microbiota have been implicated in the development of atherosclerosis and cardiovascular disease. Since the prebiotic inulin is thought to beneficially affect gut microbiota, we aimed to determine the effect of inulin supplementation on atherosclerosis development in APOE*3-Leiden.CETP (E3L.CETP) mice. Female E3L.CETP mice were fed a western-type diet containing 0.1% or 0.5% cholesterol with or without 10% inulin. The effects of inulin were determined on: microbiota composition, cecal short-chain fatty acid (SCFA) levels, plasma lipid levels, atherosclerosis development, hepatic morphology and hepatic inflammation. Inulin with 0.5% dietary cholesterol increased specific bacterial genera and elevated levels of cecal SCFAs, but did not affect plasma cholesterol levels or atherosclerosis development. Surprisingly, inulin resulted in mild hepatic inflammation as shown by increased expression of inflammation markers. However, these effects were not accompanied by increased hepatic macrophage number. Analogously, inulin induced mild steatosis and increased hepatocyte size, but did not affect hepatic triglyceride content. Inulin with 0.1% dietary cholesterol did not affect hepatic morphology, nor hepatic expression of inflammation markers. Overall, inulin did not reduce hypercholesterolemia or atherosclerosis development in E3L.CETP mice despite showing clear prebiotic activity, but resulted in manifestations of hepatic inflammation when combined with a high percentage of dietary cholesterol.
Asunto(s)
Apolipoproteína E3/genética , Aterosclerosis/inmunología , Bacterias/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , Hipercolesterolemia/inmunología , Inulina/administración & dosificación , Prebióticos/administración & dosificación , Animales , Apolipoproteína E3/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , ADN Bacteriano/genética , ADN Ribosómico/genética , Dieta Occidental/efectos adversos , Modelos Animales de Enfermedad , Ácidos Grasos/química , Femenino , Hipercolesterolemia/inducido químicamente , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Inulina/farmacología , Lípidos/sangre , Ratones , Ratones Transgénicos , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV-/- mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV-/- mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity.
RESUMEN
For genetically heterogeneous diseases a better understanding of how the underlying gene defects are functionally interconnected will be important for dissecting disease etiology. The Immunodeficiency, Centromeric instability, Facial anomalies (ICF) syndrome is a chromatin disorder characterized by mutations in DNMT3B, ZBTB24, CDCA7 or HELLS Here, we generated a Zbtb24 BTB domain deletion mouse and found that loss of functional Zbtb24 leads to early embryonic lethality. Transcriptome analysis identified Cdca7 as the top down-regulated gene in Zbtb24 homozygous mutant mESCs, which can be restored by ectopic ZBTB24 expression. We further demonstrate enrichment of ZBTB24 at the CDCA7 promoter suggesting that ZBTB24 can function as a transcription factor directly controlling Cdca7 expression. Finally, we show that this regulation is conserved between species and that CDCA7 levels are reduced in patients carrying ZBTB24 nonsense mutations. Together, our findings demonstrate convergence of the two ICF genes ZBTB24 and CDCA7 at the level of transcription.
Asunto(s)
Cara/anomalías , Síndromes de Inmunodeficiencia/genética , Proteínas Nucleares/genética , Proteínas Represoras/genética , Transcripción Genética , Animales , Codón sin Sentido/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Helicasas/genética , Cara/fisiopatología , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Síndromes de Inmunodeficiencia/fisiopatología , Masculino , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Nucleares/biosíntesis , Enfermedades de Inmunodeficiencia Primaria , Transcriptoma/genética , ADN Metiltransferasa 3BRESUMEN
The number of reports of paternal epigenetic influences on the phenotype of offspring in rodents is increasing but the molecular events involved remain unclear. Here, we show that haploinsufficiency for the histone 3 lysine 9 methyltransferase Setdb1 in the sire can influence the coat colour phenotype of wild type offspring. This effect occurs when the allele that directly drives coat colour is inherited from the dam, inferring that the effect involves an "in trans" step. The implication of this finding is that epigenetic state of the sperm can alter the expression of genes inherited on the maternally derived chromosomes. Whole genome bisulphite sequencing revealed that Setdb1 mutant mice show DNA hypomethylation at specific classes of transposable elements in the sperm. Our results identify Setdb1 as a paternal effect gene in the mouse and suggest that epigenetic inheritance may be more likely in individuals with altered levels of epigenetic modifiers.