RESUMEN
CHARGE syndrome is a neural crest-related disorder mainly caused by mutation of the chromatin remodeler-coding gene CHD7 Alternative causes include mutation of other chromatin and/or splicing factors. One of these additional players is the poorly characterized FAM172A, which we previously found in a complex with CHD7 and the small RNA-binding protein AGO2 at the chromatin-spliceosome interface. Focusing on the FAM172A-AGO2 interplay, we now report that FAM172A is a direct binding partner of AGO2 and, as such, one of the long sought-after regulators of AGO2 nuclear import. We show that this FAM172A function mainly relies on its classical bipartite nuclear localization signal and associated canonical importin-α/ß pathway, being enhanced by CK2-induced phosphorylation and abrogated by a CHARGE syndrome-associated missense mutation. Overall, this study thus strengthens the notion that noncanonical nuclear functions of AGO2 and associated regulatory mechanisms might be clinically relevant.
Asunto(s)
Síndrome CHARGE , Humanos , Transporte Activo de Núcleo Celular , Cromatina , Mutación Missense , ProteínasRESUMEN
About half of the mammalian genome is constituted of repeated elements, among which endogenous retroviruses (ERVs) are known to influence gene expression and cancer development. The HP1 (Heterochromatin Protein 1) proteins are known to be essential for heterochromatin establishment and function and its loss in hepatocytes leads to the reactivation of specific ERVs and to liver tumorigenesis. Here, by studying two ERVs located upstream of genes upregulated upon loss of HP1, Mbd1 and Trim24, we show that these HP1-dependent ERVs behave as either alternative promoters or as putative enhancers forming a loop with promoters of endogenous genes depending on the genomic context and HP1 expression level. These ERVs are characterised by a specific HP1-independent enrichment in heterochromatin-associated marks H3K9me3 and H4K20me3 as well as in the enhancer-specific mark H3K4me1, a combination that might represent a bookmark of putative ERV-derived enhancers. These ERVs are further enriched in a HP1-dependent manner in H3K27me3, suggesting a critical role of this mark together with HP1 in the silencing of the ERVs, as well as for the repression of the associated genes. Altogether, these results lead to the identification of a new regulatory hub involving the HP1-dependent formation of a physical loop between specific ERVs and endogenous genes.
Asunto(s)
Retrovirus Endógenos , Animales , Cromatina/genética , Homólogo de la Proteína Chromobox 5 , Retrovirus Endógenos/genética , Expresión Génica , Heterocromatina , Mamíferos/genéticaRESUMEN
Waardenburg syndrome type 4 (WS4) combines abnormal development of neural crest cell (NCC)-derived melanocytes (causing depigmentation and inner ear dysfunction) and enteric nervous system (causing aganglionic megacolon). The full spectrum of WS4 phenotype is present in Spot mice, in which an insertional mutation close to a silencer element leads to NCC-specific upregulation of the transcription factor-coding gene Nr2f1. These mice were previously found to develop aganglionic megacolon because of NR2F1-induced premature differentiation of enteric neural progenitors into enteric glia. Intriguingly, this prior work also showed that inner ear dysfunction in Spot mutants specifically affects balance but not hearing, consistent with the absence of melanocytes in the vestibule only. Here, we report an analysis of the effect of Nr2f1 upregulation on the development of both inner ear and skin melanocytes, also taking in consideration their origin relative to the dorsolateral and ventral NCC migration pathways. In the trunk, we found that NR2F1 overabundance in Spot NCCs forces dorso-laterally migrating melanoblasts to abnormally adopt a Schwann cell precursor (SCP) fate and conversely prevents ventrally migrating SCPs to normally adopt a melanoblast fate. In the head, Nr2f1 upregulation appears not to be uniform, which might explain why SCP-derived melanocytes do colonize the cochlea while non-SCP-derived melanocytes cannot reach the vestibule. Collectively, these data point to a key role for NR2F1 in the control of SCP-vs-melanocyte fate choice and unveil a new pathogenic mechanism for WS4. Moreover, our data argue against the proposed existence of a transit-amplifying compartment of melanocyte precursors in hair follicles.
Asunto(s)
Enfermedad de Hirschsprung , Síndrome de Waardenburg , Animales , Diferenciación Celular/genética , Enfermedad de Hirschsprung/metabolismo , Enfermedad de Hirschsprung/patología , Melanocitos/metabolismo , Ratones , Cresta Neural/metabolismo , Células de Schwann , Síndrome de Waardenburg/genéticaRESUMEN
Many population-based methods investigating chromatin dynamics and organization in eukaryotes are based on the chromosome conformation capture (3C) method. Here, we provide an updated version of the quantitative 3C (3C-qPCR) protocol for improved and simplified quantitative analyses of intra-chromosomal contacts.
Asunto(s)
Cromatina , Cromosomas , Cromatina/genética , Cromosomas/genética , Conformación Molecular , Conformación de Ácido Nucleico , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The mammalian cell nucleus contains numerous discrete suborganelles named nuclear bodies. While recruitment of specific genomic regions into these large ribonucleoprotein (RNP) complexes critically contributes to higher-order functional chromatin organization, such regions remain ill-defined. We have developed the high-salt-recovered sequences-sequencing (HRS-seq) method, a straightforward genome-wide approach whereby we isolated and sequenced genomic regions associated with large high-salt insoluble RNP complexes. By using mouse embryonic stem cells (ESCs), we showed that these regions essentially correspond to the most highly expressed genes, and to cis-regulatory sequences like super-enhancers, that belong to the active A chromosomal compartment. They include both cell-type-specific genes, such as pluripotency genes in ESCs, and housekeeping genes associated with nuclear bodies, such as histone and snRNA genes that are central components of Histone Locus Bodies and Cajal bodies. We conclude that HRSs are associated with the active chromosomal compartment and with large RNP complexes including nuclear bodies. Association of such chromosomal regions with nuclear bodies is in agreement with the recently proposed phase separation model for transcription control and might thus play a central role in organizing the active chromosomal compartment in mammals.