RESUMEN
In mammalian embryos, DNA methylation is initialized to maximum levels in the epiblast by the de novo DNA methyltransferases DNMT3A and DNMT3B before gastrulation diversifies it across regulatory regions. Here we show that DNMT3A and DNMT3B are differentially regulated during endoderm and mesoderm bifurcation and study the implications in vivo and in meso-endoderm embryoid bodies. Loss of both Dnmt3a and Dnmt3b impairs exit from the epiblast state. More subtly, independent loss of Dnmt3a or Dnmt3b leads to small biases in mesoderm-endoderm bifurcation and transcriptional deregulation. Epigenetically, DNMT3A and DNMT3B drive distinct methylation kinetics in the epiblast, as can be predicted from their strand-specific sequence preferences. The enzymes compensate for each other in the epiblast, but can later facilitate lineage-specific methylation kinetics as their expression diverges. Single-cell analysis shows that differential activity of DNMT3A and DNMT3B combines with replication-linked methylation turnover to increase epigenetic plasticity in gastrulation. Together, these findings outline a dynamic model for the use of DNMT3A and DNMT3B sequence specificity during gastrulation.
Asunto(s)
ADN Metiltransferasa 3A , Gastrulación , Animales , Ratones , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , Embrión de Mamíferos/metabolismo , ADN/metabolismo , Mamíferos/genéticaRESUMEN
Standardized lab tests are central for patient evaluation, differential diagnosis and treatment. Interpretation of these data is nevertheless lacking quantitative and personalized metrics. Here we report on the modeling of 2.1 billion lab measurements of 92 different lab tests from 2.8 million adults over a span of 18 years. Following unsupervised filtering of 131 chronic conditions and 5,223 drug-test pairs we performed a virtual survey of lab tests distributions in healthy individuals. Age and sex alone explain less than 10% of the within-normal test variance in 89 out of 92 tests. Personalized models based on patients' history explain 60% of the variance for 17 tests and over 36% for half of the tests. This allows for systematic stratification of the risk for future abnormal test levels and subsequent emerging disease. Multivariate modeling of within-normal lab tests can be readily implemented as a basis for quantitative patient evaluation.
Asunto(s)
Técnicas de Laboratorio Clínico/normas , Voluntarios Sanos , Medicina de Precisión , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Adulto JovenRESUMEN
Children with Down syndrome (DS) are prone to development of high-risk B-cell precursor ALL (DS-ALL), which differs genetically from most sporadic pediatric ALLs. Increased expression of cytokine receptor-like factor 2 (CRLF2), the receptor to thymic stromal lymphopoietin (TSLP), characterizes about half of DS-ALLs and also a subgroup of sporadic "Philadelphia-like" ALLs. To understand the pathogenesis of relapsed DS-ALL, we performed integrative genomic analysis of 25 matched diagnosis-remission and -relapse DS-ALLs. We found that the CRLF2 rearrangements are early events during DS-ALL evolution and generally stable between diagnoses and relapse. Secondary activating signaling events in the JAK-STAT/RAS pathway were ubiquitous but highly redundant between diagnosis and relapse, suggesting that signaling is essential but that no specific mutations are "relapse driving." We further found that activated JAK2 may be naturally suppressed in 25% of CRLF2pos DS-ALLs by loss-of-function aberrations in USP9X, a deubiquitinase previously shown to stabilize the activated phosphorylated JAK2. Interrogation of large ALL genomic databases extended our findings up to 25% of CRLF2pos, Philadelphia-like ALLs. Pharmacological or genetic inhibition of USP9X, as well as treatment with low-dose ruxolitinib, enhanced the survival of pre-B ALL cells overexpressing mutated JAK2. Thus, somehow counterintuitive, we found that suppression of JAK-STAT "hypersignaling" may be beneficial to leukemic B-cell precursors. This finding and the reduction of JAK mutated clones at relapse suggest that the therapeutic effect of JAK specific inhibitors may be limited. Rather, combined signaling inhibitors or direct targeting of the TSLP receptor may be a useful therapeutic strategy for DS-ALL.
Asunto(s)
Síndrome de Down/complicaciones , Quinasas Janus/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Factores de Transcripción STAT/metabolismo , Adolescente , Línea Celular Tumoral , Niño , Preescolar , Femenino , Humanos , Masculino , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Citocinas/genética , Recurrencia , Transducción de Señal , Ubiquitina Tiolesterasa/genética , Adulto JovenRESUMEN
Chromosomes are folded into highly compacted structures to accommodate physical constraints within nuclei and to regulate access to genomic information. Recently, global mapping of pairwise contacts showed that loops anchoring topological domains (TADs) are highly conserved between cell types and species. Whether pairwise loops synergize to form higher-order structures is still unclear. Here we develop a conformation capture assay to study higher-order organization using chromosomal walks (C-walks) that link multiple genomic loci together into proximity chains in human and mouse cells. This approach captures chromosomal structure at varying scales. Inter-chromosomal contacts constitute only 7-10% of the pairs and are restricted by interfacing TADs. About half of the C-walks stay within one chromosome, and almost half of those are restricted to intra-TAD spaces. C-walks that couple 2-4 TADs indicate stochastic associations between transcriptionally active, early replicating loci. Targeted analysis of thousands of 3-walks anchored at highly expressed genes support pairwise, rather than hub-like, chromosomal topology at active loci. Polycomb-repressed Hox domains are shown by the same approach to enrich for synergistic hubs. Together, the data indicate that chromosomal territories, TADs, and intra-TAD loops are primarily driven by nested, possibly dynamic, pairwise contacts.
Asunto(s)
Paseo de Cromosoma , Cromosomas/química , Cromosomas/genética , Sitios Genéticos , Conformación de Ácido Nucleico , Animales , Cromatina/química , Cromatina/genética , Regulación de la Expresión Génica , Genes Homeobox , Sitios Genéticos/genética , Humanos , Imagenología Tridimensional , Ratones , Proteínas del Grupo Polycomb/metabolismo , Procesos Estocásticos , Transcripción GenéticaRESUMEN
The development of niches for tissue-specific stem cells is an important aspect of stem cell biology. Determination of niche size and niche numbers during organogenesis involves precise control of gene expression. How this is achieved in the context of a complex chromatin landscape is largely unknown. Here we show that the nuclear protein Combgap (Cg) supports correct ovarian niche formation in Drosophila by controlling ecdysone-Receptor (EcR)- mediated transcription and long-range chromatin contacts in the broad locus (BR-C). Both cg and BR-C promote ovarian growth and the development of niches for germ line stem cells. BR-C levels were lower when Combgap was either reduced or over-expressed, indicating an intricate regulation of the BR-C locus by Combgap. Polytene chromosome stains showed that Cg co-localizes with EcR, the major regulator of BR-C, at the BR-C locus and that EcR binding to chromatin was sensitive to changes in Cg levels. Proximity ligation assay indicated that the two proteins could reside in the same complex. Finally, chromatin conformation analysis revealed that EcR-bound regions within BR-C, which span ~30 KBs, contacted each other. Significantly, these contacts were stabilized in an ecdysone- and Combgap-dependent manner. Together, these results highlight Combgap as a novel regulator of chromatin structure that promotes transcription of ecdysone target genes and ovarian niche formation.
Asunto(s)
Cromatina/genética , Proteínas de Drosophila/genética , Receptores de Esteroides/genética , Factores de Transcripción/genética , Transcripción Genética , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Ecdisona/biosíntesis , Ecdisona/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Especificidad de Órganos , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Cromosomas Politénicos/genética , Nicho de Células Madre/genéticaRESUMEN
We developed a targeted chromosome conformation capture (4C) approach that uses unique molecular identifiers (UMIs) to derive high-complexity quantitative chromosome contact profiles with controlled signal-to-noise ratios. UMI-4C detects chromosomal interactions with improved sensitivity and specificity, and it can easily be multiplexed to allow robust comparison of contact distributions between loci and conditions. This approach may open the way to the incorporation of contact distributions into quantitative models of gene regulation.
Asunto(s)
Mapeo Cromosómico/métodos , Cromosomas Humanos/química , Cromosomas Humanos/metabolismo , Regulación de la Expresión Génica , Genómica/métodos , Leucemia/genética , Cromatina/genética , Interpretación Estadística de Datos , Sitios Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Conformación de Ácido Nucleico , Relación Señal-Ruido , Programas Informáticos , Células Tumorales Cultivadas , Globinas beta/genéticaRESUMEN
Epigenomics is the study of the physical modifications, associations and conformations of genomic DNA sequences, with the aim of linking these with epigenetic memory, cellular identity and tissue-specific functions. While current techniques in the field are characterizing the average epigenomic features across large cell ensembles, the increasing interest in the epigenetics within complex and heterogeneous tissues is driving the development of single-cell epigenomics. We review emerging single-cell methods for capturing DNA methylation, chromatin accessibility, histone modifications, chromosome conformation and replication dynamics. Together, these techniques are rapidly becoming a powerful tool in studies of cellular plasticity and diversity, as seen in stem cells and cancer.
Asunto(s)
Ensamble y Desensamble de Cromatina , Metilación de ADN , Replicación del ADN , Epigenómica/métodos , Histonas , Procesamiento Proteico-Postraduccional , Animales , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , HumanosRESUMEN
Children with Down syndrome (DS) are at increased risk for acute myeloid leukemias (ML-DS) characterized by mixed megakaryocytic and erythroid phenotype and by acquired mutations in the GATA1 gene resulting in a short GATA1s isoform. The chromosome 21 microRNA (miR)-125b cluster has been previously shown to cooperate with GATA1s in transformation of fetal hematopoietic progenitors. In this study, we report that the expression of miR-486-5p is increased in ML-DS compared with non-DS acute megakaryocytic leukemias (AMKLs). miR-486-5p is regulated by GATA1 and GATA1s that bind to the promoter of its host gene ANK1. miR-486-5p is highly expressed in mouse erythroid precursors and knockdown (KD) in ML-DS cells reduced their erythroid phenotype. Ectopic expression and KD of miR-486-5p in primary fetal liver hematopoietic progenitors demonstrated that miR-486-5p cooperates with Gata1s to enhance their self renewal. Consistent with its activation of AKT, overexpression and KD experiments showed its importance for growth and survival of human leukemic cells. Thus, miR-486-5p cooperates with GATA1s in supporting the growth and survival, and the aberrant erythroid phenotype of the megakaryocytic leukemias of DS.