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1.
Curr Opin Cell Biol ; 61: 1-8, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31233905

RESUMO

In mammals, germline specification is induced during early embryogenesis when competent cells respond to extrinsic signals and form primordial germ cells (PGCs), the precursors of the gametes. The fusion of the two types of gametes, the egg and the sperm, gives rise to a new organism and closes the germline cycle. With the entry of the germline, the PGCs are separated from the soma and thus ensure the self-perpetuation of the species. Using the mouse as a model of mammalian embryogenesis, in this review we will focus on the transcriptional and epigenetic changes that regulate the initial steps of germline development, namely germline competence and PGC specification.

2.
Cell Stem Cell ; 24(5): 736-752.e12, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-30982769

RESUMO

The pathological consequences of structural variants disrupting 3D genome organization can be difficult to elucidate in vivo due to differences in gene dosage sensitivity between mice and humans. This is illustrated by branchiooculofacial syndrome (BOFS), a rare congenital disorder caused by heterozygous mutations within TFAP2A, a neural crest regulator for which humans, but not mice, are haploinsufficient. Here, we present a BOFS patient carrying a heterozygous inversion with one breakpoint located within a topologically associating domain (TAD) containing enhancers essential for TFAP2A expression in human neural crest cells (hNCCs). Using patient-specific hiPSCs, we show that, although the inversion shuffles the TFAP2A hNCC enhancers with novel genes within the same TAD, this does not result in enhancer adoption. Instead, the inversion disconnects one TFAP2A allele from its cognate enhancers, leading to monoallelic and haploinsufficient TFAP2A expression in patient hNCCs. Our work illustrates the power of hiPSC differentiation to unveil long-range pathomechanisms.

3.
Stem Cell Reports ; 12(5): 861-868, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31006630

RESUMO

The neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1-900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.

4.
Cell Stem Cell ; 24(2): 318-327.e8, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30554961

RESUMO

Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs are preferentially encoded from the genomic loci of key developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns in vivo in mouse and in human embryos. Signifying their developmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, which localizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncT depletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions.

5.
Hum Mol Genet ; 27(23): 4117-4134, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30452683

RESUMO

Pluripotent stem cells are invaluable resources to study development and disease, holding a great promise for regenerative medicine. Here we use human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) from patients with Huntington's disease (HD-iPSCs) to shed light into the normal function of huntingtin (HTT) and its demise in disease. We find that HTT binds ATF7IP, a regulator of the histone H3 methyltransferase SETDB1. HTT inhibits the interaction of the ATF7IP-SETDB1 complex with other heterochromatin regulators and transcriptional repressors, maintaining low levels of H3K9 trimethylation (H3K9me3) in hESCs. Loss of HTT promotes global increased H3K9me3 levels and enrichment of H3K9me3 marks at distinct genes, including transcriptional regulators of neuronal differentiation. Although these genes are normally expressed at low amounts in hESCs, HTT knockdown (KD) reduces their induction during neural differentiation. Notably, mutant expanded polyglutamine repeats in HTT diminish its interaction with ATF7IP-SETDB1 complex and trigger H3K9me3 in HD-iPSCs. Conversely, KD of ATF7IP in HD-iPSCs reduces H3K9me3 alterations and ameliorates gene expression changes in their neural counterparts. Taken together, our results indicate ATF7IP as a potential target to correct aberrant H3K9me3 levels induced by mutant HTT.

6.
Cell Stem Cell ; 23(2): 149-151, 2018 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-30075122

RESUMO

The transitions between different pluripotency states are regulated by large changes in enhancer landscapes. Two publications in this issue of Cell Stem Cell suggest that the pioneering and subsequent activation of silent enhancers by transcription factors (i.e., ESRRB and GRHL2) is a crucial event during these transitions (Adachi et al., 2018; Chen et al., 2018).

7.
Mol Syst Biol ; 14(6): e8214, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29858282

RESUMO

The last decade has radically renewed our understanding of higher order chromatin folding in the eukaryotic nucleus. As a result, most current models are in support of a mostly hierarchical and relatively stable folding of chromosomes dividing chromosomal territories into A- (active) and B- (inactive) compartments, which are then further partitioned into topologically associating domains (TADs), each of which is made up from multiple loops stabilized mainly by the CTCF and cohesin chromatin-binding complexes. Nonetheless, the structure-to-function relationship of eukaryotic genomes is still not well understood. Here, we focus on recent work highlighting the biophysical and regulatory forces that contribute to the spatial organization of genomes, and we propose that the various conformations that chromatin assumes are not so much the result of a linear hierarchy, but rather of both converging and conflicting dynamic forces that act on it.

8.
Nucleic Acids Res ; 46(7): 3351-3365, 2018 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-29438503

RESUMO

During neurogenesis, dynamic developmental cues, transcription factors and histone modifying enzymes regulate the gene expression programs by modulating the activity of neural-specific enhancers. How transient developmental signals coordinate transcription factor recruitment to enhancers and to which extent chromatin modifiers contribute to enhancer activity is starting to be uncovered. Here, we take advantage of neural stem cells as a model to unravel the mechanisms underlying neural enhancer activation in response to the TGFß signaling. Genome-wide experiments demonstrate that the proneural factor ASCL1 assists SMAD3 in the binding to a subset of enhancers. Once located at the enhancers, SMAD3 recruits the histone demethylase JMJD3 and the remodeling factor CHD8, creating the appropriate chromatin landscape to allow enhancer transcription and posterior gene activation. Finally, to analyze the phenotypical traits owed to cis-regulatory regions, we use CRISPR-Cas9 technology to demonstrate that the TGFß-responsive Neurog2 enhancer is essential for proper neuronal polarization.

9.
Nat Genet ; 50(1): 4-5, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29273804
11.
J Vis Exp ; (126)2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28872116

RESUMO

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.


Assuntos
Imunoprecipitação da Cromatina/métodos , Epigenômica/métodos , Biblioteca Gênica , Código das Histonas/genética , Embrião de Mamíferos
12.
Cell Stem Cell ; 20(5): 689-705.e9, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28285903

RESUMO

Poised enhancers marked by H3K27me3 in pluripotent stem cells have been implicated in the establishment of somatic expression programs during embryonic stem cell (ESC) differentiation. However, the functional relevance and mechanism of action of poised enhancers remain unknown. Using CRISPR/Cas9 technology to engineer precise genetic deletions, we demonstrate that poised enhancers are necessary for the induction of major anterior neural regulators. Interestingly, circularized chromosome conformation capture sequencing (4C-seq) shows that poised enhancers already establish physical interactions with their target genes in ESCs in a polycomb repressive complex 2 (PRC2)-dependent manner. Loss of PRC2 does not activate poised enhancers or induce their putative target genes in undifferentiated ESCs; however, loss of PRC2 in differentiating ESCs severely and specifically compromises the induction of major anterior neural genes representing poised enhancer targets. Overall, our work illuminates an unexpected function for polycomb proteins in facilitating neural induction by endowing major anterior neural loci with a permissive regulatory topology.


Assuntos
Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Imunoprecipitação da Cromatina , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Imunofluorescência , Camundongos , Complexo Repressor Polycomb 2/genética , Reação em Cadeia da Polimerase
13.
Hum Mol Genet ; 26(4): 829-842, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28087736

RESUMO

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signals within this high-density dataset are enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinement approach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation.

14.
Hum Mol Genet ; 26(4): 742-752, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28007912

RESUMO

Genome-wide association studies (GWAS) have emerged as a powerful tool to uncover the genetic basis of human common diseases, which often show a complex, polygenic and multi-factorial aetiology. These studies have revealed that 70-90% of all single nucleotide polymorphisms (SNPs) associated with common complex diseases do not occur within genes (i.e. they are non-coding), making the discovery of disease-causative genetic variants and the elucidation of the underlying pathological mechanisms far from straightforward. Based on emerging evidences suggesting that disease-associated SNPs are frequently found within cell type-specific regulatory sequences, here we present GARLIC (GWAS-based Prediction Toolkit for Connecting Diseases and Cell Types), a user-friendly, multi-purpose software with an associated database and online viewer that, using global maps of cis-regulatory elements, can aetiologically connect human diseases with relevant cell types. Additionally, GARLIC can be used to retrieve potential disease-causative genetic variants overlapping regulatory sequences of interest. Overall, GARLIC can satisfy several important needs within the field of medical genetics, thus potentially assisting in the ultimate goal of uncovering the elusive and complex genetic basis of common human disorders.


Assuntos
Biologia Computacional/instrumentação , Bases de Dados Genéticas , Doenças Genéticas Inatas/genética , Estudo de Associação Genômica Ampla , Software , Humanos
15.
Cell Rep ; 17(11): 3062-3076, 2016 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-27974217

RESUMO

Cellular heterogeneity within embryonic and adult tissues is involved in multiple biological and pathological processes. Here, we present a simple epigenomic strategy that allows the functional dissection of cellular heterogeneity. By integrating H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data, we demonstrate that the presence of broad H3K27me3 domains at transcriptionally active genes reflects the heterogeneous expression of major cell identity regulators. Using dorsoventral patterning of the spinal neural tube as a model, the proposed approach successfully identifies the majority of previously known dorsoventral patterning transcription factors with high sensitivity and precision. Moreover, poorly characterized patterning regulators can be similarly predicted, as shown for ZNF488, which confers p1/p2 neural progenitor identity. Finally, we show that, as our strategy is based on universal chromatin features, it can be used to functionally dissect cellular heterogeneity within various organisms and tissues, thus illustrating its potential applicability to a broad range of biological and pathological contexts.


Assuntos
Padronização Corporal/genética , Linhagem da Célula/genética , Epigenômica , Heterogeneidade Genética , Animais , Cromatina/genética , Imunoprecipitação da Cromatina/métodos , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Tubo Neural/crescimento & desenvolvimento , Tubo Neural/metabolismo , Análise de Sequência de RNA/métodos , Coluna Vertebral/crescimento & desenvolvimento , Coluna Vertebral/metabolismo
16.
Cell Stem Cell ; 18(1): 118-33, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26748758

RESUMO

Following implantation, mouse epiblast cells transit from a naive to a primed state in which they are competent for both somatic and primordial germ cell (PGC) specification. Using mouse embryonic stem cells as an in vitro model to study the transcriptional regulatory principles orchestrating peri-implantation development, here we show that the transcription factor Foxd3 is necessary for exit from naive pluripotency and progression to a primed pluripotent state. During this transition, Foxd3 acts as a repressor that dismantles a significant fraction of the naive pluripotency expression program through decommissioning of active enhancers associated with key naive pluripotency and early germline genes. Subsequently, Foxd3 needs to be silenced in primed pluripotent cells to allow re-activation of relevant genes required for proper PGC specification. Our findings therefore uncover a cycle of activation and deactivation of Foxd3 required for exit from naive pluripotency and subsequent PGC specification.


Assuntos
Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/fisiologia , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Separação Celular , Imunoprecipitação da Cromatina , Células-Tronco Embrionárias/citologia , Citometria de Fluxo , Regulação da Expressão Gênica , Inativação Gênica , Células Germinativas/citologia , Mutação em Linhagem Germinativa , Camundongos , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de RNA , Transcrição Genética
17.
Biol Chem ; 395(12): 1453-60, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25205712

RESUMO

Common human pathologies have a complicated etiology involving both genetic and environmental risk factors. Moreover, the genetic basis of these disorders is also complex, with multiple and weak genetic variants contributing to disease susceptibility. In addition, most of these risk genetic variants occur outside genes, within the vast non-coding human genomic space. In this review I first illustrate how large-scale genomic studies aimed at mapping cis-regulatory elements in the human genome are facilitating the identification of disease-causative non-coding genetic variation. I then discuss some of the challenges that remain to be solved before the pathological consequences of non-coding genetic variation can be fully appreciated. Ultimately, revealing the genetics of human complex disease can be a critical step towards more personalized and effective diagnosis and treatments.


Assuntos
Variação Genética , Elementos Reguladores de Transcrição , Epigênese Genética , Predisposição Genética para Doença , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Polimorfismo de Nucleotídeo Único
18.
Philos Trans R Soc Lond B Biol Sci ; 368(1620): 20120360, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23650634

RESUMO

Developmental gene expression programmes are coordinated by the specialized distal cis-regulatory elements called enhancers, which integrate lineage- and signalling-dependent inputs to guide morphogenesis. In previous work, we characterized the genome-wide repertoire of active enhancers in human neural crest cells (hNCC), an embryonic cell population with critical roles in craniofacial development. We showed that in hNCC, co-occupancy of a master regulator TFAP2A with nuclear receptors NR2F1 and NR2F2 correlates with the presence of permissive enhancer chromatin states. Here, we take advantage of pre-existing human genetic variation to further explore potential cooperation between TFAP2A and NR2F1/F2. We demonstrate that isolated single nucleotide polymorphisms affecting NR2F1/F2-binding sites within hNCC enhancers can alter TFAP2A occupancy and overall chromatin features at the same enhancer allele. We propose that a similar strategy can be used to elucidate other cooperative relationships between transcription factors involved in developmental transitions. Using the neural crest and its major contribution to human craniofacial phenotypes as a paradigm, we discuss how genetic variation might modulate the molecular properties and activity of enhancers, and ultimately impact human phenotypic diversity.


Assuntos
Variação Genética , Crista Neural/citologia , Fenótipo , Fator de Transcrição AP-2/metabolismo , Alelos , Animais , Fator I de Transcrição COUP/genética , Fator I de Transcrição COUP/metabolismo , Montagem e Desmontagem da Cromatina , Elementos Facilitadores Genéticos , Epigênese Genética , Face/anatomia & histologia , Humanos , Morfogênese , Crista Neural/metabolismo , Ligação Proteica , Fator de Transcrição AP-2/genética
19.
Dev Cell ; 24(4): 342-4, 2013 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-23449470

RESUMO

Genome condensation during mitosis presents a chromatin landscape largely inaccessible to RNA polymerase II and most transcription factors. Caravaca et al. (2013) now report in Genes and Development that the pioneer transcription factor FOXA1 is retained at mitotic chromosomes, bookmarking the genome to enable gene expression reestablishment upon mitotic exit.

20.
Plant Mol Biol ; 80(4-5): 405-18, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22968620

RESUMO

Plants perceiving drought activate multiple responses to improve survival, including large-scale alterations in gene expression. This article reports on the roles in the drought response of two Arabidopsis thaliana homeodomain-leucine zipper class I genes; ATHB7 and ATHB12, both strongly induced by water-deficit and abscisic acid (ABA). ABA-mediated transcriptional regulation of both genes is shown to depend on the activity of protein phosphatases type 2C (PP2C). ATHB7 and ATHB12 are, thus, targets of the ABA signalling mechanism defined by the PP2Cs and the PYR/PYL family of ABA receptors, with which the PP2C proteins interact. Our results from chromatin immunoprecipitation and gene expression analyses demonstrate that ATHB7 and ATHB12 act as positive transcriptional regulators of PP2C genes, and thereby as negative regulators of abscisic acid signalling. In support of this notion, our results also show that ATHB7 and ATHB12 act to repress the transcription of genes encoding the ABA receptors PYL5 and PYL8 in response to an ABA stimulus. In summary, we demonstrate that ATHB7 and ATHB12 have essential functions in the primary response to drought, as mediators of a negative feedback effect on ABA signalling in the plant response to water deficit.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Receptores de Superfície Celular/genética , Transdução de Sinais , Fatores de Transcrição/fisiologia , Proteínas de Arabidopsis/genética , Regulação para Baixo/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/genética , Regulação para Cima/fisiologia
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