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1.
Nat Commun ; 11(1): 5434, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116143

RESUMO

The interfollicular epidermis (IFE) forms a water-tight barrier that is often disrupted in inflammatory skin diseases. During homeostasis, the IFE is replenished by stem cells in the basal layer that differentiate as they migrate toward the skin surface. Conventionally, IFE differentiation is thought to be stepwise as reflected in sharp boundaries between its basal, spinous, granular and cornified layers. The transcription factor GRHL3 regulates IFE differentiation by transcriptionally activating terminal differentiation genes. Here we use single cell RNA-seq to show that murine IFE differentiation is best described as a single step gradualistic process with a large number of transition cells between the basal and spinous layer. RNA-velocity analysis identifies a commitment point that separates the plastic basal and transition cell state from unidirectionally differentiating cells. We also show that in addition to promoting IFE terminal differentiation, GRHL3 is essential for suppressing epidermal stem cell expansion and the emergence of an abnormal stem cell state by suppressing Wnt signaling in stem cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Epidérmicas/citologia , Células Epidérmicas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular , Linhagem da Célula , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Epiderme/embriologia , Epiderme/metabolismo , Feminino , Perfilação da Expressão Gênica , Idade Gestacional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Análise de Célula Única , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
2.
Nat Commun ; 11(1): 4989, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020476

RESUMO

We postulate that exit from pluripotency involves intermediates that retain pluripotency while simultaneously exhibiting lineage-bias. Using a MIXL1 reporter, we explore mesoderm lineage-bias within the human pluripotent stem cell compartment. We identify a substate, which at the single cell level coexpresses pluripotent and mesodermal gene expression programmes. Functionally these cells initiate stem cell cultures and exhibit mesodermal bias in differentiation assays. By promoting mesodermal identity through manipulation of WNT signalling while preventing exit from pluripotency using lysophosphatidic acid, we 'trap' and maintain cells in a lineage-biased stem cell state through multiple passages. These cells correspond to a normal state on the differentiation trajectory, the plasticity of which is evidenced by their reacquisition of an unbiased state upon removal of differentiation cues. The use of 'cross-antagonistic' signalling to trap pluripotent stem cell intermediates with different lineage-bias may have general applicability in the efficient production of cells for regenerative medicine.


Assuntos
Reprogramação Celular , Mesoderma/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula , Plasticidade Celular/genética , Autorrenovação Celular , Meios de Cultura , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais/genética
3.
Nat Commun ; 11(1): 5499, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127892

RESUMO

The epiblast, which provides the foundation of the future body, is actively reshaped during early embryogenesis, but the reshaping mechanisms are poorly understood. Here, using a 3D in vitro model of early epiblast development, we identify the canonical Wnt/ß-catenin pathway and its central downstream factor Esrrb as the key signalling cascade regulating the tissue-scale organization of the murine pluripotent lineage. Although in vivo the Wnt/ß-catenin/Esrrb circuit is dispensable for embryonic development before implantation, autocrine Wnt activity controls the morphogenesis and long-term maintenance of the epiblast when development is put on hold during diapause. During this phase, the progressive changes in the epiblast architecture and Wnt signalling response show that diapause is not a stasis but instead is a dynamic process with underlying mechanisms that can appear redundant during transient embryogenesis.


Assuntos
Diapausa/fisiologia , Células-Tronco Embrionárias/metabolismo , Receptores Estrogênicos/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Desenvolvimento Embrionário , Feminino , Camadas Germinativas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Estrogênicos/genética , beta Catenina/genética
4.
PLoS One ; 15(9): e0238742, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32911515

RESUMO

It has been suggested that DNA methylation can work in concert with other epigenetic factors, leading to changes in cellular phenotypes. For example, DNA demethylation modifications producing 5-hydroxymethylcytosine (5hmC) are thought to interact with histone modifications to influence the acquisition of embryonic stem cell (ESC) potency. However, the mechanism by which this occurs is still unknown. Thus, we systematically analysed the co-occurrence of DNA and histone modifications at genic regions as well as their relationship with ESC-specific expression using a number of heterogeneous public datasets. From a set of 19 epigenetic factors, we found remarkable co-occurrence of 5hmC and H4K8ac, accompanied by H3K4me1. This enrichment was more prominent at gene body regions. The results were confirmed using data obtained from different detection methods and species. Our analysis shows that these marks work cooperatively to influence ESC-specific gene expression. We also found that this trivalent mark is relatively enriched in genes related with immunity, which is a bit specific in ESCs. We propose that a trivalent epigenetic mark, composed of 5hmC, H4K8ac and H3K4me1, regulates gene expression and modulates the nature of human ESCs as a novel epigenetic code.


Assuntos
Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Genômica , Algoritmos , Metilação de DNA , Histonas/metabolismo , Humanos
5.
Proc Natl Acad Sci U S A ; 117(36): 22331-22340, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32839322

RESUMO

The chromatin remodeler CHD8 is among the most frequently mutated genes in autism spectrum disorder (ASD). CHD8 has a dosage-sensitive role in ASD, but when and how it becomes critical to human social function is unclear. Here, we conducted genomic analyses of heterozygous and homozygous Chd8 mouse embryonic stem cells and differentiated neural progenitors. We identify dosage-sensitive CHD8 transcriptional targets, sites of regulated accessibility, and an unexpected cooperation with SOX transcription factors. Collectively, our findings reveal that CHD8 negatively regulates expression of neuronal genes to maintain pluripotency and also during differentiation. Thus, CHD8 is essential for both the maintenance of pluripotency and neural differentiation, providing mechanistic insight into its function with potential implications for ASD.


Assuntos
Proteínas de Ligação a DNA , Dosagem de Genes/genética , Neurogênese/genética , Animais , Transtorno do Espectro Autista , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Camundongos , Camundongos Knockout
6.
Proc Natl Acad Sci U S A ; 117(34): 20625-20635, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788350

RESUMO

Profilin2 (PFN2) is a target of the embryonic stem cell (ESC)-enriched miR-290 family of microRNAs (miRNAs) and an actin/dynamin-binding protein implicated in endocytosis. Here we show that the miR-290-PFN2 pathway regulates many aspects of ESC biology. In the absence of miRNAs, PFN2 is up-regulated in ESCs, with a resulting decrease in endocytosis. Reintroduction of miR-290, knockout of Pfn2, or disruption of the PFN2-dynamin interaction domain in miRNA-deficient cells reverses the endocytosis defect. The reduced endocytosis is associated with impaired extracellular signal-regulated kinase (ERK) signaling, delayed ESC cell cycle progression, and repressed ESC differentiation. Mutagenesis of the single canonical conserved 3' UTR miR-290-binding site of Pfn2 or overexpression of the Pfn2 open reading frame alone in otherwise wild-type cells largely recapitulates these phenotypes. Taken together, these findings define an axis of posttranscriptional control, endocytosis, and signal transduction that is important for ESC proliferation and differentiation.


Assuntos
Células-Tronco Embrionárias/metabolismo , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo , Células-Tronco Pluripotentes/metabolismo , Profilinas/metabolismo , Regiões 3' não Traduzidas , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/citologia , Endocitose/fisiologia , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/genética , Células-Tronco Pluripotentes/citologia , Profilinas/genética , Transdução de Sinais/genética
7.
Nat Commun ; 11(1): 4118, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807789

RESUMO

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers such as PRC2 and DNA methyl-transferases, and proteins governing chromosome architecture. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , Imunofluorescência , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Proteômica
9.
Gene ; 758: 144958, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32683073

RESUMO

Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.


Assuntos
Metilação de DNA/genética , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Azacitidina/farmacologia , Linhagem Celular , Citomegalovirus/genética , Fator de Iniciação 1 em Eucariotos/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Transdução Genética
10.
Mol Cell Biol ; 40(18)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661120

RESUMO

The DNA and protein complex known as chromatin is subject to posttranslational modifications (PTMs) that regulate cellular functions such that PTM dysregulation can lead to disease, including cancer. One critical PTM is acetylation/deacetylation, which is being investigated as a means to develop targeted cancer therapies. The histone acetyltransferase (HAT) family of proteins performs histone acetylation. In humans, MOF (hMOF), a member of the MYST family of HATs, acetylates histone H4 at lysine 16 (H4K16ac). MOF-mediated acetylation plays a critical role in the DNA damage response (DDR) and embryonic stem cell development. Functionally, MOF is found in two distinct complexes: NSL (nonspecific lethal) in humans and MSL (male-specific lethal) in flies. The NSL complex is also able to acetylate additional histone H4 sites. Dysregulation of MOF activity occurs in multiple cancers, including ovarian cancer, medulloblastoma, breast cancer, colorectal cancer, and lung cancer. Bioinformatics analysis of KAT8, the gene encoding hMOF, indicated that it is highly overexpressed in kidney tumors as part of a concerted gene coexpression program that can support high levels of chromosome segregation and cell proliferation. The linkage between MOF and tumor proliferation suggests that there are additional functions of MOF that remain to be discovered.


Assuntos
Dano ao DNA , Células-Tronco Embrionárias/citologia , Histona Acetiltransferases/metabolismo , Acetilação , Carcinogênese/metabolismo , Diferenciação Celular/fisiologia , Núcleo Celular/metabolismo , Proliferação de Células/fisiologia , Transformação Celular Neoplásica/metabolismo , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional
11.
Nat Commun ; 11(1): 3603, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681107

RESUMO

Members of the PR/SET domain-containing (PRDM) family of zinc finger transcriptional regulators play diverse developmental roles. PRDM10 is a yet uncharacterized family member, and its function in vivo is unknown. Here, we report an essential requirement for PRDM10 in pre-implantation embryos and embryonic stem cells (mESCs), where loss of PRDM10 results in severe cell growth inhibition. Detailed genomic and biochemical analyses reveal that PRDM10 functions as a sequence-specific transcription factor. We identify Eif3b, which encodes a core component of the eukaryotic translation initiation factor 3 (eIF3) complex, as a key downstream target, and demonstrate that growth inhibition in PRDM10-deficient mESCs is in part mediated through EIF3B-dependent effects on global translation. Our work elucidates the molecular function of PRDM10 in maintaining global translation, establishes its essential role in early embryonic development and mESC homeostasis, and offers insights into the functional repertoire of PRDMs as well as the transcriptional mechanisms regulating translation.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Camundongos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Desenvolvimento Embrionário , Células-Tronco Embrionárias/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos/embriologia , Camundongos/genética , Biossíntese de Proteínas , Fatores de Transcrição/genética
12.
Mol Cell ; 79(1): 167-179.e11, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32497496

RESUMO

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.


Assuntos
Proteínas Argonauta/fisiologia , Células-Tronco Embrionárias/metabolismo , Glioma/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Feminino , Regulação da Expressão Gênica , Glioma/genética , Glioma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Hidrolases/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , Ligação Proteica , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética
13.
Proc Natl Acad Sci U S A ; 117(27): 15702-15711, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576691

RESUMO

Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol IIIα) and the related POLR3GL in the other form (Pol IIIß). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.


Assuntos
Cerebelo/crescimento & desenvolvimento , Desenvolvimento Embrionário/genética , Neurônios/metabolismo , RNA Polimerase III/genética , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Cerebelo/patologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Camundongos Knockout , Neurônios/patologia , Ligação Proteica/genética , Isoformas de Proteínas/genética , Subunidades Proteicas/genética
14.
Nat Commun ; 11(1): 2118, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32355211

RESUMO

ScRNA-seq has the ability to reveal accurate and precise cell types and states. Existing scRNA-seq platforms utilize bead-based technologies uniquely barcoding individual cells, facing practical challenges for precious samples with limited cell number. Here, we present a scRNA-seq platform, named Paired-seq, with high cells/beads utilization efficiency, cell-free RNAs removal capability, high gene detection ability and low cost. We utilize the differential flow resistance principle to achieve single cell/barcoded bead pairing with high cell utilization efficiency (95%). The integration of valves and pumps enables the complete removal of cell-free RNAs, efficient cell lysis and mRNA capture, achieving highest mRNA detection accuracy (R = 0.955) and comparable sensitivity. Lower reaction volume and higher mRNA capture and barcoding efficiency significantly reduce the cost of reagents and sequencing. The single-cell expression profile of mES and drug treated cells reveal cell heterogeneity, demonstrating the enormous potential of Paired-seq for cell biology, developmental biology and precision medicine.


Assuntos
RNA Mensageiro/química , Análise de Sequência de RNA , Análise de Célula Única/métodos , Células-Tronco/citologia , Células 3T3 , Animais , Antineoplásicos/farmacologia , Diferenciação Celular , Sistema Livre de Células , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células K562 , Camundongos , Medicina de Precisão , Reprodutibilidade dos Testes
15.
Proc Natl Acad Sci U S A ; 117(22): 12182-12191, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414917

RESUMO

In multicellular organisms, paralogs from gene duplication survive purifying selection by evolving tissue-specific expression and function. Whether this genetic redundancy is also selected for within a single cell type is unclear for multimember paralogs, as exemplified by the four obligatory Lef/Tcf transcription factors of canonical Wnt signaling, mainly due to the complex genetics involved. Using the developing mouse lung as a model system, we generate two quadruple conditional knockouts, four triple mutants, and various combinations of double mutants, showing that the four Lef/Tcf genes function redundantly in the presence of at least two Lef/Tcf paralogs, but additively upon losing additional paralogs to specify and maintain lung epithelial progenitors. Prelung-specification, pan-epithelial double knockouts have no lung phenotype; triple knockouts have varying phenotypes, including defective branching and tracheoesophageal fistulas; and the quadruple knockout barely forms a lung, resembling the Ctnnb1 mutant. Postlung-specification deletion of all four Lef/Tcf genes leads to branching defects, down-regulation of progenitor genes, premature alveolar differentiation, and derepression of gastrointestinal genes, again phenocopying the corresponding Ctnnb1 mutant. Our study supports a monotonic, positive signaling relationship between CTNNB1 and Lef/Tcf in lung epithelial progenitors as opposed to reported repressor functions of Lef/Tcf, and represents a thorough in vivo analysis of cell-type-specific genetic redundancy among the four Lef/Tcf paralogs.


Assuntos
Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Pulmão/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/fisiologia , Células-Tronco/metabolismo , beta Catenina/metabolismo , Animais , Diferenciação Celular , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Feminino , Fator 1-alfa Nuclear de Hepatócito/fisiologia , Pulmão/citologia , Camundongos , Camundongos Knockout , Análise de Célula Única , Células-Tronco/citologia , Proteína 1 Semelhante ao Fator 7 de Transcrição/fisiologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética
16.
Nat Med ; 26(6): 869-877, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32461697

RESUMO

Human genetic variants predicted to cause loss-of-function of protein-coding genes (pLoF variants) provide natural in vivo models of human gene inactivation and can be valuable indicators of gene function and the potential toxicity of therapeutic inhibitors targeting these genes1,2. Gain-of-kinase-function variants in LRRK2 are known to significantly increase the risk of Parkinson's disease3,4, suggesting that inhibition of LRRK2 kinase activity is a promising therapeutic strategy. While preclinical studies in model organisms have raised some on-target toxicity concerns5-8, the biological consequences of LRRK2 inhibition have not been well characterized in humans. Here, we systematically analyze pLoF variants in LRRK2 observed across 141,456 individuals sequenced in the Genome Aggregation Database (gnomAD)9, 49,960 exome-sequenced individuals from the UK Biobank and over 4 million participants in the 23andMe genotyped dataset. After stringent variant curation, we identify 1,455 individuals with high-confidence pLoF variants in LRRK2. Experimental validation of three variants, combined with previous work10, confirmed reduced protein levels in 82.5% of our cohort. We show that heterozygous pLoF variants in LRRK2 reduce LRRK2 protein levels but that these are not strongly associated with any specific phenotype or disease state. Our results demonstrate the value of large-scale genomic databases and phenotyping of human loss-of-function carriers for target validation in drug discovery.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação com Perda de Função/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Bancos de Espécimes Biológicos , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Feminino , Mutação com Ganho de Função/genética , Heterozigoto , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Longevidade/genética , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Fenótipo
17.
Nat Cell Biol ; 22(6): 651-662, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393886

RESUMO

BMP4 regulates a plethora of developmental processes, including the dorsal-ventral axis and neural patterning. Here, we report that BMP4 reconfigures the nuclear architecture during the primed-to-naive transition (PNT). We first established a BMP4-driven PNT and show that BMP4 orchestrates the chromatin accessibility dynamics during PNT. Among the loci opened early by BMP4, we identified Zbtb7a and Zbtb7b (Zbtb7a/b) as targets that drive PNT. ZBTB7A/B in turn facilitate the opening of naive pluripotent chromatin loci and the activation of nearby genes. Mechanistically, ZBTB7A not only binds to chromatin loci near to the genes that are activated, but also strategically occupies those that are silenced, consistent with a role of BMP4 in both activating and suppressing gene expression during PNT at the chromatin level. Our results reveal a previously unknown function of BMP4 in regulating nuclear architecture and link its targets ZBTB7A/B to chromatin remodelling and pluripotent fate control.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular , Células Cultivadas , Cromatina/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética
18.
Nat Cell Biol ; 22(5): 534-545, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32367046

RESUMO

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.


Assuntos
Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Blastocisto/metabolismo , Blastocisto/fisiologia , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese/fisiologia , Fatores de Transcrição Otx/metabolismo , Células-Tronco Pluripotentes/metabolismo
19.
PLoS Comput Biol ; 16(4): e1007195, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275652

RESUMO

DNA methylation is a heritable epigenetic modification that plays an essential role in mammalian development. Genomic methylation patterns are dynamically maintained, with DNA methyltransferases mediating inheritance of methyl marks onto nascent DNA over cycles of replication. A recently developed experimental technique employing immunoprecipitation of bromodeoxyuridine labeled nascent DNA followed by bisulfite sequencing (Repli-BS) measures post-replication temporal evolution of cytosine methylation, thus enabling genome-wide monitoring of methylation maintenance. In this work, we combine statistical analysis and stochastic mathematical modeling to analyze Repli-BS data from human embryonic stem cells. We estimate site-specific kinetic rate constants for the restoration of methyl marks on >10 million uniquely mapped cytosines within the CpG (cytosine-phosphate-guanine) dinucleotide context across the genome using Maximum Likelihood Estimation. We find that post-replication remethylation rate constants span approximately two orders of magnitude, with half-lives of per-site recovery of steady-state methylation levels ranging from shorter than ten minutes to five hours and longer. Furthermore, we find that kinetic constants of maintenance methylation are correlated among neighboring CpG sites. Stochastic mathematical modeling provides insight to the biological mechanisms underlying the inference results, suggesting that enzyme processivity and/or collaboration can produce the observed kinetic correlations. Our combined statistical/mathematical modeling approach expands the utility of genomic datasets and disentangles heterogeneity in methylation patterns arising from replication-associated temporal dynamics versus stable cell-to-cell differences.


Assuntos
Biologia Computacional/métodos , Metilação de DNA/fisiologia , Animais , Bromodesoxiuridina/química , Ilhas de CpG , Citosina/metabolismo , DNA/metabolismo , Metilases de Modificação do DNA/genética , Células-Tronco Embrionárias/metabolismo , Epigênese Genética/genética , Epigênese Genética/fisiologia , Epigenômica/métodos , Genoma , Genômica , Humanos , Cinética , Modelos Estatísticos , Modelos Teóricos , Processos Estocásticos
20.
Nat Commun ; 11(1): 1617, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238817

RESUMO

Translational control plays a central role in regulation of gene expression and can lead to significant divergence between mRNA- and protein-abundance. Here, we used genome-wide approaches combined with time-course analysis to measure the mRNA-abundance, mRNA-translation rate and protein expression during the transition of naïve-to-primed mouse embryonic stem cells (ESCs). We find that the ground state ESCs cultured with GSK3-, MEK-inhibitors and LIF (2iL) display higher ribosome density on a selective set of mRNAs. This set of mRNAs undergo strong translational buffering to maintain stable protein expression levels in 2iL-ESCs. Importantly, we show that the global alteration of cellular proteome during the transition of naïve-to-primed pluripotency is largely accompanied by transcriptional rewiring. Thus, we provide a comprehensive and detailed overview of the global changes in gene expression in different states of ESCs and dissect the relative contributions of mRNA-transcription, translation and regulation of protein stability in controlling protein abundance.


Assuntos
Células-Tronco Embrionárias/metabolismo , Polirribossomos/metabolismo , Proteoma/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Animais , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Redes e Vias Metabólicas , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Polirribossomos/genética , Ribossomos/genética , Transcriptoma
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