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2.
Mol Ther Nucleic Acids ; 25: 494-501, 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34589272

RESUMO

Prime editing enables efficient introduction of targeted transversions, insertions, and deletions in mammalian cells and several organisms. However, genetic disease models with base deletions by prime editing have not yet been reported in mice. Here, we successfully generate a mouse model with a cataract disorder through microinjection of prime editor 3 (PE3) plasmids to efficiently induce targeted single-base deletion. Notably, a generated mouse with a high G-deletion rate (38.2%) displays a nuclear cataract phenotype; the PE3-induced deletions in mutant mice achieve high rates of germline transmission to their progenies, with phenotypic inheritance of cataract. Our data propose that modeling a genetic disease with a single nucleotide deletion in mice can be achieved with prime genome editing in vivo.

3.
NPJ Genom Med ; 6(1): 65, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381055

RESUMO

Trimethylation of histone H3 lysine 27 trimethylation (H3K27me3) may be recruited by repressive Polycomb complexes to mediate gene silencing, which is critical for maintaining embryonic stem cell pluripotency and differentiation. However, the roles of aberrant H3K27me3 patterns in tumorigenesis are not fully understood. Here, we discovered that grand silencer domains (breadth > 50 kb) for H3K27me3 were significantly associated with epithelial cell differentiation and exhibited high gene essentiality and conservation in human esophageal epithelial cells. These grand H3K27me3 domains exhibited high modification signals involved in gene silencing, and preferentially occupied the entirety of topologically associating domains and interact with each other. We found that widespread loss of the grand H3K27me3 domains in of esophageal squamous cell carcinomas (ESCCs) were enriched in genes involved in epithelium and endothelium differentiation, which were significantly associated with overexpression with increase of active modifications of H3K4me3, H3K4me1, and H3K27ac marks, as well as DNA hypermethylation in the gene bodies. A total of 208 activated genes with loss of grand H3K27me3 domains in ESCC were identified, where the higher expression and mutation of T-box transcription factor 20 (TBX20) were associated with worse patients' outcomes. Our results showed that knockdown of TBX20 may have led to a striking defect in esophageal cancer cell growth and carcinogenesis-related pathway, including cell cycle and homologous recombination. Together, our results reveal that loss of grand H3K27me3 domains represent a catalog of remarkable activating regulators involved in carcinogenesis.

4.
Nat Commun ; 12(1): 4457, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294701

RESUMO

The role of cis-elements and their aberrations remains unclear in esophageal squamous cell carcinoma (ESCC, further abbreviated EC). Here we survey 28 H3K27ac-marked active enhancer profiles and 50 transcriptomes in primary EC, metastatic lymph node cancer (LNC), and adjacent normal (Nor) esophageal tissues. Thousands of gained or lost enhancers and hundreds of altered putative super-enhancers are identified in EC and LNC samples respectively relative to Nor, with a large number of common gained or lost enhancers. Moreover, these differential enhancers contribute to the transcriptomic aberrations in ECs and LNCs. We also reveal putative driver onco-transcription factors, depletion of which diminishes cell proliferation and migration. The administration of chemical inhibitors to suppress the predicted targets of gained super-enhances reveals HSP90AA1 and PDE4B as potential therapeutic targets for ESCC. Thus, our epigenomic profiling reveals a compendium of reprogrammed cis-regulatory elements during ESCC carcinogenesis and metastasis for uncovering promising targets for cancer treatment.


Assuntos
Elementos Facilitadores Genéticos , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Idoso , Carcinogênese/genética , Linhagem Celular Tumoral , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/secundário , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Código das Histonas/genética , Humanos , Metástase Linfática/genética , Masculino , Pessoa de Meia-Idade , Oncogenes , Fatores de Transcrição/genética
5.
Cell Prolif ; 54(8): e13096, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34240779

RESUMO

OBJECTIVES: PKM1 and PKM2, which are generated from the alternative splicing of PKM gene, play important roles in tumourigenesis and embryonic development as rate-limiting enzymes in glycolytic pathway. However, because of the lack of appropriate techniques, the specific functions of the 2 PKM splicing isoforms have not been clarified endogenously yet. MATERIALS AND METHODS: In this study, we used CRISPR-based base editors to perturbate the endogenous alternative splicing of PKM by introducing mutations into the splicing junction sites in HCT116 cells and zebrafish embryos. Sanger sequencing, agarose gel electrophoresis and targeted deep sequencing assays were utilized for identifying mutation efficiencies and detecting PKM1/2 splicing isoforms. Cell proliferation assays and RNA-seq analysis were performed to describe the effects of perturbation of PKM1/2 splicing in tumour cell growth and zebrafish embryo development. RESULTS: The splicing sites of PKM, a 5' donor site of GT and a 3' acceptor site of AG, were efficiently mutated by cytosine base editor (CBE; BE4max) and adenine base editor (ABE; ABEmax-NG) with guide RNAs (gRNAs) targeting the splicing sites flanking exons 9 and 10 in HCT116 cells and/or zebrafish embryos. The mutations of the 5' donor sites of GT flanking exons 9 or 10 into GC resulted in specific loss of PKM1 or PKM2 expression as well as the increase in PKM2 or PKM1 respectively. Specific loss of PKM1 promoted cell proliferation of HCT116 cells and upregulated the expression of cell cycle regulators related to DNA replication and cell cycle phase transition. In contrast, specific loss of PKM2 suppressed cell growth of HCT116 cells and resulted in growth retardation of zebrafish. Meanwhile, we found that mutation of PKM1/2 splicing sites also perturbated the expression of non-canonical PKM isoforms and produced some novel splicing isoforms. CONCLUSIONS: This work proved that CRISPR-based base editing strategy can be used to disrupt the endogenous alternative splicing of genes of interest to study the function of specific splicing isoforms in vitro and in vivo. It also reminded us to notice some novel or undesirable splicing isoforms by targeting the splicing junction sites using base editors. In sum, we establish a platform to perturbate endogenous RNA splicing for functional investigation or genetic correction of abnormal splicing events in human diseases.


Assuntos
Edição de Genes , Piruvato Quinase/metabolismo , Processamento Alternativo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulação para Baixo , Éxons , Feminino , Células HCT116 , Humanos , Mutagênese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Piruvato Quinase/genética , Regulação para Cima , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
6.
Nat Commun ; 12(1): 2287, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863894

RESUMO

Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.


Assuntos
Adenosina Desaminase/genética , Proteína 9 Associada à CRISPR/genética , DNA/genética , Proteínas de Escherichia coli/genética , Edição de Genes/métodos , Adenina/metabolismo , Adenosina Desaminase/metabolismo , Toxinas Bacterianas/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Citosina/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Genes Reporter , Células HEK293 , Humanos , Inosina/genética , Inosina/metabolismo , Engenharia de Proteínas , Edição de RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA-Seq , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
8.
Mol Ther ; 28(9): 2083-2095, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32526202

RESUMO

Transcription growth factor ß (TGF-ß) signaling-triggered epithelial-to-mesenchymal transition (EMT) process is associated with tumor stemness, metastasis, and chemotherapy resistance. However, the epigenomic basis for TGF-ß-induced EMT remains largely unknown. Here we reveal that HDAC1-mediated global histone deacetylation and the gain of specific histone H3 lysine 27 acetylation (H3K27ac)-marked enhancers are essential for the TGF-ß-induced EMT process. Enhancers gained upon TGF-ß treatment are linked to gene activation of EMT markers and cancer metastasis. Notably, dynamic enhancer gain or loss mainly occurs within pre-existing topologically associated domains (TADs) in epithelial cells, with minimal three-dimensional (3D) genome architecture reorganization. Through motif enrichment analysis of enhancers that are lost or gained upon TGF-ß stimulation, we identify FOXA2 as a key factor to activate epithelial-specific enhancer activity, and we also find that TEAD4 forms a complex with SMAD2/3 to mediate TGF-ß signaling-triggered mesenchymal enhancer reprogramming. Together, our results implicate that key transcription-factor (TF)-mediated enhancer reprogramming modulates the developmental transition in TGF-ß signaling-associated cancer metastasis.

9.
Nat Commun ; 11(1): 2653, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32461551

RESUMO

The transcriptome of the preimplantation mouse embryo has been previously annotated by short-read sequencing, with limited coverage and accuracy. Here we utilize a low-cell number transcriptome based on the Smart-seq2 method to perform long-read sequencing. Our analysis describes additional novel transcripts and complexity of the preimplantation transcriptome, identifying 2280 potential novel transcripts from previously unannotated loci and 6289 novel splicing isoforms from previously annotated genes. Notably, these novel transcripts and isoforms with transcription start sites are enriched for an active promoter modification, H3K4me3. Moreover, we generate a more complete and precise transcriptome by combining long-read and short-read data during early embryogenesis. Based on this approach, we identify a previously undescribed isoform of Kdm4dl with a modified mRNA reading frame and a novel noncoding gene designated XLOC_004958. Depletion of Kdm4dl or XLOC_004958 led to abnormal blastocyst development. Thus, our data provide a high-resolution and more precise transcriptome during preimplantation mouse embryogenesis.


Assuntos
Blastocisto/metabolismo , Anotação de Sequência Molecular/métodos , Transcriptoma/genética , Animais , Desenvolvimento Embrionário/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Isoformas de Proteínas/genética , Splicing de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética
10.
Cell Res ; 29(11): 911-926, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31591447

RESUMO

Primary germ layers have the potential to form all tissues in the mature organism, and their formation during gastrulation requires precise epigenetic modulation of both proximal and distal regulatory elements. Previous studies indicated that spatial and temporal patterns of gene expression in the gastrula predispose individual regions to distinct cell fates. However, the underlying epigenetic mechanisms remain largely unexplored. Here, we profile the spatiotemporal landscape of the epigenome and transcriptome of the mouse gastrula. We reveal the asynchronous dynamics of proximal chromatin states during germ layer formation as well as unique gastrula-specific epigenomic features of regulatory elements, which have strong usage turnover dynamics and clear germ layer-specific signatures. Importantly, we also find that enhancers around organogenetic genes, which are weakly expressed at the gastrulation stage, are frequently pre-marked by histone H3 lysine 27 acetylation (H3K27ac) in the gastrula. By using the transgenic mice and genome editing system, we demonstrate that a pre-marked enhancer, which is located in the intron of a brain-specific gene 2510009E07Rik, exhibits specific enhancer activity in the ectoderm and future brain tissue, and also executes important function during mouse neural differentiation. Taken together, our study provides the comprehensive epigenetic information for embryonic patterning during mouse gastrulation, demonstrates the importance of gastrula pre-marked enhancers in regulating the correct development of the mouse embryo, and thus broadens the current understanding of mammalian embryonic development and related diseases.


Assuntos
Elementos Facilitadores Genéticos/fisiologia , Epigênese Genética , Gástrula/embriologia , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/embriologia , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Células Cultivadas , Embrião de Mamíferos , Células-Tronco Embrionárias , Feminino , Gástrula/citologia , Gástrula/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurogênese/genética , Transcriptoma
11.
Mol Ther Nucleic Acids ; 17: 626-635, 2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31400605

RESUMO

The development of CRISPR/Cas9-mediated base editors (BEs) provided a versatile tool for precise genome editing. The recently developed xCas9-derived base editors (xBEs) that recognize the NG PAM substantially expand the targeting scope in the genome, while their editing efficiency needs to be improved. Here, we described an improved version of xBEs by fusing the BPNLS and Gam to the N terminus of xBEs (BPNLS-Gam-xBE3 and BPNLS-xABE), and this version of base editor displayed higher targeting efficiency for the majority of detected sites. By using this improved version of xBEs, we successfully created and corrected pathogenic mutations at genomic sites with the NGN protospacer-adjacent motif in human cells. Lastly, we used BPNLS-Gam-xBE3 to model pathogenic mutations in discarded human tripronuclear (3PN) zygotes, and no obvious off-targets and indels were detected. Taken together, the data in our study offer an efficient tool for precise genome editing and, thus, an enriched base editing toolkit.

12.
Nat Commun ; 10(1): 3733, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427575

RESUMO

Hearing loss is the most common sensory disorder. While gene therapy has emerged as a promising treatment of inherited diseases like hearing loss, it is dependent on the identification of gene delivery vectors. Adeno-associated virus (AAV) vector-mediated gene therapy has been approved in the US for treating a rare inherited eye disease but no safe and efficient vectors have been identified that can target the diverse types of inner ear cells. Here, we identify an AAV variant, AAV-inner ear (AAV-ie), for gene delivery in mouse inner ear. Our results show that AAV-ie transduces the cochlear supporting cells (SCs) with high efficiency, representing a vast improvement over conventional AAV serotypes. Furthermore, after AAV-ie-mediated transfer of the Atoh1 gene, we find that many SCs trans-differentiated into new HCs. Our results suggest that AAV-ie is a useful tool for the cochlear gene therapy and for investigating the mechanism of HC regeneration.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Dependovirus/genética , Terapia Genética/métodos , Células Ciliadas Auditivas Internas/citologia , Perda Auditiva/genética , Perda Auditiva/terapia , Animais , Células Cultivadas , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL
13.
Cancer Lett ; 451: 110-121, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30851421

RESUMO

Prostate cancer (PCa) is the most common solid organ malignancy among men, outnumbering both lung and colorectal cancer, and it is the second leading cause of male tumor-related death in the United States due to high metastasis. Recently, leukemia inhibitory factor receptor (LIFR) has been found to play roles in multiple types of cancer. However, the roles of LIFR in the progression of PCa remain to be revealed. In this study, we found that LIFR plays an oncogenic role in PCa. The phosphorylation of LIFR at S1044 contributes to subsequent activation of the AKT pathway, inducing the expression of a series of proliferation and metastatic genes. Additionally, LIFR-S1044 is phosphorylated by ERK2 but not ERK1. The signal intensity of pLIFR-S1044 and pAKT S473 in PCa tissue displays a tight positive correlation. The ERK2/LIFR/AKT axis modulates PCa progression and offers a promising therapeutic and diagnostic target for PCa.


Assuntos
Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação
14.
Cell Rep ; 26(2): 302-312.e4, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30625312

RESUMO

The coactivator-associated arginine methyltransferase CARM1 catalyzes the methylation of histone H3 arginine 17/26 (H3R17/26me) and non-histone proteins at arginine residues to regulate gene transactivation through profiling or Carm1 overexpression assays. However, the direct relationship between H3R17/26me and its causal role in mouse embryo development remains largely unclear. Here, we use rAPOBEC1-XTEN-Cas9n-UGI (BE3) to efficiently introduce a point mutation (R17H) at multiple Hist1/2H3 loci and a premature-stop codon into the catalytic domain of CARM1 in mouse embryos, resulting in remarkable downregulation of H3R17me levels and developmental defects in pre-implantation and fetal embryos. Transcriptomic analysis reveals that Yap1 and cell cycle signaling pathways are dysregulated in Carm1 truncation and H3R17H substitution embryos, and Yap1 overexpression could rescue the base-editing-elicited defects. Our data establish the direct regulatory relationship between CARM1-mediated H3R17me and early mouse embryo development and demonstrate that Yap1 acts downstream of CARM1-mediated H3R17me to regulate the mouse embryo development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Transdução de Sinais , Animais , Domínio Catalítico , Ciclo Celular , Linhagem Celular Tumoral , Código das Histonas , Histonas/química , Histonas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Mutação de Sentido Incorreto , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Transcriptoma
15.
Sci Rep ; 8(1): 7629, 2018 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-29752475

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

17.
J Mol Cell Biol ; 10(3): 216-228, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29481611

RESUMO

Clinical therapies of pluripotent stem cells (PSCs)-based transplantation have been hindered by frequent development of teratomas or tumors in animal models and clinical patients. Therefore, clarifying the mechanism of carcinogenesis in stem cell therapy is of great importance for reducing the risk of tumorigenicity. Here we differentiate Oct4-GFP mouse embryonic stem cells (mESCs) into neural progenitor cells (NPCs) and find that a minority of Oct4+ cells are continuously sustained at Oct4+ state. These cells can be enriched and proliferated in a standard ESC medium. Interestingly, the differentiation potential of these enriched cells is tightly restricted with much higher tumorigenic activity, which are thus defined as differentiation-resistant ESCs (DR-ESCs). Transcriptomic and epigenomic analyses show that DR-ESCs are characterized by primordial germ cell-like gene signatures (Dazl, Rec8, Stra8, Blimp1, etc.) and specific epigenetic patterns distinct from mESCs. Moreover, the DR-ESCs possess germ cell potential to generate Sycp3+ haploid cells and are able to reside in sperm-free spermaduct induced by busulfan. Finally, we find that TGFß signaling is overactivated in DR-ESCs, and inhibition of TGFß signaling eliminates the tumorigenicity of mESC-derived NPCs by inducing the full differentiation of DR-ESCs. These data demonstrate that these TGFß-hyperactivated germ cell-like DR-ESCs are the main contributor for the tumorigenicity of ESCs-derived target cell therapy and that inhibition of TGFß signaling in ESC-derived NPC transplantation could drastically reduce the risk of tumor development.


Assuntos
Carcinogênese/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Neurais/citologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Carcinogênese/genética , Diferenciação Celular , Linhagem Celular , Epigênese Genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Neurais/metabolismo , Transcriptoma
18.
Sci Rep ; 7(1): 5282, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28706241

RESUMO

The paraventricular nucleus of hypothalamus plays important roles in the regulation of energy balance and fetal growth. However, the molecular mechanisms underlying its formation and function have not been clearly elucidated. Various mutations in the human COUP-TFII gene, which encodes a nuclear receptor, result in growth retardation, congenital diaphragmatic hernia and congenital heart defects. Here, we show that COUP-TFII gene is expressed in the developing hypothalamus in mouse. The ventral forebrain-specific RXCre/+; COUP-TFII F/F mutant mice display growth retardation. The development of the paraventricular nucleus of hypothalamus is compromised in the COUP-TFII mutant mainly because of increased apoptosis and mis-migration of the Brn2+ neurons. Moreover, hypoplastic anterior pituitary with blood cell clusters and shrunken posterior pituitary lacking AVP/OT neuron innervations are observed in the mutant, indicating the failure of formation of the hypothalamic-pituitary axis. Mechanistic studies show that the expression of Bdnf and Nrp1 genes is reduced in the mutant embryo, and that Bdnf is a direct downstream target of the COUP-TFII protein. Thus, our findings provide a novel functional validation that COUP-TFII gene promotes the expression of Bdnf and Nrp1 genes to ensure the appropriate morphogenesis of the hypothalamic-pituitary axis, especially the paraventricular nucleus of hypothalamus, and to prevent growth retardation.


Assuntos
Fator II de Transcrição COUP/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Transtornos do Crescimento/patologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/patologia , Fatores do Domínio POU/metabolismo , Núcleo Hipotalâmico Paraventricular/patologia , Animais , Transtornos do Crescimento/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Fatores do Domínio POU/genética , Núcleo Hipotalâmico Paraventricular/metabolismo
19.
Cell Rep ; 18(4): 933-946, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28122243

RESUMO

LIF promotes self-renewal of mouse embryonic stem cells (mESCs), and in its absence, the cells differentiate. LIF binds to the LIF receptor (LIFR) and activates the JAK-STAT3 pathway, but it remains unknown how the receptor complex triggers differentiation or self-renewal. Here, we report that the LIFR cytoplasmic domain contains a self-renewal domain within the juxtamembrane region and a differentiation domain within the C-terminal region. The differentiation domain contains four SPXX repeats that are phosphorylated by MAPK to restrict STAT3 activation; the self-renewal domain is characterized by a 3K motif that is acetylated by p300. In mESCs, acetyl-LIFR undergoes homodimerization, leading to STAT3 hypo- or hyper-activation depending on the presence or absence of gp130. LIFR-activated STAT3 restricts differentiation via cytokine induction. Thus, LIFR acetylation and serine phosphorylation differentially promote stem cell self-renewal and differentiation.


Assuntos
Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Acetilação/efeitos dos fármacos , Motivos de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Autorrenovação Celular/efeitos dos fármacos , Receptor gp130 de Citocina/metabolismo , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Células HEK293 , Histona Desacetilases/química , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Fator Inibidor de Leucemia/farmacologia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/química , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Fosforilação/efeitos dos fármacos , Interferência de RNA , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
Cell Mol Life Sci ; 73(7): 1399-411, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26801220

RESUMO

Early neural fate commitment is a key process in neural development and establishment of the central nervous system, and this process is tightly controlled by extrinsic signals, intrinsic factors, and epigenetic regulation. Here, we summarize the main findings regarding the regulatory network of epigenetic mechanisms that play important roles during early neural fate determination and embryonic development, including histone modifications, chromatin remodeling, DNA modifications, and RNA-level regulation. These regulatory mechanisms coordinate to play essential roles in silencing of pluripotency genes and activating key neurodevelopmental genes during cell fate commitment at DNA, histone, chromatin, and RNA levels. Moreover, we discuss the relationship between epigenetic regulation, signaling pathways, and intrinsic factors during early neural fate specification.


Assuntos
Epigênese Genética , Animais , Diferenciação Celular , Sistema Nervoso Central/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/química , DNA/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Interferência de RNA
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