RESUMO
Period determination in the mammalian circadian clock involves the turnover rate of the repressors CRY and PER. We show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss of function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.
Assuntos
Criptocromos/metabolismo , Proteínas F-Box/metabolismo , Animais , Proteínas CLOCK/genética , Núcleo Celular/metabolismo , Cruzamentos Genéticos , Citoplasma/metabolismo , Proteínas F-Box/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , ProteóliseRESUMO
TGF-ß signaling family plays an essential role to regulate fate decisions in pluripotency and lineage specification. How the action of TGF-ß family signaling is intrinsically executed remains not fully elucidated. Here, we show that HBO1, a MYST histone acetyltransferase (HAT) is an essential cell intrinsic determinant for TGF-ß signaling in human embryonic stem cells (hESCs). HBO1-/- hESCs fail to response to TGF-ß signaling to maintain pluripotency and spontaneously differentiate into neuroectoderm. Moreover, HBO1 deficient hESCs show complete defect in mesendoderm specification in BMP4-triggered gastruloids or teratomas. Molecularly, HBO1 interacts with SMAD4 and co-binds the open chromatin labeled by H3K14ac and H3K4me3 in undifferentiated hESCs. Upon differentiation, HBO1/SMAD4 co-bind and maintain the mesoderm genes in BMP4-triggered mesoderm cells while lose chromatin occupancy in neural cells induced by dual-SMAD inhibition. Our data reveal an essential role of HBO1, a chromatin factor to determine the action of SMAD in both human pluripotency and mesendoderm specification.
Assuntos
Diferenciação Celular , Histona Acetiltransferases , Mesoderma , Transdução de Sinais , Proteína Smad4 , Humanos , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/genética , Linhagem Celular , Cromatina/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/genética , Histonas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Mesoderma/metabolismo , Mesoderma/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Proteína Smad4/metabolismo , Proteína Smad4/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Polycomb repressive complexes (PRCs) play critical roles in cell fate decisions during normal development as well as disease progression through mediating histone modifications such as H3K27me3 and H2AK119ub. How exactly PRCs recruited to chromatin remains to be fully illuminated. Here, we report that YTHDF1, the N6-methyladenine (m6 A) RNA reader that was previously known to be mainly cytoplasmic, associates with RNF2, a PRC1 protein that mediates H2AK119ub in human embryonic stem cells (hESCs). A portion of YTHDF1 localizes in the nuclei and associates with RNF2/H2AK119ub on a subset of gene loci related to neural development functions. Knock-down YTHDF1 attenuates H2AK119ub modification on these genes and promotes neural differentiation in hESCs. Our findings provide a noncanonical mechanism that YTHDF1 participates in PRC1 functions in hESCs.
Assuntos
Proteínas de Ciclo Celular , Células-Tronco Embrionárias Humanas , Proteínas de Ligação a RNA , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina , Células-Tronco Embrionárias Humanas/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Histonas/genética , Histonas/metabolismoRESUMO
CLCN2 encodes a two-pore homodimeric chloride channel protein (CLC-2) that is widely expressed in human tissues. The association between Clcn2 and the retina is well-established in mice, as loss-of-function of CLC-2 can cause retinopathy in mice; however, the ocular phenotypes caused by CLCN2 mutations in humans and the underlying mechanisms remain unclear. The present study aimed to define the ocular features and reveal the pathogenic mechanisms of CLCN2 variants associated with retinal degeneration in humans using an in vitro overexpression system, as well as patient-induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) cells and retinal organoids (ROs). A patient carrying the homozygous c.2257C > T (p.R753X) nonsense CLCN2 mutation was followed up for > 6 years. Ocular features were comprehensively characterized with multimodality imaging and functional examination. The patient presented with severe bilateral retinal degeneration with loss of photoreceptor and RPE. In vitro, mutant CLC-2 maintained the correct subcellular localization, but with reduced channel function compared to wild-type CLC-2 in HEK293T cells. Additionally, patient iPSC-derived RPE cells carrying the CLCN2 mutation exhibited dysfunctional ClC-2 chloride channels and outer segment phagocytosis. Notably, these functions were rescued following the repair of the CLCN2 mutation using the CRISPR-Cas9 system. However, this variant did not cause significant photoreceptor degeneration in patient-derived ROs, indicating that dysfunctional RPE is likely the primary cause of biallelic CLCN2 variant-mediated retinopathy. This study is the first to establish the confirmatory ocular features of human CLCN2-related retinal degeneration, and reveal a pathogenic mechanism associated with biallelic CLCN2 variants, providing new insights into the cause of inherited retinal dystrophies.
Assuntos
Células-Tronco Pluripotentes Induzidas , Distrofias Retinianas , Animais , Humanos , Camundongos , Canais de Cloreto/genética , Códon sem Sentido , Células HEK293 , Mutação , Fagocitose/genética , Espécies Reativas de Oxigênio/metabolismo , Distrofias Retinianas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologiaRESUMO
Nongenetic cellular heterogeneity is associated with aging and disease. However, the origins of cell-to-cell variability are complex and the individual contributions of different factors to total phenotypic variance are still unclear. Here, we took advantage of clear phenotypic heterogeneity of circadian oscillations in clonal cell populations to investigate the underlying mechanisms of cell-to-cell variability. Using a fully automated tracking and analysis pipeline, we examined circadian period length in thousands of single cells and hundreds of clonal cell lines and found that longer circadian period is associated with increased intercellular heterogeneity. Based on our experimental results, we then estimated the contributions of heritable and nonheritable factors to this variation in circadian period length using a variance partitioning model. We found that nonheritable noise predominantly drives intercellular circadian period variation in clonal cell lines, thereby revealing a previously unrecognized link between circadian oscillations and intercellular heterogeneity. Moreover, administration of a noise-enhancing drug reversibly increased both period length and variance. These findings suggest that circadian period may be used as an indicator of cellular noise and drug screening for noise control.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Modelos Biológicos , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Circadianas Period/metabolismo , Análise de Célula Única/métodos , Animais , Células Cultivadas , Medições Luminescentes , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteínas Circadianas Period/genética , Processos EstocásticosRESUMO
Hematopoietic stem cells (HSCs)/progenitor cells (HPCs) are generated from hemogenic endothelial cells (HECs) during the endothelial-to-hematopoietic transition (EHT); however, the underlying mechanism remains poorly understood. Here, using an array of approaches, including CRSPR/Cas9 gene knockouts, RNA-Seq, ChIP-Seq, ATAC-Seq etc., we report that vitamin C (Vc) is essential in HPC generation during human pluripotent stem cell (hPSC) differentiation in defined culture conditions. Mechanistically, we found that the endothelial cells generated in the absence of Vc fail to undergo the EHT because of an apparent failure in opening up genomic loci essential for hematopoiesis. Under Vc deficiency, these loci exhibited abnormal accumulation of histone H3 trimethylation at Lys-27 (H3K27me3), a repressive histone modification that arose because of lower activities of demethylases that target H3K27me3. Consistently, deletion of the two H3K27me3 demethylases, Jumonji domain-containing 3 (JMJD3 or KDM6B) and histone demethylase UTX (UTX or KDM6A), impaired HPC generation even in the presence of Vc. Furthermore, we noted that Vc and jmjd3 are also important for HSC generation during zebrafish development. Together, our findings reveal an essential role for Vc in the EHT for hematopoiesis, and identify KDM6-mediated chromatin demethylation as an important regulatory mechanism in hematopoietic cell differentiation.
Assuntos
Ácido Ascórbico/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Histona Desmetilases/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Cromatina/fisiologia , Desmetilação , Células Endoteliais/metabolismo , Histona Desmetilases/genética , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Metilação , Células-Tronco Pluripotentes/metabolismo , Peixe-Zebra/genéticaRESUMO
During neurogenesis, neural patterning is a critical step during which neural progenitor cells differentiate into neurons with distinct functions. However, the molecular determinants that regulate neural patterning remain poorly understood. Here we optimized the "dual SMAD inhibition" method to specifically promote differentiation of human pluripotent stem cells (hPSCs) into forebrain and hindbrain neural progenitor cells along the rostral-caudal axis. We report that neural patterning determination occurs at the very early stage in this differentiation. Undifferentiated hPSCs expressed basal levels of the transcription factor orthodenticle homeobox 2 (OTX2) that dominantly drove hPSCs into the "default" rostral fate at the beginning of differentiation. Inhibition of glycogen synthase kinase 3ß (GSK3ß) through CHIR99021 application sustained transient expression of the transcription factor NANOG at early differentiation stages through Wnt signaling. Wnt signaling and NANOG antagonized OTX2 and, in the later stages of differentiation, switched the default rostral cell fate to the caudal one. Our findings have uncovered a mutual antagonism between NANOG and OTX2 underlying cell fate decisions during neural patterning, critical for the regulation of early neural development in humans.
Assuntos
Diferenciação Celular , Linhagem da Célula , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neurais/citologia , Neurônios/citologia , Fatores de Transcrição Otx/metabolismo , Células-Tronco Pluripotentes/citologia , Padronização Corporal , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Colículos Inferiores/citologia , Colículos Inferiores/metabolismo , Núcleos da Rafe do Mesencéfalo/citologia , Núcleos da Rafe do Mesencéfalo/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Rombencéfalo/citologia , Rombencéfalo/metabolismoRESUMO
Dissociation-induced apoptosis is a striking phenomenon in human embryonic stem cells (hESCs), but not in naive mouse ESCs. Rho-associated kinase-dependent actin-myosin hyperactivation is an underlying mechanism that triggers apoptosis in dissociated hESCs; however, in this study, we show that the Ink4A-ARF-mediated senescence pathway is another mechanism to cause apoptosis in individualized hESCs. We show that P16INK4A and P14ARF are immediately induced in hESCs upon dissociation, but not in mouse ESCs. Overexpression of BMI1, a suppressor for Ink4A-ARF, greatly promotes survival and cloning efficiency of individualized hESCs mechanistically via direct binding the H3K27me3-marked Ink4A-ARF locus. Forced expression of BMI1 in hESCs does not reduce the actin-myosin activation that is triggered by dissociation, which indicates it is an independent pathway for hESC survival. Furthermore, dual inhibition of both Ink4A-ARF and actin-myosin hyperactivation enables successful passaging of hESCs via gelatin, a nonbioactive matrix. In sum, we provide an additional mechanism that underlies cell death in individualized hESCs that might help to fully understand the differential cell characteristics between naive and primed ESCs.-Wang, W., Zhu, Y., Huang, K., Shan, Y., Du, J., Dong, X., Ma, P., Wu, P., Zhang, J., Huang, W., Zhang, T., Liao, B., Yao, D., Pan, G., Liu, J. Suppressing P16Ink4a and P14ARF pathways overcomes apoptosis in individualized human embryonic stem cells.
Assuntos
Apoptose , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Células-Tronco Embrionárias/fisiologia , Humanos , Camundongos , Miosinas/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteína Supressora de Tumor p14ARF/genéticaRESUMO
It is known that there are mechanistic links between circadian clocks and metabolic cycles. Reduced nicotinamide adenine dinucleotide (NADH) is a key metabolic cofactor in all living cells; however, it is not known whether levels of NADH oscillate or not. Here we employed REX, a bacterial NADH-binding protein, fused to the VP16 activator to convert intracellular endogenous redox balance into transcriptional readouts by a reporter gene in mammalian cells. EMSA results show that the DNA binding activity of both T- and S-REX::VP16 fusions is decreased with a reduced-to-oxidized cofactor ratio increase. Transient and stabilized cell lines bearing the REX::VP16 and the REX binding operator (ROP) exhibit two circadian luminescence cycles. Consistent with these results, NADH oscillations are observed in host cells, indicating REX can act as a NADH sensor to report intracellular dynamic redox homeostasis in mammalian cells in real time. NADH oscillations provide another metabolic signal for coupling the circadian clock and cellular metabolic states.
Assuntos
Proteínas de Bactérias , Técnicas Biossensoriais , Relógios Circadianos , Proteína Vmw65 do Vírus do Herpes Simples , NAD/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Células HEK293 , Proteína Vmw65 do Vírus do Herpes Simples/biossíntese , Proteína Vmw65 do Vírus do Herpes Simples/genética , Humanos , Oxirredução , Proteínas Recombinantes de Fusão/genéticaRESUMO
The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However, it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps, we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in ß-hemoglobin gene (HBB) that cause severe ß-thalassemia (ß-Thal), corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting, and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing, we uncovered seven copy number variations, five small insertions/deletions, and 64 single nucleotide variations (SNVs) in ß-Thal iPSCs before the gene targeting step and found a single small copy number variation, 19 insertions/deletions, and 340 single nucleotide variations in the final gene-corrected ß-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps, suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting.
Assuntos
Reprogramação Celular , Endonucleases/metabolismo , Endorribonucleases/metabolismo , Marcação de Genes , Instabilidade Genômica , Células-Tronco Pluripotentes Induzidas/citologia , Alelos , Animais , Diferenciação Celular , Cromossomos/ultraestrutura , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Eritroblastos/citologia , Exoma , Deleção de Genes , Variação Genética , Humanos , Camundongos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Dedos de Zinco/genética , Globinas beta/genética , Talassemia beta/genéticaRESUMO
ß-Thalassemia (ß-Thal) is a group of life-threatening blood disorders caused by either point mutations or deletions of nucleotides in ß-globin gene (HBB). It is estimated that 4.5% of the population in the world carry ß-Thal mutants (1), posing a persistent threat to public health. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations offer an ideal therapeutic solution to this problem. However, homologous recombination-based gene correction in human iPSCs remains largely inefficient. Here, we describe a robust process combining efficient generation of integration-free ß-Thal iPSCs from the cells of patients and transcription activator-like effector nuclease (TALEN)-based universal correction of HBB mutations in situ. We generated integration-free and gene-corrected iPSC lines from two patients carrying different types of homozygous mutations and showed that these iPSCs are pluripotent and have normal karyotype. We showed that the correction process did not generate TALEN-induced off targeting mutations by sequencing. More importantly, the gene-corrected ß-Thal iPS cell lines from each patient can be induced to differentiate into hematopoietic progenitor cells and then further to erythroblasts expressing normal ß-globin. Our studies provide an efficient and universal strategy to correct different types of ß-globin mutations in ß-Thal iPSCs for disease modeling and applications.
Assuntos
Desoxirribonucleases/genética , Desoxirribonucleases/uso terapêutico , Terapia Genética , Globinas beta/genética , Talassemia beta/genética , Diferenciação Celular , Células Cultivadas , Desoxirribonucleases/metabolismo , Endonucleases/genética , Recombinação Homóloga , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mutação , Globinas beta/metabolismo , Talassemia beta/patologia , Talassemia beta/terapiaRESUMO
The hypothalamic suprachiasmatic nucleus (SCN) is the central pacemaker for mammalian circadian rhythms. As such, this ensemble of cell-autonomous neuronal oscillators with divergent periods must maintain coordinated oscillations. To investigate ultrastructural features enabling such synchronization, 805 coronal ultrathin sections of mouse SCN tissue were imaged with electron microscopy and aligned into a volumetric stack, from which selected neurons within the SCN core were reconstructed in silico. We found that clustered SCN core neurons were physically connected to each other via multiple large soma-to-soma plate-like contacts. In some cases, a sliver of a glial process was interleaved. These contacts were large, covering on average â¼21% of apposing neuronal somata. It is possible that contacts may be the electrophysiological substrate for synchronization between SCN neurons. Such plate-like contacts may explain why the synchronization of SCN neurons is maintained even when chemical synaptic transmission or electrical synaptic transmission via gap junctions is blocked. Such ephaptic contact-mediated synchronization among nearby neurons may therefore contribute to the wave-like oscillations of circadian core clock genes and calcium signals observed in the SCN.
Threedimensional reconstruction of SCN tissue via serial electron microscopy revealed a novel structural feature of SCN neurons that may account for interneuronal synchronization that persists even when the predominant mechanisms of neuronal communication are blocked. We found that SCN core neurons are connected by multiple somasoma contact specializations, ultrastructural elements that could enable synchronization of tightly packed neurons organized in clustered networks. This extensive network of platelike somasoma contacts among clustered SCN neurons may provide insight into how â¼20,000 autonomous neuronal oscillators with a broad range of intrinsic periods remain synchronized in the absence of ordinary communication modalities, thereby conferring the resilience required for the SCN to function as the mammalian circadian pacemaker.
Assuntos
Camundongos Endogâmicos C57BL , Animais , Camundongos , Neurônios do Núcleo Supraquiasmático/fisiologia , Masculino , Núcleo Supraquiasmático/fisiologia , Núcleo Supraquiasmático/citologia , Neurônios/fisiologiaRESUMO
Nucleoli are fundamentally essential sites for ribosome biogenesis in cells and formed by liquid-liquid phase separation (LLPS) for a multilayer condensate structure. How the nucleoli integrity is maintained remains poorly understood. Here, we reveal that METTL3/METTL14, the typical methyltransferase complex catalyzing N6-methyladnosine (m6A) on mRNAs maintain nucleoli integrity in human embryonic stem cells (hESCs). METTL3/METTL14 deficiency impairs nucleoli and leads to the complete loss of self-renewal in hESCs. We further show that SUV39H1/H2 protein, the methyltransferases catalyzing H3K9me3 were dramatically elevated in METTL3/METTL14 deficient cells, which causes an accumulation and infiltration of H3K9me3 across the whole nucleolus and impairs the LLPS. Mechanistically, METTL3/METTL14 complex serves as an essential adapter for CRL4 E3 ubiquitin ligase targeting SUV39H1/H2 for polyubiquitination and proteasomal degradation and therefore prevents H3K9me3 accumulation in nucleoli. Together, these findings uncover a previously unknown role of METTL3/METTL14 to maintain nucleoli integrity by facilitating SUV39H1/H2 degradation in human cells.
Assuntos
Nucléolo Celular , Metiltransferases , Proteínas Repressoras , Humanos , Metiltransferases/metabolismo , Metiltransferases/genética , Nucléolo Celular/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Histonas/metabolismo , Ubiquitinação , Células-Tronco Embrionárias Humanas/metabolismo , Proteólise , Células HEK293 , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Histona-Lisina N-MetiltransferaseRESUMO
Emergency myelopoiesis (EM) is essential in immune defense against pathogens for rapid replenishing of mature myeloid cells. During the EM process, a rapid cell-cycle switch from the quiescent hematopoietic stem cells (HSCs) to highly proliferative myeloid progenitors (MPs) is critical. How the rapid proliferation of MPs during EM is regulated remains poorly understood. Here, we reveal that ATG7, a critical autophagy factor, is essential for the rapid proliferation of MPs during human myelopoiesis. Peripheral blood (PB)-mobilized hematopoietic stem/progenitor cells (HSPCs) with ATG7 knockdown or HSPCs derived from ATG7-/- human embryonic stem cells (hESCs) exhibit severe defect in proliferation during fate transition from HSPCs to MPs. Mechanistically, we show that ATG7 deficiency reduces p53 localization in lysosome for a potential autophagy-mediated degradation. Together, we reveal a previously unrecognized role of autophagy to regulate p53 for a rapid proliferation of MPs in human myelopoiesis.
Assuntos
Mielopoese , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células Mieloides , Autofagia/genéticaRESUMO
In contrast to rodents, the mechanisms underlying human trophectoderm and early placenta specification are understudied due to ethical barriers and the scarcity of embryos. Recent reports have shown that human pluripotent stem cells (PSCs) can differentiate into trophectoderm (TE)-like cells (TELCs) and trophoblast stem cells (TSCs), offering a valuable in vitro model to study early placenta specification. Here, we demonstrate that the VGLL1 (vestigial-like family member 1), which is highly expressed during human and non-human primate TE specification in vivo but is negligibly expressed in mouse, is a critical regulator of cell fate determination and self-renewal in human TELCs and TSCs derived from naïve PSCs. Mechanistically, VGLL1 partners with the transcription factor TEAD4 (TEA domain transcription factor 4) to regulate chromatin accessibility at target gene loci through histone acetylation and acts in cooperation with GATA3 and TFAP2C. Our work is relevant to understand primate early embryogenesis and how it differs from other mammalian species.
Assuntos
Células-Tronco Pluripotentes , Fatores de Transcrição , Gravidez , Feminino , Humanos , Camundongos , Animais , Linhagem da Célula/genética , Fatores de Transcrição/genética , Trofoblastos/fisiologia , Diferenciação Celular/genética , Mamíferos , Primatas , Proteínas de Ligação a DNA/genética , Fatores de Transcrição de Domínio TEARESUMO
Post-translational modifications (PTMs) on histones play essential roles in cell fate decisions during development. However, how these PTMs are recognized and coordinated remains to be fully illuminated. Here, we show that BRPF1, a multi-histone binding module protein, is essential for pluripotency in human embryonic stem cells (ESCs). BRPF1, H3K4me3, and H3K23ac substantially co-occupy the open chromatin and stemness genes in hESCs. BRPF1 deletion impairs H3K23ac in hESCs and leads to closed chromatin accessibility on stemness genes and hESC differentiation as well. Deletion of the N terminal or PHD-zinc knuckle-PHD (PZP) module in BRPF1 completely impairs its functions in hESCs while PWWP module deletion partially impacts the function. In sum, we reveal BRPF1, the multi-histone binding module protein that bridges the crosstalk between different histone modifications in hESCs to maintain pluripotency.
RESUMO
Programmed cell death protein 1 (PD-1) immune checkpoint blockade therapy has revolutionized cancer treatment. Although PD-1 blockade is effective in a subset of patients with cancer, many fail to respond because of either primary or acquired resistance. Thus, next-generation strategies are needed to expand the depth and breadth of clinical responses. Toward this end, we designed a human primary T cell phenotypic high-throughput screening strategy to identify small molecules with distinct and complementary mechanisms of action to PD-1 checkpoint blockade. Through these efforts, we selected and optimized a chemical series that showed robust potentiation of T cell activation and combinatorial activity with αPD-1 blockade. Target identification was facilitated by chemical proteomic profiling with a lipid-based photoaffinity probe, which displayed enhanced binding to diacylglycerol kinase α (DGKα) in the presence of the active compound, a phenomenon that correlated with the translocation of DGKα to the plasma membrane. We further found that optimized leads within this chemical series were potent and selective inhibitors of both DGKα and DGKζ, lipid kinases that constitute an intracellular T cell checkpoint that blunts T cell signaling through diacylglycerol metabolism. We show that dual DGKα/ζ inhibition amplified suboptimal T cell receptor signaling mediated by low-affinity antigen presentation and low major histocompatibility complex class I expression on tumor cells, both hallmarks of resistance to PD-1 blockade. In addition, DGKα/ζ inhibitors combined with αPD-1 therapy to elicit robust tumor regression in syngeneic mouse tumor models. Together, these findings support targeting DGKα/ζ as a next-generation T cell immune checkpoint strategy.
Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Camundongos , Animais , Humanos , Receptor de Morte Celular Programada 1/metabolismo , Proteômica , Diacilglicerol Quinase/metabolismo , Linfócitos T , LipídeosRESUMO
Adhesion and detachment are coordinated critical steps during cell migration. Conceptually, efficient migration requires both effective stabilization of membrane protrusions at the leading edge via nascent adhesions and their successful persistence during retraction of the trailing side via disruption of focal adhesions. As nascent adhesions are much smaller in size than focal adhesions, they are expected to exhibit a stronger adhesivity in order to achieve the coordination between cell front and back. Here, we show that Nudel knockdown by interference RNA (RNAi) resulted in cell edge shrinkage due to poor adhesions of membrane protrusions. Nudel bound to paxillin, a scaffold protein of focal contacts, and colocalized with it in areas of active membrane protrusions, presumably at nascent adhesions. The Nudel-paxillin interaction was disrupted by focal adhesion kinase (FAK) in a paxillin-binding-dependent manner. Forced localization of Nudel in all focal contacts by fusing it to paxillin markedly strengthened their adhesivity, whereas overexpression of structurally activated FAK or any paxillin-binding FAK mutant lacking the N-terminal autoinhibitory domain caused cell edge shrinkage. These results suggest a novel mechanism for selective reinforcement of nascent adhesions via interplays of Nudel and FAK with paxillin to facilitate cell migration.
Assuntos
Proteínas de Transporte/metabolismo , Adesão Celular , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Paxilina/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Movimento Celular , Células Epiteliais/metabolismo , Quinase 1 de Adesão Focal/genética , Células HeLa , Humanos , Rim/citologia , Microscopia Confocal/métodos , Mutação , Paxilina/genética , Interferência de RNA , Fatores de Tempo , Bexiga Urinária/citologiaRESUMO
RYBP, a critical component of polycomb repressive complex1 (PRC1), is required for the pluripotency and differentiation of mouse embryonic stem cells(mESCs). However, its function and mechanism to regulate human embryonic stem cells(hESCs) remain unknown. Here, to investigate the role of RYBP in hESCs, we generate an hESC line with FLAG-HA tag knock-in to RYBP locus through CRISPR/Cas9-mediated homologous recombination. hESC with RYBP_FLAG-HA knock-in maintains normal morphology and karyotype, while it maintains pluripotency to differentiate into three germ layers.
Assuntos
Células-Tronco Embrionárias Humanas , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Recombinação Homóloga , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Proteínas do Grupo Polycomb , Proteínas Repressoras/metabolismoRESUMO
RNF1 (RING1A) is a catalytic component of the polycomb repressive complex 1 (PRC1) involved in regulation of, among others, embryonic development and disease progression. However, the exact role of RNF1 in self-renewal and differentiation of human embryonic stem cells (ESCs) remains unknown. Here, we derive one RNF1 knockout human ESC line using CRISPR/Cas9 system. The cell line retains the canonical stem cell morphology and normal karyotype. Moreover, the cell line highly expresses pluripotency genes and has three germ-layer differentiation potential. The RNF1 -/- cell line will be useful for studies on the function and role of RNF1 in human embryonic stem cell fate decisions.