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Embryonic genome activation (EGA) occurs during preimplantation development and is characterized by the initiation of de novo transcription from the embryonic genome. Despite its importance, the regulation of EGA and the transcription factors involved in this process are poorly understood. Paired-like homeobox (PRDL) family proteins are implicated as potential transcriptional regulators of EGA, yet the PRDL-mediated gene regulatory networks remain uncharacterized. To investigate the function of PRDL proteins, we are identifying the molecular interactions and the functions of a subset family of the Eutherian Totipotent Cell Homeobox (ETCHbox) proteins, seven PRDL family proteins and six other transcription factors (TFs), all suggested to participate in transcriptional regulation during preimplantation. Using mass spectrometry-based interactomics methods, AP-MS and proximity-dependent biotin labeling, and chromatin immunoprecipitation sequencing we derive the comprehensive regulatory networks of these preimplantation TFs. By these interactomics tools we identify more than a thousand high-confidence interactions for the 21 studied bait proteins with more than 300 interacting proteins. We also establish that TPRX2, currently assigned as pseudogene, is a transcriptional activator.
Assuntos
Proteínas de Homeodomínio , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Genes Homeobox , GenomaRESUMO
By screening a CRISPR knockout library for mouse pluripotent reprogramming roadblock genes, Kaemena et al. identify the KRAB-ZFP factor Zfp266 as a suppressor of efficient reprogramming. Furthermore, by analyzing DNA binding and chromatin openness, the authors found that ZFP266 has a role in suppressing reprogramming by targeting the B1 SINE sequences for silencing.
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Reprogramação Celular , Animais , CamundongosRESUMO
The paired-like homeobox transcription factor LEUTX is expressed in human preimplantation embryos between the 4- and 8-cell stages, and then silenced in somatic tissues. To characterize the function of LEUTX, we performed a multiomic characterization of LEUTX using two proteomics methods and three genome-wide sequencing approaches. Our results show that LEUTX stably interacts with the EP300 and CBP histone acetyltransferases through its 9 amino acid transactivation domain (9aaTAD), as mutation of this domain abolishes the interactions. LEUTX targets genomic cis-regulatory sequences that overlap with repetitive elements, and through these elements it is suggested to regulate the expression of its downstream genes. We find LEUTX to be a transcriptional activator, upregulating several genes linked to preimplantation development as well as 8-cell-like markers, such as DPPA3 and ZNF280A. Our results support a role for LEUTX in preimplantation development as an enhancer binding protein and as a potent transcriptional activator.
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Embryonic genome activation (EGA) is critical for embryonic development. However, our understanding of the regulatory mechanisms of human EGA is still incomplete. Human embryonic stem cells (hESCs) are an established model for studying developmental processes, but they resemble epiblast and are sub-optimal for modeling EGA. DUX4 regulates human EGA by inducing cleavage-stage-specific genes, while it also induces cell death. We report here that a short-pulsed expression of DUX4 in primed hESCs activates an EGA-like gene expression program in up to 17% of the cells, retaining cell viability. These DUX4-induced cells resembled eight-cell stage blastomeres and were named induced blastomere-like (iBM) cells. The iBM cells showed marked reduction of POU5F1 protein, as previously observed in mouse two-cell-like cells. Finally, the iBM cells were successfully enriched using an antibody against NaPi2b (SLC34A2), which is expressed in human blastomeres. The iBM cells provide an improved model system to study human EGA transcriptome.
Assuntos
Blastômeros , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas , Animais , Blastômeros/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Genes Homeobox , Genoma Humano , Proteínas de Homeodomínio/genética , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Gravidez , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/metabolismoRESUMO
MASTL is a mitotic accelerator with an emerging role in breast cancer progression. However, the mechanisms behind its oncogenicity remain largely unknown. Here, we identify a previously unknown role and eminent expression of MASTL in stem cells. MASTL staining from a large breast cancer patient cohort indicated a significant association with ß3 integrin, an established mediator of breast cancer stemness. MASTL silencing reduced OCT4 levels in human pluripotent stem cells and OCT1 in breast cancer cells. Analysis of the cell-surface proteome indicated a strong link between MASTL and the regulation of TGF-ß receptor II (TGFBR2), a key modulator of TGF-ß signaling. Overexpression of wild-type and kinase-dead MASTL in normal mammary epithelial cells elevated TGFBR2 levels. Conversely, MASTL depletion in breast cancer cells attenuated TGFBR2 levels and downstream signaling through SMAD3 and AKT pathways. Taken together, these results indicate that MASTL supports stemness regulators in pluripotent and cancerous stem cells.
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The first lineage choice in human embryo development separates trophectoderm from the inner cell mass. Naïve human embryonic stem cells are derived from the inner cell mass and offer possibilities to explore how lineage integrity is maintained. Here, we discover that polycomb repressive complex 2 (PRC2) maintains naïve pluripotency and restricts differentiation to trophectoderm and mesoderm lineages. Through quantitative epigenome profiling, we found that a broad gain of histone H3 lysine 27 trimethylation (H3K27me3) is a distinct feature of naïve pluripotency. We define shared and naïve-specific bivalent promoters featuring PRC2-mediated H3K27me3 concomitant with H3K4me3. Naïve bivalency maintains key trophectoderm and mesoderm transcription factors in a transcriptionally poised state. Inhibition of PRC2 forces naïve human embryonic stem cells into an 'activated' state, characterized by co-expression of pluripotency and lineage-specific transcription factors, followed by differentiation into either trophectoderm or mesoderm lineages. In summary, PRC2-mediated repression provides a highly adaptive mechanism to restrict lineage potential during early human development.
Assuntos
Células-Tronco Embrionárias Humanas , Complexo Repressor Polycomb 2 , Diferenciação Celular/genética , Desenvolvimento Embrionário , Histonas/genética , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismoRESUMO
Double homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding with minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.
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OBJECTIVES: Cisplatin is combined with radiotherapy for advanced head and neck squamous cell carcinoma (HNSCC). While providing a beneficial effect on survival, it also causes side effects and thus is an important target when considering treatment de-escalation. Currently, there are no biomarkers to predict its patient-selective therapeutic utility. In this study, we examined the role of the stem cell factor OCT4 as a potential biomarker to help clinicians stratify HNSCC patients between radiotherapy and chemoradiotherapy. MATERIALS AND METHODS: OCT4 immunohistochemical staining of a population-validated tissue microarray (PV-TMA) (n = 166) representative of a standard HNSCC patients was carried out, and 5-year survival was analyzed. The results were validated using ex vivo drug sensitivity analysis of HNSCC tumor samples, and further cross-validated in independent oropharyngeal (n = 118), nasopharyngeal (n = 170), and vulvar carcinoma (n = 95) clinical datasets. In vitro, genetically modified, patient-derived HNSCC cells were used. RESULTS: OCT4 expression in HNSCC tumors was associated with radioresistance. However, combination therapy with cisplatin was found to overcome thisradioresistance in OCT4-expressing HNSCC tumors. The results were validated by using several independent patient cohorts. Furthermore, CRISPRa-based OCT4 overexpression in the HNSCC cell line resulted in apoptosis resistance, and cisplatin was found to downregulate OCT4 protein expression in vitro. Ex vivo drug sensitivity analysis of HNSCC tumors confirmed the association between OCT4 expression and cisplatin sensitivity. CONCLUSION: This study introduces OCT4 immunohistochemistry as a simple and cost-effective diagnostic approach for clinical practice to identify HNSCC patients benefitting from radiosensitization by cisplatin using either full or reduced dosing.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapiaRESUMO
Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The forced expression of transcription factors may lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSCs. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can reduce this heterogeneity. Here, we describe a high-efficiency reprogramming of human somatic cells into iPSCs using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high-quality pluripotent reprogramming of human cells.
Assuntos
Reprogramação Celular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Elementos Alu/genética , Perfilação da Expressão Gênica , Loci Gênicos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/genética , Análise de Célula ÚnicaRESUMO
Despite being a biologically fundamental question, the precise timing of lineage specification during human preimplantation development remains elusive. In this issue of Cell Stem Cell, Meistermann et al. (2021) refine our view through time-lapse embryo staging and single-cell sequencing and challenge the concept of a human inner cell mass.
Assuntos
Blastocisto , Desenvolvimento Embrionário , HumanosRESUMO
CRISPR-mediated gene activation (CRISPRa) can be used to target endogenous genes for activation. By targeting pluripotency-associated reprogramming factors, human fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs). Here, we describe a method for the derivation of iPSCs from human fibroblasts using episomal plasmids encoding CRISPRa components. This chapter also provides procedure to assemble guide RNA cassettes and generation of multiplexed guide plasmids for readers who want to design their own guide RNAs.
Assuntos
Sistemas CRISPR-Cas/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição/metabolismo , Células Cultivadas , Eletroporação/métodos , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genéticaRESUMO
CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus-host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/genética , Sarcoma de Kaposi/virologia , Transativadores/genética , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Humanos , Proteínas Imediatamente Precoces/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Ativação Viral , Latência ViralRESUMO
CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data demonstrate that human somatic cells can be reprogrammed into iPSCs using only CRISPRa. Furthermore, the results unravel the involvement of EEA-motif-associated mechanisms in cellular reprogramming.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Reprogramação Celular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Elementos Alu/genética , Sequência de Bases , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neurais/metabolismo , Motivos de Nucleotídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Transcrição GênicaRESUMO
SOX2 is an important transcription factor involved in pluripotency maintenance, pluripotent reprogramming and differentiation towards neural lineages. Here we engineered the previously described HEL24.3 hiPSC to generate a SOX2 reporter by knocking-in a T2A fused nuclear tdTomato reporter cassette before the STOP codon of the SOX2 gene coding sequence. CRISPR/SaCas9-mediated stimulation of homologous recombination was utilized to facilitate faithful targeted insertion. This line accurately reports the expression of endogenous SOX2 and therefore constitutes a useful tool to study the SOX2 expression dynamics upon hiPSC culture, differentiation and somatic cell reprogramming.
Assuntos
Sistemas CRISPR-Cas , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição SOXB1/genética , Diferenciação Celular/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição SOXB1/biossínteseRESUMO
The generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming holds great potential for modeling human diseases. However, the reprogramming process remains very inefficient and a better understanding of its basic biology is required. The mesenchymal-to-epithelial transition (MET) has been recognized as a crucial step for the successful reprogramming of fibroblasts into iPSCs. It has been reported that the p53 tumor suppressor gene acts as a barrier of this process, while its homolog p63 acts as an enabling factor. In this regard, the information concerning the role of the third homolog, p73, during cell reprogramming is limited. Here, we derive total Trp73 knockout mouse embryonic fibroblasts, with or without Trp53, and examine their reprogramming capacity. We show that p73 is required for effective reprogramming by the Yamanaka factors, even in the absence of p53. Lack of p73 affects the early stages of reprogramming, impairing the MET and resulting in altered maturation and stabilization phases. Accordingly, the obtained p73-deficient iPSCs have a defective epithelial phenotype and alterations in the expression of pluripotency markers. We demonstrate that p73 deficiency impairs the MET, at least in part, by hindering BMP pathway activation. We report that p73 is a positive modulator of the BMP circuit, enhancing its activation by DNp73 repression of the Smad6 promoter. Collectively, these findings provide mechanistic insight into the MET process, proposing p73 as an enhancer of MET during cellular reprogramming.
Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Fosfoproteínas/genética , Transativadores/genética , Proteína Tumoral p73/genética , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular , Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fosfoproteínas/deficiência , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Proteína Smad6/genética , Proteína Smad6/metabolismo , Transativadores/deficiência , Proteína Tumoral p73/deficiência , Proteína Supressora de Tumor p53/deficiênciaRESUMO
OCT4 is a crucial transcription factor in the pluripotent stem cell gene regulatory network and an essential factor for pluripotent reprogramming. We engineered the previously reported HEL24.3 hiPSC to generate an OCT4 reporter cell line by knocking-in a T2A nuclear EmGFP reporter cassette before the OCT4 gene STOP codon sequence. To enhance targeted insertion, homologous recombination was stimulated using targeted cutting at the OCT4 STOP codon with CRISPR/SpCas9. This HEL24.3-OCT4-nEmGFP cell line faithfully reports endogenous OCT4 expression, serving as a useful tool to examine temporal changes in OCT4 expression in live cells during hiPSC culture, differentiation and somatic cell reprogramming.
Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Cultura de Células/métodos , Genes Reporter , Células-Tronco Pluripotentes Induzidas/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Linhagem Celular , HumanosRESUMO
Reports on the retention of somatic cell memory in induced pluripotent stem cells (iPSCs) have complicated the selection of the optimal cell type for the generation of iPSC biobanks. To address this issue we compared transcriptomic, epigenetic, and differentiation propensities of genetically matched human iPSCs derived from fibroblasts and blood, two tissues of the most practical relevance for biobanking. Our results show that iPSC lines derived from the same donor are highly similar to each other. However, genetic variation imparts a donor-specific expression and methylation profile in reprogrammed cells that leads to variable functional capacities of iPSC lines. Our results suggest that integration-free, bona fide iPSC lines from fibroblasts and blood can be combined in repositories to form biobanks. Due to the impact of genetic variation on iPSC differentiation, biobanks should contain cells from large numbers of donors.
Assuntos
Diferenciação Celular/genética , Variação Genética , Células-Tronco Pluripotentes Induzidas/citologia , Bancos de Espécimes Biológicos , Metilação de DNA/genética , Epigênese Genética , Células Eritroides/citologia , Feminino , Fibroblastos/metabolismo , Hematopoese/genética , Humanos , Masculino , Doadores de Tecidos , Transcrição GênicaRESUMO
CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.
Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular , Técnicas de Reprogramação Celular , Reprogramação Celular , Regulação da Expressão Gênica , Adolescente , Idoso , Feminino , Células HEK293 , Humanos , MasculinoRESUMO
Human iPSC line HEL24.3 was generated from healthy human foreskin fibroblasts using non-integrative reprogramming method. Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using Sendai viruses.
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Linhagem Celular/citologia , Fibroblastos/citologia , Prepúcio do Pênis/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Saúde , Humanos , Recém-Nascido , Fator 4 Semelhante a Kruppel , MasculinoRESUMO
Human iPSC line HEL47.2 was generated from healthy 83-year old male dermal fibroblasts using non-integrative reprogramming method. Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using Sendai viruses.