RESUMO
N6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m6A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m6A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m6A sites to screen for m6A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m6A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m6A site on ADM. Our study provides a functional screening of m6A sites and paves the way for functional studies of m6A at individual m6A site level.
Assuntos
Adenosina/análogos & derivados , Diferenciação Celular , Linhagem da Célula , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Adenosina/genética , Adenosina/metabolismo , Adrenomedulina/genética , Adrenomedulina/metabolismo , Sistemas CRISPR-Cas/genética , Células Cultivadas , Endoderma/metabolismo , Humanos , Proteínas/genética , Proteínas/metabolismo , Fatores de Transcrição SOXB1/genéticaRESUMO
We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB) with an improved episomal vector (EV) system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B). Here we show an â¼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1) equimolar expression of OCT4 (O) and SOX2 (S), by using a 2A linker; (2) a higher and gradual increase in the MYC (M) to KLF4 (K) ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one. The combination of EV plasmids (OS + M + K + B) is comparable with Sendai virus in reprogramming efficiency but at a fraction of the cost. The generated iPSCs are indistinguishable from those from our previous approach in pluripotency and phenotype. This improvement lays the foundation for broad applications of episomal vectors in PB reprogramming.
Assuntos
Reprogramação Celular , Engenharia Genética/métodos , Vetores Genéticos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Plasmídeos/metabolismo , Adulto , Biomarcadores/metabolismo , Diferenciação Celular , Expressão Gênica , Engenharia Genética/economia , Vetores Genéticos/química , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Leucócitos Mononucleares/citologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Plasmídeos/química , Cultura Primária de Células , 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 , Vírus Formadores de Foco no Baço/genética , Vírus Formadores de Foco no Baço/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
SOX14 is a member of the SOXB2 subgroup of transcription factors implicated in neural development. Although the first SOX14 gene in vertebrates was cloned and characterized more than a decade ago and its expression profile during development was revealed in various animal model systems, the role of this gene during neural development is largely unknown. In the present study we analyzed the expression of SOX14 in human NT2/D1 and mouse P19 pluripotent embryonal carcinoma cells. We demonstrated that it is expressed in both cell lines and upregulated during retinoic acid induced neural differentiation. We showed that SOX14 was expressed in both neuronal and non-neuronal differentiated derivatives, as revealed by immunocytochemistry. Since it was previously proposed that increased SOXB2 proteins level interfere with the activity of SOXB1 counteracting partners, we compared expression patterns of SOXB members during retinoic acid induction of embryonal carcinoma cells. We revealed that upregulation of SOX14 expression is accompanied by alterations in the expression patterns of SOXB1 members. In order to analyze the potential cross-talk between them, we generated SOX14 expression construct. The ectopic expression of SOX14 was demonstrated at the mRNA level in NT2/D1, P19 and HeLa cells, while an increased level of SOX14 protein was detected in HeLa cells only. By transient transfection experiments in HeLa cells we showed for the first time that ectopic expression of SOX14 repressed SOX1 expression, whereas no significant effect on SOX2, SOX3 and SOX21 was observed. Data presented here provide an insight into SOX14 expression during in vitro neural differentiation of embryonal carcinoma cells and demonstrate the effect of its ectopic expression on protein levels of SOXB members in HeLa cells. Obtained results contribute to better understanding the role of one of the most conserved SOX proteins.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células-Tronco de Carcinoma Embrionário/metabolismo , Células-Tronco de Carcinoma Embrionário/patologia , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição SOXB2/genética , Tretinoína/farmacologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Biomarcadores/metabolismo , Linhagem Celular , Células-Tronco de Carcinoma Embrionário/efeitos dos fármacos , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Células HeLa , Humanos , Imuno-Histoquímica , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB2/metabolismoRESUMO
Whole gene deletions or duplications are an important cause of genetic disease and phenotypic variation. Targeted techniques for the routine testing of gross rearrangements have become essential tools for diagnostic researchers with the search for the most cost-effective and efficient tool assuming high priority. We used the new selector technique, MLGA (multiplex ligation-dependent genome amplification), to confirm deletions in two genes, SOX2 (SRY [sex determining region Y]) box 2) and OTX2 (orthodenticle homeobox 2), in individuals with developmental eye disease. We conclude that MLGA has the potential to be a useful technique in diagnostic research for the identification of deletions or duplications of known genes due to its speed and relatively low cost.
Assuntos
Anormalidades do Olho/diagnóstico , Anormalidades do Olho/genética , Deleção de Genes , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Estudos de Casos e Controles , Análise Custo-Benefício , Humanos , Fatores de Transcrição Otx/genética , Fatores de Transcrição SOXB1/genéticaRESUMO
The SRY-related, HMG box SoxB1 transcription factors are highly homologous, evolutionarily conserved proteins that are expressed in neuroepithelial cells throughout neural development. SoxB1 genes are down-regulated as cells exit the cell-cycle to differentiate and are considered functionally redundant in maintaining neural precursor populations. However, little is known about Sox3 function and its mode of action during primary neurogenesis. Using gain and loss-of-function studies, we analyzed Sox3 function in detail in Xenopus early neural development and compared it to that of Sox2. Through these studies we identified the first targets of a SoxB1 protein during primary neurogenesis. Sox3 functions as an activator to induce expression of the early neural genes, sox2 and geminin in the absence of protein synthesis and to indirectly inhibit the Bmp target Xvent2. As a result, Sox3 increases cell proliferation, delays neurogenesis and inhibits epidermal and neural crest formation to expand the neural plate. Our studies indicate that Sox3 and 2 have many similar functions in this process including the ability to activate expression of geminin in naïve ectodermal explants. However, there are some differences; Sox3 activates the expression of sox2, while Sox2 does not activate expression of sox3 and sox3 is uniquely expressed throughout the ectoderm prior to neural induction suggesting a role in neural competence. With morpholino-mediated knockdown of Sox3, we demonstrate that it is required for induction of neural tissue by BMP inhibition. Together these data indicate that Sox3 has multiple roles in early neural development including as a factor required for nogginmediated neural induction.