RESUMO
Here, we report that MYC rescues early human cells undergoing reprogramming from a proliferation pause induced by OCT3/4, SOX2, and KLF4 (OSK). We identified ESRG as a marker of early reprogramming cells that is expressed as early as day 3 after OSK induction. On day 4, ESRG positive (+) cells converted to a TRA-1-60 (+) intermediate state. These early ESRG (+) or TRA-1-60 (+) cells showed a proliferation pause due to increased p16INK4A and p21 and decreased endogenous MYC caused by OSK. Exogenous MYC did not enhance the appearance of initial reprogramming cells but instead reactivated their proliferation and improved reprogramming efficiency. MYC increased expression of LIN41, which potently suppressed p21 post-transcriptionally. MYC suppressed p16 INK4A. These changes inactivated retinoblastoma protein (RB) and reactivated proliferation. The RB-regulated proliferation pause does not occur in immortalized fibroblasts, leading to high reprogramming efficiency even without exogenous MYC.
Assuntos
Reprogramação Celular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína do Retinoblastoma/metabolismo , Antígenos de Superfície/metabolismo , Linhagem Celular , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fosforilação , Proteoglicanas/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Parkinson's disease (PD) is a progressive, age-related, neurodegenerative disorder, and oxidative stress is an important mediator in its pathogenesis. DJ-1, the product of the causative gene of a familial form of PD, plays a significant role in anti-oxidative defence to protect cells from oxidative stress. DJ-1 undergoes preferential oxidation at the cysteine residue at position 106 (Cys-106) under oxidative stress. Here, using specific antibodies against Cys-106-oxidized DJ-1 (oxDJ-1), it was found that the levels of oxDJ-1 in the erythrocytes of unmedicated PD patients (n = 88) were higher than in those of medicated PD patients (n = 62) and healthy control subjects (n = 33). Elevated oxDJ-1 levels were also observed in a non-human primate PD model. Biochemical analysis of oxDJ-1 in erythrocyte lysates showed that oxDJ-1 formed dimer and polymer forms, and that the latter interacts with 20S proteasome. These results clearly indicate a biochemical alteration in the blood of PD patients, which could be utilized as an early diagnosis marker for PD.
Assuntos
Eritrócitos/metabolismo , Doença de Parkinson/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Desglicase DJ-1/química , Proteína Desglicase DJ-1/metabolismo , Idoso , Animais , Cisteína/química , Modelos Animais de Doenças , Eritrócitos/química , Feminino , Humanos , Levodopa/uso terapêutico , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Doença de Parkinson/sangue , Doença de Parkinson/tratamento farmacológico , Multimerização ProteicaRESUMO
Pluripotency can be induced in somatic cells by overexpressing transcription factors, including POU class 5 homeobox 1 (OCT3/4), sex determining region Y-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and myelocytomatosis oncogene (c-MYC). However, some induced pluripotent stem cells (iPSCs) exhibit defective differentiation and inappropriate maintenance of pluripotency features. Here we show that dynamic regulation of human endogenous retroviruses (HERVs) is important in the reprogramming process toward iPSCs, and in re-establishment of differentiation potential. During reprogramming, OCT3/4, SOX2, and KLF4 transiently hyperactivated LTR7s--the long-terminal repeats of HERV type-H (HERV-H)--to levels much higher than in embryonic stem cells by direct occupation of LTR7 sites genome-wide. Knocking down LTR7s or long intergenic non-protein coding RNA, regulator of reprogramming (lincRNA-RoR), a HERV-H-driven long noncoding RNA, early in reprogramming markedly reduced the efficiency of iPSC generation. KLF4 and LTR7 expression decreased to levels comparable with embryonic stem cells once reprogramming was complete, but failure to resuppress KLF4 and LTR7s resulted in defective differentiation. We also observed defective differentiation and LTR7 activation when iPSCs had forced expression of KLF4. However, when aberrantly expressed KLF4 or LTR7s were suppressed in defective iPSCs, normal differentiation was restored. Thus, a major mechanism by which OCT3/4, SOX2, and KLF4 promote human iPSC generation and reestablish potential for differentiation is by dynamically regulating HERV-H LTR7s.
Assuntos
Retrovirus Endógenos/genética , Retrovirus Endógenos/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/virologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Embrionárias/virologia , Epigênese Genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes Induzidas/virologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/fisiologia , Células-Tronco Pluripotentes/fisiologia , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Viral/antagonistas & inibidores , RNA Viral/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/fisiologiaRESUMO
During mammalian embryonic development, the primitive streak initiates the differentiation of pluripotent epiblast cells into germ layers. Pluripotency can be reacquired in committed somatic cells using a combination of a handful of transcription factors, such as OCT3/4, SOX2, KLF4 and c-MYC (hereafter referred to as OSKM), albeit with low efficiency. Here we show that during OSKM-induced reprogramming towards pluripotency in human cells, intermediate cells transiently show gene expression profiles resembling mesendoderm, which is a major component of the primitive streak. Based on these findings, we discover that forkhead box H1 (FOXH1), a transcription factor required for anterior primitive streak specification during early development, significantly enhances the reprogramming efficiency of human fibroblasts by promoting their maturation, including mesenchymal to epithelial transition and the activation of late pluripotency markers. These results demonstrate that during the reprogramming process, human somatic cells go through a transient state that resembles mesendoderm.