Browse
Submit Data
Databases
API
Help

Dataset Information

32 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Heterokaryon-based reprogramming of human B lymphocytes for pluripotency requires Oct4 but not Sox2.

ABSTRACT: Differentiated cells can be reprogrammed through the formation of heterokaryons and hybrid cells when fused with embryonic stem (ES) cells. Here, we provide evidence that conversion of human B-lymphocytes towards a multipotent state is initiated much more rapidly than previously thought, occurring in transient heterokaryons before nuclear fusion and cell division. Interestingly, reprogramming of human lymphocytes by mouse ES cells elicits the expression of a human ES-specific gene profile, in which markers of human ES cells are expressed (hSSEA4, hFGF receptors and ligands), but markers that are specific to mouse ES cells are not (e.g., Bmp4 and LIF receptor). Using genetically engineered mouse ES cells, we demonstrate that successful reprogramming of human lymphocytes is independent of Sox2, a factor thought to be required for induced pluripotent stem (iPS) cells. In contrast, there is a distinct requirement for Oct4 in the establishment but not the maintenance of the reprogrammed state. Experimental heterokaryons, therefore, offer a powerful approach to trace the contribution of individual factors to the reprogramming of human somatic cells towards a multipotent state.

SUBMITTER: Pereira CF

PROVIDER: S-EPMC2527997 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

Project description:A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

| S-EPMC4489496 | biostudies-literature

Pluripotency reprogramming by competent and incompetent POU factors uncovers temporal dependency for Oct4 and Sox2.

Project description:Oct4, along with Sox2 and Klf4 (SK), can induce pluripotency but structurally similar factors like Oct6 cannot. To decode why Oct4 has this unique ability, we compare Oct4-binding, accessibility patterns and transcriptional waves with Oct6 and an Oct4 mutant defective in the dimerization with Sox2 (Oct4defSox2). We find that initial silencing of the somatic program proceeds indistinguishably with or without Oct4. Oct6 mitigates the mesenchymal-to-epithelial transition and derails reprogramming. These effects are a consequence of differences in genome-wide binding, as the early binding profile of Oct4defSox2 resembles Oct4, whilst Oct6 does not bind pluripotency enhancers. Nevertheless, in the Oct6-SK condition many otherwise Oct4-bound locations become accessible but chromatin opening is compromised when Oct4defSox2 occupies these sites. We find that Sox2 predominantly facilitates chromatin opening, whilst Oct4 serves an accessory role. Formation of Oct4/Sox2 heterodimers is essential for pluripotency establishment; however, reliance on Oct4/Sox2 heterodimers declines during pluripotency maintenance.

| S-EPMC6677745 | biostudies-literature

OCT4 and SOX2 Work as Transcriptional Activators in Reprogramming Human Fibroblasts.

Project description:SOX2 and OCT4, in conjunction with KLF4 and cMYC, are sufficient to reprogram human fibroblasts to induced pluripotent stem cells (iPSCs), but it is unclear if they function as transcriptional activators or as repressors. We now show that, like OCT4, SOX2 functions as a transcriptional activator. We substituted SOX2-VP16 (a strong activator) for wild-type (WT) SOX2, and we saw an increase in the efficiency and rate of reprogramming, whereas the SOX2-HP1 fusion (a strong repressor) eliminated reprogramming. We report that, at an early stage of reprogramming, virtually all DNA-bound OCT4, SOX2, and SOX2-VP16 were embedded in putative enhancers, about half of which were created de novo. Those associated with SOX2-VP16 were, on average, stronger than those bearing WT SOX2. Many newly created putative enhancers were transient, and many transcription factor locations on DNA changed as reprogramming progressed. These results are consistent with the idea that, during reprogramming, there is an intermediate state that is distinct from both parental cells and iPSCs.

| S-EPMC5648000 | biostudies-literature

Dissecting the role of distinct OCT4-SOX2 heterodimer configurations in pluripotency.

Project description:The transcription factors OCT4 and SOX2 are required for generating induced pluripotent stem cells (iPSCs) and for maintaining embryonic stem cells (ESCs). OCT4 and SOX2 associate and bind to DNA in different configurations depending on the arrangement of their individual DNA binding elements. Here we have investigated the role of the different OCT4-SOX2-DNA assemblies in regulating and inducing pluripotency. To this end, we have generated SOX2 mutants that interfere with specific OCT4-SOX2 heterodimer configurations and assessed their ability to generate iPSCs and to rescue ESC self-renewal. Our results demonstrate that the OCT4-SOX2 configuration that dimerizes on a Hoxb1-like composite, a canonical element with juxtaposed individual binding sites, plays a more critical role in the induction and maintenance of pluripotency than any other OCT4-SOX2 configuration. Overall, the results of this study provide new insight into the protein interactions required to establish a de novo pluripotent network and to maintain a true pluripotent cell fate.

| S-EPMC4551974 | biostudies-literature

Counteracting activities of OCT4 and KLF4 during reprogramming to pluripotency.

Project description:Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) after overexpressing four transcription factors, of which Oct4 is essential. To elucidate the role of Oct4 during reprogramming, we investigated the immediate transcriptional response to inducible Oct4 overexpression in various somatic murine cell types using microarray analysis. By downregulating somatic-specific genes, Oct4 induction influenced each transcriptional program in a unique manner. A significant upregulation of pluripotent markers could not be detected. Therefore, OCT4 facilitates reprogramming by interfering with the somatic transcriptional network rather than by directly initiating a pluripotent gene-expression program. Finally, Oct4 overexpression upregulated the gene Mgarp in all the analyzed cell types. Strikingly, Mgarp expression decreases during the first steps of reprogramming due to a KLF4-dependent inhibition. At later stages, OCT4 counteracts the repressive activity of KLF4, thereby enhancing Mgarp expression. We show that this temporal expression pattern is crucial for the efficient generation of iPSCs.

| S-EPMC3964287 | biostudies-literature

Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins OCT4, SOX2, KLF4 and c-MYC.

Project description:It has been shown that human and murine fibroblasts can be reprogrammed by ectopic expression of transcription factors using viral vectors. For the purpose of human therapeutic applications, the integration of viral transgenes into the genome is unlikely to be accepted. We therefore produced recombinant transcription factor proteins in E. coli (OCT4, SOX2, c-MYC and KLF4) carrying the cell penetrating TAT domain from HIV1. The purified proteins were able to enter into mammalian cells when added to tissue culture medium but appeared not to translocate to the nucleus. Further investigation indicated that most of the protein was tied up in the endosomes and was unavailable for reprogramming. Once this problem has been solved it seems likely that protein reprogramming will be the method of choice for clinical applications.

| S-EPMC3772671 | biostudies-literature

Enhanced OCT4 transcriptional activity substitutes for exogenous SOX2 in cellular reprogramming.

Project description:Adenoviral early region 1A (E1A) is a viral gene that can promote cellular proliferation and de-differentiation in mammalian cells, features required for the reprogramming of somatic cells to a pluripotent state. E1A has been shown to interact with OCT4, and as a consequence, to increase OCT4 transcriptional activity. Indeed, E1A and OCT4 are sufficient to revert neuroepithelial hybrids to pluripotency, as demonstrated in previous cell fusion experiments. However, the role that E1A might play in the generation of induced pluripotent stem cells (iPSCs) has not been investigated yet. In this report, we show that E1A can generate iPSCs in combination with OCT4 and KLF4, thus replacing exogenous SOX2. The generated iPSCs are bona fide pluripotent cells as shown by in vitro and in vivo tests. Overall, our study suggests that E1A might replace SOX2 through enhancing OCT4 transcriptional activity at the early stages of reprogramming.

| S-EPMC4725906 | biostudies-literature

Novel AKT phosphorylation sites identified in the pluripotency factors OCT4, SOX2 and KLF4.

Project description:The four OSKM factors OCT4, SOX2, KLF4 and c-MYC are key transcription factors modulating pluripotency, self-renewal and tumorigenesis in stem cells. However, although their transcriptional targets have been extensively studied, little is known about how these factors are regulated at the posttranslational level. In this study, we established an in vitro system to identify phosphorylation patterns of the OSKM factors by AKT kinase. OCT4, SOX2, KLF4 and c-MYC were expressed in Sf9 insect cells employing the baculoviral expression system. OCT4, SOX2 and KLF4 were localized in the nucleus of insect cells, allowing their easy purification to near homogeneity upon nuclear fractionation. All transcription factors were isolated as biologically active DNA-binding proteins. Using in vitro phosphorylation and mass spectrometry-based phosphoproteome analyses several novel and known AKT phosphorylation sites could be identified in OCT4, SOX2 and KLF4.

| S-EPMC4825772 | biostudies-literature

R-loops coordinate with SOX2 in regulating reprogramming to pluripotency.

Project description:R-loops modulate genome stability and regulate gene expression, but the functions and the regulatory mechanisms of R-loops in stem cell biology are still unclear. Here, we profiled R-loops during somatic cell reprogramming and found that dynamic changes in R-loops are essential for reprogramming and occurred before changes in gene expression. Disrupting the homeostasis of R-loops by depleting RNaseH1 or catalytic inactivation of RNaseH1 at D209 (RNaseH1D209N) blocks reprogramming. Sox2, but not any other factor in the Yamanaka cocktail, overcomes the inhibitory effects of RNaseH1 activity loss on reprogramming. Sox2 interacts with the reprogramming barrier factor Ddx5 and inhibits the resolvase activity of Ddx5 on R-loops and thus facilitates reprogramming. Furthermore, reprogramming efficiency can be modulated by dCas9-mediated RNaseH1/RNaseH1D209N targeting the specific R-loop regions. Together, these results show that R-loops play important roles in reprogramming and shed light on the regulatory module of Sox2/Ddx5 on R-loops during reprogramming.

| S-EPMC7360481 | biostudies-literature

Reprogramming towards pluripotency requires AID-dependent DNA demethylation.

Project description:Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.

| S-EPMC2906123 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data