Oct4 activates IL-17A to orchestrate M2 macrophage polarization and cervical cancer metastasis.

BACKGROUND: Cervical cancer is a common malignant tumor in the female. Interleukin (IL)-17A is a proinflammatory factor and exerts a vital function in inflammatory diseases and cancers. M2 macrophage has been confirmed to promote tumor development. Nevertheless, it is not yet known whether IL-17A facilitates cervical cancer development by inducing M2 macrophage polarization. Therefore, this study was conducted to investigate the regulatory effect of IL-17A on M2 macrophage polarization and the underlying mechanism in cervical cancer development. METHODS: RT-qPCR was utilized for testing IL-17A expression in cancer tissues and cells. Flow cytometry was applied to evaluate the M1 or M2 macrophage polarization. Cell proliferative, migratory, and invasive capabilities were measured through colony formation and transwell assays. ChIP and luciferase reporter assays were applied to determine the interaction between IL-17A and octamer-binding transcription factor 4 (OCT4). RESULTS: IL-17A expression and concentration were high in metastatic tissues and cells of cervical cancer. IL-17A was found to facilitate M2 macrophage polarization in cervical cancer. Furthermore, IL-17A facilitated the macrophage-mediated promotion of cervical cancer cell proliferative, migratory, and invasive capabilities. Mechanistic assays manifested that Oct4 binds to and transcriptionally activated IL-17A in cervical cancer cells. Furthermore, Oct4 promoted cervical cancer cell malignant phenotype and M2 macrophage polarization by activating the p38 pathway that, in turn, upregulated IL-17A. Additionally, in vivo experiments confirmed that Oct4 knockdown reduced tumor growth and metastasis. CONCLUSION: Oct4 triggers IL-17A to facilitate the polarization of M2 macrophages, which promotes cervical cancer cell metastasis.

Sox9 downregulation in non-obstructive azoospermia by UTF1 and mediator role of POU5F1.

BACKGROUND: Spermatogenesis is the process of producing mature sperm from Spermatogonial stem cells (SSCs) and this process requires a complex cooperation of different types of somatic and germ cells. Undifferentiated spermatogonia initiate the spermatogenesis and Sertoli cells as the only somatic cells inside of the seminiferous tubule play a key role in providing chemical and physical requirements for normal spermatogenesis, here, we investigated the dysfunction of these cells in non-obstructive azoospermia. MATERIAL AND METHOD: In this study, we analyzed the expression of sox9 and UTF1 in the non-obstructive human testis by immunohistochemistry. Moreover, we used the KEGG pathway and bioinformatics analysis to reveal the connection between our object genes and protein. RESULTS: The immunohistochemistry analysis of the non-obstructive human seminiferous tubule showed low expression of Sox9 and UTF1 that was detected out of the main location of the responsible cells for these expressions. Our bioinformatics analysis clearly and strongly indicated the relation between UTF1 in undifferentiated spermatogonia and Sox9 in Sertoli cells mediated by POU5F1. CONCLUSION: Generally, this study showed the negative effect of POU5F1 as a mediator between Sertoli cells as the somatic cells within seminiferous tubules and undifferentiated spermatogonia as the spermatogenesis initiator germ cells in non-obstructive conditions.

In Vivo Reprogramming Using Yamanaka Factors in the CNS: A Scoping Review.

Central nervous system diseases, particularly neurodegenerative disorders, pose significant challenges in medicine. These conditions, characterized by progressive neuronal loss, have remained largely incurable, exacting a heavy toll on individuals and society. In recent years, in vivo reprogramming using Yamanaka factors has emerged as a promising approach for central nervous system regeneration. This technique involves introducing transcription factors, such as Oct4, Sox2, Klf4, and c-Myc, into adult cells to induce their conversion into neurons. This review summarizes the current state of in vivo reprogramming research in the central nervous system, focusing on the use of Yamanaka factors. In vivo reprogramming using Yamanaka factors has shown promising results in several animal models of central nervous system diseases. Studies have demonstrated that this approach can promote the generation of new neurons, improve functional outcomes, and reduce scar formation. However, there are still several challenges that need to be addressed before this approach can be translated into clinical practice. These challenges include optimizing the efficiency of reprogramming, understanding the cell of origin for each transcription factor, and developing methods for reprogramming in non-subventricular zone areas. Further research is needed to overcome the remaining challenges, but this approach has the potential to revolutionize the way we treat central nervous system disorders.

Complex regulatory networks influence pluripotent cell state transitions in human iPSCs.

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discover 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which are highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlie the coordinated expression of genes in the GNMs. Epigenetic analyses reveal that regulatory networks underlying self-renewal and pluripotency are more complex than previously realized. Genetic analyses identify thousands of regulatory variants that overlapped predicted transcription factor binding sites and are associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network are significantly enriched for regulatory variants with large effects, suggesting that they play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work bins tens of thousands of regulatory elements in hiPSCs into discrete regulatory networks, shows that pluripotency and self-renewal processes have a surprising level of regulatory complexity, and suggests that genetic factors may contribute to cell state transitions in human iPSC lines.

Interlinked bi-stable switches govern the cell fate commitment of embryonic stem cells.

The development of embryonic stem (ES) cells to extraembryonic trophectoderm and primitive endoderm lineages manifests distinct steady-state expression patterns of two key transcription factors-Oct4 and Nanog. How dynamically such kind of steady-state expressions are maintained remains elusive. Herein, we demonstrate that steady-state dynamics involving two bistable switches which are interlinked via a stepwise (Oct4) and a mushroom-like (Nanog) manner orchestrate the fate specification of ES cells. Our hypothesis qualitatively reconciles various experimental observations and elucidates how different feedback and feedforward motifs orchestrate the extraembryonic development and stemness maintenance of ES cells. Importantly, the model predicts strategies to optimize the dynamics of self-renewal and differentiation of embryonic stem cells that may have therapeutic relevance in the future.

A positive feedback between PDIA3P1 and OCT4 promotes the cancer stem cell properties of esophageal squamous cell carcinoma.

BACKGROUND: Increasing evidence has indicated that long non-coding RNAs (lncRNAs) have been proven to regulate esophageal cancer progression. The lncRNA protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P1) has been shown to promote cancer stem cell properties; however, its mechanism of action remains unclear. In this study, we investigated the regulation of esophageal cancer stem cell properties by the interaction of PDIA3P1 with proteins. METHODS: The GEPIA2 and Gene Expression Omnibus databases were used to analyze gene expression. PDIA3P1 expression in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to determine the effects of PDIA3P1 on ESCC cell proliferation, migration, and invasion. The sphere formation assay, number of side population cells, and CD271 + /CD44 + cells were detected by flow cytometry to identify the cancer stem cell properties. RNA immunoprecipitation (RIP), RNA pull-down, co-immunoprecipitation (co-IP), dual luciferase reporter, and cleavage under targets and tagmentation (CUT&Tag) assays were performed to elucidate the underlying molecular mechanisms. RESULTS: PDIA3P1 expression was upregulated in ESCC cell lines and tissues. Functionally, higher PDIA3P1 expression promoted cell proliferation, invasion, and metastasis and inhibited apoptosis in esophageal cancer. Importantly, PDIA3P1 promoted cancer stem cell properties in ESCC. Mechanistically, PDIA3P1 interacted with and stabilized octamer-binding transcription factor 4 (OCT4) by eliminating its ubiquitination by the ubiquitinating enzyme WW domain-containing protein 2 (WWP2). Moreover, as a transcription factor, OCT4 bound to the PDIA3P1 promoter and promoted its transcription. CONCLUSIONS: Our research revealed a novel mechanism by which a positive feedback loop exists between PDIA3P1 and OCT4. It also demonstrated that the PDIA3P1-WWP2-OCT4 loop is beneficial for promoting the cancer stem cell properties of ESCC. Owing to this regulatory relationship, the PDIA3P1-WWP2-OCT4-positive feedback loop might be used in the diagnosis and prognosis, as well as in the development of novel therapeutics for esophageal cancer.

Highly cooperative chimeric super-SOX induces naive pluripotency across species.

Our understanding of pluripotency remains limited: iPSC generation has only been established for a few model species, pluripotent stem cell lines exhibit inconsistent developmental potential, and germline transmission has only been demonstrated for mice and rats. By swapping structural elements between Sox2 and Sox17, we built a chimeric super-SOX factor, Sox2-17, that enhanced iPSC generation in five tested species: mouse, human, cynomolgus monkey, cow, and pig. A swap of alanine to valine at the interface between Sox2 and Oct4 delivered a gain of function by stabilizing Sox2/Oct4 dimerization on DNA, enabling generation of high-quality OSKM iPSCs capable of supporting the development of healthy all-iPSC mice. Sox2/Oct4 dimerization emerged as the core driver of naive pluripotency with its levels diminished upon priming. Transient overexpression of the SK cocktail (Sox+Klf4) restored the dimerization and boosted the developmental potential of pluripotent stem cells across species, providing a universal method for naive reset in mammals.

A short history of pluripotent stem cells markers.

The expression of one or more of a small number of molecules, typically cell surface-associated antigens, or transcription factors, is widely used for identifying pluripotent stem cells (PSCs) or for monitoring their differentiation. However, none of these marker molecules are uniquely expressed by PSCs and all are expressed by stem cells that have lost the ability to differentiate. Consequently, none are indicators of pluripotency, per se. Here we summarize the nature and characteristics of several markers that are in wide use, including the cell surface antigens, stage-specific embryonic antigen (SSEA)-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, GCTM2, and the transcription factors POUF5/OCT4, NANOG, and SOX2, highlighting issues that must be considered when interpreting data about their expression on putative PSCs.

A paradigm for post-embryonic Oct4 re-expression: E7-induced hydroxymethylation regulates Oct4 expression in cervical cancer.

The Octamer-binding transcription factor-4 (Oct4) is upregulated in different malignancies, yet a paradigm for mechanisms of Oct4 post-embryonic re-expression is inadequately understood. In cervical cancer, Oct4 expression is higher in human papillomavirus (HPV)-related than HPV-unrelated cervical cancers and this upregulation correlates with the expression of the E7 oncogene. We have reported that E7 affects the Oct4-transcriptional output and Oct4-related phenotypes in cervical cancer, however, the underlying mechanism remains elusive. Here, we characterize the Oct4-protein interactions in cervical cancer cells via computational analyses and Mass Spectrometry and reveal that Methyl-binding proteins (MBD2 and MBD3), are determinants of Oct4-driven transcription. E7 triggers MBD2 downregulation and TET1 upregulation, thereby disrupting the methylation status of the Oct4 gene. This coincides with an increase in the total DNA hydroxymethylation leading to the re-expression of Oct4 in cervical cancer and likely affecting broader transcriptional patterns. Our findings reveal a previously unreported mechanism by which the E7 oncogene can regulate Oct4 re-expression and global transcriptional patterns by increasing DNA hydroxymethylation and lowering the barrier to cellular plasticity during carcinogenesis.

POU5F1 promotes the proliferation, migration, and invasion of gastric cancer cells by reducing the ubiquitination level of TRAF6.

POU5F1 plays an important role in maintaining the cancer stem cell (CSC) -like properties of gastric cancer (GC) cells. The impact of POU5F1 on the proliferation and metastasis of GC was examined, along with the potential of ATRA as a specific therapeutic agent for GC. The dysregulation of POU5F1 expression in GC tissues was analyzed using public databases and bioinformatics techniques, and the disparity in POU5F1 expression between normal gastric tissues and GC tissues was further assessed through western blot, RT-qPCR, and immunohistochemistry. The present study aimed to investigate the impact of POU5F1 on the proliferation, migration, and invasion of GC cells through both in vivo and in vitro experiments. Additionally, the effects of ATRA on the proliferation, migration, and invasion of GC cells were examined using in vivo and in vitro approaches. Our findings revealed a significant upregulation of POU5F1 in GC tissues, which was found to be associated with a poorer prognosis in patients with GC. Moreover, POU5F1 was observed to enhance the proliferation, migration, and invasion of GC cells in vitro, as well as promote subcutaneous tumor growth and lung metastasis of GC cells in vivo. The overexpression of POU5F1 mechanistically triggers the process of Epithelial-mesenchymal transition (EMT) by down-regulating E-Cadherin and up-regulating N-Cadherin and VIM. POU5F1 hinders the ubiquitination of TRAF6 through negative regulation of TRIM59, thereby facilitating the activation of the NF-κB pathway. Furthermore, the administration of ATRA effectively impedes the proliferation, migration, and invasion of GC cells by suppressing the expression of POU5F1. The upregulation of POU5F1 elicits EMT, fosters the initiation of the NF-κB signaling pathway in GC cells, and stimulates the proliferation, invasion, and metastasis of GC cells. All-trans retinoic acid (ATRA) can impede these POU5F1-induced effects, thereby potentially serving as an adjunctive therapeutic approach for GC.

Expression and Functional Analysis of core stemness factors OSKM (OCT4, SOX2, KLF4, and MYC) in Pan-cancer.

The dedifferentiation process of tumorigenesis and somatic cell reprogramming has some commonness and differences, which is the key question to cancer therapeutic strategy and stem cell applications. To further explore the commonalities and variance between carcinogenesis and induced pluripotent stem cell reprogramming, we investigated the role of stemness factors OSKM (OCT4, SOX2, KLF4, and MYC) in the pan-cancer process using public clinical data. Expression of OSKM in human pan-cancer was analyzed via the Genotype Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) database based on the RNA-seq data of tissues. The correlation of expression between OSKM genes was analyzed via the Tumor Immune Evaluation Resource (TIMER) database, while the STRING tool was used to construct the protein-protein interaction network for OSKM. Prognostic impact of OSKM in pan-cancer was analyzed by Cox proportional hazards regression model. The relationships between OSKM and tumor stemness, tumor microenvironment and immune checkpoint and were performed by Sangerbox platform using Pearson correlation analysis. Our results showed that OSKM were universally expressed and significantly altered in tumors compared with adjacent normal tissues in most tumor types. In addition, correlation analysis revealed the relevance of OSKM genes to patient prognosis, cancer cell stemness, tumor microenvironment or immune checkpoint. However, there is little similarity between these genes in terms of how they function in each cancer type. This study elucidates the different roles of core stemness factors OSKM in pan-cancer, offering potential therapeutic targets for novel anti-cancer strategies and knowledge to minimize the potential carcinogenic effects during stem cell transplantation.

Multifaceted SOX2-chromatin interaction underpins pluripotency progression in early embryos.

Pioneer transcription factors (TFs), such as OCT4 and SOX2, play crucial roles in pluripotency regulation. However, the master TF-governed pluripotency regulatory circuitry was largely inferred from cultured cells. In this work, we investigated SOX2 binding from embryonic day 3.5 (E3.5) to E7.5 in the mouse. In E3.5 inner cell mass (ICM), SOX2 regulates the ICM-trophectoderm program but is dispensable for opening global enhancers. Instead, SOX2 occupies preaccessible enhancers in part opened by early-stage expressing TFs TFAP2C and NR5A2. SOX2 then widely redistributes when cells adopt naive and formative pluripotency by opening enhancers or poising them for rapid future activation. Hence, multifaceted pioneer TF-enhancer interaction underpins pluripotency progression in embryos, including a distinctive state in E3.5 ICM that bridges totipotency and pluripotency.

OCT4 regulates WNT/ß-catenin signaling and prevents mesoendoderm differentiation by repressing EOMES in porcine pluripotent stem cells.

The regulatory network between signaling pathways and transcription factors (TFs) is crucial for the maintenance of pluripotent stem cells. However, little is known about how the key TF OCT4 coordinates signaling pathways to regulate self-renewal and lineage differentiation of porcine pluripotent stem cells (pPSCs). Here, we explored the function of OCT4 in pPSCs by transcriptome and chromatin accessibility analysis. The TFs motif enrichment analysis revealed that, following OCT4 knockdown, the regions of increased chromatin accessibility were enriched with EOMES, GATA6, and FOXA1, indicating that pPSCs differentiated toward the mesoendoderm (ME) lineage. Besides, pPSCs rapidly differentiated into ME when the WNT/ß-catenin inhibitor XAV939 was removed. However, the ME differentiation of pPSCs caused by OCT4 knockdown did not rely on the activation of WNT/ß-catenin signaling because the target gene of WNT/ß-catenin signaling, AXIN2 was not upregulated after OCT4 knockdown, despite significant upregulation of WLS and some WNT ligands. Importantly, OCT4 is directly bound to the promoter and enhancers of EOMES and repressed its transcription. Overexpression of EOMES was sufficient to induce ME differentiation in the presence of XAV939. These results demonstrate that OCT4 can regulate WNT/ß-catenin signaling and prevent ME differentiation of pPSCs by repressing EOMES transcription.

AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts.

AKT/PKB is a kinase crucial for pluripotency maintenance in pluripotent stem cells. Multiple post-translational modifications modulate its activity. We have previously demonstrated that AKT1 induces the expression of the pluripotency transcription factor Nanog in a SUMOylation-dependent manner in mouse embryonic stem cells. Here, we studied different cellular contexts and main candidates that could mediate this induction. Our results strongly suggest the pluripotency transcription factors OCT4 and SOX2 are not essential mediators. Additionally, we concluded that this induction takes place in different pluripotent contexts but not in terminally differentiated cells. Finally, the cross-matching analysis of ESCs, iPSCs and MEFs transcriptomes and AKT1 phosphorylation targets provided new clues about possible factors that could be involved in the SUMOylation-dependent Nanog induction by AKT.

Regulatory Elements Outside Established Pou5f1 Gene Boundaries Are Required for Multilineage Differentiation of Embryonic Stem Cells.

The transcription factor Oct4 can rightfully be considered a pivotal element in maintaining pluripotency. In addition, its ability to function as a pioneer factor enables the reprogramming of somatic cells back into a pluripotent state. To better understand the regulation of the Oct4-encoding gene (Pou5f1), the main genetic elements that regulate its expression in different states of pluripotency ought to be identified. While some elements have been well characterized for their ability to drive Pou5f1 expression, others have yet to be determined. In this work, we show that translocation of the Pou5f1 gene fragment purported to span all essential cis-elements, including the well-known distal and proximal enhancers (DE and PE), into the Rosa26 locus impairs the self-renewal of mouse embryonic stem cells (ESCs) in the naïve pluripotency state, as well as their further advancement through the formative and primed pluripotency states, inducing overall differentiation failure. These results suggest that regulatory elements located outside the previously determined Pou5f1 boundaries are critical for the proper spatiotemporal regulation of this gene during development, indicating the need for their better characterization.

Generation, characterization, and differentiation of induced pluripotent stem-like cells in the domestic cat.

Induced pluripotent stem (iPS) cells are generated from somatic cells and can differentiate into various cell types. Therefore, these cells are expected to be a powerful tool for modeling diseases and transplantation therapy. Generation of domestic cat iPS cells depending on leukemia inhibitory factor has been reported; however, this strategy may not be optimized. Considering that domestic cats are excellent models for studying spontaneous diseases, iPS cell generation is crucial. In this study, we aimed to derive iPS cells from cat embryonic fibroblasts retrovirally transfected with mouse Oct3/4, Klf4, Sox2, and c-Myc. After transfection, embryonic fibroblasts were reseeded onto inactivated SNL 76/7 and cultured in a medium supplemented with basic fibroblast growth factor. Flat, compact, primary colonies resembling human iPS colonies were observed. Additionally, primary colonies were more frequently observed in the KnockOut Serum Replacement medium than in the fetal bovine serum (FBS) medium. However, enhanced maintenance and proliferation of iPS-like cells occurred in the FBS medium. These iPS-like cells expressed embryonic stem cell markers, had normal karyotypes, proliferated beyond 45 passages, and differentiated into all three germ layers in vitro. Notably, expression of exogenous Oct3/4, Klf4, and Sox2 was silenced in these cells. However, the iPS-like cells failed to form teratomas. In conclusion, this is the first study to establish and characterize cat iPS-like cells, which can differentiate into different cell types depending on the basic fibroblast growth factor.

Hepatitis B Virus x Protein Increases Cellular OCT3/4 and MYC and Facilitates Cellular Reprogramming.

Hepatitis B virus x (HBx) is a multifunctional protein coded by the Hepatitis B virus that is involved in various cellular processes such as proliferation, cell survival/apoptosis, and histone methylation. HBx was reported to be associated with liver "cancer stem cells." The stemness inducing properties of HBx could also facilitate the generation of pluripotent stem cells from somatic cells. It is well established that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using a cocktail of transcription factors called Yamanaka's factors (YFs) (OCT4, SOX2, KLF4, and MYC). The reprogramming process proceeds step-by-step with reprogramming factor chromatin interactions, transcription, and chromatin states changing during transitions. HBx is a "broad spectrum trans-activator" and therefore could facilitate these transitions. We electroporated low passage and high passage (difficult to reprogram) fibroblasts using YFs with and without HBx and evaluated the reprogramming efficiency. We also investigated the tri-lineage and terminal differentiation potential of iPSC derived using HBx. We found that the addition of HBx to YF improves iPSC derivation, and it increases the efficiency of iPSC generation from "difficult or hard-to-reprogram samples" such as high passage/senescent fibroblasts. Further, we show that HBx can substitute the key transcription factor MYC in the YF cocktail to generate iPSC. The cellular levels of OCT3/4 and MYC were increased in HBx expressing cells. Our results have practical value in improving the efficiency of pluripotent stem cell derivation from "difficult to reprogram" somatic cells, in addition to providing some insights into the mechanisms of liver carcinogenesis in chronic hepatitis B. To conclude, HBx improves the reprogramming efficiency of YFs. HBx increases the cellular levels of OCT3/4 and MYC.

Application of the Yamanaka Transcription Factors Oct4, Sox2, Klf4, and c-Myc from the Laboratory to the Clinic.

The transcription factors Oct4, Sox2, Klf4, and c-Myc enable the reprogramming of somatic cells into induced pluripotent cells. Reprogramming generates newly differentiated cells for potential therapies in cancer, neurodegenerative diseases, and rejuvenation processes. In cancer therapies, these transcription factors lead to a reduction in the size and aggressiveness of certain tumors, such as sarcomas, and in neurodegenerative diseases, they enable the production of dopaminergic cells in Parkinson's disease, the replacement of affected neuronal cells in olivopontocerebellar atrophy, and the regeneration of the optic nerve. However, there are limitations, such as an increased risk of cancer development when using Klf4 and c-Myc and the occurrence of abnormal dyskinesias in the medium term, possibly generated by the uncontrolled growth of differentiated dopaminergic cells and the impairment of the survival of the new cells. Therefore, the Yamanaka transcription factors have shown therapeutic potential through cell reprogramming for some carcinomas, neurodegenerative diseases, and rejuvenation. However, the limitations found in the studies require further investigation before the use of these transcription factors in humans.

Evaluation of the determinants for improved pluripotency induction and maintenance by engineered SOX17.

An engineered SOX17 variant with point mutations within its DNA binding domain termed SOX17FNV is a more potent pluripotency inducer than SOX2, yet the underlying mechanism remains unclear. Although wild-type SOX17 was incapable of inducing pluripotency, SOX17FNV outperformed SOX2 in mouse and human pluripotency reprogramming. In embryonic stem cells, SOX17FNV could replace SOX2 to maintain pluripotency despite considerable sequence differences and upregulated genes expressed in cleavage-stage embryos. Mechanistically, SOX17FNV co-bound OCT4 more cooperatively than SOX2 in the context of the canonical SoxOct DNA element. SOX2, SOX17, and SOX17FNV were all able to bind nucleosome core particles in vitro, which is a prerequisite for pioneer transcription factors. Experiments using purified proteins and in cellular contexts showed that SOX17 variants phase-separated more efficiently than SOX2, suggesting an enhanced ability to self-organise. Systematic deletion analyses showed that the N-terminus of SOX17FNV was dispensable for its reprogramming activity. However, the C-terminus encodes essential domains indicating multivalent interactions that drive transactivation and reprogramming. We defined a minimal SOX17FNV (miniSOX) that can support reprogramming with high activity, reducing the payload of reprogramming cassettes. This study uncovers the mechanisms behind SOX17FNV-induced pluripotency and establishes engineered SOX factors as powerful cell engineering tools.

FACT regulates pluripotency through proximal and distal regulation of gene expression in murine embryonic stem cells.

BACKGROUND: The FACT complex is a conserved histone chaperone with critical roles in transcription and histone deposition. FACT is essential in pluripotent and cancer cells, but otherwise dispensable for most mammalian cell types. FACT deletion or inhibition can block induction of pluripotent stem cells, yet the mechanism through which FACT regulates cell fate decisions remains unclear. RESULTS: To explore the mechanism for FACT function, we generated AID-tagged murine embryonic cell lines for FACT subunit SPT16 and paired depletion with nascent transcription and chromatin accessibility analyses. We also analyzed SPT16 occupancy using CUT&RUN and found that SPT16 localizes to both promoter and enhancer elements, with a strong overlap in binding with OCT4, SOX2, and NANOG. Over a timecourse of SPT16 depletion, nucleosomes invade new loci, including promoters, regions bound by SPT16, OCT4, SOX2, and NANOG, and TSS-distal DNaseI hypersensitive sites. Simultaneously, transcription of Pou5f1 (encoding OCT4), Sox2, Nanog, and enhancer RNAs produced from these genes' associated enhancers are downregulated. CONCLUSIONS: We propose that FACT maintains cellular pluripotency through a precise nucleosome-based regulatory mechanism for appropriate expression of both coding and non-coding transcripts associated with pluripotency.