Stem cell specific interferon stimulated gene expression is regulated by the formative pluripotency network through IRF1

Stem cells intrinsically express a subset of genes which are normally associated with interferon stimulation, and thus the innate immunity response. Expression of these interferon stimulated genes (ISG) in stem cells is independent of external stimuli such as viral infection. Here we show that the interferon regulatory factor 1, Irf1, is directly controlled by the murine formative pluripotency gene regulatory network and therefore upregulated in the transition from naive to formative pluripotency. IRF1 in turn binds to regulatory regions of a conserved set of ISGs and is required for their faithful expression in formative pluripotent cells. IRF1 also binds to an enhancer of the formative pluripotency transcription factor Oct6 and is partially required for upregulation of Oct6. IRF1 therefore acts as a link between the formative pluripotency network and the regulation of innate immunity genes in formative pluripotency.

Histone Demethylase KDM5B Catalyzed H3k4me3 Demethylation to Promote Differentiation of Bone Marrow Mesenchymal Stem Cells into Cardiomyocytes

BACKGROUND: Studies have shown that histone H3 methylation is involved in regulating the differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs). KDM5B can specifically reduce the level of histone 3 lysine 4 trimethylation (H3K4me3), thereby activating the expression of related genes and participating in biological processes such as cell differentiation, embryonic development and tumor formation. Whether KDM5B is involved in the regulation of BMSCs differentiation into cardiomyocytes through the above manner has not been reported.OBJECTIVE: To investigate the effect of KDM5B on the induction and differentiation of swine BMSCs into myocardial cells in vitro.METHODS: Swine bone marrow BMSCs were isolated and cultured, and the overexpression, interference expression and blank vector of KMD5B were constructed and transfected by lentivirus. BMSCs was induced to differentiate into cardiomyocytes by 5-azacytidine (5-AZA) in vitro, and the differentiation efficiency was compared by immunofluorescence, RT-PCR, Western Blot and whole-cell patch clamp detection.RESULT: Compared with the control group, the expression levels of histone H3K4me3 and pluripotency gene Nanog in KDM5B overexpression group were significantly decreased, while the expression level of key myocardial gene HCN4 was significantly increased, and the Na+ current density on the surface of differentiated myocardial cell membrane was significantly increased. Meanwhile, the corresponding results of the KDM5B silent expression group were just opposite. CONCLUSIONS: It indicated that enhanced KDM5B expression could promote the differentiation of BMSCs into cardiomyocytes and improve the differentiation efficiency by controlling H3K4 methylation levels..

ESRRB Facilitates the Conversion of Trophoblast-Like Stem Cells From Induced Pluripotent Stem Cells by Directly Regulating CDX2

Porcine-induced pluripotent stem cells (piPSCs) could serve as a great model system for human stem cell preclinical research. However, the pluripotency gene network of piPSCs, especially the function for the core transcription factor estrogen-related receptor beta (ESRRB), was poorly understood. Here, we constructed ESRRB-overexpressing piPSCs (ESRRB-piPSCs). Compared with the control piPSCs (CON-piPSCs), the ESRRB-piPSCs showed flat, monolayered colony morphology. Moreover, the ESRRB-piPSCs showed greater chimeric capacity into trophectoderm than CON-piPSCs. We found that ESRRB could directly regulate the expressions of trophoblast stem cell (TSC)-specific markers, including KRT8, KRT18 and CDX2, through binding to their promoter regions. Mutational analysis proved that the N-terminus zinc finger domain is indispensable for ESRRB to regulate the TSC markers. Furthermore, this regulation needs the participation of OCT4. Accordingly, the cooperation between ESRRB and OCT4 facilitates the conversion from pluripotent state to the trophoblast-like state. Our results demonstrated a unique and crucial role of ESRRB in determining piPSCs fate, and shed new light on the molecular mechanism underlying the segregation of embryonic and extra-embryonic lineages.

Identifying SOX2-OT transcript that is responsible for regulating SOX2 in cancer cells and embryonic stem cells

SOX2 overlapping transcript (SOX2-OT) is an evolutionarily conserved long non-coding RNA (lncRNA) whose intronic region contains the transcript of pluripotency gene SRY-box transcription factor 2 (SOX2). It has been suggested that SOX2-OT can regulate its overlapping gene, SOX2. Studies demonstrated that elevated SOX2-OT promotes SOX2 expression in cancer cells, whereas levels of SOX2-OT are inversely correlated with levels of SOX2 in embryonic stem cells. It is not clear why there is a tremendous discrepancy in the regulation of SOX2 by SOX2-OT in cancer cells and embryonic stem cells. Due to the diversified transcription of the SOX2-OT gene, we hypothesize that differential expression of transcripts of the SOX2-OT gene in cancer cells and embryonic stem cells may contribute to the divergence in the regulatory relationship of SOX2-OT and SOX2. A CRISPR screening platform can be leveraged to systemic evaluate which transcript of the SOX2-OT gene may be responsible for upregulation or downregulation of SOX2 in cancer cells and embryonic stem cells, respectively.

Direct repression of Nanog and Oct4 by OTX2 modulates contribution of epiblast-derived cells to germline and somatic lineage

In mammals the pre-gastrula proximal epiblast gives rise to Primordial Germ Cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work showed that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here we show that OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs occurs also in mutant embryos. We propose that the OTX2 repressive control of Oct4 and Nanog is at the basis of the mechanism determining epiblast contribution to germline and somatic lineage.

A cohesin cancer mutation reveals a role for the hinge domain in genome organization and gene expression

The cohesin complex spatially organizes interphase chromatin by bringing distal genomic loci into close physical proximity, looping out the intervening DNA. Mutation of cohesin complex subunits is observed in cancer and developmental disorders, but the mechanisms through which these mutations may contribute to disease remain poorly understood. Here, we investigate a recurrent missense mutation to the hinge domain of the cohesin subunit SMC1A, observed in acute myeloid leukemia. Engineering this mutation into murine embryonic stem cells caused widespread changes in gene expression, including dysregulation of the pluripotency gene expression program. This mutation reduced cohesin levels at promoters and enhancers, decreased DNA loops and interactions across short genomic distances, and weakened insulation at CTCF-mediated DNA loops. These findings provide insight into how altered cohesin function contributes to disease and identify a requirement for the cohesin hinge domain in three-dimensional chromatin structure.

BAF Complex in Embryonic Stem Cells and Early Embryonic Development

Embryonic stem cells (ESCs) can self-renew indefinitely and maintain their pluripotency status. The pluripotency gene regulatory network is critical in controlling these properties and particularly chromatin remodeling complexes. In this review, we summarize the research progresses of the functional and mechanistic studies of BAF complex in mouse ESCs and early embryonic development. A discussion of the mechanistic bases underlying the distinct phenotypes upon the deletion of different BAF subunits in ESCs and embryos will be highlighted.

Induced in vivo transdifferentiation of vertebrate muscle into early endoderm-like cells

The extent to which differentiated cells, while remaining in their native microenvironment, can be reprogrammed to assume a different identity will reveal fundamental insight into cellular plasticity and impact regenerative medicine. To investigate in vivo cell lineage potential, we leveraged the zebrafish as a practical vertebrate platform to determine factors and mechanisms necessary to induce differentiated cells of one germ layer to adopt the lineage of another. We discovered that ectopic co-expression of Sox32 and Oct4 in several non-endoderm lineages, including skeletal muscle, can specifically trigger an early endoderm genetic program in a cell-autonomous manner. Gene expression, live imaging, and functional studies reveal that the endoderm-induced muscle cells lose muscle gene expression and morphology, while specifically gaining endoderm organogenesis lineage markers, such as the pancreatic specification genes, hhex and ptf1a, via a mechanism resembling normal development. Endoderm induction by a pluripotent defective form of Oct4, endoderm markers appearing prior to loss of muscle cell morphology, and a lack of mesoderm, ectoderm, dedifferentiation, and pluripotency gene activation, together, suggests that reprogramming is endoderm specific and occurs via direct transdifferentiation. Our work demonstrates that within a living vertebrate animal, differentiated cells can be induced to directly adopt the identity of a completely unrelated cell lineage, while remaining in a distinct microenvironment, suggesting that differentiated cells in vivo may be more amenable to lineage conversion than previously appreciated. This discovery of extensive lineage potential of differentiated cells, in vivo, challenges our understanding of cell lineage restriction and may pave the way towards new in vivo sources of replacement cells for degenerative diseases such as diabetes.

Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells

Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7–9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.