Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. Here we report that enhancing RA signaling by expressing RA receptors (RARs) or by RA agonists profoundly promoted reprogramming, but inhibiting it using a RAR-α dominant-negative form completely blocked it. Coexpressing Rarg (RAR-γ) and Lrh-1 (liver receptor homologue 1; Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells to ground-state iPSCs requires only 4 d induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous factor-independent iPSCs, which resembled ground-state mouse ES cells in growth properties, gene expression, and signaling dependency. Our findings demonstrate that signaling through RARs has critical roles in molecular reprogramming and that the synergistic interaction between Rarg and Lrh1 directs reprogramming toward ground-state pluripotency. The human iPSCs described here should facilitate functional analysis of the human genome.

https://www.pnas.org/content/pnas/108/45/18283.full.pdf

Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1

A small set of core transcription factors (TFs) dominates control of the gene expression program in embryonic stem cells and other well-studied cellular models. These core TFs collectively regulate their own gene expression, thus forming an interconnected auto-regulatory loop that can be considered the core transcriptional regulatory circuitry (CRC) for that cell type. There is limited knowledge of core TFs, and thus models of core regulatory circuitry, for most cell types. We recently discovered that genes encoding known core TFs forming CRCs are driven by super-enhancers, which provides an opportunity to systematically predict CRCs in poorly studied cell types through super-enhancer mapping. Here, we use super-enhancer maps to generate CRC models for 75 human cell and tissue types. These core circuitry models should prove valuable for further investigating cell-type-specific transcriptional regulation in healthy and diseased cells.

/pdf/models-of-human-core-transcriptional-regulatory-circuitries-4x979jjooi.pdf

Models of human core transcriptional regulatory circuitries

Pluripotency, a characteristic of cells in the inner cell mass of the mammalian preimplantation blastocyst as well as of embryonic stem cells, is defined as the ability of a cell to generate all of the cell types of an organism. A group of transcription factors is essential for the establishment and maintenance of the pluripotent state. Recent studies have demonstrated that differentiated somatic cells could be reverted to a pluripotent state by the overexpression of a set of transcription factors, further highlighting the significance of transcription factors in the control of pluripotency. Among these factors, a member of the POU transcription factor family, Oct4, is central to the machinery governing pluripotency. Oct4 is highly expressed in pluripotent cells and becomes silenced upon differentiation. Interestingly, the precise expression level of Oct4 determines the fate of embryonic stem cells. Therefore, to control the expression of Oct4 precisely, a variety of regulators function at multiple levels, including transcription, translation of mRNA and post-translational modification. Additionally, in cooperation with Sox2, Nanog and other members of the core transcriptional regulatory circuitry, Oct4 activates both protein-coding genes and noncoding RNAs necessary for pluripotency. Simultaneously, in association with transcriptional repressive complexes, Oct4 represses another set of targets involved in developmental processes. Importantly, Oct4 can re-establish pluripotency in somatic cells, and proper reprogramming of Oct4 expression is indispensable for deriving genuine induced pluripotent stem cell lines. In the past several years, genome-wide identification of Oct4 target genes and Oct4-centered protein interactomes has been reported, indicating that Oct4 exerts tight control over pluripotency regulator expression and protects embryonic stem cells in an undifferentiated state. Nevertheless, further investigation is required to fully elucidate the underlying molecular mechanisms through which Oct4 maintains and reinitiates pluripotency. Systemic and dynamic exploration of the protein complexes and target genes associated with Oct4 will help to elucidate the role of Oct4 more comprehensively.

/pdf/role-of-oct4-in-maintaining-and-regaining-stem-cell-1j8ovhs421.pdf

Role of Oct4 in maintaining and regaining stem cell pluripotency.

Metformin, a Type II diabetic treatment drug, which inhibits transcription of gluconeogenesis genes, has recently been shown to lower the risk of some diabetes-related tumors, including breast cancer. Recently, “cancer stem cells” have been demonstrated to sustain the growth of tumors and are resistant to therapy. To test the hypothesis that metformin might be reducing the risk to breast cancers, the human breast carcinoma cell line, MCF-7, grown in 3-dimensional mammospheres which represent human breast cancer stem cell population, were treated with various known and suspected breast cancer chemicals with and without non-cytotoxic concentrations of metformin. Using OCT4 expression as a marker for the cancer stem cells, the number and size were measured in these cells. Results demonstrated that TCDD (100 nM) and bisphenol A (10 µM) increased the number and size of the mammospheres, as did estrogen (10 nM E2). By monitoring a cancer stem cell marker, OCT4, the stimulation by these chemicals was correlated with the increased expression of OCT4. On the other hand, metformin at 1 and 10 mM concentration dramatically reduced the size and number of mammospheres. Results also demonstrated the metformin reduced the expression of OCT4 in E2 & TCDD mammospheres but not in the bisphenol A mammospheres, suggesting different mechanisms of action of the bisphenol A on human breast carcinoma cells. In addition, these results support the use of 3-dimensional human breast cancer stem cells as a means to screen for potential human breast tumor promoters and breast chemopreventive and chemotherapeutic agents.

/pdf/metformin-represses-self-renewal-of-the-human-breast-444rg8v67p.pdf

Metformin represses self-renewal of the human breast carcinoma stem cells via inhibition of estrogen receptor-mediated OCT4 expression.

Stem cells, stress, metabolism and cancer: a drama in two Octs

Oct4 encodes a transcription factor that is involved in the maintenance of self-renewal in stem cells. Recently, the molecular mechanisms that regulate Oct4 expression have come under investigation. In this study, we demonstrate that the orphan nuclear receptor steroidogenic factor-1 (SF-1) behaves as a transcriptional activator of human Oct4 (hOct4) through direct interaction with a SF-1 binding element in the hOct4 proximal promoter. We found that Oct4 and SF-1 were co-expressed in undifferentiated human embryonal carcinoma NCCIT cells and downregulated during retinoic acid-mediated differentiation. We examined the functional role played by SF-1 in regulation of hOct4 transcription using a luciferase reporter assay and Western blot analysis. Overexpression of SF-1 increased up to about threefold hOct4 promoter activity and endogenous hOct4 protein expression. Sequence analysis of the hOct4 promoter revealed that the transcriptional activity was closely linked to Conserved Regions 1 (CR1) and 2 (CR2), which contain three putative SF-1-binding sites (1st, 2nd, and 3rd SF-1). Binding assays and mutagenesis of binding sites indicated that the 1st and 2nd SF-1 elements (in CR1 and CR2, respectively) might be important cis-regulatory elements in hOct4 promoter activity. However, differences in response to SF-1 overexpression between wild-type and mutant hOct4 promoters revealed that the 1st SF-1 element is the key binding site for SF-1-mediated transcriptional activation. Thus, our data indicate that SF-1 plays a crucial role in the regulation of hOct4 transcription through direct binding to the 1st SF-1 in CR1 of the hOct4 proximal promoter.

Transcriptional regulation of human Oct4 by steroidogenic factor‐1

Octamer-binding transcription factor-4 (Oct4), a member of the POU domain transcription factors, is crucial for both early embryonic development and the maintenance of stem cell pluripotency. The human Oct4 (hOct4) 5' upstream sequence contains four conserved regions (CR1, 2, 3, 4) that are homologous in the murine. In this study, we constructed a series of deletion mutants of the hOct4 5' upstream region and identified cis-regulatory elements that may be important determinants for the transcriptional activity of the hOct4 promoter. Our studies showed that CR2, 3, and 4 each acted as positive cis-regulatory elements in hOct4 promoter activity. We also newly identified a putative negative cis-acting element located between CR1 and CR2. In addition, the sequence -380/-1 at CR1 that contains a GC box was sufficient to provide the minimal promoter activity. Site-directed mutagenesis and electrophoretic mobility shift assays revealed the GC box located in the -380/-1 region may play a critical role in controlling the transcriptional activity of hOct4 by the direct binding of Sp1 or Sp3 transcription factors to the GC box. An overexpression study showed that Sp1 and Sp3 positively and negatively regulate hOct4 promoter activity. Thus, the hOct4 promoter upstream region contains multiple regulatory elements, one of which, the GC box, may be an important cis-regulatory element that regulates the transcription of the hOct4 promoter by the binding of Sp family transcription factors.

Characterization of putative cis-regulatory elements that control the transcriptional activity of the human Oct4 promoter.

Triacylglycerol (TAG), a major energy reserve in lipid form, accumulates mainly in seeds. Although TAG levels are usually low in vegetative tissues because of the repression of seed maturation programs, these programs are derepressed upon the exposure of vegetative tissues to environmental stresses. Metabolic reprogramming of TAG accumulation is driven primarily by transcriptional regulation. A substantial proportion of transcription factors regulating seed TAG biosynthesis also participates in stress-induced TAG accumulation in vegetative tissues. TAG accumulation leads to the formation of lipid droplets (LDs) and plastoglobules (PGs), which play important roles in plant tolerance to environmental stresses. Toxic lipid intermediates generated from environmental stress-induced lipid membrane degradation are captured by TAG-containing LDs and PGs. This review summarizes recent advances in the transcriptional control of metabolic reprogramming underlying stress-induced TAG accumulation, and provides biological insight into the plant adaptive strategy, linking TAG biosynthesis with plant survival.

Transcriptional regulation of triacylglycerol accumulation in plants under environmental stress conditions.

Triacylglycerol (TAG), a major energy reserve in lipid form, accumulates mainly in seeds. Although TAG concentrations are usually low in vegetative tissues because of the repression of seed maturation programs, these programs are derepressed upon the exposure of vegetative tissues to environmental stresses. Metabolic reprogramming of TAG accumulation is driven primarily by transcriptional regulation. A substantial proportion of transcription factors regulating seed TAG biosynthesis also participates in stress-induced TAG accumulation in vegetative tissues. TAG accumulation leads to the formation of lipid droplets and plastoglobules, which play important roles in plant tolerance to environmental stresses. Toxic lipid intermediates generated from environmental-stress-induced lipid membrane degradation are captured by TAG-containing lipid droplets and plastoglobules. This review summarizes recent advances in the transcriptional control of metabolic reprogramming underlying stress-induced TAG accumulation, and provides biological insight into the plant adaptive strategy, linking TAG biosynthesis with plant survival.

Hyun Jin Do

Papers

Transcriptional regulation of human Oct4 by steroidogenic factor‐1

Characterization of putative cis-regulatory elements that control the transcriptional activity of the human Oct4 promoter.

Transcriptional regulation of triacylglycerol accumulation in plants under environmental stress conditions.

Transcriptional regulation of triacylglycerol accumulation in plants under environmental stress conditions.