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The proliferation and differentiation of skeletal muscle cells are usually controlled by serum components. Myogenic differentiation is induced by a reduction of serum components in vitro. It has been recently reported that serum contains not only various growth factors with specific actions on the proliferation and differentiation of myogenic cells, but also exogenous exosomes, the function of which is poorly understood in myogenesis. We have found that exosomes in fetal bovine serum are capable of exerting an inhibitive effect on the differentiation of C2C12 myogenic cells in vitro. In this process of inhibition, the downregulation of Tceal5 and Tceal7 genes was observed. Expression of these genes is specifically increased in direct proportion to myogenic differentiation. Loss- or gain- of function studies with Tceal5 and Tceal7 indicated that they have the potential to regulate myogenic differentiation via exosomes in fetal bovine serum.

In vivo reprogramming provokes a wide range of cell fate conversion. Here, we discover that in vivo induction of higher levels of OSKM in mouse somatic cells leads to increased expression of primordial germ cell (PGC)-related genes and provokes genome-wide erasure of genomic imprinting, which takes place exclusively in PGCs. Moreover, the in vivo OSKM reprogramming results in development of cancer that resembles human germ cell tumors. Like a subgroup of germ cell tumors, propagated tumor cells can differentiate into trophoblasts. Moreover, these tumor cells give rise to induced pluripotent stem cells (iPSCs) with expanded differentiation potential into trophoblasts. Remarkably, the tumor-derived iPSCs are able to contribute to non-neoplastic somatic cells in adult mice. Mechanistically, DMRT1, which is expressed in PGCs, drives the reprogramming and propagation of the tumor cells in vivo. Furthermore, the DMRT1-related epigenetic landscape is associated with trophoblast competence of the reprogrammed cells and provides a therapeutic target for germ cell tumors. These results reveal an unappreciated route for somatic cell reprogramming and underscore the impact of reprogramming in development of germ cell tumors., (© 2021. The Author(s).)

Sex determination of germ cells is vital to creating the sexual dichotomy of germ cell development, thereby ensuring sexual reproduction. However, the underlying mechanisms remain unclear. Here, we show that ZGLP1, a conserved transcriptional regulator with GATA-like zinc fingers, determines the oogenic fate in mice. ZGLP1 acts downstream of bone morphogenetic protein, but not retinoic acid (RA), and is essential for the oogenic program and meiotic entry. ZGLP1 overexpression induces differentiation of in vitro primordial germ cell-like cells (PGCLCs) into fetal oocytes by activating the oogenic programs repressed by Polycomb activities, whereas RA signaling contributes to oogenic program maturation and PGC program repression. Our findings elucidate the mechanism for mammalian oogenic fate determination, providing a foundation for promoting in vitro gametogenesis and reproductive medicine., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

During somatic cell reprogramming to induced pluripotent stem cells (iPSCs), fibroblasts undergo dynamic molecular changes, including a mesenchymal-to-epithelial transition (MET) and gain of pluripotency; processes that are influenced by Yamanaka factor stoichiometry. For example, in early reprogramming, high KLF4 levels are correlated with the induction of functionally undefined, transiently expressed MET genes. Here, we identified the cell-surface protein TROP2 as a marker for cells with transient MET induction in the high-KLF4 condition. We observed the emergence of cells expressing the pluripotency marker SSEA-1 + mainly from within the TROP2 + fraction. Using TROP2 as a marker in CRISPR/Cas9-mediated candidate screening of MET genes, we identified the transcription factor OVOL1 as a potential regulator of an alternative epithelial cell fate characterized by the expression of non-iPSC MET genes and low cell proliferation. Our study sheds light on how reprogramming factor stoichiometry alters the spectrum of intermediate cell fates, ultimately influencing reprogramming outcomes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Naive pluripotent stem cells (PSCs) utilize both glycolysis and oxidative phosphorylation (OXPHOS) to satisfy their metabolic demands. However, it is unclear how somatic cells acquire this hybrid energy metabolism during reprogramming toward naive pluripotency. Here, we show that when transduced with Oct4, Sox2, and Klf4 (OSK) into murine fibroblasts, Zic3 and Esrrb synergistically enhance the reprogramming efficiency by regulating cellular metabolic pathways. These two transcription factors (TFs) cooperatively activate glycolytic metabolism independently of hypoxia inducible factors (HIFs). In contrast, the regulatory modes of the TFs on OXPHOS are antagonistic: Zic3 represses OXPHOS, whereas Esrrb activates it. Therefore, when introduced with Zic3, Esrrb restores OXPHOS activity, which is essential for efficient reprogramming. In addition, Esrrb-mediated OXPHOS activation is critical for the conversion of primed PSCs into the naive state. Our study suggests that the combinatorial function of TFs achieves an appropriate balance of metabolic pathways to induce naive PSCs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Primordial germ cells (PGCs) are the foundation of totipotency and vital for reproduction and heredity. PGCs in mice arise from the epiblast around Embryonic Day (E) 7.0, migrate through the hindgut endoderm, and colonize and proliferate in the embryonic gonads until around E13.5 prior to their differentiation either into prospermatogonia or oogonia. PRDM1, a transcriptional repressor, plays an essential role in PGC specification that includes robustly repressing a somatic mesodermal program. Using an inducible conditional knockout system, we show here that PRDM1 is critically required throughout PGC development. When Prdm1 was deleted in migrating PGCs at E9.5 or E10.5, or in male gonadal PGCs at E11.5, PGCs were eliminated by apoptosis from around E10.5, E11.5, or E13.5, respectively. When Prdm1 was deleted in female gonadal PGCs at E11.5, PGCs progressed into the first meiotic prophase in an apparently normal fashion, but the oogonia exhibited an aberrant pachytene phenotype, undergoing abrupt apoptosis from around E16.5. The escape of a fraction of PGCs (∼10%) from the Prdm1 deletion was sufficient to recover fairly normal germ cell pools, both in male and female adults. The key targets of PRDM1 in migrating and/or gonadal PGCs, including genes for development, apoptosis, and prospermatogonial differentiation, showed only a modest overlap with those upon PGC specification, and were enriched with histone H3 lysine 27 trimethylation (H3K27me3). Our findings provide critical insight into the mechanism for maintaining the transcriptional integrity of PGCs., (© 2016 by the Society for the Study of Reproduction, Inc.)

It remains controversial whether the routes from somatic cells to induced pluripotent stem cells (iPSCs) are related to the reverse order of normal developmental processes. Specifically, it remains unaddressed whether or not the differentiated cells become iPSCs through their original tissue stem cell-like state. Previous studies analyzing the reprogramming process mostly used fibroblasts; however, the stem cell characteristics of fibroblasts made it difficult to address this. Here, we generated iPSCs from mouse astrocytes, a type of glial cells, by three (OCT3/4, KLF4, and SOX2), two (OCT3/4 and KLF4), or four (OCT3/4, KLF4, and SOX2 plus c-MYC) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently up-regulated during reprogramming, and Sox1-positive cells become iPSCs. The up-regulation of Sox1 is essential for OCT3/4- and KLF4-induced reprogramming. Genome-wide analysis revealed that the gene expression profile of Sox1-expressing intermediate-state cells resembles that of NSCs. Furthermore, the intermediate-state cells are able to generate neurospheres, which can differentiate into both neurons and glial cells. Remarkably, during fibroblast reprogramming, neither Sox1 up-regulation nor an increase in neurogenic potential occurs. Our results thus demonstrate that astrocytes are reprogrammed through an NSC-like state., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Germ cell specification is accompanied by epigenetic remodeling, the scale and specificity of which are unclear. Here, we quantitatively delineate chromatin dynamics during induction of mouse embryonic stem cells (ESCs) to epiblast-like cells (EpiLCs) and from there into primordial germ cell-like cells (PGCLCs), revealing large-scale reorganization of chromatin signatures including H3K27me3 and H3K9me2 patterns. EpiLCs contain abundant bivalent gene promoters characterized by low H3K27me3, indicating a state primed for differentiation. PGCLCs initially lose H3K4me3 from many bivalent genes but subsequently regain this mark with concomitant upregulation of H3K27me3, particularly at developmental regulatory genes. PGCLCs progressively lose H3K9me2, including at lamina-associated perinuclear heterochromatin, resulting in changes in nuclear architecture. T recruits H3K27ac to activate BLIMP1 and early mesodermal programs during PGCLC specification, which is followed by BLIMP1-mediated repression of a broad range of targets, possibly through recruitment and spreading of H3K27me3. These findings provide a foundation for reconstructing regulatory networks of the germline epigenome., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Hedgehog pathway is activated in various types of malignancies. We previously reported that inhibition of SMO or GLI prevents osteosarcoma growth in vitro and in vivo. Recently, it has been reported that arsenic trioxide (ATO) inhibits cancer growth by blocking GLI transcription. In this study, we analyzed the function of ATO in the pathogenesis of osteosarcoma. Real-time PCR showed that ATO decreased the expression of Hedgehog target genes, including PTCH1, GLI1, and GLI2, in human osteosarcoma cell lines. WST-1 assay and colony formation assay revealed that ATO prevented osteosarcoma growth. These findings show that ATO prevents GLI transcription and osteosarcoma growth in vitro. Flow cytometric analysis showed that ATO promoted apoptotic cell death. Comet assay showed that ATO treatment increased accumulation of DNA damage. Western blot analysis showed that ATO treatment increased the expression of γH2AX, cleaved PARP, and cleaved caspase-3. In addition, ATO treatment decreased the expression of Bcl-2 and Bcl-xL. These findings suggest that ATO treatment promoted apoptotic cell death caused by accumulation of DNA damage. In contrast, Sonic Hedgehog treatment decreased the expression of γH2AX induced by cisplatin treatment. ATO re-induced the accumulation of DNA damage attenuated by Sonic Hedgehog treatment. These findings suggest that ATO inhibits the activation of Hedgehog signaling and promotes apoptotic cell death in osteosarcoma cells by accumulation of DNA damage. Finally, examination of mouse xenograft models showed that ATO administration prevented the growth of osteosarcoma in nude mice. Because ATO is an FDA-approved drug for treatment of leukemia, our findings suggest that ATO is a new therapeutic option for treatment of patients with osteosarcoma.

Transcription factors (TFs) are able to regulate differentiation-related processes, including dedifferentiation and direct conversion, through the regulation of cell type-specific transcriptional profiles. However, the functional interactions between the TFs regulating different transcriptional profiles are not well understood. Here, we show that the TFs capable of inducing cell type-specific transcriptional profiles prevent the dedifferentiation induced by TFs for pluripotency. Of the large number of TFs expressed in a neural-lineage cell line, we identified a subset of TFs that, when overexpressed, strongly interfered with the dedifferentiation triggered by the procedure to generate induced pluripotent stem cells. This interference occurred through a maintenance mechanism of the cell type-specific transcriptional profile. Strikingly, the maintenance activity of the interfering TF set was strong enough to induce the cell line-specific transcriptional profile when overexpressed in a heterologous cell type. In addition, the TFs that interfered with dedifferentiation in hepatic-lineage cells involved TFs with known induction activity for hepatic-lineage cells. Our results suggest that dedifferentiation suppresses a cell type-specific transcriptional profile, which is primarily maintained by a small subset of TFs capable of inducing direct conversion. We anticipate that this functional correlation might be applicable in various cell types and might facilitate the identification of TFs with induction activity in efforts to understand differentiation.

Background: Although the HIV-1 Nef protein (27 kDa) localizes primarily in cytoplasm, there is considerable evidence suggesting its occasional localization in the nucleus. Nef is known to play an important role in transcriptional events and viral replication, but the actual target of Nef in the nucleus remains to be identified., Objective: To examine the functional roles of Nef in the nucleus and its possible interactions with other unknown factors in the nucleus., Methods: High-density microarray analysis was used to screen directly the unique functions of Nef on host gene transcription. The nuclear localization of Nef and its effects on the expression of peroxisome proliferator-activated receptors (PPAR) was examined using PPAR promoter/reporter assay and immunoblotting. A long terminal repeat/reporter assay was used to investigated the effects of Nef and PPAR on viral transcription., Results: Nef in the nucleus suppressed PPAR gamma expression and reduced fatty acid levels in human T and macrophage cell lines. Expression of Nef or PPAR suppressed viral replication; the effect of PPAR gamma or retinoid X receptor-alpha on viral replication were reduced by coexpression of Nef in MT(-)4 T cells., Conclusion: Nef may be involved in both viral replication and the wasting syndrome associated with AIDS.

The squamous–columnar junction (SCJ) is a boundary consisting of precisely positioned transitional epithelium between the squamous and columnar epithelium. Transitional epithelium is a hotspot for precancerous lesions, and is therefore clinically important; however, the origins and physiological properties of transitional epithelium have not been fully elucidated. Here, by using mouse genetics, lineage tracing, and organoid culture, we examine the development of the SCJ in the mouse stomach, and thus define the unique features of transitional epithelium. We find that two transcription factors, encoded by Sox2 and Gata4, specify primitive transitional epithelium into squamous and columnar epithelium. The proximal–distal segregation of Sox2 and Gata4 expression establishes the boundary of the unspecified transitional epithelium between committed squamous and columnar epithelium. Mechanistically, Gata4-mediated expression of the morphogen Fgf10 in the distal stomach and Sox2-mediated Fgfr2 expression in the proximal stomach induce the intermediate regional activation of MAPK/ERK, which prevents the differentiation of transitional epithelial cells within the SCJ boundary. Our results have implications for tissue regeneration and tumorigenesis, which are related to the SCJ. How boundary regions form in the epithelia of tissues, for example, the squamous–columnar junction is unclear. Here, the authors identify the transcription factors Sox2 and Gata4 as specifying the transitional epithelium in the murine squamous–columnar junction region in the stomach epithelia. [ABSTRACT FROM AUTHOR]

Muscle satellite cells (SCs) are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs) isolated from Pax3GFP/+ embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3GFP/+ mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease. [ABSTRACT FROM AUTHOR]

Summary: Intermittent fasting is one of the most effective dietary restriction regimens that extend life span in C. elegans and mammals. Fasting-stimulus responses are key to the longevity response; however, the mechanisms that sense and transduce the fasting stimulus remain largely unknown. Through a comprehensive transcriptome analysis in C. elegans, we find that along with the FOXO transcription factor DAF-16, AP-1 (JUN-1/FOS-1) plays a central role in fasting-induced transcriptional changes. KGB-1, one of the C. elegans JNKs, acts as an activator of AP-1 and is activated in response to fasting. KGB-1 and AP-1 are involved in intermittent fasting-induced longevity. Fasting-induced upregulation of the components of the SCF E3 ubiquitin ligase complex via AP-1 and DAF-16 enhances protein ubiquitination and reduces protein carbonylation. Our results thus identify a fasting-responsive KGB-1/AP-1 signaling pathway, which, together with DAF-16, causes transcriptional changes that mediate longevity, partly through regulating proteostasis. [Copyright &y& Elsevier]

Summary: In skeletal muscle differentiation, muscle-specific genes are regulated by two groups of transcription factors, the MyoD and MEF2 families, which work together to drive the differentiation process. Here, we show that ERK5 regulates muscle cell fusion through Klf transcription factors. The inhibition of ERK5 activity suppresses muscle cell fusion with minimal effects on the expression of MyoD, MEF2, and their target genes. Promoter analysis coupled to microarray assay reveals that Klf-binding motifs are highly enriched in the promoter regions of ERK5-dependent upregulated genes. Remarkably, Klf2 and Klf4 expression are also upregulated during differentiation in an ERK5-dependent manner, and knockdown of Klf2 or Klf4 specifically suppresses muscle cell fusion. Moreover, we show that Sp1 transcription factor links ERK5 to Klf2/4, and that nephronectin, a Klf transcriptional target, is involved in muscle cell fusion. Therefore, an ERK5/Sp1/Klf module plays a key role in the fusion process during skeletal muscle differentiation. [Copyright &y& Elsevier]

Background: The cellular and molecular mechanisms of tumour response following chemotherapy are largely unknown. We found that low dose anti-tumour agents up-regulate early growth response 1 (EGR1) expression. EGR1 is a member of the immediate-early gene group of transcription factors which modulate transcription of multiple genes involved in cell proliferation, differentiation, and development. It has been reported that EGR1 act as either tumour promoting factor or suppressor. We therefore examined the expression and function of EGR1 in osteosarcoma. Methods: We investigated the expression of EGR1 in human osteosarcoma cell lines and biopsy specimens. We next examined the expression of EGR1 following anti-tumour agents treatment. To examine the function of EGR1 in osteosarcoma, we assessed the tumour growth and invasion in vitro and in vivo. Results: Real-time PCR revealed that EGR1 was down-regulated both in osteosarcoma cell lines and osteosarcoma patients' biopsy specimens. In addition, EGR1 was up-regulated both in osteosarcoma patient' specimens and osteosarcoma cell lines following anti-tumour agent treatment. Although forced expression of EGR1 did not prevent osteosarcoma growth, forced expression of EGR1 prevented osteosarcoma cell invasion in vitro. In addition, forced expression of EGR1 promoted down-regulation of urokinase plasminogen activator, urokinase receptor, and urokinase plasminogen activity. Xenograft mice models showed that forced expression of EGR1 prevents osteosarcoma cell migration into blood vessels. Conclusions: These findings suggest that although chemotherapy could not prevent osteosarcoma growth in chemotherapy-resistant patients, it did prevent osteosarcoma cell invasion by down-regulation of urokinase plasminogen activity via up-regulation of EGR1 during chemotherapy periods. [ABSTRACT FROM AUTHOR]

Nuclear factor-κB (NF-κB) is an important transcription factor involved in various biological functions, including tumorigenesis. Hence, NF-κB has attracted attention as a target factor for cancer treatment, leading to the development of several inhibitors. However, existing NF-κB inhibitors do not discriminate between its subunits, namely, RelA, RelB, cRel, p50, and p52. Conventional methods used to evaluate interactions between transcription factors and DNA, such as electrophoretic mobility shift assay and luciferase assays, are unsuitable for high-throughput screening (HTS) and cannot distinguish NF-κB subunits. We developed a HTS method named DNA strand exchange fluorescence resonance energy transfer (DSE-FRET). This assay is suitable for HTS and can discriminate a NF-κB subunit. Using DSE-FRET, we searched for RelA-specific inhibitors and verified RelA inhibition for 32,955 compounds. The compound A55 (2-(3-carbamoyl-6-hydroxy-4-methyl-2-oxopyridin-1(2H)-yl) acetic acid) selectively inhibited RelA–DNA binding. We propose that A55 is a seed compound for RelA-specific inhibition and could be used in clinical applications. [ABSTRACT FROM AUTHOR]

ES cell (ESC) identity is stably maintained through the coordinated regulation of transcription factors and chromatin structure. SMARCB1, also known as INI1, SNF5, BAF47, is one of the subunits of SWI/SNF (BAF) complexes that play a crucial role in regulating gene expression by controlling chromatin dynamics. Genetic ablation of Smarcb1 in mice leads to embryonic lethality at the peri-implantation stage, indicating that Smarcb1 is important for the early developmental stages. However, the role of SMARCB1 in the maintenance of the ESC identity remains unknown. Here we established mouse ESCs lacking Smarcb1 and investigated the effect of Smarcb1 ablation on the differentiation propensity of ESCs. We found an increased expression of trophectoderm-related genes including Cdx2 in Smarcb1 -deficient ESCs. Consistently, they exhibited an extended differentiation propensity into the trophectoderm lineage cells in teratomas. However, although Smarcb1 -deficient cells were infrequently incorporated into the trophectoderm cell layer of blastocysts, they failed to contribute to mature placental tissues in vivo. Furthermore, Smarcb1 -deficient cells exhibited a premature differentiation in the neural tissue of E14.5 chimeric embryos. Notably, we found that binding motifs for CTCF, which is involved in the maintenance of genomic DNA architecture was significantly enriched in chromatin regions whose accessibility was augmented in Smarcb1 -deficient cells, while those for pluripotency factors were overrepresented in regions which have more closed structure in those cells. Collectively, we propose that SMARCB1-mediated remodeling of chromatin landscapes is important for the maintenance and differentiation of ESCs. • Smarcb1 -deficient ES cells exhibit an increased expression of trophectoderm-related genes including Cdx2. • Smarcb1 -deficient ES cells retain pluripotency but exhibit an impaired differentiation in vivo. • SMARCB1-mediated remodeling of chromatin landscapes may be important for the maintenance and differentiation of ESCs. [ABSTRACT FROM AUTHOR]