The pluripotency of embryonic stem (ES) cells is thought to be maintained by a few key transcription factors, including Oct3/4 and Sox2. The function of Oct3/4 in ES cells has been extensively characterized, but that of Sox2 has yet to be determined. Sox2 can act synergistically with Oct3/4 in vitro to activate Oct-Sox enhancers, which regulate the expression of pluripotent stem cell-specific genes, including Nanog, Oct3/4 and Sox2 itself. These findings suggest that Sox2 is required by ES cells for its Oct-Sox enhancer activity. Using inducible Sox2-null mouse ES cells, we show that Sox2 is dispensable for the activation of these Oct-Sox enhancers. In contrast, we demonstrate that Sox2 is necessary for regulating multiple transcription factors that affect Oct3/4 expression and that the forced expression of Oct3/4 rescues the pluripotency of Sox2-null ES cells. These results indicate that the essential function of Sox2 is to stabilize ES cells in a pluripotent state by maintaining the requisite level of Oct3/4 expression.
Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4) as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3) and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21) as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.
Sox-2 is a transcriptional cofactor expressed in embryonic stem (ES) cells as well as in neuronal cells. It has been demonstrated that Sox-2 plays an important role in supporting gene expression in ES cells, especially by forming a complex with embryonic Octamer factor, Oct-3/4. Here, we have analyzed the regulatory regions of the Sox-2 gene and identified two enhancers which stimulate transcription in ES cells as well as in embryonal carcinoma cells. These regulatory regions, which we termed Sox regulatory regions (SRR) 1 and 2, exert their function specifically when cells are in an undifferentiated state. Interestingly, like the regulatory elements of FGF-4 and UTF1 genes, combinatorial action of Octamer and Sox-2 binding sites support the SRR2 activity. However, biochemical analyses reveal that, due to the unique sequence and/or its organization, the SRR2 bears distinct characteristics from those of FGF-4 and UTF1 regulatory elements. That is, unlike the FGF-4 gene enhancer, the SRR2 precludes the binding of the Oct-1-Sox-2 complex. The difference between the SRR2 and UTF1 regulatory element is in the ability of SRR2 to recruit the Oct-6-Sox-2 complex as well as the Oct-3/4-Sox-2 complex. Co-transfection analyses confirm that both complexes are able to stimulate transcription through the SRR2 element.
The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of Pcgf6 in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells.DOI:
http://dx.doi.org/10.7554/eLife.21064.001
UTF1 is a transcriptional coactivator which has recently been isolated and found to be expressed mainly in pluripotent embryonic stem (ES) cells (A. Okuda, A. Fukushima, M. Nishimoto, et al., EMBO J. 17:2019-2032, 1998). To gain insight into the regulatory network of gene expression in ES cells, we have characterized the regulatory elements governing UTF1 gene expression. The results indicate that the UTF1 gene is one of the target genes of an embryonic octamer binding transcription factor, Oct-3/4. UTF1 expression is, like the FGF-4 gene, regulated by the synergistic action of Oct-3/4 and another embryonic factor, Sox-2, implying that the requirement for Sox-2 by Oct-3/4 is not limited to the FGF-4 enhancer but is rather a general mechanism of activation for Oct-3/4. Our biochemical analyses, however, also reveal one distinct difference between these two regulatory elements: unlike the FGF-4 enhancer, the UTF1 regulatory element can, by its one-base difference from the canonical octamer-binding sequence, selectively recruit the complex comprising Oct-3/4 and Sox-2 and preclude the binding of the transcriptionally inactive complex containing Oct-1 or Oct-6. Furthermore, our analyses reveal that these properties are dictated by the unique ability of the Oct-3/4 POU-homeodomain that recognizes a variant of the Octamer motif in the UTF1 regulatory element.
Corresponding author A.Okuda and A.Fukushima contributed equally to this workWe have obtained a novel transcriptional cofactor, termed undifferentiated embryonic cell transcription factor 1 (UTF1), from F9 embryonic carcinoma (EC) cells. This protein is expressed in EC and embryonic stem cells, as well as in germ line tissues, but could not be detected in any of the other adult mouse tissues tested. Furthermore, when EC cells are induced to differentiate, UTF1 expression is rapidly extinguished. In normal mouse embryos, UTF1 mRNA is present in the inner cell mass, the primitive ectoderm and the extra-embryonic tissues. During the primitive streak stage, the induction of mesodermal cells is accompanied by the down-regulation of UTF1 in the primitive ectoderm. However, its expression is maintained for up to 13.5 days post-coitum in the extra-embryonic tissue. Functionally, UTF1 boosts the level of transcription of the adenovirus E2A promoter. However, unlike the pluripotent cell-specific E1A-like activity, which requires the E2F sites of the E2A promoter for increased transcriptional activation, UTF1-mediated activation is dependent on the upstream ATF site of this promoter. This result indicates that UTF1 is not a major component of the E1A-like activity present in pluripotent embryonic cells. Further analyses revealed that UTF1 interacts not only with the activation domain of ATF-2, but also with the TFIID complex in vivo. Thus, UTF1 displays many of the hallmark characteristics expected for a tissue-specific transcriptional coactivator that works in early embryogenesis.
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