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The transcription factor Sox2 is a core component of the pluripotency control circuits in the early embryo, and later controls many aspects of neural development. Here, we demonstrate that Sox2 expression in the epiblast (mouse blastoderm) and anterior neural plate (ANP) is determined by the upstream enhancer N2. The mouse enhancer N2 exhibits strong activity in mouse ES cells, epiblast and ANP, and is regulated correctly in chicken and zebrafish embryos. Targeted deletion of this enhancer in mouse embryos caused a large reduction of Sox2 expression to 10% of that of wild-type levels in epiblast and ANP. However, this was tolerated by mouse embryo, probably due to functional compensation by Sox3. The activity of enhancer N2 depends on phylogenetically conserved bipartite POU factor-binding motifs in a 73-bp core sequence that function synergistically, but this activation does not involve Sox2. The major POU factor expressed at the epiblastic stage is Pou5f1 (Oct3/4), while those in the anterior neural plate are Pou3f factors (Oct6, Brn2 etc.). These factors are gradually exchanged during the transition from epiblast to ANP stages in mouse embryos and epiblast stem cells (EpiSC). Consistently, enhancer N2 activity changes from full Pou5f1 dependence to Pou3f dependence during the development of neural plate cells (NPC) from EpiSC, as assessed by specific POU factor knockdown in these cells. Zebrafish mutant embryos completely devoid of Pou5f1 activity failed to activate enhancer N2 and to express Sox2 in the blastoderm and ANP, and these defects were rescued by exogenous supply of pou5f1. Previously, Pou5f1-Sox2 synergism-dependent Sox2 activation through enhancer SRR2 in ES cells has been highlighted, but this mechanism is limited to ES cells and amniotes. In contrast, the enhancer N2-mediated, POU factor-dependent activation of Sox2, without involvement of Sox2, is a phylogenetically conserved core mechanism that functions in gene regulatory networks at early embryonic stages.

Hemoprotein H-450 was purified from rat liver cytosol to homogeneity by an improved procedure. The purified H-450 showed a subunit molecular weight of 64,000 daltons and contained 0.7-0.9 mol of protoheme per mol subunit. Among rat tissues examined, liver and kidney contained significant amounts of H-450 in the cytosol. Oxidized H-450 showed a Soret peak at 428 nm and a broad beta band at around 550 nm. Reduced H-450 was found to exist in two interconvertible forms, alkaline and acid forms. The alkaline form showed Soret, beta, and alpha peaks at 448, 540, and 571 nm, whereas the acid form showed Soret, beta, and alpha peaks at 425, 530, and 558 nm. The spectral properties of both oxidized and reduced H-450 in alkaline medium resemble those of cytochrome P-450 having a nitrogenous ligand at the 6th coordination position of the heme. Upon addition of low concentrations of HgCl2, H-450 was converted to a denatured form both in the oxidized and the reduced states and lost its unique spectral characteristics. Addition of carbon monoxide to reduced H-450 produced a new spectral species which resembled that of the reduced carbon monoxide complex of P-420, a denatured form of cytochrome P-450. Comparison of the EPR signal of oxidized H-450 with those of a cytochrome P-450, P-450(PB-1), and several model compounds indicated the presence of a thiolate anion at the 5th coordination position of the heme of H-450. Judging from EPR data, oxidized H-450 also converts between acid and alkaline forms, whose signals were observed at g = 1.867, 2.31, and 2.507 and at g = 1.910, 2.28, and 2.424, respectively. These lines of evidence indicate that the 5th and 6th coordination positions of the heme of H-450 are a thiolate and a nitrogenous group, respectively. With respect to the heme environments, H-450 is a member of the cytochrome P-450 family, and has a nitrogenous ligand at the 6th coordination position of the heme.

To determine the orthology of sterol 14-demethylase (P45014DM), the only known P450 enzyme distributed widely in eukaryotes with a conserved metabolic role, the full-length amino acid sequences of rat and human P45014DMs were determined from the cloned cDNA sequences, and compared with those of the corresponding fungal proteins (CYP51). The amino acid identity value between given pairs of P45014DMs ranged from 93% (human/rat) to 39% (human or rat/Saccharomyces cerevisiae). All the P45014DMs formed a single cluster in a phylogenetic tree constructed from representative P450 protein sequences currently available. The nearest neighbors to the P45014DM cluster in the phylogenetic tree were CYP7 (cholesterol 7 alpha-hydroxylase) and CYP8 (prostacyclin synthase), and the divergence point of fungal and mammalian P45014DMs was clearly more recent than that of P45014DM and CYP7/CYP8. These lines of evidence show that fungal and mammalian P45014DMs are really orthologous. This is the first example of orthologous P450s occurring in distinct kingdoms. P45014DM may be an ancient P450 which arose before the divergence of major eukaryotic branches and has been conserved throughout evolution. The amino acid identity value (93%) between human and rat P45014DMs was comparable to those observed for some housekeeping enzymes. In addition, a processed pseudogene of P45014DM was found in a rat genomic DNA library, suggesting the expression of P45014DM in germ line cells. These facts suggest that P45014DM may be a housekeeping enzyme essential for the viability of mammals.

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