New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2

Abstract

The POU transcription factor Oct-4 is expressed specifically in the germ line, pluripotent cells of the pregastrulation embryo and stem cell lines derived from the early embryo. Osteopontin (OPN) is a protein secreted by cells of the preimplantation embryo and contains a GRGDS motif that can bind to specific integrin subtypes and modulate cell adhesion/migration. We show that Oct-4 and OPN are coexpressed in the preimplantation mouse embryo and during differentiation of embryonal cell lines. Immunoprecipitation of the first intron of OPN (i-opn) from covalently fixed chromatin of embryonal stem cells by Oct-4-specific antibodies indicates that Oct-4 binds to this fragment in vivo. The i-opn fragment functions as an enhancer in cell lines that resemble cells of the preimplantation embryo. Furthermore, it contains a novel palindromic Oct factor recognition element (PORE) that is composed of an inverted pair of homeodomain-binding sites separated by exactly 5 bp (ATTTG +5 CAAAT). POU proteins can homo-and heterodimerize on the PORE in a configuration that has not been described previously. Strong transcriptional activation of the OPN element requires an intact PORE. In contrast, the canonical octamer overlapping with the downstream half of the PORE is not essential. Sox-2 is a transcription factor that contains an HMG box and is coexpressed with Oct-4 in the early mouse embryo. Sox-2 represses Oct-4 mediated activation of i-opn by way of a canonical Sox element that is located close to the PORE. Repression depends on a carboxy-terminal region of Sox-2 that is outside of the HMG box. Expression, DNA binding, and transactivation data are consistent with the hypothesis that OPN expression is regulated by Oct-4 and Sox-2 in preimplantation development.[Key Words: POU; Oct; Sox; osteopontin; preimplantation embryo] Received March 9, 1998; revised version accepted April 14, 1998.The fertilized oocyte undergoes cleavage until a uniform cluster of cells, the morula, is formed. The first apparent differentiation occurs as the inner cell mass (ICM) separates from the trophectoderm during blastocoel formation (Gardner 1983). Trophectoderm refers to the epithelial cell layer that encloses the ICM and blastocoel. Subsequently, cells dissociate from the ICM and cover its blastocoelic surface to form the hypoblast (also called primitive endoderm). These cells do not form a welldefined polarized epithelium, eventually loose cell contacts, and contain an extensive rough endoplasmatic reticulum, which is often swollen with secretory material (Nadijcka and Hillman 1974). The hypoblast differentiates into the parietal and visceral endoderms (Gardner 1983). Parietal endoderm cells form from hypoblast precursors that migrate and adhere to the thin basal lamina on the inner surface of the trophectoderm. In contrast, visceral endoderm cells do not migrate and consists of a columnar epithelial layer surrounding the late ICM or early epiblast (also called primitive endoderm). Relatively little is known about the molecular signals guiding ce...

Establishment of a new embryonic stem cell line derived from C57BL/6 mouse expressing EGFP ubiquitously

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Distinction between AP1 and NF-E2 factor-binding at specific chromatin regions in mammalian cells

“…Furthermore, Drosphila or yeast cells were analysed in most cases (Orlando and Paro, 1993;Walter and Biggin, 1996;Rundlett et al, 1998), which have much smaller genome complexities than the mammals. More recently, the above approach has been used for the study of Oct-4 factor binding in the Osteopontin enhancer (Botquin et al, 1998), and for the demonstration of association of transcriptional activators with virus-induced promoters in vivo (Wathelet et al, 1998). The reciprocal binding patterns of the AP1 and NF-E2 factors in the c-jun promoter and the b-globin-LCR in K562 cells (Figures 2 and 3) have clearly demonstrated the feasibility of the strategy outlined in Figure 2A for determining the identity of nuclear factor(s) bound in vivo at speci®c motif(s) to which a family of related DNA-binding factors could recognize and interact with.…”

Oct4 distribution and level in mouse clones: consequences for pluripotency

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