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...

Oct3/4, a hallmark of the earliest stages of embryogenesis, is expressed in undifferentiated embryonal carcinoma (EC) and embryonic stem (ES) cells. Oct3/4 gene expression is dependent on the promoter region, the proximal enhancer and the newly identified distal enhancer. We have analysed in vivo occupancy of these elements. In undifferentiated EC and ES cells, strong footprints were detected at specific sites of all three regulatory elements. These were promptly lost upon RA treatment in ES cells and in P19 EC cells, in parallel with sharply reduced Oct3/4 mRNA levels. Thus, the occupancy of regulatory elements is coupled with Oct3/4 expression, and RA treatment causes coordinated factor displacement, leading to extinction of gene activity. In F9 EC cells, footprint was first abolished at the proximal enhancer. However, this loss of binding site occupancy did not result in a decrease in Oct3/4 mRNA levels. The partial factor displacement seen in F9 EC cells, combined with the observation that EC and ES cells utilize the proximal and distal enhancers in differential manner, indicate the complex pattern of Oct3/4 gene regulation, which could reflect a cell type‐ and lineage‐specific expression of the gene in vivo.

Activation of transcription by Oct-4 from remote binding sites requires a cofactor that is restricted to embryonal stem cells. The adenovirus E1A protein can mimic the activity of this stem cell-specific factor and stimulates Oct-4 activity in differentiated cells. Here we characterize the Oct-4-E1A interaction and show that the E1A 289R protein harbors two independent Oct-4 binding sites, both of which specifically interact with the POU domain of Oct-4. Furthermore, we demonstrate that, like E1A, the human papillomavirus E7 oncoprotein also specifically binds to the Oct-4 POU domain. E7 and Oct-4 can form a complex both in vitro and in vivo. Expression of E7 in differentiated cells stimulates Oct-4-mediated transactivation from distal binding sites. Moreover, Oct-4, but not other Oct factors, is active when expressed in cells transformed by human papillomavirus. Our results suggest that different viruses have evolved oncoproteins that share the ability to target Oct-4 and to mimic a stem cell-specific activity.

The human helper-dependent adeno-associated virus type 2 (AAV-2) has been shown to induce differentiation in various cell types in culture including pluripotent embryonic cells, in the absence of helper virus. To assess whether induction of differentiation may influence developmental processes we analysed the effect of AAV-2 on developing mouse embryos. In vitro infection of fertilized eggs induced arrest of development at the twocell stage. Moreover, micro injection of AAV-2 DNA (comprising either the complete AAV-2 genome or a fragment containing the P5 promoter region) into onecell embryos, blocked development at the morula stage. In vivo, AAV-2 infection of pregnant mice led to fetal death and early abortion. These results demonstrate that the human adeno-associated virus, which is thought to be non-pathogenic, can perturb embryonic development in mice. This may provide a suitable animal model system to further elucidate the biological significance of the recent detection of adeno-associated virus DNA in human abortion material.