The Carboxy-Terminal Transactivation Domain of Oct-4 Acquires Cell Specificity through the POU Domain

Abstract

The POU transcription factor Oct-4 is expressed in totipotent and pluripotent cells of the early mouse embryo and the germ cell lineage. Transactivation capacities of regions flanking the DNA binding domain of Oct-4 were analyzed in undifferentiated and differentiated cell lines. The amino-and carboxy-terminal regions (N domain and C domain) fused to the Gal4 DNA binding domain both functioned as transactivation domains in all cell lines tested. However, the C domain failed to activate transcription in some cell lines in the context of the native protein. The underlying regulatory mechanism appears to involve the POU domain of Oct-4 and can discriminate between different POU domains, since constructs in which the C domain was instead fused to the POU domain of Pit-1 were again equally active in all cell lines. These results indicate that the C domain is subject to cell-type-specific regulation mediated by the Oct-4 POU domain. Phosphopeptide analysis revealed that the cell-type-specific difference of C-domain activity correlates with a difference in Oct-4 phosphorylation status. Since Oct-4 is expressed in a variety of distinct cell types during murine embryogenesis, these results suggest an additional regulatory mechanism for determining Oct-4 function in rapidly changing cell types during development.The octamer motif is a regulatory DNA element involved in both ubiquitous and cell-type-specific gene expression and is recognized by a family of eukaryotic transcription factors that share a homologous bipartite DNA binding domain, the POU domain (13,44,52,55). The POU domain consists of two structurally independent subdomains: a 75-amino-acid aminoterminal region specific for this class of transcription factors (POU S ) and a 60-amino-acid carboxy-terminal homeodomain (POU H ). Both subdomains make specific contacts with DNA through a helix-turn-helix structure (17) and are connected by a variable linker that can vary from 15 to 56 amino acids (aa) in length.Regions outside the POU domain are not critical for DNA binding and show little sequence conservation. In many cases,

Stem cell pluripotency and transcription factor Oct4

“…The N-terminal domain (N domain) is rich in proline and acidic residues, while the C-terminal domain (C domain) is rich in proline, serine and threonine residues [7]. The N domain has traditionally been accepted for its role in transactivation [16]. More recent data suggest that the C domain also plays a role in transactivation [17].…”

“…More recent data suggest that the C domain also plays a role in transactivation [17]. Brehm et al replaced the POU DNA binding domain with those from other transcription factors, for example, the heterologous yeast Gal4 DNA binding domain [16]. This replacement did not affect its transactivation function, suggesting that general transactivation function can be transferred to unrelated DNA binding domains.…”

“…This replacement did not affect its transactivation function, suggesting that general transactivation function can be transferred to unrelated DNA binding domains. It was subsequently demonstrated that the activity of Oct4 C domain is cell type specific and is regulated through phosphorylation, whereas the N domain is not [16], [18], [19]. The cell type specificity was observed only if the C domain was linked to the POU domains of Oct4 and Oct-2, but not to Pit-1 or the Gal4 DNA binding domain [16].…”

“…It was subsequently demonstrated that the activity of Oct4 C domain is cell type specific and is regulated through phosphorylation, whereas the N domain is not [16], [18], [19]. The cell type specificity was observed only if the C domain was linked to the POU domains of Oct4 and Oct-2, but not to Pit-1 or the Gal4 DNA binding domain [16]. This finding suggests that Oct4 POU-domain may function differently by serving as interaction sites for cell type-specific regulatory factors.…”

“…The fact that the N domain differs from the C domain in activity and cell type specificity may help explain the functional diversity for Oct4. Furthermore, the C domain may activate certain targets, which do not respond to the N domain during development [16]. Examination of Oct4 structure with respect to its diverse biological function is only the starting point in unraveling the regulatory circuits responsible for maintaining stem cell pluripotency and controlling the differentiation of stem cells to various cell types.…”