Transcription Factor AP-2γ Regulates Murine Adenosine Deaminase Gene Expression during Placental Development

Abstract

Trophoblast cells are specialized extra-embryonic cells present only in eutherian mammals. They play a major role in the implantation and placentation processes. To understand better the molecular mechanisms that control the development and function of trophoblast cells, we sought to identify the transcription factors that regulate murine adenosine deaminase (ADA) gene expression in the placenta. Here we report a detailed characterization of a placenta-specific footprinting region (FP1) in the Ada placental regulatory element. The sequence of FP1 was mapped by DNase I footprinting and was found to match a consensus AP-2 transcription factor-binding site. Electrophoretic mobility shift assays demonstrated that FP1 interacted with AP-2-like proteins. Further analysis using AP-2 antibody confirmed that AP-2 protein was indeed present in the placenta and bound to FP1. Mutation at the AP-2 site in FP1 abolished the ability of the Ada placental regulatory element to bind AP-2 proteins and failed to target chloramphenicol acetyltransferase reporter gene expression to placentas in transgenic mice, indicating that AP-2 is required for Ada expression in the placenta. In addition, RNase protection assays demonstrated that AP-2␥ was the predominant AP-2 family member expressed in the placenta. In situ hybridization analysis revealed that AP-2␥ expression was enriched in the trophoblast lineage throughout development, suggesting that AP-2␥ may be critical for trophoblast development and differentiation.

A GCM Motif Protein Is Involved in Placenta-specific Expression of Human Aromatase Gene

“…As we demonstrated previously (11), the core element in C4 is similar to the TSE originally described in the ␣-hCG promoter that is recognized by putative TSEBP (12). The binding activity to TSE has been attributed to members of the AP2 family transcription factor (27), of which AP2␥ is the major AP-2 transcript in mouse placenta (34). TSE2, the second element we located in the placenta-specific enhancer of the aromatase gene, is not a known recognition sequence for transcription factors.…”

“…A variety of cis-elements for known and unidentified trans-factors have been described in the placentaspecific enhancers that have been analyzed, but not a single element has been found in common. TSE is associated with several genes like those for the ␣-and ␤-subunits, chorionic somatomammotropin, aromatase, and adenosine deaminase (34) and therefore could be a candidate for the master switch for placental cell differentiation. But there are exceptions such as leukemia inhibitory factor receptor and the leptin gene.…”

A 914-bp promoter is sufficient to reproduce the endogenous prolyl oligopeptidase gene localization in the mouse placenta if not subject to position effect

“…6B). Although we used whole placentas for this analysis, AP-2probably bound to the POP promoter in the cells that expressed POP, because the two proteins were expected to be co-localized in the placenta (Matsubara et al, 2011;Sapin et al, 2000;Shi and Kellems, 1998). This suggests that AP-2 may play some role in the POP gene activation.…”

“…A placenta-specific transcription factor, AP-2, could be a candidate because it was expressed in the same manner as POP (Sapin et al, 2000;Shi and Kellems, 1998) and the POP promoter had several AP-2 binding sites (Kimura et al, 1999). This time, we searched for the candidate AP-2 binding sites again by using the TRANSFAC software (http://www.gene-regulation.com/pub/databases.html) and found nine sites in the 914-bp POP promoter, one of which was the sequence for AP-2 (Fig.…”

Conditional inactivation of transcription factor AP‐2γ by using the Cre/loxP recombination system

“…AP-2␣ is predominantly essential for craniofacial morphogenesis and ventral body wall closure (Schorle et al, 1996;Zhang et al, 1996), while the lack of AP-2␤ causes polycystic kidney disease (Moser et al, 1997). AP-2␥ expression is detected as early as day 3.5 of murine development in the trophoblast lineage and persists in all of its derivatives (Shi and Kellems, 1998). In the embryo proper, AP-2␥ expression starts at day 7.5 of development and can be detected in neural, neural crest, and epithelial cells .…”