Regulation of the ovine interferon‐tau gene by a blastocyst‐specific transcription factor, Cdx2

Abstract

Expression of ovine interferon-tau (oIFNtau), a factor essential for the process of maternal recognition of pregnancy in ruminant ungulates, is restricted to the trophoblast. However, the molecular mechanisms by which oIFNtau expression is restricted to the trophectoderm have not been fully elucidated. The objective of this study was to determine whether oIFNtau gene transcription could be regulated through Cdx2 expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Human choriocarcinoma JEG3 cells were co-transfected with an oIFNtau (-654 base pair, bp)-luciferase reporter (-654-oIFNtau-Luc) construct and several transcription factor expression plasmids. Compared to -654-oIFNtau-Luc alone, transcription of the -654-oIFNtau-Luc increased more than 30 times when this construct was co-transfected with Cdx2, Ets-2, and c-jun. The degree of transcription decreased to 1/4 levels when the upstream region was reduced to -551 bp, and became minimal with further deletions; this was confirmed with the use of the reporter constructs with mutated c-jun, Ets-2, and/or Cdx2 sites. In trophoblast unrelated NIH3T3 cells, which do not support IFNtau gene transcription, the oIFNtau-Luc transcription was enhanced approximately eightfold when the cells were co-transfected with the Cdx2/Ets-2 or Cdx2/Ets-2/c-jun expression plasmids. These findings were confirmed by gel-shift assays examining Cdx binding site on the oIFNtau gene's upstream region, by immunohistochemical study identifying the presence of Cdx2 in day 15 and 17 ovine conceptuses, and by Western blot detecting Cdx2 in day 17 conceptuses. Our results indicate that oIFNtau gene transcription is regulated by Cdx2, and suggest that Cdx2 could be a key molecule in determining oIFNtau gene transcription by the trophectoderm.

Blastocyst-Derived Trophoblast Stem Cells from the Rhesus Monkey

“…Therefore, Cdx2 expression is thought to be associated with the initiation of TE fate. In ovine embryos, Cdx2 was localized to TE, but staining intensity appeared to decrease after implantation (45). Human trophoblasts derived from EBs showed increased expression of CDX2 (13).…”

Blastocyst elongation, trophoblastic differentiation, and embryonic pattern formation

“…One of the most highly expressed genes during the gastrulation is TKDP4 and it may be key factor in early trophoblast proliferation (MacLean et al silences trophoblast IFNT transcription in concert with ETS-2 and CDX2 in TMCs, suggesting that these genes are also involved in regulating trophoblast cell proliferation and IFNT expression (Ezashi et al 2001, Imakawa et al 2006. On the other hand, the AP-2 family and Sp-1 may participate in the regulation of TGC-specific gene expression, even though the details of this regulation pathway remain to be studied (Limesand & Anthony 2001, Spencer et al 2004).…”

Expression and Potential Role of GATA6 in Ruminant Trophoblasts during Peri-Implantation Periods

“…3). The numerous transcription factors found thus far as regulators of IFNT genes include ETS2 [30,31], activating protein 1 (AP-1; official symbol JUN) [32], CDX2 [24,27], homeobox protein distal-less 3 (DLX3) [33], and co-activators cAMP-response element binding protein (CREB)-binding protein (CREBBP) [34] and p300 [35]. Recently, we found that GATA2 and GATA3 could also increase IFNT transcription in our transient transfection system [15,16].…”

“…The Renilla luciferase-pRL-TK vector (Promega), driven by the herpes simplex virus-thymidine kinase (HSV-TK) promoter, was used to normalize the transfection efficiency [27]. Amount of reporter construct used relative to that of the internal control vector pRL-TK was 20:1.…”

“…At 48 h after transfection, the cells were lysed by adding 100 µl Passive Lysis Buffer (Promega). A luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega) as described previously [27]. …”