Submitted to: Molecular Reproduction and Development
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/16/2005
Publication Date: 5/20/2006
Citation: Imakawa, I., Kim, M.-S., Matsuda-Minehata, F., Ishida, S., Iizuka, M., Suzuki, M., Chang, K.-T., Echternkamp, S.E., Christenson, R.K. 2006. Regulation of the ovine interferon-tau gene by a blastocyst-specific transcription factor, Cdx2. Molecular Reproduction and Development. 73(5):559-567. Interpretive Summary: Pregnancy in sheep relies on the continued production by the ovary of the hormone progesterone. When a sheep is not pregnant, the uterus produces a factor, prostaglandin, that eliminates ovarian progesterone production allowing the sheep to come back into heat. When a sheep is pregnant, the developing embryo secretes a protein, interferon, that blocks uterine production of prostaglandin, and the ovary continues to secrete progesterone. Interferon production by the embryo is transient and occurs only in specific embryonic cells. The objectives of the current study was to determine how the transient, cell-specific expression of interferon is controlled. Portions of a region of an interferon gene that controls expression were tested to determine which known gene transcription factors affect expression. The region of the interferon gene required and the interaction of this region with the transcription factors Cdx2, Ets-2, and c-jun were demonstrated. It was proposed that these and other transcription factors are responsible for controlling the transient expression of interferon by the developing sheep embryo.
Technical Abstract: Expression of ovine interferon-tau (oIFNt), 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 oIFNt expression is restricted to the trophectoderm have not been fully elucidated. The objective of this study was to determine whether oIFNt 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 oIFNt (-654 base pair, bp)-luciferase reporter (-654-oIFNt-Luc) construct and several transcription factor expression plasmids. Compared to -654-oIFNt-Luc alone, transcription of the -654-oIFNt-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 IFNt gene transcription, the oIFNt-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 oIFNt 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 oIFNt gene transcription is regulated by Cdx2, and suggest that Cdx2 could be a key molecule in determining oIFNt gene transcription by the trophectoderm.