Transcriptional Regulation of Two Conceptus Interferon Tau Genes Expressed in Japanese Black Cattle during Peri-Implantation Period

Abstract

Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants. However, its expression in vivo and its transcriptional regulation are not yet well characterized. Objectives of this study were to determine conceptus IFNT gene isoforms expressed in the bovine uterus and to identify differences in promoter sequences of IFNT genes that differ in their expression. RNA-seq data analysis of bovine conceptuses on days 17, 20, and 22 (day 0 = day of estrus) detected the expression of two IFNT transcripts, IFNT1 and IFNTc1, which were indeed classified into the IFNT gene clade. RNA-seq and quantitative RT-PCR analyses also revealed that the expression levels of both IFNT mRNAs were highest on day 17, and then decreased on days 20 and 22. Bovine ear-derived fibroblast (EF) cells, a model system commonly used for bovine IFNT gene transcription study in this laboratory, were cotransfected with luciferase reporter constructs carrying upstream (positions −637 to +51) regions of IFNT1 or IFNTc1 gene and various transcription factor expression plasmids including CDX2, AP-1 (Jun) and ETS2. CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. The upstream regions of IFNT1 and IFNTc1 loci were then serially deleted or point-mutated at potential CDX-, AP-1-, and ETS-binding sites. Compared to the wild-type constructs, deletion or mutation at CDX2 or ETS2 binding sites similarly reduced the luciferase activities of IFNT1- or IFNTc1-promoter constructs. However, with the AP-1 site mutated construct, IFNT1- and IFNTc1-reporters behaved differently. These results suggest that two forms of bovine conceptus IFNT genes are expressed in utero and their transcriptional regulations differ.