STUDY ON THE TEMPORAL EXPRESSION OF INTERFERON-TAU GENE USING CHROMATIN IMMUNOPRECIPITATION (CHIP)

Abstract

Interferon-tau (IFNT), the conceptus protein that signals maternal recognition of pregnancy in ruminants, is secreted transiently during early pregnancy. In ewes, IFNT secretion is highest, on a cellular basis, around day 14 and as the conceptus grows and elongates total IFNT production peaks at day 16 and diminishes through day 21. To elucidate molecular mechanisms for the temporal expression of interferon-tau (IFNT) genes, chromatin immunoprecipitation was performed on the upstream region of IFNT (oIFNτ o10) gene using ovine conceptuses and antibodies against histone H3 acetyl K18 (acetyl H3K18), histone H3 mono-methyl K9 (mono-methyl H3K9) and histone H3 di-methyl K9 (dimethyl H3K9.). Days 14.5 (n=7), 16 (n=6) and 20 (n=2) ovine conceptuses (day 0 = day of estrus) were obtained via hysterectomy at University of Tennessee Experimental Station. After removal from the uterus, conceptuses were treated with formaldehyde, sonicated, followed by overnight immunoprecipitation with antibodies or rabbit IgG. Precipitates were then subjected to PCR analysis examining the upstream regions, − 866 to −655 base pair (bp) and −238 to −46 bp, of the oIFNτ o10 gene. Experiments were extended to examine the same regions of oIFNT gene in Madin-Darby Bovine Kidney (MDBK) cells. Extensive signals were found for acetyl H3K18 on day 14.5, which gradually decreased toward day 20. Very low signals for mono-methyl H3K9 were found on day 14.5, however, strong signals were detected on day 20. However, no signals from the dimethyl H3K9 treatment were found in any conceptuses. In addition, the distal region (−866 to −655 bp) appeared to be more acetylated than the proximal one on day 14. 5. In MDBK cells, weak, but detectable signals were found for acetyl H3K18, mono-methyl H3K9 and di-methyl H3K9. These data indicated that a high acetylation/low methylation status was found when oIFNτnmRNA was expressed extensively. As the conceptus development proceded toward day 20 and IFNT expression diminished, a low acetylation/high methylation status was observed. These data represent the first demonstration of the acetylation/methylation status for IFNT genes, and indicate that changes in acetylation and methylation status in the chromatin structure of IFNT genes may be molecular events that influence subsequent activation or inactivation of various transcription factors. (poster)