284 IDENTIFICATION AND CHARACTERIZATION OF THE 5′-FLANKING REGION OF THREE MOUSE MATERNAL GENES (HISTONE H1OO, NUCLEOPLASMIN 2, AND ZYGOTE ARREST 1): TRANSCRIPTIONAL ACTIVITY IN MOUSE OOCYTES

Abstract

Maternal transcripts that accumulate during oocyte growth are involved in the meiotic maturation, the initiation of the first mitosis, and the later pre-implantation development. Although the conserved E-box sequences in promoter region of some maternal genes (for example, Zp3 and Gdf-9), which are important in regulating gene transcripts as binding sites of transcriptional factors, may play a role in the oocyte-specific expression in ovary, the molecular mechanism that regulates the expression of the maternal genes is still not known. In this study, we have focused on the transcriptional activity of promoter regions to clarify the molecular mechanism of transcriptional regulation of these maternal genes [Histone H1oo (H1oo), Nucleoplasmin 2 (Npm2), and Zygote arrest 1 (Zar1)]. First, we observed the expression of firefly luciferase expression vectors under promoter regions of 3 maternal genes in oocytes isolated from 10- to 12-day-old mice, which is mainly NSN-type, transcriptionally active form (Bouniol-Baly et al. 1999 Biol. Reprod. 60, 580–587). Transcriptional activities of H1oo (-3975), Npm2 (-2610), and Zar1 (-5187) promoters were detected in oocytes, the relative luciferase activities being an average of 70, 130, and 12, respectively. On the other hand, these promoter activities were not detected in embryos at the 2-cell stage. Furthermore, deletion analysis of promoter elements (E-boxes) of H1oo and Npm2 was done by microinjecting deletion constructs into oocytes. In the H1oo promoter, deletion of sequences between -3975 and -72 bp from the transcription start site resulted in one-third of the level obtained in the full H1oo (-3975) promoter. In addition, deletion of sequence -68 bp resulted in no detection of luciferase activity. These findings indicate that the putative distal promoter sequences exist at the 52-flanking region (-3975 to -759) of the H1oo gene and that the region (-314 to -68) including the E-box region (-72) may be required for high-level transcriptional activity of the H1oo promoter. In the Npm2 promoter, deletion of sequences between -2610 and -180 bp from the transcription start site resulted in one-third of the level of wild-type activity of the Npm2 (-2610) promoter. In addition, deletion of sequence -101 bp resulted in no detection of luciferase activity. These findings also indicate that 3 putative distal promoter sequences exist at the 52-flanking region (-2610 to -210) of the Npm2 gene and that the region (-210 to -101) that includes the E-box region (-180) is crucial for high-level transcriptional activity of the Npm2 promoter. In conclusion, the E-box may be a key regulatory region for the expression of two of the maternal genes (H1oo and Npm2) examined. Currently, we are attempting to identify the transcriptional factor binding sites by DNase I footprint analysis and gel mobility shift assay.