Results And Discussion

The expression of the ovalbumin gene is induced in accordance with the development of oviduct tissue, which in turn is developmentally regulated but is also possible to be induced artificially by administering steroid hormones to immature female chickens.6'7 We confirmed that laying hens and 12-day estradiol-treated chickens expressed ovalbumin either in the level of mRNA or of protein (data not shown). Untreated immature chickens did not express ovalbumin as expected. Oviducts from these chickens were used for the following experiments.

The length between the transcription start site of the ovalbumin and the end of the neighboring gene (T) is about 8 kb. Within this region, there are four DNase I hypersensitive sites (DHSs).8 The first DHS includes the transcription start site and extends up to 300 bp upstream and three other DHSs are centered at -0.80, -3.3 and -6.0 kb upstream (Fig. 1). It has been reported that the appearance of these regions was associated with ovalbumin expression: these were present in laying hens and in chickens artificially induced by steroids while being absent from erythrocytes in which the ovalbumin gene was not expressed.9 It is generally postulated that DHSs contain important regions for transcriptional regulation. There are several reported regulatory elements which regulate the transcription of the ovalbumin located inside DHSs. DHS I contains a proximal promoter about 90 bp in the length and a negative regulatory region spanning -90 to -300 bp.10 DHS II is reported to be responsible for the induction of steroid hormones (usually used as a mixture of estradiol and glucocorticoid) using primary culture of oviduct cells obtained from estradiol-induced immature oviducts.9-12 An estrogen-responsive enhancer element is present in DHS III.13 There is also a tissue-specific silencer element that overlaps with DHS III. This represses the expression of reporter gene in the liver but not in the oviduct.14 There is no evidence for function or binding proteins on DHS IV.

Figure 1. Putative regulatory elements in the 5'-flaking region of the chicken ovalbumin gene. 1/2 EREDR: Half estrogen response element direct repeat; NF1BE: Nuclear factor 1 binding element; SDRE: Steroid-dependent response element; NRE: Negative regulatory element; COUP: Chicken ovalbumin upstream promoter; TSE: Tissue specific element; Modified from Muramatsu and Sanders. 15(Figure is not drawn to scale).

Figure 1. Putative regulatory elements in the 5'-flaking region of the chicken ovalbumin gene. 1/2 EREDR: Half estrogen response element direct repeat; NF1BE: Nuclear factor 1 binding element; SDRE: Steroid-dependent response element; NRE: Negative regulatory element; COUP: Chicken ovalbumin upstream promoter; TSE: Tissue specific element; Modified from Muramatsu and Sanders. 15(Figure is not drawn to scale).

Within the 6.3 kb upstream of the ovalbumin promoter (including trancription start site), there are 36 CpG sites which are distributed almost uniformly in the promoter region and do not form a CpG island (Fig. 1). First, we analyzed the methylation status of immature chickens (Fig. 2). Most CpG sites were methylated throughout the promoter-enhancer region. Some CpG sites near DHS II and the transcription start site were not methylated even in the absence of expression. These were not mehylated in erythrocytes either (data not shown), suggesting that demethylation of these sites is not related to tissue specific expression of the ovalbumin.

Several regions of CpGs are selectively unmethylated in both estradiol-treated chickens and laying hens as shown in Fig. 2. First three CpGs in the transcribed region were demethylated. The first is inside the first exon and the other two sites are in the first intron. Such selective demethylation of the 5'-region was reported for other genes.16 Inside DHS II, a CpG site was specifically demethylated upon ovalbumin expression. This specific demethylation might be related with the gene transcription and also this CpG site is located very near to SDRE which is steroid inducible element. Sanders group proved that the promoter region from -0.9 kb to +1 (DHS II CpG site and SDRE are included in this promoter region) is sufficient for steroid induction when transfected in primary cultures of oviduct cells.10

In our analysis, partial demethylation for DHS III was observed in the ovalbumin expressing chickens. But the downstream and upstream regions of DHS III were strongly methylated even in laying hens. We analyzed more than ten clones of bisulfite modified DNA of the DHS III region and the results showed that about half were methylated while the others were unmethylated. But we did not obtain any partial methylation state in the case of DHS II of estradiol-treated chickens and laying hens and there was also a clear change in the methylation pattern between immature chickens, estradiol-treated chickens and laying hens (Fig. 2). Our results suggest that some regulatory mechanisms induce demethylation in some regions of this promoter and further DNA methylation is an epigenetic process, cells existed in a mixed population of different methylation states.

The CpG site in DHS IV was completely methylated in both estradiol-treated chickens and laying hens (Fig. 2). This could be a reflection of the lack of functional importance of this region. In fact, the expression of the reporter CAT gene was sufficient by the ovalbumin promoter -3.2 kb to +1 in the oviduct and was not affected by the existence of -4 kb to -8 kb covering DHS IV.14 These results suggest that our methylation analysis is in good accordance with other experimental methods which indicate the importance of gene regulation. Further analysis would be helpful to fully understand the biochemical and functional features of the ovalbumin promoter.

* Methylated

Unmethylated $ Mixed (Methylated/Unmethylated)

* Methylated

Unmethylated $ Mixed (Methylated/Unmethylated)

Figure 2. CpG methylation pattern of ovalbumin promoter of immature chickens, estradiol-treated chickens and laying hens. (Figure is not drawn to scale).

0 0

Post a comment