Expression and Methylation of the β-Subunit Gene of Prolyl 4-Hydroxylase: In Erythrocytes, Tendon and Cornea of Chick Embryos

Abstract

It has recently been demonstrated that the β-subunit of prolyl 4-hydroxylase (E.C. 1.14.11.2) is the same gene product as protein disulfide isomerase (PDI) and cellular thyroid hormone binding protein (THP). Therefore, it is very likely that the β-subunit of the prolyl 4-hydroxylase gene serves as a house keeping gene in most cell types. In the present study, we examined the distribution of the chicken β-subunit of prolyl 4-hydroxylase/protein disulfide isomerase (CPHβ/PDI) in erythrocytes, corneas and tendons of 13-, 17-, and 19-day-old chick embryos by immunohistochemistry using antibodies against CPHβ/PDI. Our data indicate that erythrocytes do not express the CPHβ/PDI gene whereas tendon cells express CPHβ/PDI at all developmental stages examined. The basal cells of corneal epithelium express CPHβ/PDI, but the superficial cell layers of stratified corneas of 19-day-old chick embryos do not. The expression of the CPHβ/PDI gene is also confirmed by in situ hybridization with cDNA encoding CPHβ/PDI. The results indicate that the expression of CPHβ/PDI in cornea is probably developmentally regulated. It has been suggested that methylation of genomic DNA is one of many possible regulatory mechanisms for gene expression. In order to examine whether methylation of genomic DNA may play any role in the expression of the β-subunit gene, genomic DNA was isolated from corneas, tendons, and erythrocytes of individual 13-, 17-, and 19-day-old chick embryos. DNA samples were digested with Sma I and Eco RI, or Pst I and Sma I and followed by either Msp I., Hpa II, or Hha I and were then subjected to Southern hybridization with ³²P-labeled genomic DNA fragments of CPHβ/PDI. Our results indicate that the CPHβ/PDI gene is methylated at the Hha I site in the 4th exon in erythrocytes whereas the same sites in tendon and cornea are hypomethylated. Examination of 5′-end flanking sequences of exon 1 of the CPHβ/PDI gene with the methylation sensitive endonucleases, Hha I and Hpa II did not reveal any difference in erythrocyte, cornea and tendon cells. Thus, our results indicated that DNA methylation may not play an important role in the expression of CPHβ/PDI.

Key Words:

• prolyl 4-hydroxylase
• β-subunit of prolyl 4-hydroxylase
• protein disulfide isomerase
• DNA methylation
• gene expression