Hypomethylation of the interphotoreceptor retinoid-binding protein (IRBP) promotor and first exon is linked to expression of the gene.
The interphotoreceptor retinoid-binding protein (IRBP) is limited in expression to retinal photoreceptor cells and a subset of pinealocytes. We have obtained a genomic clone containing the entire coding region and 7 kb of 5' flanking sequence. As a first step in studying IRBP gene regulation we have examined the CpG methylation patterns of the entire IRBP gene in expressing and non-expressing human cells. This has been done by isolation of high molecular weight DNA from Y-79 cells grown in suspension or attached to poly-D-lysine, which synthesize IRBP at different levels, and from human lymphocytes, which were shown by northern analysis to lack IRBP message. The DNA was digested by either Hpa II, Msp I, or Hha I. Southern blots were prepared with these digests and hybridized with probes made from fragments covering the complete genomic clone. Probes from the first exon, the introns and the 3' end gave banding patterns which showed no differences between the expressing cells and the lymphocytes. A probe from the very 5' end did not give a clear banding pattern, probably due to the presence of repetitive elements in the probe. However, a Hind III probe covering the 5' flanking 3 kb and the beginning of the first exon hybridized with a 1.8 kb band in Hpa II digests of Y-79 cells which was not present in Hpa II digests of lymphocyte DNA. In addition, a 2.1-2.3 kb Hha I band was found only in the Y-79 DNA digests. Sequence analysis of the promoter region indicated that these bands were due to hypomethylation of sites within a CpG rich island from -1578 to -1108 in the promoter and hypomethylation of sites in the beginning of the first exon. A Hha I site between the CpG island and the first exon was not hypomethylated in the expressing Y-79 cells. We propose that hypomethylation of the CpG rich island of the IRBP promoter and the first exon is linked to the expression of this gene.
Albini
A
A
National Eye Institute, National Institutes of Health, Bethesda, MD.
Toffenetti
J
J
Zhu
Z
Z
Chader
G J
GJ
Noonan
D M
DM
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005091:Exons; D005136:Eye Proteins; D005786:Gene Expression Regulation; D006801:Humans; D008214:Lymphocytes; D008745:Methylation; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012177:Retinol-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D000099214:Retinol-Binding Proteins, Interstitial
D004247:DNA; D005136:Eye Proteins; D012177:Retinol-Binding Proteins; D000099214:Retinol-Binding Proteins, Interstitial