Alternative Promoter Identified between a Hypermethylated Upstream Region of Repetitive Elements and a CpG Island in Human ABO Histo-blood Group Genes

Abstract

We have studied the expression of human histo-blood group ABO genes during erythroid differentiation, using an ex vivo culture of AC133 ؊ CD34 ؉ cells obtained from peripheral blood. 5-Rapid amplification of cDNA ends analysis of RNA from those cells revealed a novel transcription start site, which appeared to mark an alternative starting exon (1a) comprising 27 bp at the 5-end of a CpG island in ABO genes. Results from reverse transcription-PCR specific to exon 1a indicated that the cells of both erythroid and epithelial lineages utilize this exon as the transcription starting exon. Transient transfection experiments showed that the region just upstream from the transcription start site possesses promoter activity in a cell type-specific manner when placed 5 adjacent to the reporter luciferase gene. Results from bisulfite genomic sequencing and reverse transcription-PCR analysis indicated that hypermethylation of the distal promoter region correlated with the absence of transcripts containing exon 1a, whereas hypermethylation in the interspersed repeats 5 adjacent to the distal promoter was commonly observed in all of the cell lines examined. These results suggest that a functional alternative promoter is located between the hypermethylated region of repetitive elements and the CpG island in the ABO genes.In 1900 Karl Landsteiner discovered the ABO blood group system, which is important in blood transfusions and personal identification in criminal investigations (1). Two carbohydrate antigens, A and B, and their antibodies constitute this system. The functional A and B alleles at the ABO genetic locus encode glycosyltransferases ␣133GalNAc transferase (A-transferase) and ␣133Gal transferase (B-transferase), respectively. A-transferase transfers a GalNAc residue from UDP-GalNAc to the precursor H substrate, producing A antigens as defined by the trisaccharide determinant structure GalNAc␣133-(Fuc␣132)Gal␤13 R. Similarly, B-transferase catalyzes the transfer of a Gal from UDP-Gal to the same H substrate, producing B antigens defined by Gal␣133(Fuc␣132)-Gal␤13 R (2-5). Molecular genetic studies of human ABO genes have demonstrated that ABO genes consist of at least seven exons spanning over 18 kb of genomic DNA and that two critical single base substitutions in the last coding exon result in amino acid substitutions responsible for the different donor nucleotide sugar substrate specificity between A-and B-transferases. A single base deletion in exon 6 was ascribed to shift the reading frame of codons and to abolish the transferase activity of A-transferase in most O alleles (6 -9).The ABO antigens are expressed in a cell type-specific manner; the isoantigens A, B, and H of blood groups A, B, and O are not confined to red cells but are also found in most secretions and on some epithelial cells. However, they are absent in connective tissues, muscles, and the central nervous system (10). Moreover, ABH antigens are known to undergo drastic changes during development, differentiation, and maturation of cells in epithe...

Loss of blood group A antigen expression in bladder cancer caused by allelic loss and/or methylation of the ABO gene

“…Reportedly, the promoter region of the ABO gene is located between À117 and þ 31 from the translation start site, of which hypermethylation regulates gene expression. 19,20 In the present study, we divided CpG island spanning À789 to þ 6 into six regions and examined the methylation status by BiPS analysis (Figure 2). In the preliminary experiment, methylated DNA could be identified as the extra band, if more than 25% of the template DNA was methylated (data not shown).…”

“…Recent advance in cancer epigenetics shed light on the reduced expression of A antigen in malignant cells. Kominato et al 19,20 reported that hypermethylation of the promoter region of the ABO gene induced ABO gene silencing in their study using a human stomach carcinoma cell line. Iwamoto et al 21 established subclones with positive or negative expression of the A antigen from parental colonic cancer cell lines and reported a distinct difference in the methylation pattern of the CpG island of the promoter region of the ABO glycosyltransferase, that is densely methylated in a subclone lacking the expression of the A antigen.…”

Epithelial Expression of Human ABO Blood Group Genes Is Dependent upon a Downstream Regulatory Element Functioning through an Epithelial Cell-specific Transcription Factor, Elf5

“…On the other hand, the distribution of the A-and B-antigens is cell type-specific; for example, the antigens are expressed on red blood cells and epithelial cells, as well as in salivary glands, although they are absent from the central nervous system, muscle, and connective tissue. Moreover, ABH antigens are known to be expressed during the maturation of erythroid and epithelial cells; for example, when erythroid cells differentiate in vitro, ABO is expressed at an undetectable level in the early phase, increases subsequently, and then decreases later (3,4). In addition to the normal cell differentiation process, changes in ABH antigen expression have also been documented in abnormal processes such as tumorigenesis (1).…”

“…A proximal promoter has been found within the ABO CpG island (CGI) (9,10). However, a cell type-specific, distal promoter has been demonstrated at the 5Ј boundary of the CGI, although the amount of transcript from this promoter was very small (3). Because the ABO proximal promoter showed constitutive activity regardless of the cells examined in transfection experiments (3), it had been assumed that some cell-specific regulatory elements are involved in cell type-specific ABO expression.…”

“…However, a cell type-specific, distal promoter has been demonstrated at the 5Ј boundary of the CGI, although the amount of transcript from this promoter was very small (3). Because the ABO proximal promoter showed constitutive activity regardless of the cells examined in transfection experiments (3), it had been assumed that some cell-specific regulatory elements are involved in cell type-specific ABO expression. Recently, a candidate for erythroid cell-specific regulatory element, named the ϩ5.8-kb site, has been proposed in the first intron of ABO (11).…”