Human ABO gene transcriptional regulation

The ABO blood group system, discovered by Karl Landsteiner in 1900, 1 is fundamental to the safety of blood transfusion, which requires identification of weak phenotypes or subgroups. The system is composed of two carbohydrate antigens, A and B, and their antibodies. The International Society of Blood Transfusion designates four antigens—A; B; A, B; and A1—based on the specificity of the antibodies. Biochemical and molecular genetic studies have clarified the molecular basis of the histo-blood group ABO system. 2-4 The functional A and B alleles at the ABO genetic locus encode two glycosyltransferases, α-1-3-N-acetylgalactosaminyltransferase (A-transferase) and α-1-3-galactosyltransferase (B-transferase), respectively. Initial molecular genetic studies demonstrated that ABO is composed of seven exons spanning approximately 19. 5 kb of genomic DNA (Fig. 1A) 5 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 the A- and B-transferases. 6-8 A single-base deletion in Exon 6 is considered to shift the reading frame of codons and to abolish the transferase activity of A-transferase in most O alleles. 6 On the other hand, some aspects remain to be explored. 9, 10 The ABO antigens are expressed in a cell type–specific manner9; the isoantigens A, B, and H of blood groups A, B, and O are not confined to red blood cells (RBCs) but are also found in most secretions and on some epithelial cells. However, they are absent in connective tissue, muscle, and the central nervous system. Moreover, ABH antigens are known to undergo drastic changes during the development, differentiation, and maturation of cells in the epithelial and erythroid lineages. 10 For example, studies of A-antigen expression during the maturation of erythroid progenitors in a two-phase liquid culture system showed that A-positive cells gradually increased during erythroid maturation. 11-13 Fluorescence-activated cell sorting analysis with monoclonal antibodies has demonstrated the expression of A-antigens on colony cells derived from blast-forming units–erythroid and colony-forming units–erythroid. 14 In addition to these physiological processes, profound changes have also been documented in pathological conditions such as tumorigenesis. Reduction or complete deletion of A- or B-antigen expression in bladder and oral cancers has been documented. 15-17 Moreover, the loss of ABH antigens has been correlated with the progression of various cancers, including lung and bladder carcinomas. 18 Finally, since Yamamoto and colleagues6 clarified the molecular genetic basis of the ABO system, a number of weak phenotypes have been found to be attributable to single nucleotide polymorphisms in the coding exons and splicing sites and hybrid formation between common alleles, 19, 20 although other weak phenotypes for which no variant apparently exists in the coding exons and splicing sites have also been reported. 21-23 Therefore, to understand the molecular mechanism responsible for the control of ABO gene expression in a cell type–specific manner, during normal cell differentiation, in cancer cells lacking A or B antigens, and in weak phenotypes, it is essential to grasp how ABO gene transcription is regulated. The DNA sequences in and around specific genes provide the code that dictates when, where, and at what level specific genes are transcribed. 24, 25 This code comprises three parts: the core promoter where RNA production initiates and is directed toward Exon 2, the region proximal to the core promoter, and the more distant regulatory sequences that enhance RNA production. It has become obvious that enhancers usually work in groups (i. e. , the locus control region and super-enhancers), each being bound by several transcription factors (TFs), forming a so-called enhanceosome. These enhanceosomes are nucleated by pioneer TFs early during differentiation, and these TFs are subsequently replaced by other TFs that trigger recruitment of the preinitiation complex, involving RNA polymerase II, to the promoter. Enhancers also interact with each other through a multilooped structure. Thus, for elucidation of ABO regulation, it is important to reveal regulatory regions such as the core promoter, the region proximal to it, and the more distant enhancer, as well as the TFs that bind to those regions. Although A- or B-antigen expression is dependent upon many steps including the structure of ABO, transcriptional regulation of ABO, translational regulation, modification or localization of A- or B-transferase, and H antigen expression, in this review we focus on the transcriptional regulation of the ABO gene through regulatory regions and TFs, and outline the molecular basis for weak phenotypes with variants in those regions. ABO gene regulatory regions have been identified by in vitro studies and genetic studies of weak phenotypes. The variants are described according to the HGVS nomenclature using the nucleotide sequences of accession numbers NG₀06669. 1 and NM₀20469. 1 as a reference in the genetic study sections. The relationship between the descriptions of variants in Intron 1 according to the Human Genome Variation Society (HGVS) nomenclature and those in the original reports is shown in Table 1. However, the positions that were used in the in vitro experiments described in the original papers remain unchanged. The positions reported in the original papers have been described according to the nucleotide sequences of accession number NT₀35014. 4 as a reference. Initially, Yamamoto et al. 5 demonstrated two transcription initiation sites upstream of the ATG translation start site in ABO using human pancreatic cDNA. Consistently, similar transcription initiation sites were found upstream of the translation start site using erythroid cells differentiated in vitro from AC133−CD34+ cells and K562 cells by us and others. 35, 36 For demonstrating the proximal promoter of ABO, we carried out transfection experiments into gastric cancer KATOIII cells and erythroleukemia HEL cells using luciferase reporter plasmids prepared from a genomic clone of human ABO. Those experiments defined the proximal ABO promoter between −150 and −2 relative to the translation start site in those cells (Figs. 1A and 2), and the promoter showed constitutive activity regardless of the cell types examined. 37, 38 Electrophoretic mobility shift assays (EMSAs) demonstrated that the GC box at −56 to −44 in the promoter bound a ubiquitous TF Sp1 or Sp1-like protein (s) (Fig. 1A), whereas mutations of the recognition motif that abrogated binding of those factors reduced the promoter activity in both cell types. 38 Thus, Sp1 or Sp1-like protein (s) seemed to be important for proximal promoter activity. The nucleotide sequence in ABO reveals two CpG islands (CGIs) (Fig. 1A), one extending from the immediate 5′ flanking region through the first exon and into Intron 1, and the other extending from Intron 6 to Exon 7. Thus, the ABO proximal promoter is located within a CGI. Because a promoter within a CGI would include a few transcription initiation sites, the presence of several transcription initiation sites in ABO is relevant. Cai et al. 39 reported a nucleotide deletion between −35 and −18, i. e. , c. −35_−18del, in the proximal ABO promoter in the B3 phenotype (Fig. 2), which reduced the promoter activity in a plasmid-based reporter assay. Similarly, three single-nucleotide substitutions at −77, −76, or −68 in the ABO promoter, i. e. , c. −77C > G, c. −76G > C, or c. −68G > T, respectively, have been reported in A3 and B3, each substitution reducing the promoter activity in luciferase reporter assays. 32, 40 Recently, another single-nucleotide substitution at −72 in the ABO promoter, i. e. , c. −72G > A, was reported in B3. 41 These genetic variants confirm the functional significance of the proximal ABO promoter in vivo. ABO transcription is likely regulated by the proximal promoter and cell-specific regulatory regions (Fig. 1B), because the proximal promoter shows constitutive activity regardless of cell type. 35 For delineation of distal regulatory regions involved in ABO regulation in erythroid cells, we used both in vitro experiments and genetic approaches. First, because DNase I–hypersensitive sites (DHSs) are associated with transcriptional regulatory regions including the promoter and distal enhancer, we prepared luciferase reporter plasmids on the basis of DHSs within a 15-kb region of genomic DNA in and around ABO in erythroleukemia K562 cells with publicly available data from DNase-Seq and FAIRE-seq on the University of California, Santa Cruz (UCSC) Genome Browser. Subsequent plasmid-based reporter assays demonstrated a distal regulatory region between +5653 and +6154 relative to the translation start site, named the +5. 8-kb site (Fig. 1A), in K562 cells, and the regulatory activity of this region was specific to erythroid cells. 27 According to the HGVS nomenclature, the +5. 8-kb site corresponds to c. 28 + 5624₆125. The same site was referred to the GATA binding site by the others. 41 EMSAs and chromatin immunoprecipitation (ChIP) assays demonstrated that the region bound hematopoietic TF GATA-1 or -2, and RUNX1 (Fig. 1A), whereas mutations of the recognition motifs that abrogated binding of those factors reduced the regulatory activities of the +5. 8-kb site in K562 cells. 27, 33, 42 Thus, binding of GATA-1 or -2, and RUNX1 to the +5. 8-kb site seemed to be crucial for the erythroid cell–specific activity of the region (Fig. 1B). Comparison of the genomic DNA in the ABO gene between humans and these primate species demonstrated high conservation between the ATG translation start codon and the stop codon of the ABO gene, except for a few regions. The +5. 8-kb site is conserved among humans, chimpanzees, and orangutans, showing similar expression of the A and B antigens on RBCs. However, it is intriguing to note that the site is not conserved in rhesus monkeys and marmosets, in which the A and B antigens are expressed only slightly on RBCs. Therefore, a comparative approach could indicate involvement of the site in ABO expression in human erythroid lineage cells. Genetic studies demonstrated a 5. 8-kb deletion of c. 28 + 5110₁0889del in Intron 1 of ABO and a 3. 0-kb deletion of c. 28 + 4077₇107del in individuals with the Bm phenotype, termed B m5. 8 and B m3. 0, respectively (Fig. 1C). 26, 27 These deletions involved the +5. 8-kb site. The Bm phenotype is characterized by the discrepancy of B antigen expression between RBCs and secretions43: Bm RBCs are not agglutinated by anti-B or anti-A, B antibody, whereas the saliva of Bm secretors contains about as much B substance as that of a normal B secretor. However, the B antigens on RBCs can only be detected by sensitive techniques such as adsorption and elution of anti-B. Bm erythrocytes contain abundant H sites, which can be converted into B sites by in vitro treatment with B-transferase derived from normal B individuals. B-transferase activity was detected in serum of Bm individuals, although the activity was distinctly reduced in all cases. The Bm trait is inherited as a rare allele at the ABO locus, although a few nonhereditary cases have also been reported. 44, 45 Thus, deletion of the erythroid cell–specific regulatory region or the +5. 8-kb site on the B m allele could explain the discrepancy of B-antigen expression between RBCs and secretions in Bm. Further genetic studies found variants in the GATA motif of c. 28 + 5861 T > G or c. 28 + 5859G > C in Bm or Am, respectively (Fig. 3). 29, 30 The former was termed B mGAGA. Am is analogous to Bm in blood group A. Moreover, deletion of the RUNX1 binding motif of c. 28 + 5865₅887del was revealed in Am (Fig. 3). 33 A similar discrepancy of blood antigen expression between RBCs and secretions is also observed in A3 and B3 phenotypes, where single nucleotide substitutions of c. 28 + 5864G > A and c. 28 + 5880A > G were found around the GATA and RUNX1 motifs in the +5. 8-kb site (Fig. 3). 32 Similarly, single-nucleotide substitution of c. 28 + 5885C > T was reported around the RUNX1 motif in an individual with B3. 34 These genetic studies confirmed the regulatory significance of the +5. 8-kb site for erythroid cell–specific expression of ABO in vivo. It appears that the proximal promoter and the +5. 8-kb site are required for ABO expression in an erythroid cell–specific manner (Fig. 1B), although it remains to be explored whether regions other than the +5. 8-kb site might affect ABO expression in erythroid cells. For delineation of ABO regulation in epithelial cells, we prepared luciferase reporter plasmids on the estimation of enhancers within a 50-kb region of genomic DNA in and around ABO in epithelial cells by publicly available data from DNase-Seq and chromatin state segmentation (ChromHMM) on the UCSC Genome Browser. Subsequent plasmid-based reporter assays indicated a distal regulatory region between +22563 and +22781 relative to the translation start site of ABO, termed the +22. 6-kb site, in KATOIII cells (Fig. 1A), and the regulatory activity of the region was specific to epithelial cells. 48 Subsequently, we validated the significance of the +22. 6-kb site with use of KATOIII cells with homozygote deletion of the site constructed by the CRISPR/Cas9 system. EMSAs and ChIP assays demonstrated that the region bound an epithelial cell–specific TF, Elf5 (Fig. 1A), whereas variant of the recognition motif that abrogated binding of the factor reduced the regulatory activity of the site in KATOIII cells. Thus, binding of Elf5 seemed to be crucial for the epithelial cell–specific activity of the region. It is likely that the proximal promoter and the +22. 6-kb site are required for ABO expression in epithelial cells (Fig. 1B), although regions other than the +22. 6-kb site might also have some influence. The initial luciferase reporter assays with KATOIII cells demonstrated that a positive element was located between −3931 and −3650 from the translation start site where four tandem copies of 43-bp repeat units bound a positive TF, CCAAT-binding factor/NF-Y, through the CCAAT motif (Fig. 1A). 37 However, similar regulatory activity was not observed in K562 cells and HEL cells of erythroid origin. 18, 23 Thus, it was likely that the minisatellite was not involved in transcriptional regulation of ABO in erythroid cells. Genetic population studies revealed that both the B and O alleles are linked via four tandem copies of a 43-bp repeat unit, and that the A1 allele is linked in the absence of this tandemly repetitive element. 49-51 Seltsam et al. 52 observed unexpected variations in the CCAAT-binding factor/NF-Y enhancer region including the repeat units in four individuals with weak B phenotypes, suggesting that those weak phenotypes might be caused by sequence variations in the enhancer region. On the other hand, Thuresson et al. 53 reported a hybrid allele between O 2 and B which lacked three repeat units, although the B transcript level was similar to that in fresh peripheral blood samples from normal controls. Thus, it remains controversial whether ABO transcription is influenced by the CCAAT-binding factor/NF-Y enhancer region in erythroid cells. 36, 52, 53 At the 5′ end of the CGI involving the ABO proximal promoter in cultured cells expressing ABO, an alternative starting Exon 1a comprising 27 base pairs was found by 5′-RACE (Fig. 1A). The level of transcription from Exon 1a was much lower than that from Exon 1. 35 The luciferase reporter assays demonstrated that the sequence located between −864 and −699 was responsible for transcription from Exon 1a in both erythroid and epithelial cell lineages. However, significance of Exon 1a remained elusive. The plasmid-based reporter assays demonstrated a negative regulatory element just upstream from the proximal ABO promoter in KATOIII cells and HEL cells, 54 suggesting that ABO transcription is regulated by negative elements in the −307 to −150 region from the translation start site. EMSAs indicated that this region bound to a nuclear factor from KATOIII cells. However, we have not identified this factor. ABO transcription is regulated by a constitutive proximal promoter and a cell-specific regulatory region such as the +5. 8-kb site or the +22. 6-kb site (Fig. 1B). Luciferase reporter assays showed that the erythroid cell-specific regulatory activity of the +5. 8-kb site was dependent upon binding of the erythroid cell–specific TF GATA-1 or -2. 27 In addition, variants in the GATA motif were found in Bm and Am, 29, 30 in which B- or A-antigen expression is reduced on RBCs, while a large amount of B or A substance is present in the saliva of secretors. Similarly, plasmid-based reporter assays demonstrated that the epithelial cell–specific regulatory activity of the +22. 6-kb site was dependent upon binding of the epithelial cell–specific TF Elf5. 48 In fibroblasts not expressing GATA-1 or -2, or Elf5, it is plausible that abrogation of the siteʼs cell-specific regulatory activity contributes to lack of ABO expression. 48 Therefore, it is likely that the cell type–specific expression of ABO is dependent upon expression of cell-specific TFs binding to those cell-specific regulatory elements. In vitro erythroid differentiation of CD34+ cells and AC133−CD34+ cells from peripheral blood mononuclear cells indicated that ABO was expressed at an early stage and disappeared later, 35, 42 and that the period when ABO was expressed at a higher level preceded that of FUT1 expression (Fig. 4). Expression of RUNX1 and GATA-2 characteristically decreases during erythroid differentiation of CD34+ cells. 42 Thus, it seems likely that down regulation of ABO expression might be ascribed to reduction of RUNX1 in the later phase of erythroid differentiation. However, the mechanism of ABO expression at an early stage of erythroid differentiation remains to be explored. ABH antigens are often absent from glycoproteins and glycolipids of malignant tissue in the gastrointestinal tract, oral cavity, uterine cervix, lung, prostate, breast, and bladder. 19, 43 ABO antigen expression decreases as a result of down regulated transcription of ABO, which is ascribed to at least two different mechanisms: allelic loss and hypermethylation of the ABO promoter region in a CGI. 55-58 There is abundant evidence that methylated CGIs at transcription start sites are associated with some silent genes. We and others have also demonstrated that hypermethylation of the ABO promoter could be responsible for absence of the ABO transcript and A-antigen in gastric and colon cancer cell lines. 56, 57 Using clinical samples of oral squamous cell cancer, Gao et al. 58 showed that loss of A/B antigens was responsible for molecular events such as loss of the A/B allele or ABO promoter hypermethylation in two-thirds of tissue samples they examined. Thus, an additional mechanism for loss of A/B antigens other than allelic loss or promoter hypermethylation remained to be clarified in one-third of such cases. The origin of DNA hypermethylation is currently being clarified, and mutations in such genes as Ten-eleven-translocation 2, DNA and 1 and 2 are known to be involved in DNA mutations in splicing factors also through splicing of and down regulation of transcription is associated with DNA hypermethylation of the promoter. Therefore, the and of DNA hypermethylation to be The of weak A expression with is well 43 et have reported that loss of ABH antigens from RBCs of with is a and that DNA of the ABO promoter the loss of ABO allelic expression in a of include and including Recently, we have reported a with in blood demonstrated us to the mechanism blood group A-antigen reduction on of mutations using cells demonstrated mutations in and involving transfection into K562 cells showed that the expression of ABO was by expression of the the of RUNX1 could encode an a that might as a negative it was plausible that this RUNX1 might be a genetic factor to A-antigen loss on RBCs. This is another of a factor other than DNA that could be responsible for ABO antigen reduction in with Therefore, is a for of with and ABO antigen reduction on RBCs to which events are attributable to loss on RBCs in The of ABO was among in in The of the Bm and was at the Blood that both are the most ABO blood group variants in the A study demonstrated B m5. 8 in individuals, B in and B in one among individuals with Bm and with polymerase B m5. 8 (Fig. In Bm is by and B m5. 8 at blood in Genetic of B m5. 8 is because Bm for of all weak phenotypes in the on the it seems likely that B m5. 8 might have been inherited a period and the the variant has not been reported in and from where to B m5. 8 could be specific to the Recently, the GATA site in B has been reported in individuals with and Moreover, a deletion of Intron 1 including the +5. 8-kb site has been found in an individual with It seems intriguing to the that the same or a similar deletion of the +5. 8-kb site could result in different phenotypes. shown ABO transcription is regulated by the proximal promoter and cell-specific regulatory regions. However, ABO transcription was not in the cells with deletions of the +22. 6-kb reporter assays demonstrated transcriptional activity in the region (Fig. which was to interact with the ABO transcription start site on the basis of publicly available data from and on the UCSC Genome Browser. 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