No abstract

Interleukin-1␤ (IL-1␤) is produced primarily by stimulated monocytes, suggesting that the IL1B gene, which codes for this protein, depends upon at least one cell-type-specific factor. Our previous characterization of the IL1B promoter indicated that the region between ؊131 and ؉12 is sufficient to direct cell-type-specific expression of a reporter gene (F. Shirakawa, K. Saito, C. A. Bonagura, D. L. Galson, M. J. Fenton, A. C. Webb, and P. E. Auron, Mol. Cell. Biol. 13:1332-1344, 1993). We now show that a sequence located between positions ؊50 and ؊39 of the IL1B promoter binds the tissue-restricted Ets domain transcription factor Spi-1/PU.1 (Spi-1). Mutation of this site abrogates binding of this factor and reduces the ability of the IL1B promoter to function in macrophages. A second Spi-1 binding site located between positions ؊115 and ؊97 also is required for maximal IL1B promoter activity in the presence of the proximal Spi-1 binding site. In addition, an activation domain-deficient Spi-1 expression vector acts as a dominant-negative inhibitor of reporter gene expression in a monocyte cell line. Finally, the IL1B promoter, which is inactive in Spi-1-deficient HeLa cells, is activated in these cells by cotransfection with a Spi-1 expression vector. Thus, the cell-type-specific expression of the IL1B promoter appears to be dependent on the binding of Spi-1.Interleukin-1␤ (IL-1␤), a 17-kDa polypeptide derived from the proteolytic processing of a 31-kDa prointerleukin 1␤ precursor (proIL-1␤), possesses a wide spectrum of inflammatory, metabolic, physiologic, hematopoietic, and immunological properties (for a review, see reference 8). Although IL-1␤ is produced by many different types of cells in response to a broad range of stimuli, it is most abundantly expressed by activated monocytes, in which the proIL-1␤ transcript can account for as much as 5% of the poly(A) ϩ mRNA (38). The proIL-1␤ gene (referred to here by its genomic locus name, IL1B) is not normally transcribed in competent cells until induced by stimuli commonly used for induction in model systems, such as lipopolysaccharide (LPS), phorbol 12-myristate acetate (PMA), or the IL-1␤ protein itself. In recent studies, the IL1B gene-inducible enhancer was located between Ϫ3134 and Ϫ2729 (3, 31). This enhancer appears to function in a cell-type-independent fashion when ligated to the murine c-fos promoter and functions well in either HeLa or monocyte cells. However, when either this enhancer region or the simian virus 40 (SV40) enhancer was ligated to the IL1B promoter, located between Ϫ131 and ϩ12, each was demonstrated to function more efficiently in monocytes than in HeLa cells (31). Since HeLa cells do not produce IL-1␤ (3), the IL1B promoter-proximal region appears to be required for tissue specificity. Therefore, expression of the IL1B gene is controlled by two independent elements, one providing cell type specificity and the other responsible for signal-dependent induction. Although recent studies have begun to characterize the regulatory pathways fo...

The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABO gene expression remain obscure. On the basis of DNase I-hypersensitive sites in and upstream of ABO in K562 cells, in the present study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1. This element was shown to enhance ABO promoter activity in an erythroid cellspecific manner. Electrophoretic mobilityshift assays demonstrated that it bound to the tissue-restricted transcription factor GATA-1. Mutation of the GATA motifs to abrogate binding of this factor reduced the regulatory activity of the element. Therefore, GATA-1 appears to be involved in the cell-specific activity of the element.

We have studied the transcriptional regulatory mechanism of the human histo-blood group ABO genes, and identified DNA cis-elements and trans-activating protein that control the expression of these genes which are important in blood transfusion and organ transplantation. We introduced the 5 -upstream sequence of ABO genes into the promoterless reporter vector and characterized the promoter activity of deletion constructs using transient transfection assays with gastric cancer cell line KATO III cells. The sequence just upstream of the transcription start site (cap site), and an enhancer element, which is located further upstream (between ؊3899 and ؊3618 base pairs ( Histo-blood group ABH(O) antigens, the major alloantigens in humans (1), have been characterized as defined trisaccharide determinants GalNAc␣133(Fuc␣132)Gal␤13 R, Gal␣133(Fuc␣132)Gal␤13 R, and disaccharide determinant Fuc␣132Gal␤13 R for A, B, and H, respectively (2, 3). These structures represent the secondary gene products which are synthesized from the precursor H substrate by ␣133GalNAc (A transferase) and ␣133Gal transferase (B transferase), the primary gene products coded by the functional alleles at the ABO locus (4, 5). Molecular genetic studies of the ABO genotypes have identified two critical single-base substitutions between A and B genes, the resultant 2-amino acid substitutions being responsible for the different donor nucleotide-sugar substrate specificity between A and B transferases. A single base deletion, which shifts the codon reading frame and abolishes the function of A transferase, has been identified in O allelic cDNAs (6, 7).ABH antigens are known to undergo drastic changes during development, differentiation, and maturation. Studies of these antigens in stratified squamous epithelia provided one of the clearest examples of differential expression during cell maturation (8). In non-keratinized stratified squamous epithelia, the immature cells in the basal layers are characterized by the expression of sialylated or unsubstituted precursor peripheral cores, while differentiated and mature cells in the upper layers sequentially express ␣132-fucosylated H structures, and A and B antigens depending on the ABO genotype of the individual. This sequential expression of carbohydrate antigens is associated with the differentiation pattern of the epithelium. An interesting question is how these changes are controlled during cell differentiation. Since keratinocytes are known to greatly change their gene expression during terminal cell differentiation (9), the switch-on of the ABO genes during the maturation may be governed by the same factor(s). To fill in the gap between the expression of the ABO genes and the appearance of the ABO phenotypes in the terminal differentiation of epithelial cells, it is essential to understand the transcriptional regulatory mechanism of the ABO genes. In addition to the normal cell differentiation process, the changes of ABH antigen expression have also been documented in abnormal processes such as tumorig...

We have investigated the regulatory role of DNA methylation in the expression of the human histo-blood group ABO genes. The ABO gene promoter region contains a CpG island whose methylation status correlates well with gene expression in the cell lines tested. The CpG island was found hypomethylated in some cell lines that expressed ABO genes, whereas the other cell lines that did not express ABO genes were hypermethylated. The ABO blood group system discovered by Karl Landsteiner (1) at the beginning of this century is of great importance in blood transfusions and organ transplantations. Two carbohydrate antigens, A-and B-antigens, and their antibodies constitute this system. The A and B functional alleles at the ABO genetic locus encode glycosyltransferases ␣133GalNAc transferase (designated A-transferase) and ␣133Gal transferase (designated 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 the human ABO genes have identified two critical single base substitutions that result in amino acid substitutions responsible for the different donor nucleotidesugar substrate specificity between A-and B-transferases. A single base deletion, which shifts the reading frame of codons and abolishes the function of A-transferase, has been identified in most O alleles (6, 7).The ABO genes 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 only but are also found in most secretions and on some epithelial cells. However, they are absent in connective tissues and the central nervous system (8). ABH antigens are known to undergo drastic changes during development, differentiation, and maturation of normal cells (9). In addition to these physiological processes, profound changes have also been documented in pathological processes such as tumorigenesis. Reduction or complete deletion of A/B antigen expression in bladder and oral cancers has been documented, as well as the apparent onco-developmental expression of the ABH antigens in gastric and distal colon tumors (10 -12). Moreover, the loss of ABH antigens has been correlated with tumor progression of various carcinomas including lung and bladder carcinomas (13-16). Thus, delineation of regulatory mechanism is essential to understand these complicated expression patterns of the ABO genes.In an initial attempt to elucidate the molecular mechanism controlling the expression of the human ABO genes, we isolated several genomic clones that covered the ABO genes over 18 kb 1 (17). A 4.7-kb EcoRI/NcoI 5Ј-upstream fragment flanking the coding sequence in exon 1 of the human ABO gene was subcloned into the promoterless pGL3-basic vector upstream of the lucifer...

These observations suggest that the mutation in the GATA motif of the erythroid-specific regulatory element may diminish the binding of GATA transcription factors and down regulate transcriptional activity of the element on the B allele, leading to reduction of B antigen expression in erythroid lineage cells of the Bm individual.

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...

The expression of the ABO promoter appears to be influenced by the binding of Sp1 or Sp1-like protein(s) in both erythroid and epithelial cell lineages.