Phosphorylation status of transcription factor C/EBPalpha determines cell-surface poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis.

The cell-surface straight and branched repeats of N-acetyllactosamine (LacNAc) units, called poly-LacNAc chains, characterize the histo-blood group i and I antigens, respectively. The transition of straight to branched poly-LacNAc chain (i to I) is determined by the I locus, which expresses 3 IGnT transcripts, IGnTA, IGnTB, and IGnTC. Our previous investigation demonstrated that the i-to-I transition in erythroid differentiation is regulated by the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). In the present investigation, the K-562 cell line was used as a model to show that the i-to-I transition is determined by the phosphorylation status of the C/EBPalpha Ser-21 residue, with dephosphorylated C/EBPalpha Ser-21 stimulating the transcription of the IGnTC gene, consequently resulting in I branching. Results from studies using adult erythropoietic and granulopoietic progenitor cells agreed with those derived using the K-562 cell model, with lentiviral expression of C/EBPalpha in CD34(+) hematopoietic cells demonstrating that the dephosphorylated form of C/EBPalpha Ser-21 induced the expression of I antigen, granulocytic CD15, and also erythroid CD71 antigens. Taken together, these results demonstrate that the regulation of poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis share a common mechanism, with dephosphorylation of the Ser-21 residue on C/EBPalpha playing the critical role.

Expression of the human Sd(a) beta-1,4-N-acetylgalactosaminyltransferase II gene is dependent on the promoter methylation status.

It has been noted that the expression of Sd(a), including its antigenic structure, the beta-1,4-N-acetylgalactosyltransferase II (beta4GalNAcT-II) activity responsible for its formation, and the Sd(a) beta4GalNAcT-II mRNA transcript, is drastically reduced in oncogenetic processes in gastrointestinal tissues. Markedly reduced metastatic potential has been demonstrated in colon and gastric cancer cells transfected with the Sd(a) beta4GalNAcT-II gene. In this study, a putative CpG island encompassing the promoter and exon 1 regions in the human Sd(a) beta4GalNAcT-II gene was identified, and the investigation of DNA methylation of the Sd(a) gene promoter region demonstrated a clear association between the methylation status of the CpG island promoter and expression of the Sd(a) gene in gastrointestinal cancer cell lines. Hypomethylation of the promoter region of the Sd(a) gene was shown in cells where this gene was expressed. By contrast, there was significant hypermethylation of the Sd(a) gene promoter in cells that did not express the gene. A specific methylation profile in the Sd(a) gene CpG island was demonstrated in KATO III gastric cancer cells. In colon cancer cells with the hypermethylated Sd(a) gene promoter, treatment with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine, resulted in demethylation of the promoter region and substantially induced the expression of the Sd(a) gene and the Sd(a) antigenic structure. These results strongly suggest that promoter DNA methylation plays a crucial role in the regulation of the Sd(a) beta4GalNAcT-II gene and Sd(a) antigen expression.

I branching formation in erythroid differentiation is regulated by transcription factor C/EBPalpha.

The histo-blood group i and I antigens have been characterized as straight and branched repeats of N-acetyllactosamine, respectively, and the conversion of the straight-chain i to the branched-chain I structure on red cells is regulated to occur after birth. It has been demonstrated that the human I locus expresses 3 IGnT transcripts, IGnTA, IGnTB, and IGnTC, and that the last of these is responsible for the I branching formation on red cells. In the present investigation, the K-562 cell line was used as a model to show that the i-to-I transition in erythroid differentiation is determined by the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha), which enhances transcription of the IGnTC gene, consequently leading to formation of the I antigen. Further investigation suggested that C/EBPalpha IGnTC-activation activity is modulated at a posttranslational level, and that the phosphorylation status of C/EBPalpha may have a crucial effect. Results from studies using adult and cord erythropoietic cells agreed with those derived using the K-562 cell model, with lentiviral expression of C/EBPalpha in CD34(+) hemopoietic cells demonstrating the determining role of C/EBPalpha in the induction of the IGnTC gene as well as in I antigen expression.

Expression of the histo-blood group B gene predominates in AB-genotype cells.

BACKGROUND: It has been demonstrated that the 43-bp minisatellite sequence in the 5' region of the ABO gene plays an important role in its transcriptional regulation. It was determined in previous investigations that the structure of the minisatellite enhancer was specific to A, B, and O alleles. STUDY DESIGN AND METHODS: Real-time polymerase chain reaction (PCR) detection and a PCR-restriction fragment length polymorphism (RFLP) strategy were used to compare the quantities of the A and B transcripts in AB-genotype cells, including peripheral blood cells and cancer cell line with the group AB phenotype. The 5' 3.7-kb regions of the A and B genes were cloned and the sequences compared. The transcriptional activities of the 5' segments of the A and B genes were compared with luciferase reporter assay. RESULTS: Both real-time PCR and PCR-RFLP analyses show that there is evidently more of the B transcript in the AB-genotype cells. It was demonstrated that the 5' segment of the B gene had a markedly higher transcription-activation activity relative to the A gene. This difference in transcription capability appears to result from the variation in minisatellite-enhancer structures in the A and B genes, which contain one and four repeats of the 43-bp enhancer unit, respectively. CONCLUSION: Our study indicates that the majority of steady-state mRNA within AB-genotype cells is composed of the B transcript and that this phenomenon is due to the predominant expression of the B gene relative to the A gene.