Transcriptional regulatory mechanisms of the A4GALT

P1 antigen is glycosphingolipid in human erythrocyte membrane. Erythrocytes are classified according to the expression of P1 antigen: P1-positive (P1+) phenotype and P1-negative (P1-) phenotype. P1 antigenic structure is Galα1-4Galβ1-4GlcNAc and the enzyme responsible for synthesis P1 antigen would be α1,4-galactosyltransferase (A4GALT). The difference between P1+/- erythrocytes might result from the difference of A4GALT transcript levels. Hence, the different transcriptional regulation of the A4GALT gene might be a major factor determining P1+/- phenotypes.
In order to prove this speculation, we try to prove the transcriptional regulatory mechanisms of the A4GALT and identify transcription factors which are required for A4GALT expression. In previous studies, 8 single-nucleotide polymorphisms (SNP) in A4GALT intron 1 were found with correlation to the P1+/- phenotypes. We speculated that those different eight transcription factors might recognize and bind to these eight SNPs. We used the expression vector to overexpress these transcription factors in K562 cells, and then quantify the expression level of A4GALT by qPCR.
Overexpression of the transcription factor Ikaros or Aiolos increased the abundance of A4GALT transcripts (5 times). Then, we used the reporter assay to observe the difference caused by Ikaros or Aiolos between the P1+/- alleles in order to know whether Ikaros or Aiolos could modulate A4GALT expression. We found that Ikaros and Aiolos might not involve in the allele-specific modulation of A4GALT. We suggest that the significant difference between P1+ and P1- allele requires the cooperation of various transcription factors. We hope to understand the transcriptional regulatory mechanisms of A4GALT expression and the relation between A4GALT and P1 antigen polymorphism.