The Xg blood group is differentially expressed on erythrocytes from men and women. The underlying gene, , was identified in 1994, but the molecular background for Xg expression remains undefined. This gene, now designated , partly resides in pseudoautosomal region 1 and encodes a protein of unknown function from the X chromosome. By comparing calculated allele frequencies in different populations with 2612 genetic variants in the region, rs311103 showed the strongest correlation to the expected distribution. The same single-nucleotide polymorphism (SNP) had the most significant impact on transcript levels in whole blood ( = 2.0 × 10). The minor allele, rs311103C, disrupts a GATA-binding motif 3.7 kb upstream of the transcription start point. This silences erythroid messenger RNA expression and causes the Xg(a-) phenotype, a finding corroborated by SNP genotyping in 158 blood donors. Binding of GATA1 to biotinylated oligonucleotide probes with rs311103G but not rs311103C was observed by electrophoretic mobility shift assay and proven by mass spectrometry. Finally, a luciferase reporter assay indicated this GATA motif to be active for rs311103G but not rs311103C in HEL cells. By using an integrated bioinformatic and molecular biological approach, we elucidated the underlying genetic basis for the last unresolved blood group system and made Xg genotyping possible.
The bactericidal/permeability-increasing protein (BPI), stored in human neutrophil granulocytes, is cytotoxic against Gram-negative bacteria. Several genes related to BPI cluster on human chromosome 20 and on mouse chromosome 2, but expression and characterization of a BPI ortholog in the mouse have not been reported. We asked whether BPI is structurally and functionally conserved between humans and mice and whether murine BPI might be synthesized in neutrophils as well as in other tissues. We report the isolation of a murine full-length cDNA encoding a 54-kDa protein, showing 53% amino acid identity and 71% similarity, to human BPI. The murine BPI and human BPI genes show a similar exon-intron organization. Murine BPI mRNA was detected in testis, epididymis, and bone marrow, as well as in Sertoli and promyelocytic cell lines. Although levels of BPI mRNA in human and murine testis were comparable, expression in murine bone marrow cells was low as compared with that in human bone marrow. BPI protein showed a cytoplasmic, granular localization in mature neutrophils. BPI gene expression in Sertoli and promyelocytic cells was enhanced several-fold by all-trans retinoic acid. Overexpression of murine BPI in human embryonic kidney 293 cells resulted in antibacterial activity against Escherichia coli, comparable with that obtained with human BPI. In conclusion, it was demonstrated that mouse neutrophils store BPI with antibacterial activity and that murine BPI is also expressed in testis and epididymis.
P1 and P are glycosphingolipid antigens synthesized by the -encoded α1,4-galactosyltransferase, using paragloboside and lactosylceramide as acceptor substrates, respectively. In addition to the compatibility aspects of these histo-blood group molecules, both constitute receptors for multiple microbes and toxins. Presence or absence of P1 antigen on erythrocytes determines the common P (P1P) and P (P1P) phenotypes. transcript levels are higher in P individuals and single-nucleotide polymorphisms (SNPs) in noncoding regions of , particularly rs5751348, correlate with P/P status. Despite these recent findings, the molecular mechanism underlying these phenotypes remains elusive. The In(Lu) phenotype is caused by Krüppel-like factor 1 () haploinsufficiency and shows decreased P1 levels on erythrocytes. We therefore hypothesized KLF1 regulates expression. Intriguingly, -specific sequences including rs5751348 revealed potential binding sites for several hematopoietic transcription factors, including KLF1. However, KLF1 binding did not explain -specific shifts in electrophoretic mobility-shift assays and small interfering RNA silencing of did not affect transcript levels. Instead, protein pull-down experiments using but not oligonucleotide probes identified runt-related transcription factor 1 (RUNX1) by mass spectrometry. Furthermore, RUNX1 binds alleles selectively, and knockdown of significantly decreased transcription. These data indicate that RUNX1 regulates and thereby the expression of clinically important glycosphingolipids implicated in blood group incompatibility and host-pathogen interactions.
The Wilms' tumour gene 1 (WT1) protein is highly expressed in most leukaemias. Co-expression of WT1 and the fusion protein AML1-ETO in mice rapidly induces acute myeloid leukaemia (AML). Mechanisms behind expression of WT1, as well as consequences thereof, are still unclear. Here, we report that the fusion protein BCR/ABL1 increases expression of WT1 mRNA and protein via the phosphatidylinositol-3 kinase (PI3K)-Akt pathway. Inhibition of BCR/ABL1 or PI3K activity strongly suppressed transcription from WT1 promoter/enhancer reporters. Forced expression of BCR/ABL1 in normal human progenitor CD34 þ cells increased WT1 mRNA and protein, further supporting the notion of BCR/ABL1-driven expression of WT1 in human haematopoietic cells. Forced expression of WT1 in K562 cells provided protection against cytotoxic effects of the ABL1 tyrosine kinase inhibitor imatinib, as judged by effects on viability measured by trypan blue exclusion, metabolic activity, annexin V and DAPI (4 0 , 6-diamidino-2-phenylindole) staining. None of the isoforms provided any detectable protection against apoptosis induced by arsenic trioxide and only very weak protection against etoposide, indicating that WT1 interferes with specific apoptotic signalling pathways. Our data demonstrate that WT1 expression is induced by oncogenic signalling from BCR/ABL1 and that WT1 contributes to resistance against apoptosis induced by imatinib.
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