Inherited glycosylphophatidylinositol (GPI) deficiency (IGD), characterized by thrombosis and epilepsy but minimal hemolysis, is caused by a c>g mutation at position −270 that disrupts binding of the transcription factor (TF) Sp1 to the core promoter of the mannosyltransferase-encoding gene PIGM. In IGD, the level of GPI expression varies considerably between hematopoietic cells of different lineages; for example, while in erythrocytes it is near normal, accounting thus for the absence of significant intravascular hemolysis, GPI synthesis in granulocytes is severely deficient suggesting erythroid-specific transcriptional regulation of PIGM. We tested the hypothesis that the erythroid-megakaryocytic-specific TF GATA-1 is critical regulator of PIGM transcription and it can overcome the transcriptional block imposed by the c>g mutation. Consistent with this hypothesis, we found that in luciferase assays a 2kb PIGM promoter construct had at least 3-fold higher activity when transfected in erythroid (K562) rather than myeloid (HL60), B lymphoid or epithelial (HeLA) cell lines. Co-transfection of the 2kb WT or c>g mutated construct with GATA-1 resulted in proportional increase of the transcriptional activity which was completely abolished when shorter, 0.6kb WT or c>g constructs were used, implying the presence of GATA-1 binding elements (BE) between −0.6 and −2kb. The functional significance of 3 such GATA-1 BE initially identified by bioinformatic analysis was investigated next. In vitro binding to each of these BE was shown in electromobility shift assays using appropriate oligos, nuclear extracts from K562 cells and anti-GATA-1 antibody while in vivo binding was demonstrated by chromatin immune-precipitation assays. The relative transcriptional activity of the 3 GATA-1 BE was tested in luciferase assays by co-transfecting into HeLA cells WT or c>g 2 kb constructs with single, double or triple BE disrupted by site-directed mutagenesis. Overall the results suggested that the proximal of the 3 GATA-1 BE is the most active and that the transcription-enhancing effect of GATA-1 is diminished in the presence of the disease causing c>g mutation. Thus, as well as explaining the near normal synthesis of GPI in erythrocytes in IGD, our results also provide an important paradigm of co-operative function of a generic (Sp1) and tissue-specific (GATA-1) TF in the transcriptional regulation of a house-keeping gene such as PIGM.

Disclosures: Almeida:Celgene: Consultancy, Research Funding.

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