Differential binding of Sp1 to the −270GC-rich box in erythroid and B cells. (A) Schematic of PIGM promoter. a1 to a8 are amplicons of the PIGM promoter used in chromatin immunoprecipitation analysis. The gray box denotes the position of the experimentally validated Sp1 binding site mutated in IGD at position −270 from ATG. Red and blue boxes correspond to predicted GATA1 and Sp1 binding sites, respectively. (B) Sp1 binding at the length of the PIGM promoter in WT LBCL and K562 cells. DHFR and α-globin represent amplicons in the promoter region of the respective genes and are shown as positive controls. +6000bp represents an amplicon 6 kbp downstream of the ATG start codon of PIGM and is shown as a negative control. n = 5 to 6 independent experiments. (C) Sp1 binding at the PIGM promoter in primary erythroid precursor cells. Sp1 binding was assessed in d5 and d7 cord blood–derived erythroid precursors by chromatin immunoprecipitation quantitative polymerase chain reaction. Results are presented as fold enrichment over immunoglobulin G control (n = 1). (D) The impact of a dominant-negative (DN) form of Sp1 on PIGM transcription in K562 cells. Cells were transduced either with a green fluorescent protein (GFP)-expressing retrovirus containing a complementary DNA encoding a DN form of Sp1 or with a no-insert GFP control. mRNA levels were assessed by qRT-PCR in flow-sorted purified cells 48 hours later and normalized against GAPDH. The graph shows PIGM and DHFR mRNA levels in cells transduced with the GFP-DN Sp1 construct relative to GFP-only control. Two independent experiments are shown.