PIGM transcription and chromatin accessibility in erythroid and B cells. (A) Fluorescence-activated cell sorter analysis of cell-surface expression of CD59 in primary erythroid precursor cells from a normal wild-type (WT) donor (green), heterozygous (Het1; blue) parent, and patient with IGD (P1; red). Erythroid precursor cells were generated from peripheral blood mononuclear cells, and flow cytometric analysis was performed on day 7 of the culture. Histograms show CD59 expression on erythroid precursors identified as CD36⁺GlyA+ cells (upper panel). Gray line: isotypic control. (B) PIGM mRNA expression in normal, Het1, and P1 primary erythroid precursor cells. PIGM mRNA levels were assessed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in flow-sorted CD36⁺GlyA+ erythroid precursor cells as shown in (A). Data were normalized to GAPDH. Het1 and P1, n = 1; normal, n = 4. (C) PIGM mRNA expression in Het1 and P1 B cells. PIGM mRNA levels were assessed by qRT-PCR in sorted primary CD19⁺GPI+ and CD19⁺GPI− B cells (see supplemental Figure 1A). GPI expression was assessed after staining with flourescent aerolysin. Note that heterozygous B cells are all GPI+ . Data were normalized to GAPDH. Het1, n = 2; P1, n = 2. (D) WT and mutated (Mut) PIGM promoter transcriptional activity in vitro. Luciferase reporter assays were performed in K562 cells. Cell lysates were assayed for luminescence 48 hours after transfection. Luminescence values were normalized to renilla, and results are presented relative to the activity of the WT promoter construct. pGL3 represents a promoter-less control construct. Mean values of 2 independent experiments, each performed in triplicate assays, are shown. (E) Nucleosome mapping by MNase protection assay at the length of the PIGM promoter in WT LBCL, HeLa, and K562 cells. The abundance of eleven 100-bp amplicons that overlap by 50 bp (shown below the graph) was quantified by quantitative polymerase chain reaction using equal amounts of MNase-digested or undigested DNA, as described the supplemental Methods. The gray box indicates the −270GC-rich box. Letters represent amplicons upstream of the transcription start site (TSS; shown as an arrow), whereas numbers denote regions downstream of the TSS. (F) PIGM mRNA expression in 2 WT LBCL, HeLa, and K562 cells. PIGM mRNA levels were assessed by qRT-PCR. Data were normalized to GAPDH and are shown as mean ± standard error of the mean (n = 3).