Erythroid-specific promoter activates a novel ZBTB7A transcript. (A) Messenger RNA from CD34⁺ hematopoietic stem cells differentiated toward the erythroid lineage for 1 (HSPC) and 3 (Ret) weeks was subjected to 5′ RACE using a reverse primer specific for exon 2 of ZBTB7A and analyzed by agarose gel electrophoresis. The larger bands present in the reticulocyte samples were sequenced and revealed a novel transcriptional start site upstream of the previously annotated ZBTB7A RefSeq transcriptional start site. (B) Polymerase chain reaction quantification revealing that transcripts containing exon 0 are lowly expressed in HSPC cells but highly upregulated during differentiation (n = 3 per time point). Values were normalized to 18S ribosomal RNA levels. Error bars represent the standard error of the mean (P = .068; paired Student 2-tailed t test). (C) Nuclear extracts were harvested from COS cells and COS cells overexpressing either KLF1 or GATA-1. Binding of these proteins to wild-type (WT) and mutant (Mut) KLF1 and GATA-1 consensus sequences of the ZBTB7A promoter were analyzed by electrophoretic mobility shift assay using radiolabeled probes. Free-probe, KLF1:DNA, and GATA-1:DNA complexes are as indicated. The identities of these complexes were confirmed by supershifting (*) with antibodies specific for KLF1 and GATA-1.