treated KLF1 transgenic cells only. As relevant controls, KLF1 was also detected at the b-globin promoter, but did not occupy the non-erythroid amylase gene promoter, nor a region located approximately 1 kb upstream of the b-globin promoter. As the rate of transcription of the Alad1b gene gradually decreased over time, we assessed the stability of KLF1 binding to the Alad1b promoter. Examination of the kinetics of KLF1 recruitment to the Alad1b promoter at multiple time points post 4-OHT exposure revealed maximal binding levels within the first 30 min of induction, correlating with the measured kinetic response in Alad1b transcription. KLF1 occupancy remained maximal for the duration of the analysis. These findings suggest that the decrease in the transcriptional rate of Alad1b observed previously is not a consequence of a decrease in KLF1 promoter occupancy. To assess KLF1’s ability to transactivate the Alad erythroid promoter, we employed a dual-luciferase reporter assay. The Alad promoter was cloned into pGL3-Basic vector and co-transfected into K562 cells, with a renilla luciferase expression construct and either a KLF1 expression vector or a vector control. The HS2-b-Luc construct was used as a positive control for KLF1-dependent transactivation. HS2-b-Luc and the Alad1b promoter were activated only in the presence of KLF1. Importantly, KLF1 did not activate the minimal pGL3Basic regulatory sequences, nor an Alad1b promoter with CACC mutations, indicating that transactivation requires an intact binding motif. Cell cycle monitoring via p18 expression analysis indicated that KLF1 over-expression did not induce the differentiation of K562 cells . This data suggests that KLF1 is able to modulate transactivation of the Alad1b promoter independent of erythroid differentiation. Results Alad is Induced Specifically by KLF1 in K1-ERp Cells Examination of global gene expression in KLF1-null murine fetal liver erythroblasts revealed that mRNA of the first three enzymes of the heme 
biosynthesis pathway was underrepresented, consistent with KLF1-regulated expression of these genes. Alad gene transcription in erythroid cells is regulated by a tissuespecific promoter harboring consensus-binding sequences for GATA1 as well as a conserved CACC element. Similar motifs exist in the Alas-2 and Pbgd gene promoters. ChIP-Seq analysis using a KLF-specific antibody demonstrated an enrichment of these promoters in fetal erythroid progenitor cells. Interestingly, the binding to Alas-2 and Pbgd promoters was weaker, suggesting that KLF1 may play a more prominent role in the regulation of Alad1b-mediated transcription. To explore these differences, we evaluated KLF1-dependent Alad, Alas-2 and Pbgd transcription in K1-ERp cells. This erythroid model, derived from KLF1-null fetal erythroblasts, expresses a transgenic KLF1 cDNA, fused in frame to sequences encoding a hemagglutinin epitope, and regulated by an estrogen receptor-dependent regulatory sequence. Exposure of KLF1-transduced cells to tamoxifen results in rapid translocation of KLF1 to the nucleus. Associated with this change, we observed an induction of Alad1b mRNA levels over a 6 h time period as measured by semi-quantitative real-time reverse transcription PCR . This buy JW 55 increase in transcript levels phenocopies that observed between KLF1-null and wild type primary murine erythroblasts. Interestingly, we noted no significant change in Alad1b mRNA levels between K1-ERp cells and the parental KLF1 null ce