KLF1 Modulates Delta-Aminolevulinic Acid Dehydratase (ALAD) Gene Transcription in a Context-Specific Manner

Abstract 2094

Krüppel-like factor 1 (KLF1) is a zinc finger-encoding transcription factor that recognizes CACC elements, and is essential for maximal erythroid-specific gene transcription. Several critical mechanisms dependent on KLF1 and required for gene activation have been elucidated, predominantly using the beta-globin locus. KLF1 has been associated with the ordered recruitment of SWI/SNF and RNA polymerase-II complexes, necessary for chromatin remodeling and gene transcription respectively. KLF1 has also been reported to influence erythroid-specific heme biosynthesis. Studies in KLF1-null fetal erythroblasts and a KLF-1 deficient cell line have demonstrated that mRNA levels of the first three enzymes of the biosynthetic pathway are underrepresented. However, although in vitro studies of the rate-limiting enzymes ALAS2 and PBGD suggested a potential regulatory role for KLF1, in vivo studies failed to validate these findings. ALAD is the second enzyme of the pathway. Complete loss of ALAD expression in erythroid cells results in catastrophic events during zebrafish ontogeny. Interestingly, no human erythropoietic defect has been reported as a consequence of aberrant ALAD expression. To extend the analysis of KLF1's regulation of heme biosynthesis, we evaluated KLF1 binding of enzyme regulatory sequences by EMSA and ChIP studies, identifying a KLF1 binding CACC element in the erythroid-specific ALAD promoter. This regulatory element was transactivated specifically by a KLF1 transgene in KLF1-deficient cells. Using a unique 4-OH-Tamoxifen (4-OHT) mediated KLF1-inducible erythroid cell line (K1-ERp), we identified KLF1 as an essential, and early (within 2 hours of induction) activator of transcription of the endogenous ALAD, but not ALAS2 or PBGD genes. Further studies in K1-ERp cells, including DNAseI hypersensitivity and ChIP assays revealed that KLF1 occupancy at the erythroid-specific ALAD promoter triggers a series of molecular events including histone modifications, and enhanced recruitment of the sequence-specific transcription factors, GATA-1, NF-E2 and the TAL-1/SCL multiprotein complex. Importantly, we identified differences in the kinetics of recruitment of the closely related histone acetyltransferases proteins CBP and p300 and the SWI/SNF ATPase Brg1. The latter complex was recruited subsequent to KLF1 binding, although the ALAD promoter was already DNAseI hypersensitive. These results suggest strongly that KLF1 plays a major role in the regulation of heme biosynthesis in erythroid cells. Furthermore, our data challenges a model in which an identical temporal cascade of molecular events are required for transcription at KLF1-dependent promoters.