Altered Regulation of β like Globin Genes by a Redesigned Erythroid Transcription Factor.

The well characterized switch during ontogeny of globin gene expression from embryonic/ fetal to adult type is a result of a complex interplay between cis and trans acting regulatory elements at the beta globin locus. Trans acting elements include tissue specific transcription factors that bind specific motifs within the beta globin gene cluster with high specificity. Erythroid Kruppel like factor (EKLF) is one such erythroid specific, zinc finger transcription factor that is critical for the activation of the beta globin promoter and for consolidating the switch from gamma to beta globin during development. The ability to willfully regulate the expression of endogenous genes using redesigned zinc finger transcription factors is an emerging field. There is tremendous appeal in utilizing the understanding of transcriptional control pathways to design tools that will elucidate molecular mechanisms and provide potential therapeutic tools. To this end we redesigned Erythroid Kruppel Like Factor (EKLF) as a transcriptional repressor. The zinc finger DNA binding domain was linked to the repressor domain from the Drosophila Engrailed protein with the prediction that this construct (ENG/ZNF) would bind the beta globin promoter and repress it. It was hypothesized that embryonic/fetal globin activation would result by a competitive mechanism. When introduced transiently into cells these transcription factors are effective in repressing the adult beta globin promoter CACCC element, the natural target for EKLF. In stable MEL clones, repression of the adult beta globin gene is accompanied by a reactivation of the endogenous embryonic globin gene. In order to study this effect in the context of a whole animal we generated transgenic mice expressing ENG/ZNF. A 271 bp region 5′of the ANK-1 gene was chosen to drive expression in transgenic mice as it provides erythroid specific expression with copy number dependence and minimal position dependence. D13.5 fetal livers were subject to RT-PCR analysis in the linear range to quantitate the ratios of BH1 to alpha globin transcripts. The 9 ENG/ZNF transgenic embryos express BH1 mRNa in a range of values that is statistically higher than in 9 control littermates (Mann Whitney U test, p value 0.02) and beta major globin mRNA at lower levels. We further studied ENG/ZNF in the developmentally plastic environment of differentiating murine embryonic stem cells. The construct was stably integrated into a targeting site upstream of the HPRT locus under the control of a tetracycline inducible promoter. The Doxycycline induction of ENG/ZNF transgene expression results in a 4 fold activation of embryonic globin at day 6 of embryoid body development; however there is no evidence of beta globin repression. Since at this stage of embryoid body development, primitive erythroid cells are 100–500 fold more abundant than definitive erythroid cells, this may reflect a differential effect of EKLF in primitive erythroid cells. To evaluate this further, we are currently performing analyses in primitive versus definitive erythroid colonies. In conclusion, our studies support the competitive model of globin switching and may contribute to the delineation of a stage specific role of EKLF. In addition, transcriptional reagents that augment gamma globin expression hold promise as novel therapeutic agents for sickle cell disease and other hemoglobinopathies.