EKLF/Klf1 Regulates Erythroid Transcription By Its Pioneering Activity and Subsequent Control of RNA Pol II Pause-Release

EKLF/Klf1 is a master transcriptional activator of critical genes that regulate both erythroid fate specification and terminal erythroid maturation. EKLF binds to DNA using three Zn-fingers at its C-terminus while the N-terminus constitutes a transcription activation domain (TAD) that interacts with various transcription co-factors including the protein acetylase CBP. An autosomal semi-dominant mutation at a single residue (E339D) in the mouse EKLF Zn-finger leads to Neonatal anemia (Nan). A mutation at the same residue in human EKLF (E325K) causes Congenital Dyserythropoietic Anemia type IV (CDA IV). Nan/Nan mice show lethality at embryonic day E10-11, in contrast to EKLF-/- homozygotes that survive until E15. Nan/+ heterozygotes survive to adulthood but are severely anemic, unlike EKLF+/- heterozygotes that display no aberrant phenotypes. The Nan-EKLF protein has an altered DNA binding specificity leading to a vastly altered transcriptome by two mechanisms. First, Nan-EKLF binding causes ectopic gene expression that significantly contributes to the severe anemia in Nan/+. Second, a subset of EKLF targets is downregulated in heterozygous Nan/+ mutants despite the presence of one copy of wild type EKLF, exacerbating the anemia. Thus, uncovering the mechanism by which gene expression is altered in Nan/+ may illuminate how EKLF normally activates transcription of its targets in vivo. To this end, we first examined the global occupancy of RNA Pol II phospho-Ser5 (as a paused mark) and phospho-Ser2 (as an elongation mark) in the mouse E13.5 fetal liver as a source of primary definitive erythroid cells. At promoters of ectopically expressed genes, where only Nan-EKLF (but not WT) binding is expected, we predominantly find increased levels of both paused and elongating RNA Pol II suggesting that Nan-EKLF binding activates transcription at ectopic genes by RNA Pol II recruitment and promoter proximal pausing. Further, we find increased levels of H3K27ac and CBP occupancy at these sites indicating that the mechanism of Pol II recruitment relies on CBP-mediated H3K27 acetylation and increased chromatin accessibility. Overall, this suggests robust pioneering activity of Nan-EKLF likely mediated by the interaction of its TAD with the CBP/p300 acetylase complex. At genes downregulated in Nan/+ we find two major patterns of Pol II occupancy. One is the converse of that seen at ectopic genes wherein there is a concomitant decrease in both Pol II p-Ser5 and p-Ser2 levels