Abstract
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, along with lower H3K27ac and CBP levels suggesting EKLF gene activation has been lost at these sites in Nan/+. This includes cell cycle EKLF targets such as E2f2 and Rgcc. The second set of genes have comparable levels of p-Ser5 (paused) Pol II in Nan/+ and WT, but lower levels of p-Ser2 (elongating) Pol II in Nan/+. This suggests that although Pol II is being recruited to the TSS and pauses effectively, the pause-release step leading to effective transcription elongation is impaired. This subset includes important EKLF targets such as Bcl11a, Pax7, Xpo7, and several membrane transporters. As expected, CBP and H3K27ac levels are similar in WT and Nan/+ at these sites.
To determine the cause of impaired RNA Pol II pause-release we examined the global occupancies of key transcription elongation factors such as P-TEFb and NELF. We find that levels of NELF, a negative elongation factor, remain unchanged in WT and Nan/+. However, levels of the P-TEFb subunit Cdk9, a positive elongation factor that facilitates release of paused RNA Pol II, is significantly lower at the TSS of these genes in Nan/+. This suggests that in Nan/+, possible reduction or loss of EKLF binding at some EKLF target promoters impairs effective recruitment of positive transcription elongation factors, resulting in a failure to efficiently release paused RNA Pol II. This causes downregulation of these EKLF target genes and contributes to the severe anemic phenotypes of the Nan mouse.
We conclude that: EKLF exhibits expression control of its target genes at both the transcriptional initiation and elongation steps in vivo; EKLF can act as a pioneer transcription factor and increase chromatin accessibility through H3K27 acetylation by CBP leading to recruitment and pausing of RNA Pol II; and EKLF recruits the positive transcription elongation complex P-TEFb, enabling the controlled release of paused RNA Pol II at transcription start sites of a select group of its targets.
Disclosures
No relevant conflicts of interest to declare.
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