Altered Erythroid and Megakaryocytic Differentiation in Mice Expressing a Unique Chromatin Remodeling Domain of Erythroid Krüppel-Like Factor (EKLF)

The zinc finger-encoding transacting factor EKLF binds key regulatory elements of many erythroid-specific genes, and is essential for definitive erythropoiesis. Mice lacking this factor (EKLF^−/−) die of anemia by E15.5 of gestation, failing to activate β-globin gene transcription, and demonstrating a block in the erythroid differentiation program at the primitive erythroblast stage. In contrast, megakaryocytic progenitors are amplified in EKLFnull embryos, with increased Fli-1 gene expression (a marker of early megakaryocytic differentiation), consistent with the idea that EKLF modulates the megakaryocyticerythroid (M-E) differentiation switch. We have demonstrated that an amino terminal mutant of EKLF (Δ221EKLF), is required to induce chromatin remodeling at the β-globin promoter in an EKLF-null erythroid cell line, but additional amino terminal sequences are required for initiation of β-globin gene transcription (Brown et al., 2002). To evaluate the role of this chromatin remodeling (CR) domain in erythroid and megakaryocytic differentiation in vivo, we have generated a knock-in allele of EKLF^CR allele. Similar to EKLF-null embryos, mice homozygous for this mutant allele die of anemia by E15.5 of gestation. In contrast to erythroid cells lacking EKLF, EKLF^CR/CR progenitors demonstrate

• appropriate binding of the CR encoding domain to all EKLF-regulatory sequences;

• a block in erythropoiesis at a more a mature stage in differentiation

• a chromatin architecture and histone modification pattern at erythroid-specific genes that recapitulates the events observed in EKLF^+/+ erythroblasts at a similar stage of erythroid ontogeny;

• a failure of terminal erythroid gene transcription.

Examining the role of EKLF^CR in megakaryopoiesis, we observed

• inhibition of megakaryocytic progenitor amplification in EKLF^CR/CR fetal hematopoietic cell populations when compared to EKLF-null embryos;

• loss of Fli-1 gene expression in EKLF^CR expressing cells;

• binding of the EKLF^CR mutant protein to the Fli-1 promoter with inhibition of gene transcription;

• a repressed chromatin architecture at megakaryocytic gene loci.

In contrast to these results, mice homozygous for a knockin allele encoding the zinc finger DNA binding domain alone (Δ253EKLF), a region shown previously to be sufficient for chromatin remodeling in vitro, demonstrate erythroid and megakaryocytic phenotypes that resemble those observed in EKLF-null hematopoietic progenitors. Taken together, our results suggest strongly that the unique EKLF^CR domain is necessary and sufficient to modulate the chromatin-specific roles of EKLF at erythroid- and megakaryocytic-specific loci in definitive hematopoietic cells in vivo.