Date of Award
Doctor of Philosophy (PhD)
John M. Cunningham, M.D.
Linda M. Hendershot, Ph.D. Satoru K. Nishimoto, Ph.D. Derek A. Persons, M.D., Ph.D. Gerard P. Zambetti, Ph.D.
β-globin, chromatin, EKLF, erythropoiesis, megakaryopoiesis
The zinc finger-encoding transacting factor EKLF, or erythroid Krüppel-like factor, binds key regulatory elements of many erythroid-specific genes, and is essential for definitive erythropoiesis. Mice lacking this factor 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 EKLF-null embryos, with increased Fli-1 gene expression, a marker of early megakaryocytic differentiation. These observations are consistent with the idea that EKLF modulates the megakaryocytic-erythroid (M-E) differentiation switch.
Our laboratory has previously demonstrated that an amino terminal sequence of EKLF (D221EKLF) is required to induce chromatin remodeling at the β-globin promoter in an EKLF-null erythroid cell line. However, additional amino terminal sequences are required for initiation of β-globin gene transcription. To evaluate the role of this chromatin remodeling domain in erythroid and megakaryocytic differentiation in vivo, I have generated a knock-in allele of D221EKLF. Using the recombineering method, a lambda phage-based homolgous recombination method in E. coli, cDNA encoding theD221EKLF domain has been inserted into the endogenous initiation site, thus placing the mutant protein under the cis-regulatory elements of the endogenous murine EKLF locus. Subsequently, D221EKLF alleles have been generated by gene targeting in ES cells. I have used the mice to probe the in vivo role of D221EKLF in definitive hematopoietic cells.
Similar to EKLF-null embryos, mice homozygous for the D221EKLF mutant allele die of anemia by E15.5 of gestation. Molecular analysis ofD221EKLF erythroblasts reveals i) a failure to activate β-globin gene transcription; ii) lack of GATA-1 and NF-E2 recruitment to the β-globin promoter; iii) a block in terminal erythroid differentiation. In contrast to erythroid cells lacking EKLF, D221EKLF erythroid progenitors demonstrate appropriate binding of the D221EKLF encoding domain to all EKLF-regulatory sequences and a chromatin architecture and histone modification pattern at erythroid-specific genes that recapitulate the events observed in wild-type EKLF erythroblasts at a similar stage of erythroid ontogeny.
Examining the role of D221EKLF in megakaryopoiesis, I observed inhibition of megakaryocytic progenitor expansion in D221EKLF fetal hematopoietic cell populations when compared to EKLF-null embryos. Molecular analysis of D221EKLF erythroblasts reveals i) binding of theD221EKLF mutant protein to the Fli-1 promoter with inhibition of gene transcription; ii) hypoacetylation of histone H3 at the Fli-1 promoter; iii) recruitment of a Sin3A-containing corepressor complex to the Fli-1 promoter. Taken together, my results suggest strongly that the unique D221EKLF domain is sufficient to modulate the chromatin-specific roles of EKLF at erythroid- and megakaryocytic-specific loci in definitive hematopoietic cells in vivo.
Jansen, Valerie Malyvanh , "Generation and Characterization of a Knock-In Allele of EKLF: Probing the in vivo Role of the Chromatin Remodeling Domain in Definitive Hematopoietic Cells" (2009). Theses and Dissertations (ETD). Paper 139. http://dx.doi.org/10.21007/etd.cghs.2009.0152.