Generation and Characterization of a Knock-In Allele of EKLF: Probing the in vivo Role of the Chromatin Remodeling Domain in Definitive Hematopoietic Cells

<p>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.</p> <p> 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 <em>in vivo</em>, I have generated a knock-in allele of D221EKLF. Using the recombineering method, a lambda phage-based homolgous recombination method in <em>E. coli,</em> cDNA encoding theD221EKLF domain has been inserted into the endogenous initiation site, thus placing the mutant protein under the <em>cis-</em>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 <em>in vivo </em>role of D221EKLF in definitive hematopoietic cells.</p> <p> 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.</p> <p> 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 theD221EKLFmutant 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 <a></a>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 <em>in vivo</em>.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Molecular Sciences

Year dc:date.available

2009

Author dc:creator

• Jansen, Valerie Malyvanh

Contributors dc:contributor

• John M. Cunningham, M.D.

• β-globin
• chromatin
• EKLF
• erythropoiesis
• megakaryopoiesis
• Medical Molecular Biology
• Medical Sciences
• Medicine and Health Sciences

Repository record dc:identifier

https://dc.uthsc.edu/dissertations/139

OAI identifier oai:identifier

oai:dc.uthsc.edu:dissertations-1127