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.
","abstract_html":"<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 theD221EKLF<sup> </sup>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 <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>","abstract_has_math":false,"creators":["Jansen, Valerie Malyvanh"],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Dissertation","degree_discipline":"Molecular Sciences","degree_department":null,"school":null,"contributors":["John M. Cunningham, M.D."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2009,"date_issued":"2009-05-01T07:00:00Z","date_published":"2009-05-01T07:00:00Z","updated_at":"2026-07-24T05:00:11Z","subjects":["β-globin","chromatin","EKLF","erythropoiesis","megakaryopoiesis","Medical Molecular Biology","Medical Sciences","Medicine and Health Sciences"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://dc.uthsc.edu/dissertations/139","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["John M. Cunningham, M.D."]},{"key":"dc:creator","label":"Author","values":["Jansen, Valerie Malyvanh"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2016-06-08T07:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Molecular Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["β-globin","chromatin","EKLF","erythropoiesis","megakaryopoiesis","Medical Molecular Biology","Medical Sciences","Medicine and Health Sciences"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://dc.uthsc.edu/dissertations/139"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:title","label":"Title","values":["Generation and Characterization of a Knock-In Allele of EKLF: Probing the in vivo Role of the Chromatin Remodeling Domain in Definitive Hematopoietic Cells"]}]}],"canonical_facts":{"dc:contributor":["John M. Cunningham, M.D."],"dc:creator":["Jansen, Valerie Malyvanh"],"dc:date.available":["2016-06-08T07:00:00Z"],"dc:description.abstract":["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.
"],"dc:identifier":["https://dc.uthsc.edu/dissertations/139"],"dc:subject":["β-globin","chromatin","EKLF","erythropoiesis","megakaryopoiesis","Medical Molecular Biology","Medical Sciences","Medicine and Health Sciences"],"dc:title":["Generation and Characterization of a Knock-In Allele of EKLF: Probing the in vivo Role of the Chromatin Remodeling Domain in Definitive Hematopoietic Cells"],"thesis:degree_discipline":["Molecular Sciences"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T05:00:11Z"}