Defense Date

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Human Genetics

First Advisor

Joyce A. Lloyd

Abstract

Krüppel-like factors KLF1 and KLF2 are closely related transcription factors with three zinc finger domains in their carboxy-termini. KLF1 (erythroid Krüppel-like factor, or EKLF) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β- globin genes. KLF1 and KLF2 have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double knockout (KO) mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, and double KO embryos are more anemic and die sooner than either single KO. We have shown that KLF1 and KLF2 positively regulate the human ϵ- (embryonic) and γ-globin (fetal) genes during embryonic erythropoiesis. Chromatin immunoprecipitation assays (ChIP) show that KLF1 and KLF2 bind to the promoters of the human ϵ- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region (LCR) in mouse embryonic erythroid cells. ChIP assays show that KLF1 but not KLF2 ablation results in abnormal histone modifications in the β-globin locus in mouse embryonic erythroid cells. H3K9Ac and H3K4me3, which correlate with open chromatin and active transcription, are both reduced in KLF1-/- primitive erythroid cells. Human CD34+ hematopoietic stem cells obtained from umbilical cord blood were in vitro differentiated along the erythroid lineage. ChIP assays indicate that both KLF1 and KLF2 bind to the promoter of γ-globin gene in this fetal erythroid model. KLF1 knockdown in these cells affects mainly adult β- globin gene expression. However, the decrease in β- globin gene expression in KLF1 knockdown also affects the ratio of γ- to β- globin in these cells. H3K9Ac and H3K4me3 were decreased only at the β- globin gene which coincides with lower recruitment of RNA polymerase II and its active form, RNA polymerase II phospho-serine 2. In conclusion, we showed using mouse primitive erythroid cells and cord blood definitive cells that KLF1 and KLF2 coordinate the regulation of the mouse and human β- globin genes by direct binding to the promoters and LCR in the β- globin locus. In conclusion, cord blood hematopoietic cells could serve as a complimentary system in addition to the transgenic mouse models to study the regulation of γ- globin gene expression.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

December 2012

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