The regulation of alpha-like globin gene expression throughout development and differentiation

Thesis

A key question in molecular biology is how genes are turned on and off throughout development and differentiation? Precise spatio-temporal gene regulation involves complex interplay between cis- and trans-acting regulatory factors. Cis-acting elements include enhancers, promoters, and insulators. Trans-acting factors include transcription factors (TFs), co-factors, chromatin remodelling factors, and ncRNAs. In this thesis, I use the well characterised ⍺-globin gene locus to study the roles of the CTCF-binding site (CBS) insulator elements at the 3ʹ of the ⍺-globin sub-TAD in normal erythropoiesis and the transcription factor KLF1 in developmental gene switching of the ⍺-like globin genes.

First, I characterise a mouse model and show that deletion of the 3ʹ CBSs (θ1, θ2, HS+44, and HS+48) has a surprisingly minimal role in formation or maintenance of the ⍺-globin sub-TAD. I find no significant changes to the enhancer interaction, chromatin accessibility, or gene expression profiles of the ⍺-globin locus. I discuss, instead, the possibility that the ⍺-globin genes themselves may delimit the ⍺-globin sub-TAD.

Next, I characterise a novel KLF1StrepII-FKBP mouse model. A major difficulty when studying KLF1, particularly for low cell-input assays, is limited availability of suitable antibodies for chromatin immunoprecipitation (ChIP). I surprisingly find that the Strep-II tag is insufficient for robust chipmentation (low-input ChIP) of KLF1StrepII-FKBP. I discuss the factors that might make KLF1 difficult to immunoprecipitate and suggest future optimisations to the chipmentation protocol.

Finally, I investigate the role of KLF1 in ⍺-like globin gene switching. Some humans with KLF1 mutations upregulate embryonic 𝜁-globin in definitive erythroblasts. Understanding mechanisms of 𝜁-globin (de)repression has important therapeutic implications for treatment of severe ⍺-thalassemia. I use the FKBP-V degron tag to quickly and effectively degrade KLF1StrepII-FKBP protein in primary definitive erythroblasts to test whether KLF1 depletion recapitulates 𝜁-globin de-repression in mouse. I observe many of the expected cellular and gene expression effects, including modest upregulation of 𝜁-globin at the RNA but not protein level. I discuss some discrepancies between the human patients and mouse model that may explain this. Overall, I present the KLF1StrepII-FKBP model as a novel and powerful tool for future use beyond globin switching to investigate the numerous erythroid functions of KLF1.

Authors: Holliman, SR

• DOI: 10.5287/ora-go4pe4zv7
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: haemoglobin; HBZ; hemoglobin; gene regulation; gene repression; gene expression; 𝜁-globin; insulator; globin; gene switching; Hba-x; promoter; gene silencing; LRF; erythropoiesis; BCL11A; enhancer; zeta-globin; ⍺-globin; erythroblast; erythroid; KLF1; alpha-globin
• Subjects: Gene silencing; Genetic regulation