The transcriptional regulation of the zeta-globin gene

Thesis

The ζ-globin gene (HBZ in humans and Hba-x in mice) is the embryonic α-like globin gene that is expressed at the early stages of primitive erythropoiesis but silenced in the definitive erythroid lineage. Understanding the regulation of ζ-globin transcription, especially the mechanisms by which it is silenced, would shed light on gene regulation and offer therapeutic opportunities for ζ-globin induction in patients with severe α-thalassemia.

Previous studies in the laboratory have excluded many common mechanisms of gene silencing, here, Polycomb repression mediated by PRC1 has also been excluded as a participant in ζ-globin silencing. Of interest, histone deacetylation mediated via the NuRD complex appears to play a role in ζ-globin silencing in definitive erythropoiesis. However, the constantly hypoacetylated ζ-globin locus shows no enrichment of NuRD complex in definitive erythroblasts by ChIP-seq, suggesting the potential role of NuRD in ζ-globin silencing may rely on its nucleosome remodelling activity rather than its histone deacetylase components. In contrast to the definitive erythroid lineage, ζ-globin repression is independent of histone deacetylation during the primitive intra-lineage switch.

A comprehensive CRISPR screen across the ζ-globin promoter has identified two repressive cis-acting elements, which are predicted binding motifs of LRF and BCL11A; two transcription factors known to be responsible for ζ-globin repression. Perturbation of the LRF motif has a greater effect than altering the BCL11A motif in definitive erythroblasts from mice and humans. Base editing of both elements increases ζ-globin to 20% of total α-like globin transcription and a pan-cellular ζ-globin induction at the protein level. This effect size is similar to that seen in the LRF and BCL11A double knockout model, showing that these two factors repress ζ-globin transcription by direct promoter binding.

It has previously been shown that individuals with a common deletion of the α-globin genes, termed Southeast Asian deletion (--SEA), which leaves the embryonic ζ-gene intact, have a raised level of ζ-globin expression. To gain insight into the mechanisms of ζ-globin gene expression in such cases, a mouse model that recapitulates the --SEA mutation in humans has been generated and characterised. This model has shown that promoter competition within the α-globin cluster plays an important part in ζ-globin silencing during the primitive intra-lineage switch, suggesting that in the definitive erythroid lineage, in addition to trans-acting factors, promoter competition may also play a role in determining the level of ζ-globin reactivation.

Authors: Liu, S

• DOI: 10.5287/bodleian:z6kG4RqOk
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: gene regulation; thalassaemia; genome-editing