imprint:
[Erscheinungsort nicht ermittelbar]: University of New South Wales. School of Biotechnology & Biomolecular Sciences, 2021
Language:
English
Origination:
University thesis:
Dissertation, University of New South Wales. School of Biotechnology & Biomolecular Sciences, 2021
Footnote:
Description:
β-Haemoglobinopathies are a group of inherited monogenic disorders resulting from mutations in the β-globin gene that disrupt the production of the β-globin subunits of adult haemoglobin. β-Haemoglobinopathies include sickle cell anaemia and β-thalassemia. Existing treatments for these diseases predominantly involve short term symptomatic relief and are of limited effectiveness. As the prevalence of these disorders is high and rising, there is a need for readily available, effective long term treatments. Reversal of the foetal to adult globin switch is an attractive therapeutic approach for β-haemoglobinopathies as elevated levels of foetal haemoglobin (HbF) in adult red blood cells reduce the clinical severity of these diseases.The transcription factor Leukaemia/Lymphoma-Related Factor (LRF), also known as Zinc Finger and BTB Domain Containing 7A (ZBTB7A), is one of two major repressors of the foetal γ-globin genes. While LRF has been shown to bind to a site 200 bp upstream of the transcriptional start site (TSS) of the γ-globin genes to repress foetal γ-globin, little is known about whether LRF binds to other sites in β-globin locus to regulate globin gene expression. Efforts to identify such sites have been hampered by ambiguities in published LRF consensus motifs. In this thesis, we aimed to better define the LRF binding consensus and to use this information to identify LRFs binding sites throughout the β-globin locus. We discovered a novel LRF binding site 60 bp upstream of the foetal γ-globin TSS, with sequence similarity to the 200 site, that bound LRF in vitro. By systematically mutating individual bases within these binding sites and investigating the impact of these mutations on LRF binding in vitro we precisely defined the LRF consensus motif at the -200 and -60 sites as GGNNNNGG. Additionally, we demonstrated that a single nucleotide polymorphism in intron 2 of the β-globin gene creates a de novo LRF binding site with a second binding consensus, GACCC. We used LRF zinc finger (ZF) truncation constructs to show that LRF uses different combinations of ZFs to bind to the two consensus motifs: ZF1-2 to bind to GACCC sites and ZF1, 2 and 4 to bind GGNNNNGG sites.We identified sites throughout the β-globin locus matching these two LRF consensus motifs and tested whether LRF bound to these sites in vitro. Two sites were selected for further functional analysis – one within the β-globin promoter and the other within first intron of the γ-globin gene. We introduced mutations aimed to disrupt LRF binding at these sites into an adult erythroid cell line using CRISPR-Cas9 genome editing and demonstrated that both mutations altered globin gene expression.Our insights into the two LRF binding consensus motifs and the additional roles of LRF binding and regulating gene expression at other sites in the β-globin locus may allow targeted approaches to be developed for disrupting LRF binding to upregulate HbF expression.