| Summary: | <p>The expression of the human α-globin locus is regulated by a set of well characterized enhancer elements. Mutations in the α-globin protein coding genes or their regulatory elements can cause a form of anaemia called α-thalassemia which results from the downregulation of α-globin in red blood cells. There are rare cases where this downregulation is caused by genetic defects outside the know elements.</p> <p>This thesis deals with a genetic mutation found in an intergenic region in the α-globin locus outside of any know regulatory site. The individuals with the mutation show the typical features of α-thalassemia but have also acquired the expression of a new transcript in their red blood cells. This transcript originates near a single base pair substitution which has been associated with the phenotype and is located upstream of the α-globin genes. The mutation is a transition from the wild type Thymidine to a Cytosine, which generates a new binding motif for the key erythroid transcription factor GATA1. Previous studies of an affected individual’s primary erythroid cells demonstrate enrichment of GATA1 in the region of the mutation, along with the recruitment of TAL1 and RNA Polymerase II and an increase in acetylation of histone H3.</p> <p>The thesis tests the hypothesis that the new transcriptional unit may utilise the established transcription regulatory landscape of the α-globin locus for its own expression. This would result in competition with the α-globin promoters for enhancer activity, thereby causing a downregulation of α-globin transcription. The first objective of this thesis is to recapitulate the previously observed phenotype in a cell line system. This model is then used to address the question of how the normally un-transcribed sequence has acquired the ability to be expressed and lastly, this thesis explores the molecular mechanisms that cause the downregulation of α-globin. </p> <p>To this end an iPSC-based erythroid differentiation system was characterized. This system appears to recapitulate key aspects of erythropoiesis and generates a highly pure culture containing a large number of erythroid-like cells. Next, patient iPSC lines containing the mutation were generated and differentiated along the erythroid pathway. These cells faithfully express the novel transcript and show a reduction in α-globin mRNA levels. Expression of the new transcript is accompanied by the emergence of a new region of open chromatin at the site of the mutation along with the recruitment of GATA1, KLF1 and RNA Polymerase II and the enrichment of the promoter mark H3K4me3.</p> <p>Chromatin conformation analysis was used to assess the proximity between the elements in the α-globin locus in the presence of the mutation. This demonstrates increased interaction between the enhancers and the site of the new transcriptional unit and reduced interaction of the α-globin promoters with their enhancers, suggesting competition for enhancer activity.</p> <p>Finally, to demonstrate that the variant is necessary for the observed phenotype a patient line was genetically engineered to correct the mutation. This rescues the phenotype by reducing the expression of the new transcript, abolishing the accessible region and upregulating α-globin transcription. In addition, the mutation was introduced in a wild type iPSC line to recreate the phenotype. This results in the emergence of the patient-specific region of accessible chromatin, expression of the novel transcript and downregulation of α-globin. These observations prove that the single base pair change is sufficient to cause the formation of the new transcriptional unit and that it is the causal mutation for this form of α-thalassemia.</p>
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