Medical Biology - Theses
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ItemCharacterising the molecular regulation of erythroferrone( 2023-10)ERFE encodes the hormone erythroferrone which is secreted from erythroblasts in response to increased erythroid drive. ERFE protein suppresses hepcidin, the master iron regulator, which allows iron to be released from body-iron stores and used for red blood cell production. In erythropoietic disorders such as thalassaemia, ineffective red blood cell production results in reduced tissue-oxygen levels, increased erythroid drive, and chronic hepcidin suppression. However, despite representing a possible drug target, the regulation of ERFE has not been well studied. This work has identified a putative ERFE control locus comprising an enhancer and several key transcription factors using in vitro differentiated Human Umbilical Cord Blood-derived Erythroid Progenitor (HUDEP-2) cells. ERFE transcription and chromatin accessibility were tracked during four stages of terminal erythroblast maturation using quantitative PCR and Assay for Transposase-Accessible Chromatin-sequencing (ATAC-seq). These data demonstrated a dynamic chromatin accessibility landscape with distinct erythroid maturation stages and an expression profile that peaked in intermediate erythroblasts (p<0.001). Capture-C then demonstrated contact between ERFE’s 5’ promoter and a putative enhancer that also aligns with trimethylation of lysine 4 and acetylation of lysine 4 on histone 3 (promoter marks), monomethylation of lysine 4 and aceytylation of lysine 27 on histone 3 (enhancer marks) Cleavage Under Targets & Release Using Nuclease (CUT&RUN). Moreover, when ERFE expression is at its highest, CUT&RUN showed that response elements in the enhancer are bound by master erythroid regulators GATA1, KLF1 and TAL1, and the stress erythroid response factor, STAT5, suggesting a role for multiple signalling pathways in ERFE activation. These pathways, and ERFE’s place within them, were further explored using weighted gene correlation network analysis on RNA sequencing from the four progenitor stages, where gene set enrichment analysis demonstrated that ERFE is co-expressed alongside genes that are highly associated with haem metabolism (p=3.65x10-30). Overall, this data provides new insights into the regulation of erythroferrone and may contribute valuable details for identifying therapeutic targets in iron-loading anaemias.