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Title: Elucidating the role of MOZ and its implications for KAT6A Global Developmental Delay syndrome
Author: Whitchurch, Jonathan B.
ISNI:       0000 0004 7971 4152
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2019
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The histone acetyltransferase Monocytic Leukaemia Zinc Finger Protein (MOZ) was originally identified at the breakpoint of a chromosomal translocation (inv8) associated with acute myeloid leukaemia (AML). MOZ, encoded by the KAT6A gene, belongs to the MYST family of histone acetyltransferases (HATs) and is expressed in a range of human tissues. It is required for the transcriptional regulation of HOX genes during development and is indispensable for the self-renewal ability and differentiation of haematopoietic stem cells. In mouse studies, KAT6A inactivation results in cardiac development abnormalities and impaired B lymphocyte maturation. Furthermore, this HAT is required for dentinogenesis and the inhibition of cell senescence. Initially this protein was primarily studied in the context of cancer, leading to advances in the understanding of the structure and function of this epigenetic regulator, for example the DPF domain which confers histone substrate specificity. MOZ functions as a component of a chromatin regulatory complex containing ING5, EAF6 and the bromodomain protein BRPF1, catalysing H3K9, H3K14 and H3K23 acetylation. Recently, pathogenic sequence variants in KAT6A, clustering in exons 17 & 18 have been implicated in global developmental delay (GDD) and intellectual disability syndromes. Most of these arise as de novo heterozygous mutations, resulting most commonly in premature termination codons (PTCs). Whether these variants result in nonsense-mediated mRNA decay (NMD), or the production of dominant-negative truncated MOZ proteins, is unknown. In this study, we used CRISPR-Cas9 nickase to generate PTCs in the KAT6A gene in erythroleukemia K562 cells, to resemble those found in individuals displaying KAT6A GDD. Clones were generated in which 2 or 3 alleles of KAT6A were targeted (K562 is triploid for this gene). We observed a reduction in spliced mRNA transcript levels of KAT6A harbouring PTCs in exon 17, indicating the action of transcript degradation by NMD, as no change in pre-mRNA levels were observed. Inhibitors of NMD were able to rescue the expression of KAT6A and MOZ target gene transcripts. In contrast, no evidence of NMD was observed in clones harbouring PTCs in KAT6A exon 18, which expressed truncated MOZ proteins. MTT assays indicated defects in all KAT6A PTC clones with regard to their rates of increase in the total number of viable, metabolically active cells over time, accompanied by changes in mRNA expression levels of selected MOZ target genes, as determined by qRT-PCR. Analysis of publically available RNA-seq data from two individuals with this syndrome indicated significant changes in HOX gene expression. Gene Ontology terms identified from differentially expressed gene lists indicates transcriptional changes in an enrichment of genes involved in cell differentiation and Wnt signalling. Taken together, our results offer a clearer insight into the molecular mechanisms behind the KAT6A GDD syndrome, demonstrating that C-terminal regions of the MOZ protein are indeed essential for some normal molecular functions of this HAT. This paves the way for potential therapies in the future.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH426 Genetics