Transcriptional and epigenetic basis for the treatment of inherited disorders of the glycolytic and pentose phosphate pathways using histone deacetylase inhibitor
File(s)
Author(s)
Okoli, Steven
Type
Thesis
Abstract
Glucose-6-phosphate dehydrogenase is a housekeeping enzyme responsible for the first and rate-limiting catalytic step in the pentose phosphate pathway. This pathway generates NADPH which, in recycling glutathione, neutralises reactive oxygen species a process essential to maintaining oxidative homeostasis.
G6PD deficiency affects approximately four hundred million people worldwide. While the majority are asymptomatic, some experience episodic haemolysis precipitated by physical stressors such as birth, infection and oxidants. A small but significant group with severe (Class I) mutations develop a chronic haemolytic anaemia. Histone acetylase (HDAC) inhibitors selectively enhance transcriptional output of the mutant G6PD allele that increase G6PD protein expression and enzyme activity to clinical significance. These findings provide proof-of-concept, but the underlying molecular mechanisms are unclear.
This project employed two complementary chromatin chemo-precipitation techniques to decipher these mechanisms. Click ChIP utilised suberoylanilide hydroxamic acid (SAHA) as a genetic probe to determine its interactors - protein complexes, histone marks, chromatin binding proteins, and other genomic regions - on a genome-wide level. The second technique (CAPTURE) examined the interactions of SAHA on a locus-specific level using modified K562 cells to define the molecular changes that result in the selective induction of G6PD activity following treatment with a HDAC inhibitor.
Data from these two techniques indicated that SAHA activates cellular stress response pathways which either directly or indirectly induce G6PD expression. Histone proteomics showed that the G6PD promoter exists in a poised state with both activating and repressive post translational modifications (PTMs) occurring at H3K9 and H4K20 on the same nucleosome. The asymmetric configuration of the nucleosome associated with the G6PD locus is unique amongst enzymes of the glycolytic and pentose phosphate pathways, preparing the gene for rapid activation, and that SAHA increases activating PTMs which in turn selectively increases gene expression...
G6PD deficiency affects approximately four hundred million people worldwide. While the majority are asymptomatic, some experience episodic haemolysis precipitated by physical stressors such as birth, infection and oxidants. A small but significant group with severe (Class I) mutations develop a chronic haemolytic anaemia. Histone acetylase (HDAC) inhibitors selectively enhance transcriptional output of the mutant G6PD allele that increase G6PD protein expression and enzyme activity to clinical significance. These findings provide proof-of-concept, but the underlying molecular mechanisms are unclear.
This project employed two complementary chromatin chemo-precipitation techniques to decipher these mechanisms. Click ChIP utilised suberoylanilide hydroxamic acid (SAHA) as a genetic probe to determine its interactors - protein complexes, histone marks, chromatin binding proteins, and other genomic regions - on a genome-wide level. The second technique (CAPTURE) examined the interactions of SAHA on a locus-specific level using modified K562 cells to define the molecular changes that result in the selective induction of G6PD activity following treatment with a HDAC inhibitor.
Data from these two techniques indicated that SAHA activates cellular stress response pathways which either directly or indirectly induce G6PD expression. Histone proteomics showed that the G6PD promoter exists in a poised state with both activating and repressive post translational modifications (PTMs) occurring at H3K9 and H4K20 on the same nucleosome. The asymmetric configuration of the nucleosome associated with the G6PD locus is unique amongst enzymes of the glycolytic and pentose phosphate pathways, preparing the gene for rapid activation, and that SAHA increases activating PTMs which in turn selectively increases gene expression...
Version
Open Access
Date Issued
2022-07
Date Awarded
2023-07
License URL
Advisor
Karadimitris, Anastasios
Layton, Mark
Caputo, Valentina
Sponsor
British Society for Haematology
Imperial Health Charity
Publisher Department
Department of Immunology and Inflammation
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)