Investigation into the role of heterochromatin protein 1 gamma (HP1γ) in gene regulation in mammals

Heterochromatin protein 1 (HP1) was identified as a key component of the condensed DNA surrounding centromeres in most eukaryotic cells. Genes placed in close proximity to such constitutively dense heterochromatin regions were found to be stochastically silenced in a proportion of the cells leading to variegation of expression, a classical epigenetic phenomenon known as position effect variegation (PEV). Mutagenesis screens identified HP1 and SUV39H as powerful suppressors of variegation which were necessary for heterochromatin-mediated PEV. SUV39H was found to methylate the histone H3 tail at lysine 9 at heterochromatin providing a binding site for HP1. This discovery provided the first direct evidence for a histone or epigenetic code in which the ‘writer’ of the code on chromatin would be the SUV39H and the ‘reader’ HP1. This mechanism was subsequently found to be conserved from S. pombe to humans. A mechanism which shares features with PEV has also been implicated in the pathogenesis of repeat-expansion diseases, such as Friedreich’s ataxia, in which the affected genes are aberrantly silenced. In mice there are 3 highly conserved HP1 isoforms, HP1α, HP1β and HP1γ. HP1α and β are found by microscopy to be localised to constitutive heterochromatic condensed regions in the nucleus whereas HP1γ has a pan-nuclear distribution and is therefore implicating in regulating euchromatic genes. There is a paucity of data examining how and where HP1γ regulates gene expression in vivo at the genome-wide level. This thesis employed knockout and knockdown strategies to investigate this. In view of the early lethality of mice in which HP1γ has been deleted by homologous recombination, the function of HP1γ was studied here by establishing mouse embryonic fibroblast cell lines. Immunoflourescent microscopy revealed for the first time that HP1γ was necessary for the localisation of HP1β at constitutive heterochromatic regions in the nucleus in most cells. This delocalisation of HP1β was associated with aberrant upregulation of the repetitive major satellite DNA associated with pericentromeric heterochromatin implying for the first time that HP1γ plays an important role in maintaining centromeric heterochromatin in a silenced state thought to be important for the maintenance of genome integrity. Strikingly, analysis of the transcriptome revealed a large number of genes (4293) to be dysregulated in male cells compared to females (1186) where the effect of HP1γ deficiency resulted in aberrant expression of immune related genes that would normally be repressed. This sexually dimorphic effect was investigated further by studying the effect of HP1γ deficiency on the subset of 176 genes found to differ in expression between normal males and females. Moreover, HP1γ in males was found essential for maintaining the relative repression of 114 genes in males compared to females, suggesting that the Y chromosome interacts with HP1γ to reduce the expression of these genes. In summary, a novel function for HP1γ in repressing pericentromeric DNA and in maintaining sexually dimorphic gene expression was discovered.Open Acces

Epigenetic and neurological effects and safety of high-dose nicotinamide in patients with Friedreich's ataxia: an exploratory, open-label, dose-escalation study

Background: Friedreich's ataxia is a progressive degenerative disorder caused by deficiency of the frataxin protein. Expanded GAA repeats within intron 1 of the frataxin (FXN) gene lead to its heterochromatinisation and transcriptional silencing. Preclinical studies have shown that the histone deacetylase inhibitor nicotinamide (vitamin B3) can remodel the pathological heterochromatin and upregulate expression of FXN. We aimed to assess the epigenetic and neurological effects and safety of high-dose nicotinamide in patients with Friedreich's ataxia. Methods: In this exploratory, open-label, dose-escalation study in the UK, male and female patients (aged 18 years or older) with Friedreich's ataxia were given single doses (phase 1) and repeated daily doses of 2–8 g oral nicotinamide for 5 days (phase 2) and 8 weeks (phase 3). Doses were gradually escalated during phases 1 and 2, with individual maximum tolerated doses used in phase 3. The primary outcome was the upregulation of frataxin expression. We also assessed the safety and tolerability of nicotinamide, used chromatin immunoprecipitation to investigate changes in chromatin structure at the FXN gene locus, and assessed the effect of nicotinamide treatment on clinical scales for ataxia. This study is registered with ClinicalTrials.gov, number NCT01589809. Findings: Nicotinamide was generally well tolerated; the main adverse event was nausea, which in most cases was mild, dose-related, and resolved spontaneously or after dose reduction, use of antinausea drugs, or both. Phase 1 showed a dose-response relation for proportional change in frataxin protein concentration from baseline to 8 h post-dose, which increased with increasing dose (p=0·0004). Bayesian analysis predicted that 3·8 g would result in a 1·5-times increase and 7·5 g in a doubling of frataxin protein concentration. Phases 2 and 3 showed that daily dosing at 3·5–6 g resulted in a sustained and significant (p<0·0001) upregulation of frataxin expression, which was accompanied by a reduction in heterochromatin modifications at the FXN locus. Clinical measures showed no significant changes. Interpretation: Nicotinamide was associated with a sustained improvement in frataxin concentrations towards those seen in asymptomatic carriers during 8 weeks of daily dosing. Further investigation of the long-term clinical benefits of nicotinamide and its ability to ameliorate frataxin deficiency in Friedreich's ataxia is warranted

Identification of a novel distal regulatory element of the human Neuroglobin gene by the chromosome conformation capture approach

Neuroglobin (NGB) is predominantly expressed in the brain and retina. Studies suggest that NGB exerts protective effects to neuronal cells and is implicated in reducing the severity of stroke and Alzheimer's disease. However, little is known about the mechanisms which regulate the cell type-specific expression of the gene. In this study, we hypothesized that distal regulatory elements (DREs) are involved in optimal expression of the NGB gene. By chromosome conformation capture we identified two novel DREs located -70 kb upstream and +100 kb downstream from the NGB gene. ENCODE database showed the presence of DNaseI hypersensitive and transcription factors binding sites in these regions. Further analyses using luciferase reporters and chromatin immunoprecipitation suggested that the -70 kb region upstream of the NGB gene contained a neuronalspecific enhancer and GATA transcription factor binding sites. Knockdown of GATA-2 caused NGB expression to drop dramatically, indicating GATA-2 as an essential transcription factor for the activation of NGB expression. The crucial role of the DRE in NGB expression activation was further confirmed by the drop in NGB level after CRISPR-mediated deletion of the DRE. Taken together, we show that the NGB gene is regulated by a cell type-specific loop formed between its promoter and the novel DRE

Genetic variation at mouse and human ribosomal DNA influences associated epigenetic states

Background: Ribosomal DNA (rDNA) displays substantial inter-individual genetic variation in human and mouse. A systematic analysis of how this variation impacts epigenetic states and expression of the rDNA has thus far not been performed. Results: Using a combination of long- and short-read sequencing, we establish that 45S rDNA units in the C57BL/6J mouse strain exist as distinct genetic haplotypes that influence the epigenetic state and transcriptional output of any given unit. DNA methylation dynamics at these haplotypes are dichotomous and life-stage specific: at one haplotype, the DNA methylation state is sensitive to the in utero environment, but refractory to post-weaning influences, whereas other haplotypes entropically gain DNA methylation during aging only. On the other hand, individual rDNA units in human show limited evidence of genetic haplotypes, and hence little discernible correlation between genetic and epigenetic states. However, in both species, adjacent units show similar epigenetic profiles, and the overall epigenetic state at rDNA is strongly positively correlated with the total rDNA copy number. Analysis of different mouse inbred strains reveals that in some strains, such as 129S1/SvImJ, the rDNA copy number is only approximately 150 copies per diploid genome and DNA methylation levels are < 5%. Conclusions: Our work demonstrates that rDNA-associated genetic variation has a considerable influence on rDNA epigenetic state and consequently rRNA expression outcomes. In the future, it will be important to consider the impact of inter-individual rDNA (epi)genetic variation on mammalian phenotypes and diseases

Deciphering the Role of the Non-Coding Genome in Regulating Gene-Diet Interactions

Protein encoding genes constitute a small fraction of mammalian genomes. In addition to the protein coding genes, there are other functional units within the genome that are transcribed, but not translated into protein, the so called non-coding RNAs. There are many types of non-coding RNAs that have been identified and shown to have important roles in regulating gene expression either at the transcriptional or post-transcriptional level. A number of recent studies have highlighted that dietary manipulation in mammals can influence the expression or function of a number of classes of non-coding RNAs that contribute to the protein translation machinery. The identification of protein translation as a common target for nutritional regulation underscores the need to investigate how this may mechanistically contribute to phenotypes and diseases that are modified by nutritional intervention. Finally, we describe the state of the art and the application of emerging &#8216;-omics&#8217; technologies to address the regulation of protein translation in response to diet

Deciphering the role of the non-coding genome in regulating gene-diet interactions

Protein encoding genes constitute a small fraction of mammalian genomes. In addition to the protein coding genes, there are other functional units within the genome that are transcribed, but not translated into protein, the so called non-coding RNAs. There are many types of non-coding RNAs that have been identified and shown to have important roles in regulating gene expression either at the transcriptional or post-transcriptional level. A number of recent studies have highlighted that dietary manipulation in mammals can influence the expression or function of a number of classes of non-coding RNAs that contribute to the protein translation machinery. The identification of protein translation as a common target for nutritional regulation underscores the need to investigate how this may mechanistically contribute to phenotypes and diseases that are modified by nutritional intervention. Finally, we describe the state of the art and the application of emerging &#8216;-omics&#8217; technologies to address the regulation of protein translation in response to diet

Investigation into the role of heterochromatin protein 1 gamma (HP1[gamma]) in gene regulation in mammals

Heterochromatin protein 1 (HP1) was identified as a key component of the condensed DNA surrounding centromeres in most eukaryotic cells. Genes placed in close proximity to such constitutively dense heterochromatin regions were found to be stochastically silenced in a proportion of the cells leading to variegation of expression, a classical epigenetic phenomenon known as position effect variegation (PEV). Mutagenesis screens identified HP1 and SUV39H as powerful suppressors of variegation which were necessary for heterochromatin-mediated PEV. SUV39H was found to methylate the histone H3 tail at lysine 9 at heterochromatin providing a binding site for HP1. This discovery provided the first direct evidence for a histone or epigenetic code in which the ‘writer’ of the code on chromatin would be the SUV39H and the ‘reader’ HP1. This mechanism was subsequently found to be conserved from S. pombe to humans. A mechanism which shares features with PEV has also been implicated in the pathogenesis of repeat-expansion diseases, such as Friedreich’s ataxia, in which the affected genes are aberrantly silenced. In mice there are 3 highly conserved HP1 isoforms, HP1α, HP1β and HP1γ. HP1α and β are found by microscopy to be localised to constitutive heterochromatic condensed regions in the nucleus whereas HP1γ has a pan-nuclear distribution and is therefore implicating in regulating euchromatic genes. There is a paucity of data examining how and where HP1γ regulates gene expression in vivo at the genome-wide level. This thesis employed knockout and knockdown strategies to investigate this. In view of the early lethality of mice in which HP1γ has been deleted by homologous recombination, the function of HP1γ was studied here by establishing mouse embryonic fibroblast cell lines. Immunoflourescent microscopy revealed for the first time that HP1γ was necessary for the localisation of HP1β at constitutive heterochromatic regions in the nucleus in most cells. This delocalisation of HP1β was associated with aberrant upregulation of the repetitive major satellite DNA associated with pericentromeric heterochromatin implying for the first time that HP1γ plays an important role in maintaining centromeric heterochromatin in a silenced state thought to be important for the maintenance of genome integrity. Strikingly, analysis of the transcriptome revealed a large number of genes (4293) to be dysregulated in male cells compared to females (1186) where the effect of HP1γ deficiency resulted in aberrant expression of immune related genes that would normally be repressed. This sexually dimorphic effect was investigated further by studying the effect of HP1γ deficiency on the subset of 176 genes found to differ in expression between normal males and females. Moreover, HP1γ in males was found essential for maintaining the relative repression of 114 genes in males compared to females, suggesting that the Y chromosome interacts with HP1γ to reduce the expression of these genes. In summary, a novel function for HP1γ in repressing pericentromeric DNA and in maintaining sexually dimorphic gene expression was discovered.published_or_final_versionPaediatrics and Adolescent MedicineDoctoralDoctor of Philosoph