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Genome-wide mapping of differentially modified chromatin modifications in response to histone deacetylase inhibitors and implications for spinal muscular atrophy

thesis
posted on 06.02.2017, 02:15 by Lunke, Sebastian
Spinal muscular atrophy (SMA) is one of the leading genetic causes for death in infancy and childhood. The disease is characterized by severe muscle wasting as a result of degeneration of alpha motor neurons in the anterior horns of the spinal cord, a result of homozygous loss of function of the survival of motor neuron gene 1 (SMN1). The resulting reduction in the essential SMN protein leads to the disruption of a number of vital cellular functions. Unique to SMA, a second, almost identical copy of SMN1 exists in the genome. Due to a splice defect, only 10% of the transcripts derived from this SMN2 gene encode for functional SMN protein. The resulting low levels of functional protein are able to uphold its essential housekeeping functions in the cell, but are insufficient to ensure motor neuron survival. Although today no cure or effective treatment is available, up-regulation of the SMN2 gene has been shown to rescue SMA phenotype. Among the most promising candidates for treatment of SMA are histone de-acetylase inhibitors (HDACi), including valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, also known as vorinostat). Both drugs have been shown to up-regulate SMN2 gene expression and rescue SMA phenotype in mice. This increase in gene expression is believed to be a result of increasing the levels of histone acetylation, an epigenetic mark correlated with gene expression. Although HDACi are today widely used in clinical trials in a broad spectrum of diseases, their genome wide effects on epigenetic modifications are scarcely studied. In particular the correlation between genomic localization of differential epigenetic modification and changes in gene expression in response to HDACi treatment is not well defined. In this study, chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) was employed to study the response in modification levels of a number of epigenetic marks, including histone acetylation, repressive and permissive histone lysine methylation and DNA methylation. These changes were subsequently correlated with differential expression in response to HDACi treatment, as determined by next generation RNA sequencing (RNA-Seq). Detailed bioinformatics analysis of the resulting data reveals large scale changes in all studied epigenetic modifications, especially in response to SAHA treatment, with surprisingly little correlation to changes in gene expression. Secondly, the association of the methyl CpG binging protein 2 (MeCP2) and components of the SWI/SNF chromatin-remodeling complex with the SMN promoter were analyzed. MeCP2 and SWI/SNF have been shown in the past to play an essential role in neurological disorder Rett syndrome, where they are involved in the transcriptional repression of the FMR1 gene. Due to its abundance and importance in neuronal tissues, it was speculated that MeCP2 is involved in the transcriptional regulation of SMN. ChIP experiments identified an enrichment of MeCP2 and Brg1, a SWI/SNF subunit, on the SMN promoter. Both protein were released in response to treatment with the HDACi trichostatin A, though not VPA. The role of MeCP2 in transcriptional regulation of SMN, as well as genome-wide mapping of MeCP2 binding sites, however remained inconclusive.

History

Principal supervisor

Assam El-Osta

Year of Award

2012

Department, School or Centre

Medicine Alfred Hospital

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences