Global Analysis of Histone Post-translational Modifications Specific to Rett Syndrome using 2D RPLC/WCX-HILIC for Online Top-down Mass Spectrometry
EMSL Project ID
46890
Abstract
Epigenetics is a rapidly expanding field in biology, due to many technological breakthroughs and a greater understanding of the mechanisms of gene regulation. The location of epigenetic markers such as histone acetylation, DNA and histone methylation as well as nucleosome positioning appear to play critical roles in cell cycle progression. Abnormalities in these markers have been implicated with the development of many diseases, yet unanswered questions remain regarding the specific interactions between various epigenetic modifications and the appearance of a disease phenotype. Rett syndrome is a post-natal neural disorder which almost exclusively affects females and as yet is untreatable. Symptoms include a period of apparently normal early development followed by a plateau and then regression. The cause of this syndrome, mutations in the methyl CpG binding protein 2 (MeCP2), has been implicated in over 90% of Rett cases. Experimental mouse models, based on the loss of function of the Mecp2 gene, have revealed changes in the morphology and function of brain cells. MeCP2 is a known transcriptional repressor with a high affinity for binding methylated DNA and once bound is thought to affect chromatin structure through recruitment of the histone deacetylase complex (HDAC). [1, 2] Rett syndrome is a good model for studying epigenetic markers for several reasons. The coordinated interactions between MeCP2, HDACs and DNA methylation indicate a mechanistic link between chromatin structure and DNA methylation, and histone proteins are present in large amounts in neural tissue. Furthermore, while the link between the Rett phenotype and MeCP2 mutation is well established, including the milder forms of Rett syndrome associated with MeCP2 truncations, a clear correlation between genotype and phenotype has yet to be uncovered - in part due to the many types of mutation which can result in Rett syndrome. [3, 4] We are interested in characterising the histone post-translational modifications (PTM's) of wild type and MeCP2 null mutants using a mouse model, specifically searching for PTM profiles indicative of MeCP2 deficiency. To date, there is conflicting evidence in the literature concerning the effect of MeCP2 gene deletion on histone methylation and acetylation. [5] Our preliminary data, using 1-D LC-FT-ICR MS on acid extracted histones from whole mouse brain, shows a clear difference between wild type and MeCP2 knockout histone H4 acetylation profiles. We wish to extend this analysis by employing 2-D RPLC/HILIC top-down mass spectrometry for high throughput, high sensitivity global profiling of extracts from specific brain regions (cortex, midbrain and cerebellum) to search for undiscovered histone modifications. We will then pursue targeted analysis by using single cell types (neurons and glia). Our aim is to clarify the limited and contradictory information in the literature regarding MeCP2, DNA methylation and histone PTM's.
Project Details
Project type
Exploratory Research
Start Date
2012-01-09
End Date
2013-02-12
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members