title: Molecular Characterization of mutated Histone H3.3 in childhood Glioblastoma 
creator: Bender, Sebastian
subject: ddc-570
subject: 570 Life sciences
subject: ddc-610
subject: 610 Medical sciences Medicine
description: In recent years, affordable costs of next-generation sequencing technologies set the basis for the characterization of a multitude of cancer genomes. In doing so, scientists all over the world reconfirmed that cancer is a disease of the (epi)genome. The growing number of genetic abnormalities found in chromatin remodeling factors in various cancer entities produced evidence that aberrant chromatin modification plays an essential role in cancer initiation and progression.   Besides numerous mutations of histone modifying factors in different cancer types, two recurrent mutations within the histone H3.3 coding gene H3F3A have been identified in about 50% of pediatric glioblastoma. Both mutations result in amino acid substitutions at two critical residues of the histone tail of H3.3 (K27M or G34R/V). Since glioblastomas harboring one of these H3.3 mutations are characterized by a complex but stereotypic pattern of genetic and epigenetic alterations, the project presented here uses a variety of molecular techniques to study the functional mechanisms associated with both H3.3 mutations.   By using confocal imaging of fluorescently labeled H3.3 mutants and genome-wide sequencing-based chromatin immunoprecipitation (ChIP-Seq) of ectopically expressed H3.3 mutants, it was demonstrated that deposition and incorporation of histone H3.3 into chromatin is not affected by the mutations. Furthermore, experiments with different cell lines, which were genetically modified to stably overexpress mutant H3.3, provide evidence that   (at least) some of the genome-wide changes that were originally found in H3.3 mutated glioblastomas, can be faithfully recapitulated in vitro.    The investigation of several epigenetic marks by Western blot analysis and immunohistochemistry identified a strong dominant negative effect of the K27M mutant H3.3 protein, which causes global reduction of the key epigenetic mark H3K27me3. Moreover, this epigenetic alteration found in primary tumors, was induced in vitro by overexpression of K27M mutant H3.3 in different cell lines.    By using mass spectrometry to dissect the pattern of posttranslational modifications (PTM) at the histone tail of G34R mutant H3.3, a new PTM has been identified, namely dimethylation of arginine 34. Furthermore, first evidence is provided that this new PTM is causative for the downregulation of K36 trimethylation at G34R mutant H3.3.  Finally, overexpression of mutant H3.3 in the brain of neonatal mice was performed by retroviral gene transfer. In doing so, this study aims at the elucidation of the in vivo tumorigenic potential of both histone H3.3 mutations alone or in combination with impaired TP53 function.   In summary, the findings of this thesis provide novel insights into the comprehensive epigenetic changes caused by mutant histone H3.3 in pediatric GBMs. These results might provide the basis for the development of novel therapeutic strategies targeting the epigenetic changes induced by H3.3 mutations, which have been identified recently in nearly half of the tumors in children suffering from this devastating brain tumor.    
date: 2013-10-01
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/14876/1/PhD-thesis_SBe2013_FINAL2.pdf
identifier: DOI:10.11588/heidok.00014876
identifier: urn:nbn:de:bsz:16-heidok-148760
identifier:   Bender, Sebastian  (2013) Molecular Characterization of mutated Histone H3.3 in childhood Glioblastoma.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/14876/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng