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Practical Course HP-F10: Epigenomics and Cancer

Type: Practical Course with Student Seminars

Date: 16. May - 3. June 2022

Hosts/Supervisors: Clarissa Gerhäuser (responsible organizer contact: c.gerhaeuser@dkfz.de), Ali Bakr, Ashish Goyal, Mark Hartmann, Daniel Lipka, Maria Llamazares Prada, Pavlo Lutsik, Christoph Plass, Peter Schmezer, Dieter Weichenhan

Topics:

Several mechanisms have been identified that are involved in altered expression of cancer related genes in tumours. Genetic alterations, including mutations and deletions, have been known to be involved in tumorigenesis for many years. More recently, DNA methylation has been recognized as an additional mechanism for gene silencing. Aberrant silencing of tumour suppressor genes by DNA methylation is an early event in tumorigenesis and a major contributor in tumour development. In addition, post-translational histone modifications are involved in the regulation of gene expression. Recent experimental evidence demonstrated that histone modifications may be altered in malignancies on both a genome-wide and a discrete gene loci level. As a consequence, this leads to altered gene expression patterns and phenotype (e.g. DNA repair) that contribute to tumorigenesis. These epigenetic mechanisms, altered DNA methylation and histone modifications, lead to disturbed gene expression without changing the genomic sequence. They thus offer the exciting possibility for new therapeutic interventions, e.g. the inhibition of DNA methyltransferases (DNMTs) to inhibit aberrant DNA methylation.

The training program will introduce important features of these mechanisms and will demonstrate new analytical methods specific for the elucidation of epigenetic aberrations.

Content:

  • Determination of differential histone modification changes in DNMT double-knockout vs DNMT wildtype cells by antibody-guided chromatin tagmentation (ACT-seq) and qPCR
  • Bioinformatics tools and techniques for the analysis of genome-wide DNA methylation and chromatin data
  • Characterization and quantification of specific methylation sites by multiplexed targeted bisulfite-sequencing (experimental procedure and bioinformatic analysis)
  • CRISPR/Cas9 – mediated knockout of chromatin-remodelling-related genes (CRISPR/Cas9 Ribonucleoprotein complex electroporation, determination of knockout efficiency by TIDE analysis and western blotting)
  • Alterations in chromatin remodelling activity and its consequences on DNA repair will be analysed (induction of defined double strand breaks via site-specific nuclease, quantification of DNA damage-related post-translational modifications of histones by ChIP/qRT-PCR, analysis of H2AX foci as marker of repair efficiency via immuno-fluorescence microscopy)
  • Identification of DNA methyltransferase inhibitors (M.Sss1 methyltransferase assay)

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