- Author
-
D.W. Poramba-Liyanage
- Title
- Expanding the Epi-discovery tool box
- Subtitle
- Systematic and direct screening for chromatin regulators and chromatin binders using DNA barcoding
- Supervisors
- Co-supervisors
-
M.M.S. van Lohuizen
- Award date
- 9 October 2020
- Number of pages
- 251
- ISBN
- 9789463808118
- Document type
- PhD thesis
- Faculty
- Faculty of Medicine (AMC-UvA)
- Abstract
-
Eukaryotic genomes are packaged in chromatin, which consists of DNA, histones, and associated proteins. Chapter 1 describes the challenges this brings in understanding general principles of chromatin such as regulation of the gene expression, and the maintenance of chromatin states during transcription, DNA repair and DNA replication. To understand the dynamics of chromatin at a detailed level, this thesis has focused on dissecting locus-specific chromatin regulatory pathways and protein binding events. Chapter 2 and Chapter 3 describes Epi-ID, a screening strategy in yeast that enables the direct assessment of locus specific chromatin status in thousands of gene mutants in parallel. Epi-ID takes advantage of short DNA sequences called DNA barcodes that are introduced into an array of yeast knock-out mutants, at a common chromosomal location in the genome. Chromatin immunoprecipitation on pools of barcoded mutant strains followed by barcode counting by high throughput sequencing reports on the abundance of the chromatin mark of interest in each mutant strain. Further, transcription, replication, and repair processes all involve interactions of many different proteins and how these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown. To acquire this type of direct evidence Chapter 4 and Chapter 5 describes the development and application of Epi-Decoder, a Tag-ChIP-Barcode-Seq technology in yeast. Epi-Decoder is orthogonal to proteomics approaches and does not rely on MS but instead takes advantage of DNA sequencing. Chapter 6 further summarizes the findings and focusses on their limitations and horizons.
- Persistent Identifier
- https://hdl.handle.net/11245.1/b5036475-7b8a-4fcc-804a-53c36301e1cb
- Downloads
-
Thesis (complete)
Front matter
Chapter 1: General introduction: Understanding the dynamic nature of chromatin and insights into technologies for decoding the epigenome
Chapter 2: Epi-ID: Systematic and direct screening for chromatin regulators in yeast by Barcode-ChIP-Seq
Chapter 3: Swr1 controls H2A.Z levels in S. cerevisiae by incorporation of new and maintenance of old histones
Chapter 4: Decoding the chromatin proteome of a single genomic locus by DNA sequencing
Chapter 5: Inhibition of transcription leads to rewiring of locus-specific chromatin proteomes
Chapter 6: Summary and general discussion
Bibliography; Nederlandse samenvatting (Summary in Dutch language); PhD portfolio; Acknowledgements; Curriculum vitae
- Supplementary materials
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