In order to understand gene regulatory networks (GRNs) in mammalian cells, it is pivotal to assess the interaction between proteins and DNA. In particular, the specific DNA binding activity of transcription factors (TFs) determines the expression of target genes and in general the overall connectivity of the GRN. However, the genomic location of TF binding cannot be predicted just from the DNA sequence, and functional assays are required to detect this interaction. The investigation of the binding of TF to DNA is usually accomplished by chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). While in the last 10 years this method enabled a better understanding of how transcription is regulated in living cells, it does have some drawbacks. In particular, the need for very highly specific antibodies and the large amount of starting material limit the ability of ChIP-seq to address biological questions when dealing with samples of small quantity. A technique called DNA Adenine Methyltransferase Identification (DamID) was developed in Drosophila as an alternative method for the detection of protein- DNA interactions and it is based on the fusion of a protein of interest (POI) with the DNA adenine methyltransferase (Dam). This fusion causes DNA methylation of adenines surrounding the sites where POI binds and the subsequent identification of the methylation sites allows mapping of the binding event without antibodies and using less cells as starting material. While this technology was successful in detecting the interaction between nuclear lamina and DNA in mammalian cells, to date little reports are present in the literature about TF DamID. This is mainly due to the different nature of TF binding compared to Lamin (punctuated instead of broad) and to the elevated intrinsic activity of Dam that makes the detection of real signal above the noise challenging. I here demonstrate a step-by-step optimization of the DamID technology coupled to next-generation sequencing (DamID-seq) that I used to map the binding of the mouse embryonic stem cell master regulator Oct4 in as few as 1,000 cells. This new technology paves the way for exciting new experiments where the number of cells is scarce such as in vitro cell state change or in vivo processes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:735740 |
| Date | January 2017 |
| Creators | Tosti, Luca |
| Contributors | Kaji, Keisuke ; Tomlinson, Simon |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/28700 |
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