John Arne Dahl successfully defends PhD thesis on epigenetics of pluripotency (2010)

John Arne Dahl
John Arne Dahl

7 January 2010

John Arne Dahl (Collas Lab) defended his PhD thesis entitled "Chromatin immunoprecipitation assays to investigate the epigenetics of pluripotency".

Interactions of proteins with DNA are essential to maintain genome stability and to control DNA replication and repair, chromosome segregation and gene expression. Chromatin immunoprecipitation (ChIP) is a powerful technique for studying protein-DNA interactions within the cell. ChIP assays have been widely used in the past decade to map the location of post-translationally modified histones and transcription factors in the genome.

In a typical ChIP assay, DNA and proteins are cross-linked to maintain association of proteins with their target DNA sequence. Chromatin is then fragmented and used for immunoprecipitation of protein-DNA complexes using antibodies specific for the protein of interest. The precipitated (ChIP) DNA is purified and DNA sequences associated with the precipitated protein are identified by polymerase chain reaction, sequencing or hybridization to microarrays (ChIP-on-chip).

In spite of the versatility in the nature of DNA-bound proteins and cell types that can be examined, ChIP assays have notoriously been known to be cumbersome, time-consuming and to require large numbers of cells (in the tens of millions).This has hampered applications to rare cell samples such as small stem cell populations or embryonic cells.

John Arne Dahl’s Ph.D. thesis work consisted in drastically simplifying the ChIP assay by reducing the number of steps, and time to completion from 3-4 days down to a few hours. The work also entailed refining the assay to make mapping of protein-DNA interactions in the genome amenable to as few as 100 or 1,000 cells – even for genome-scale mapping (a first).
A consequence of this work has been a world-wide spreading of the miniaturized ChIP assay – which is no long longer “taboo” – and applications to small tissue and cancer biopsies, small purified stem cell populations, and mammalian embryos.


Publications:

1. Dahl. J.A., and Collas, P. 2007. Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay applicable to small cell samples unravels epigenetic dynamics of pluripotency marker genes in differentiating human carcinoma cells. Stem Cells 25, 1037-1046.
2. Freberg, C.K., Dahl, J.A., Timoskainen, S., and Collas, P. 2007. Epigenetic reprogramming of OCT4 and NANOG by embryonal carcinoma cell extract. Mol. Biol. Cell. 18, 1543-1553.
3. Dahl, J.A., and Collas, P. 2007. A quick and quantitative chromatin immunoprecipitation assay for small cell samples. Front. Biosciences 12, 4925-4931.
4. Dahl, J.A., and Collas, P. 2008. MicroChIP: a rapid micro-chromatin immunoprecipitation assays for small cell numbers and biopsies. Nucleic Acids Res. 36, e15.
5. Dahl, J.A., and Collas, P. 2008. A rapid micro chromatin immunoprecipitation assay (µChIP). Nature Protocols 3, 1032-1045.
6. Lund-Olesen, T., Dufva, M., Dahl, J.A., Collas, P., and Fougt-Hansen, M. 2008. Sensitive on-chip quantitative real-time PCR performed on an adaptable and robust platform. Biomedical Microdevices 10, 769-776.
7. Dahl, J.A., Duggal, S., Coulston, N., Millar, D., Melki, J., Shahdafar, A., Brinchmann, J.E., and Collas, P. 2008. Genomic and epigenetic instability of human bone marrow mesenchymal stem cells cultured in autologous serum or fetal calf serum. Int. J. Dev. Biol. 52, 1033-1042.
8. Dahl, J.A., and Collas, P. 2009. µChIP: immunoprecipitation for small cell numbers. Meth. Mol. Biol. 567, 59-74.
9. Dahl, J.A., Reiner, A.H., and Collas, P. 2009. Fast genomic µChIP-chip from 1,000 cells. Genome Biol. 10, R13.
10.Collas, P., and Dahl, J.A. 2008. Chop it, ChIP it., check it: the status of chromatin immunoprecipitation. Front. Bioscience 13:929-943.
11. Dahl, Reiner, A.H., Klungland, A., Wakayama, T., and Collas, P. 2010. Histone H3 lysine 27 methylation asymmetry on developmentally-regulated promoters distinguish the first two lineages in mouse preimplantation embryos. PLoS ONE, 5, e9150.