NET-Seq
DamID
DamID allows the identification of protein-binding sites in living cells without the need for crosslinking or immunoprecipitation. It was developed in 2006 as a microarray method before it was adapted to NGS. DamID involves the low-level expression of a fusion protein consisting of DNA adenine methyltransferase (Dam) and a chromatin protein of interest. This fusion protein is targeted to the native binding sites of the chromatin protein, where Dam methylates adenines in the surrounding DNA. Subsequently, the methylated DNA fragments are isolated, amplified by selective PCR, and sequenced.
Pros:
- Allows the identification of protein-binding sites in living cells without the need for crosslinking or immunoprecipitation
- Dedicated algorithms are available1
Cons:
- Dam can be toxic
- Limited to kilobase-sized regions
- Pindyurin A. V., Pagie L., Kozhevnikova E. N., van Arensbergen J. and van Steensel B. Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila. Nucleic Acids Res. 2016;44:5646-5657
- Mitchell A. C., Javidfar B., Bicks L. K., et al. Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex. Nat Commun. 2016;7:12743
- Perovanovic J., Dell'Orso S., Gnochi V. F., et al. Laminopathies disrupt epigenomic developmental programs and cell fate. Sci Transl Med. 2016;8:335ra358
- McCann T. S., Guo Y., McDonald W. H. and Tansey W. P. Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing. Proc Natl Acad Sci U S A. 2016;113:1309-1314
- Carl S. H. and Russell S. Common binding by redundant group B Sox proteins is evolutionarily conserved in Drosophila. BMC Genomics. 2015;16:292
- Kind J., Pagie L., de Vries S. S., et al. Genome-wide maps of nuclear lamina interactions in single human cells. Cell. 2015;163:134-147
- Klocko A. D., Rountree M. R., Grisafi P. L., Hays S. M., Adhvaryu K. K. and Selker E. U. Neurospora importin alpha is required for normal heterochromatic formation and DNA methylation. PLoS Genet. 2015;11:e1005083
- Steglich B., Stralfors A., Khorosjutina O., et al. The Fun30 chromatin remodeler Fft3 controls nuclear organization and chromatin structure of insulators and subtelomeres in fission yeast. PLoS Genet. 2015;11:e1005101