High-resolution mapping of transcription factor binding sites on native chromatin
By Sivakanthan Kasinathan, Steven Henikoff. Epigenetics & Chromatin, 2013
97 citations recorded by the source corpus.
Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) is widely used for profiling of TF binding, but is limited by low resolution and poor specificity and sensitivity. We present a simple protocol that starts with micrococcal nuclease-digested uncross-linked chromatin and is followed by affinity purification of TFs and paired-end sequencing. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide highly accurate base-pair resolution maps that are not biased toward accessible chromatin, and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila melanogaster GAGA Factor and Pipsqueak. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.
View Paper: https://www.semanticscholar.org/paper/f5e3a4d3bfc1e6b54bd444bb169b0b3f3c444173
Citations in the corpus
- An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites (2016, methodology): “cerevisiae strains carrying Flag-tagged H2A (SBY2688), Cse4 (SBY5146), Abf1 and Reb1 under the control of their respective endogenous promoters were previously described (10, 27, 46).”
- Targeted in situ genome-wide profiling with high efficiency for low cell numbers (2018, unknown): “In contrast to standard cross-linking ChIP, in which sonication is used to fragment the chromatin to a minimum of ~200-bp fragments, exonuclease treatment in ChIP-exo or MNase digestion in high-resolution X-ChIP-seq or native ChIP approaches allows limit or near-limit digestion 6–8, 23, 39 .”