Dataset Information

Protocol for fractionation-assisted native ChIP (fanChIP) to capture protein-protein/DNA interactions on chromatin.

ABSTRACT: Conventional chromatin immunoprecipitation (ChIP) includes many steps that need to be optimized. Here, we have described a protocol of fractionation-assisted native ChIP (fanChIP) which combines subfractionation and native ChIP to purify protein/chromatin complexes applicable for analyses of both protein-protein and protein-DNA interactions within a short period of time. fanChIP is advantageous as subcellular fractionation removes chromatin-unbound materials before immunoprecipitation, and the chromatin fragmentation by micrococcal nuclease (MNase) in a mild condition enables one-step purification of intact protein/chromatin complexes. For complete details on the use and execution of this protocol, please refer to Miyamoto et al. (2020).

SUBMITTER: Miyamoto R

PROVIDER: S-EPMC8024768 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Protocol for fractionation-assisted native ChIP (fanChIP) to capture protein-protein/DNA interactions on chromatin.

Miyamoto Ryo R Yokoyama Akihiko A

STAR protocols 20210323 2

Conventional chromatin immunoprecipitation (ChIP) includes many steps that need to be optimized. Here, we have described a protocol of fractionation-assisted native ChIP (fanChIP) which combines subfractionation and native ChIP to purify protein/chromatin complexes applicable for analyses of both protein-protein and protein-DNA interactions within a short period of time. fanChIP is advantageous as subcellular fractionation removes chromatin-unbound materials before immunoprecipitation, and the c ...[more]

PMID: 33855306

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Project description:BackgroundChIP-chip and ChIP-seq are widely used methods to map protein-DNA interactions on a genomic scale in vivo. Waldminghaus and Skarstad recently reported, in this journal, a modified method for ChIP-chip. Based on a comparison of our previously-published ChIP-chip data for Escherichia coli ?32 with their own data, Waldminghaus and Skarstad concluded that many of the ?32 targets identified in our earlier work are false positives. In particular, we identified many non-canonical ?32 targets that are located inside genes or are associated with genes that show no detectable regulation by ?32. Waldminghaus and Skarstad propose that such non-canonical sites are artifacts, identified due to flaws in the standard ChIP methodology. Waldminghaus and Skarstad suggest specific changes to the standard ChIP procedure that reportedly eliminate the claimed artifacts.ResultsWe reanalyzed our published ChIP-chip datasets for ?32 and the datasets generated by Waldminghaus and Skarstad to assess data quality and reproducibility. We also performed targeted ChIP/qPCR for ?32 and an unrelated transcription factor, AraC, using the standard ChIP method and the modified ChIP method proposed by Waldminghaus and Skarstad. Furthermore, we determined the association of core RNA polymerase with disputed ?32 promoters, with and without overexpression of ?32. We show that (i) our published ?32 ChIP-chip datasets have a consistently higher dynamic range than those of Waldminghaus and Skarstad, (ii) our published ?32 ChIP-chip datasets are highly reproducible, whereas those of Waldminghaus and Skarstad are not, (iii) non-canonical ?32 target regions are enriched in a ?32 ChIP in a heat shock-dependent manner, regardless of the ChIP method used, (iv) association of core RNA polymerase with some disputed ?32 target genes is induced by overexpression of ?32, (v) ?32 targets disputed by Waldminghaus and Skarstad are predominantly those that are most weakly bound, and (vi) the modifications to the ChIP method proposed by Waldminghaus and Skarstad reduce enrichment of all protein-bound genomic regions.ConclusionsThe modifications to the ChIP-chip method suggested by Waldminghaus and Skarstad reduce rather than increase the quality of ChIP data. Hence, the non-canonical ?32 targets identified in our previous study are likely to be genuine. We propose that the failure of Waldminghaus and Skarstad to identify many of these ?32 targets is due predominantly to the lower data quality in their study. We conclude that surprising ChIP-chip results are not artifacts to be ignored, but rather indications that our understanding of DNA-binding proteins is incomplete.

Dataset Information

Protocol for fractionation-assisted native ChIP (fanChIP) to capture protein-protein/DNA interactions on chromatin.

Publications

Protocol for fractionation-assisted native ChIP (fanChIP) to capture protein-protein/DNA interactions on chromatin.

Similar Datasets

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