Going Beyond ChIP-seq

‘Tis the holiday season and whether or not you’re awaiting a visit from that tubby fella with the white beard, you may have added ChIP-seq to your holiday wish list. ChIP-seq, of course, has become an indispensable tool for investigating transcriptional regulation and, most notably, deciphering transcription factor function. Its power lies in its ability to map transcription factor binding sites genome wide without any prior knowledge of the binding site location or sequence and, thus, has been broadly adopted within the scientific community.

 Publications by year that reference the use of ChIP-seq revealing over 10,000 ChIP-seq publications in 2019. (Source: Google Scholar)

Transcriptional regulation, however, is not limited to discrete sites in the genome; instead relying on interactions between promoter and enhancer DNA elements carefully orchestrated by trans-acting protein factors. These interactions bridge between ssmall regulatory elements separated by hundreds or even thousands of bases of sequence forming large DNA loops. Underscoring the importance of these 3D interactions, loop disruption or new loop formation has been observed to contribute to various forms of human disease¹.

While ChIP-seq data has been invaluable to teasing apart complex regulatory networks and developing transcriptional models, it is blind to this 3D genome architecture, essentially treating each protein binding site as an isolated event. These 3D structures are detectable using orthogonal technologies, such as Hi-C; however, this requires additional time and cost. Up until recently, a unified assay capable of detecting both protein binding and long-range interactions mediated by the bound protein has been a notable gap in the molecular biology toolbox.

The Dovetail^® HiChIP MNase Kit combines the benefits of ChIP-seq with Hi-C, a proximity ligation method that captures long-range interactions using standard Illumina paired-end sequencing. The protocol can be completed in three days, sample to sequence, by any lab proficient in standard molecular biology practices and access to an Illumina instrument.

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 Day one consists of cross-linking, fragmentation and addition of ChIP antibody for O/N incubation. Day two includes chromatin immunoprecipitation and proximity ligation, during which incorporation of a biotinylated bridge at ligation junctions mark chimeric molecules. Finally, cross-links are reversed, and chimeric molecules are enriched and made ready for NGS library preparation and sequencing on day three.

Akin to ChIP-seq, the Dovetail HiChIP data captures sequences directly bound by the protein of interest. However, highlighting the added value of HiChIP over ChIP-seq, additional long-range protein-mediated interactions are captured in the same sequencing run. Signal peaks corresponding to positions of protein binding are viewable just as one would with ChIP-seq with the ability to overlay the long-range interaction information.

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 Comparison of HiChIP and ChIP-seq data using the same anti-CTCF antibody. The ChIP-seq data is preserved in the HiChIP dataset with the addition of long-range CTCF interactions as viewed in the arc track.

As such, this new tool enables researchers to go beyond the current limitations of ChIP-seq and query both protein-DNA binding and protein-directed chromatin conformation (including enhancer/promoter interactions) in a single assay. To learn more about the Dovetail HiChIP MNase Kit, I encourage you to download our technical note. Perhaps you’ll want to take a moment to update that holiday wish list?

¹Flavahan et al. (2019) Nature, 575: 229