ISDH‐seq: a robust methodology for profiling and characterization of open chromatin

SummaryProfiling of open chromatin is essential for understanding regulatory mechanisms that govern DNA‐templated events. However, methodology‐dependent detection of open chromatin sites (OCSs) has been reported in both humans and plants. Therefore, there is a pressing need for any reliable orthogonal methodologies to broaden the identification of open chromatin across the genome, particularly in plants. We here report the development of an in situ DNase I hypersensitivity sequencing (ISDH‐seq) for the efficient characterization of open chromatin, which can be applicable to 50–200 K nuclei. This technique identifies 72% and 120% more OCSs than newly generated DNase‐ and ATAC‐seq from the same batch of tissues as ISDH, respectively, in the rice genome. As compared to DH‐specific OCSs, ISDH‐specific OCSs exhibit distinct epigenetic features, including relative hypomethylation, a higher frequency of OCSs associated with H3K27me3 and increased associations with spatial chromatin interactions. Genes with H3K27me3‐enriched ISDH‐specific OCSs tend to be more expressed in a stress‐ and tissue‐dependent manner, which have significant biological implications. The functions of ISDH‐specific OCSs may be mediated by TF‐centred networks or through chromatin loops. Importantly, a subset of genes co‐regulated by more ISDH‐specific distal OCSs (dOCSs) through chromatin loops are more evolutionarily conserved than genes regulated by a single ISDH‐specific trans‐OCS, including domesticated genes responsible for important agronomic traits. Thus, ISDH‐seq can enrich and expand regulatory landscapes, facilitating a better understanding of their biological implications across plant genomes. It could serve as a reliable method that complements existing techniques and can be adapted for use in non‐plant systems.