Rapid and Low-Input Profiling of Histone Marks in Plants Using Nucleus CUT&Tag

Abstract

Characterizing genome-wide histone posttranscriptional modifications and transcriptional factor occupancy is crucial for deciphering their biological functions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for genome-wide profiling of histone modifications and transcriptional factor-binding sites. However, the current ChIP-seq experimental procedure in plants requires significant material and several days for completion. CUT&Tag is an alternative method of ChIP-seq for low-sample and single-cell epigenomic profiling using protein A-Tn5 transposase fusion proteins (PAT). In this study, we developed a nucleus CUT&Tag (nCUT&Tag) protocol based on the live-cell CUT&Tag technology. Our results indicate that nCUT&Tag could be used for histone modifications profiling in both monocot rice and dicot rapeseed using crosslinked or fresh tissues. In addition, both active and repressive histone marks such as H3K4me3 and H3K9me2 can be identified using our nCUT&Tag. More importantly, all the steps in nCUT&Tag can be finished in only 1 day, and the assay can be performed with as little as 0.01 g of plant tissue as starting materials. Therefore, our results demonstrate that nCUT&Tag is an efficient alternative strategy for plant epigenomic studies.

Biotinylated Tn5 transposase‐mediated CUT&Tag efficiently profiles transcription factor‐DNA interactions in plants

“…We have developed B‐CUT&Tag‐seq and B‐CUT&Tag‐qPCR procedures that are likely to be widely applied in profiling plant TF‐DNA interactions. CUT&Tag provides outstanding performance with low chromatin input even at the single‐cell level (Bartosovic et al ., 2021; Ouyang et al ., 2021); however, for most scenarios involving plant tissues such as leaves, stems, roots, inflorescences, fruits and seeds, the amount of input sample does not appear to be the limiting factor. We have employed CUT&Tag assays in plants for years and identified the most likely limiting factors for its successful application as the isolation of intact nuclei and the abundance of target TFs.…”

“…Since its invention, CUT&Tag has provided an alternative method for profiling protein‐DNA interactions in both animals and plants. CUT&Tag for bulk histone modification has been successfully applied to investigate the epigenomic landscapes of monocot rice and dicot rapeseed and cotton (Ouyang et al ., 2021; Tao et al ., 2020). In addition to the bulk histone modification states that reflect chromatin accessibility, annotation of biologically significant cis ‐regulatory elements relating to yield, quality and stress tolerance is of great importance in crop production.…”

Removal of H3K27me3 by JMJ Proteins Controls Plant Development and Environmental Responses in Arabidopsis

“…Hence, spatial information is completely lost. Recently, low-input binding tests in plants, such as CUT&Tag, CUT&RUN, nCUT&Tag, and ChIL, have been developed to study interactions between DNA and proteins using low-input samples or single live cells (Zheng and Gehring, 2019;Sakamoto et al, 2020;Tao et al, 2020;Ouyang et al, 2021). By combining these with cell sorting or laser microdissection techniques, cell type-specific JMJ protein binding and histone modification data can be obtained in the future.…”

Multi-Omics Approach Reveals OsPIL1 as a Regulator Promotes Rice Growth, Grain Development, and Blast Resistance

“…22 Compared to ChIP-seq, the CUT&Tag procedure involves fewer steps, which facilitates the processing of multiple low-input samples or even single cells to query their chromatin state; importantly, the entire experimental procedure can be completed in only 1 day. 23 An integration of CUT&Tag and RNA-seq results can help in identifying important transcription factors and revealing insights into complex biological networks. Further downstream of transcription factors, metabolomics can examine the metabolites in distinct tissues at chosen time points, and untargeted metabolomics achieves close to globalized metabolomics, characterizing most, if not all, chemical compounds in a given sample.…”