Cell type‐specific epigenome profiling using affinity‐purified nuclei

Chitikova, Zhanna; Steiner, Florian

doi:10.1002/dvg.22919

Cited by 4 publications

(4 citation statements)

References 57 publications

(94 reference statements)

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“…Coupling INTACT and ATAC-seq in a single pipeline enables profiling of cell-type-specific chromatin accessibility. Given the wide implementation of the INTACT method in plant research, tools for characterizing tens of different cell types from various plant species are readily available [24, 48–50].…”

Section: Discussionmentioning

confidence: 99%

Regulatory chromatin landscape in Arabidopsis thaliana roots uncovered by coupling INTACT and ATAC-seq

et al. 2018

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BackgroundThere is a growing interest in the role of chromatin in acquiring and maintaining cell identity. Despite the ever-growing availability of genome-wide gene expression data, understanding how transcription programs are established and regulated to define cell identity remains a puzzle. An important mechanism of gene regulation is the binding of transcription factors (TFs) to specific DNA sequence motifs across the genome. However, these sequences are hindered by the packaging of DNA to chromatin. Thus, the accessibility of these loci for TF binding is highly regulated and determines where and when TFs bind. We present a workflow for measuring chromatin accessibility in Arabidopsis thaliana and define organ-specific regulatory sites and binding motifs of TFs at these sites.ResultsWe coupled the recently described isolation of nuclei tagged in specific cell types (INTACT) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) as a genome-wide strategy to uncover accessible regulatory sites in Arabidopsis based on their accessibility to nuclease digestion. By applying this pipeline in Arabidopsis roots, we revealed 41,419 accessible sites, of which approximately half are found in gene promoters and contain the H3K4me3 active histone mark. The root-unique accessible sites from this group are enriched for root processes. Interestingly, most of the root-unique accessible sites are found in nongenic regions but are correlated with root-specific expression of distant genes. Importantly, these gene-distant sites are enriched for binding motifs of TFs important for root development as well as motifs for TFs that may play a role as novel transcriptional regulators in roots, suggesting that these accessible loci are functional novel gene-distant regulatory elements.ConclusionsBy coupling INTACT with ATAC-seq methods, we present a feasible pipeline to profile accessible chromatin in plants. We also introduce a rapid measure of the experiment quality. We find that chromatin accessibility at promoter regions is strongly associated with transcription and active histone marks. However, root-specific chromatin accessibility is primarily found at intergenic regions, suggesting their predominance in defining organ identity possibly via long-range chromatin interactions. This workflow can be rapidly applied to study the regulatory landscape in other cell types, plant species and conditions.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0381-9) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Regulatory chromatin landscape in Arabidopsis thaliana roots uncovered by coupling INTACT and ATAC-seq

et al. 2018

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“…In the meantime, wash buffer (0.4% Igepal in homogenization buffer, [ 28 ]) was added to the nuclei solution, collected from the 30–40% iodixanol gradient layer (2:1). This was necessary to reduce the solution’s viscosity and remove the outer nuclear membrane before affinity purification (as mentioned in [ 60 ]) to facilitate anti-GFP binding to sun1sfGFP in the peri-nuclear membrane. The wash buffer-diluted nuclei solution was incubated with anti-GFP bead solution in the ratio of 40:1.…”

Section: Methodsmentioning

confidence: 99%

INTACT vs. FANS for Cell-Type-Specific Nuclei Sorting: A Comprehensive Qualitative and Quantitative Comparison

Chongtham

Butto

Mungikar

et al. 2021

IJMS

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Increasing numbers of studies seek to characterize the different cellular sub-populations present in mammalian tissues. The techniques “Isolation of Nuclei Tagged in Specific Cell Types” (INTACT) or “Fluorescence-Activated Nuclei Sorting” (FANS) are frequently used for isolating nuclei of specific cellular subtypes. These nuclei are then used for molecular characterization of the cellular sub-populations. Despite the increasing popularity of both techniques, little is known about their isolation efficiency, advantages, and disadvantages or downstream molecular effects. In our study, we compared the physical and molecular attributes of sfGFP+ nuclei isolated by the two methods—INTACT and FANS—from the neocortices of Arc-CreERT2 × CAG-Sun1/sfGFP animals. We identified differences in efficiency of sfGFP+ nuclei isolation, nuclear size as well as transcriptional (RNA-seq) and chromatin accessibility (ATAC-seq) states. Therefore, our study presents a comprehensive comparison between the two widely used nuclei sorting techniques, identifying the advantages and disadvantages for both INTACT and FANS. Our conclusions are summarized in a table to guide researchers in selecting the most suitable methodology for their individual experimental design.

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Section: Introductionmentioning

confidence: 99%

“…To overcome these limitations, alternative techniques have been developed based around epitope labeling and immunoprecipitation of nuclei (Chitikova & Steiner, 2016). These nuclei tagging approaches, such as the "Isolation of Nuclei Tagged in specific Cell Types" (INTACT) method (Deal & Henikoff, 2010) have been applied to tissue specific experiments in Arabidopsis (Maher et al, 2018;Sijacic et al, 2018), Drosophila (Agrawal et al, 2019;Bozek et al, 2019;Henry et al, 2012;Jones et al, 2018), Xenopus (Amin et al, 2014), and mice (Ambati et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

In vivotissue-specific chromatin profiling inDrosophila melanogasterusing GFP-tagged nuclei

Jauregui-Lozano

Bakhle

Weake

2021

Preprint

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The chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each cell type. Thus, profiling the chromatin state of different cell types in an intact organism under disease or physiological conditions can provide insight into how chromatin regulates cell homeostasis in vivo. To overcome the many challenges associated with characterizing chromatin state in specific cell types, we developed an improved approach to isolate Drosophila nuclei tagged with GFP expressed under Gal4/UAS control. Using this protocol, we profiled chromatin accessibility using Omni-ATAC, and examined the distribution of histone marks using ChIP-seq and CUT&Tag in adult photoreceptor neurons. We show that the chromatin landscape of photoreceptors reflects the transcriptional state of these cells, demonstrating the quality and reproducibility of our approach for profiling the transcriptome and epigenome of specific cell types in Drosophila.

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Cell type‐specific epigenome profiling using affinity‐purified nuclei

Cited by 4 publications

References 57 publications

Regulatory chromatin landscape in Arabidopsis thaliana roots uncovered by coupling INTACT and ATAC-seq

Regulatory chromatin landscape in Arabidopsis thaliana roots uncovered by coupling INTACT and ATAC-seq

INTACT vs. FANS for Cell-Type-Specific Nuclei Sorting: A Comprehensive Qualitative and Quantitative Comparison

In vivotissue-specific chromatin profiling inDrosophila melanogasterusing GFP-tagged nuclei

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