NuclampFISH enables cell sorting based on nuclear RNA expression for chromatin analysis

Abstract

Background: Transcriptional bursts are periods when RNA polymerase interacts with a DNA locus, leading to active gene transcription. This bursting activity can vary across individual cells, and analyzing the differences in transcription sites can help identify key drivers of gene expression for a specific target RNA. Scaffolding methods based on fluorescence in situ hybridization (FISH) have been widely used to amplify the fluorescent signal of RNAs and sort cells based on RNA expression levels. Examples include click-amplifying FISH (clampFISH) and hybridization chain reaction (HCR). However, these methods are limited in their ability to target and amplify transcription sites, due to the long probes' hindered accessibility through cellular compartment membranes and crosslinked proteins. Thus, sorting based on transcriptional bursting has not been achieved. Additionally, the required formaldehyde fixation interferes with downstream analysis of chromatin and protein-binding interactions.

Results: To address these challenges, we developed a platform, nuclear clampFISH (nuclampFISH) that integrates click-amplified FISH with reversible crosslinkers and allows access to the nucleus. We demonstrate that with optimized parameters and by eliminating the cytosol and cell membrane, this method enables the amplification of fluorescent signal for RNAs using a reversible crosslinker, enabling the sorting of cells based on nuclear RNA expression and is compatible with downstream biochemical analysis including chromatin conformation assays. We applied this assay to demonstrate that transcriptionally active cells have more accessible chromatin for a respective gene.

Conclusions: This new method enables the sorting of cells based on transcriptional bursts. This method combines the specificity of a single-cell assay for detecting transcription sites with the throughput of flow cytometry to enable bulk assays such as chromatin conformation or other biochemical assays. Notably, the tools developed are highly accessible and do not require specialized computation or equipment.

Keywords: Chromatin conformation; ClampFISH; RNA FISH amplification; SmFISH; Transcription site; Transcriptional burst.

Fig. 1

FISH-based methods to detect transcription sites include a. smFISH, b. clampFISH, and c. HCR FISH. d Overlaid FISH spots in mRNA exon and intron indicate transcription (txn) sites. e Images of EEF2 exons and smFISH images of EEF2 introns were used to show the colocalization and identify transcription sites. f Overlaid density plot of exon RNA count distribution for clampFISH (n=158 cells), HCR FISH (n=160 cells), and smFISH (n=157 cells) for two biological replicates. g quantification of transcription sites (txn sites) detected by method. A transcription site is defined as colocalization of exon probe with smFISH intron probe. Error bars represent the standard deviation from the mean. Statistical analysis was performed using a paired t-test. ***p < 0.0001, ****p < 0.00001

Fig. 2

Method development and workflow of nuclampFISH. a clampFISH images after 2 rounds of amplification of EEF2 exon and smFISH images of EEF2 intron without Pla B treatment. The arrow indicates the transcription sites. b clampFISH images of EEF2 exon and smFISH images of EEF2 intron after Pla B treatment. c NuclampFISH images of EEF2 exon after nucleus isolation and smFISH images of EEF2 intron. d NuclampFISH images of EEF2 exon after nucleus isolation and modified hybridization parameters, and smFISH images of EEF2 intron. e Workflow of nuclampFISH methods. f Timeline of nuclampFISH process. g Amplification efficiency of nuclampFISH, two rounds, four rounds, six rounds, eight rounds of nuclampFISH images were shown. h Box and whisker plot of nuclampFISH spots intensities in each round of amplification. n = 3 biological replicates

Fig. 3

Application of reversible crosslinker to nuclampFISH. a HeLa cells stained for EEF2 exon mRNA using clampFISH after DSP (left) or formaldehyde (right) crosslinking. b HeLa cells processed with clampFISH secondary probes only, following DSP (left) or formaldehyde (right) crosslinking. c Density plot showing percell EEF2 mRNA counts in DSP (n = 100 cells) versus formaldehydetreated (n = 100 cells) samples. d NuclampFISH (left) and smFISH (right) images of EEF2 intron in isolated nuclei following Pladienolide B treatment under DSP crosslinking. e NuclampFISH (left) and smFISH (right) images of EEF2 intron in isolated nuclei following Pladienolide B treatment under formaldehyde crosslinking

Fig. 4

NuclampFISH workflow, sorting, and chromatin accessibility analysis. a Schematic of the nuclampFISH protocol, fluorescence-activated nuclear sorting, and downstream chromatin accessibility assay. b Pseudocolor flow‐cytometry plot of EEF2 intron fluorescence in HeLa nuclei. Gates for low (G1), medium (G2), and high (G3) expression define sorting populations. c Representative fluorescence micrographs of sorted G1, G2, and G3 nuclei. d Left: Bar graph of mean nuclampFISH signal intensity in each sorted population. Right: Correlation between qRTPCR–measured intron abundance and nuclampFISH fluorescence intensity; equal numbers of nuclei were analyzed per group. e Chromatin accessibility quantified by log₁₀ relative quantity (RQ) from 5 × 10⁵ sorted nuclei per group, indicating open‐chromatin levels for EEF2 intron regions in G1, G2, and G3