Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Summary

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

Abstract

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation.

Introduction

Mammalian X-chromosome inactivation (XCI) is a strategy to compensate for the imbalance in X-linked gene dosage between XX and XY, wherein one of the two X-chromosomes in females is transcriptionally inactivated¹. X-chromosome inactivation is a great model system for long non-coding RNA (lncRNA) research. X-chromosome inactivation is regulated by multiple lncRNAs, and has been extensively studied over the past few decades to uncover the crosstalk mechanisms between lncRNAs, transcription, chromatin structure and nuclear organization^2,3.

The X inactivation center (XIC) located on the X-chromosome i....

Protocol

1. Probe Preparation

1. Obtain multiple unique oligonucleotides in Xist (20-30 nucleotide-length oligonucleotides, 63-65 ºC melting temperature, Table 1) with a 5’-amino modification and suspend in water.
2. Pool equimolar amounts of oligonucleotides with 5’-amino modification (total 4.5 µg) and label with amine-reactive fluorescent dye following the manufacturer’s instruction.
   1. Dissolve the pooled oligonucleotides in final 5 µl of nuclease-free water and add 3 µl of 1 M sodium bicarbonate to the oligonucleotide solution.
   2. Add 2 µl DMSO to the vial of amine-reactive dye an....

Results

Representative images of quick immuno-FISH are shown in Figure 1A. Co-localization of the Xist RNA cloud and H3K27me3 signal on the Xi was detected in differentiating female cells. At day 12 upon differentiation, more than 90% of EB cells had an Xist cloud (Figure 1B). Short oligonucleotide probes efficiently penetrated into the nuclei, leading to a visualization of almost all H3K27me3 signals co-localized with Xist RNA (Figure 1C; 97%, n = 150).

Discussion

In this paper, we have presented a quick immuno-FISH protocol that takes less than 5 hr to complete slide preparation, Xist RNA FISH, and immunofluorescence for H3K27me3. In comparison with the general immuno-FISH approach for Xist RNA detection, which usually needs overnight incubation for RNA FISH, this protocol not only significantly reduces time spent but also improves the sensitivity of immune-FISH using oligonucleotide probes.

Since Xist is highly transcribed during X-chromosome inactiva.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
oligonucleotides with 5’-amino modification	IDT
Alexa Fluor 488 Reactive Dye	Life Technologies	A32750
MicroSpin G-25 columns	GE Healthcare	27-5325-01
Cytospin 2	Shandon	59900102
Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)	Roche	10715859103
Histone H3K27me3 antibody	Active Motif	61017
Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed	Life Technologies	A21424
ProLong Gold antifade reagent	Life Technologies	P36931