Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Summary

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Abstract

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories ^1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Introduction

Epigenetic mechanisms trigger establishment and inheritance of developmental and cell-type specific transcriptional profiles. At one level this involves modulation of chromatin packaging that defines active or silent genomic regions. On a larger scale, global 3D organization of the genome and nuclear architecture also play a role in the control of transcriptional patterns. Thus, dissection of these epigenomic features is essential for a full understanding of how genes are regulated ^6-11.

Combined immunofluorescence and 3D DNA FISH provide a unique opportunity to complement molecular and biochemical analyses by assessing ....

Protocol

1. DNA Probe Labeling with Fluorophores: Nick Translation (~ 6 hr)

1. Clean BAC DNA (prepared by maxi-prep) or plasmids or PCR products, all resuspended in H₂O, can be used for labeling. Note that for a robust FISH signal, probes should span at least 10 kb.
2. Incubate DNA in RNase A for 30 min at 37 °C (All reagents are listed in Table 1).
3. Incubate the nick translation reaction for 2 hr at 16 °C (see Tables 2 and 3). Alternative methods of direct labeling may be used (Example: FISH Tag, Invitrogen).
4. Inactivate the reaction for one hr at -80 °C ....

Results

DNA and immuno-FISH are used in the Skok lab to study the changes in nuclear organization associated with the process of V(D)J recombination of antigen receptor loci during B and T lymphocyte development. The techniques detailed above enable us to i) measure distances between the two ends of a locus (contraction) ii) measure distances between alleles or loci (pairing), iii) analyze the DNA damage occurring within loci, iv) assess the location of alleles and loci relative to nuclear sub-compartments (repressive per.......

Discussion

The techniques detailed above were used in our lab to analyze the regulation of V(D)J recombination of the Immunoglobulin and Tcra/d loci in developing lymphocytes ^30,31 . We are confident that this technique can be adapted for detection of various nuclear proteins, nuclear compartments and loci, in different cell-types. Modifications of the protocol may be necessary, and in this case the major steps to focus on are the following. First, the length of permeabilization can be adjusted de.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
H2O	Fisher	# BP2470
RNase A	Sigma	# R4642
dNTP	Sigma	# DNTP100
Alexa dUTP	Invitrogen	# C11397 to C-11401
Cy3 or Cy5 dUTP	Fisher	# 45-001-xxx
DNase I	Roche	# 04536282001
DNA Pol I	Biolabs	# M0209
0.025 μm filters	Millipore	# VSWP02500
Cot-1 DNA 1 mg/ml	Invitrogen	# 18440
Hybloc DNA 1 mg/ml	Applied Genetics	# MHB
Salmon sperm	Sigma	# D1626	powder to be resuspended at 10 mg/ml in H2O
NaAc (Sodium Acetate, pH 5.2, buffer solution)	Sigma	# S7899
Ficoll 400 (Mol Biol grade)	Fisher	# 525
Polyvinylpyrrolidone (Mol Biol grade)	Fisher	# BP431
Dextran sulfate powder	Sigma	# D8906
SSPE (Saline-Sodium Phosphate-EDTA) 20x solution	Fisher	# BP1328
Formamide	Fisher	# BP227
Coverslips	Fisher	# 12-548-B
Slides	Fisher	# 12-550
6-well plates	Fisher	# 0720080
PBS, 10x	Fisher	# MT-46-013-CM
Poly-L-lysine solution	Sigma	# P8920
Paraformaldehyde, prills, 95%	Sigma	# 441244
Triton-X-100, Mol Biol grade	Sigma	# T8787
BSA (Bovine Serum Albumin) Fraction V	Fisher	# BP 1600
Normal goat serum	Vector Labs	# S-1000
Tween-20, Mol Biol grade	Sigma	# P9416
SSC (Saline Sodium Citrate) 20x solution	Fisher	# BP1325
ProLong Gold antifade reagent	Invitrogen	# P36930
DAPI (4',6-diamidino-2-phenylindole)	Sigma	# D9542
Best test one coat rubber cement	Art or office supply stores
Table 1. Specific reagents and small equipment.