3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

Summary

3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

Abstract

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene expression, cell identity and differentiation. Many approaches developed to study the 3D architecture of the genome can be divided into two complementary categories: chromosome conformation capture based technologies (C-technologies) and imaging. While the former is based on capturing the chromosome conformation and proximal DNA interactions in a population of fixed cells, the latter, based on DNA fluorescence in situ hybridization (FISH) on 3D-preserved nuclei, allows contemporary visualization of multiple loci at a single cell level (multicolor), examining their interactions and distribution within the nucleus (3D multicolor DNA FISH). The technique of 3D multicolor DNA FISH has a limitation of visualizing only a few predetermined loci, not permitting a comprehensive analysis of the nuclear architecture. However, given the robustness of its results, 3D multicolor DNA FISH in combination with 3D-microscopy and image reconstruction is a possible method to validate C-technology based results and to unambiguously study the position and organization of specific loci at a single cell level. Here, we propose a step by step method of 3D multicolor DNA FISH suitable for a wide range of human primary cells and discuss all the practical actions, crucial steps, notions of 3D imaging and data analysis needed to obtained a successful and informative 3D multicolor DNA FISH within different biological contexts.

Introduction

Higher eukaryotes need to systematically condense and compact a huge amount of genetic information in the minute 3D space of the nucleus^1,2,3,4. Today, we know that the genome is spatially ordered in compartments and topologically associated domains⁵ and that the multiple levels of DNA folding generate contacts between different genomic regions that may involve chromatin loop formation^6,7. The 3D dynamic looping of chromatin can influence many different biolog....

Protocol

1. DNA probe preparation and labelling procedures with nick translation

1. Purify and clean bacterial artificial chromosomes (BACs), plasmids or PCR products with specific kits (Table of Materials), resuspend in ddH₂O, check by electrophoresis on an agarose gel and quantify.
2. Perform nick translation on 1.5−2 µg of DNA from step 1.1 in a final volume of 50 µL by mixing all the reagents in a 0.5 mL low binding DNA tube according to Table 1.
   NOTE: Directly labelled probes can improve the signal to noise ratio. Kits to produce indirectly and directly labelled probes with various Alexa f....

Results

The method of 3D multicolor DNA FISH described in this article allows contemporary visualization of different genomic loci within preserved 3D nuclei (Figure 1B). This protocol permits the measurement of distances between alleles, and different genomic loci in order to evaluate their spatial proximity, and to assess their location within the nuclear space (e.g., loci distance from the centroid or the periphery of the nuclei)¹⁶. However, there are many.......

Discussion

The current method describes a step by step protocol to perform 3D multicolor DNA FISH on a wide range of human primary cells. Although DNA FISH is a technology in wide use, 3D multicolor DNA FISH on preserved 3D interphase nuclei is still difficult to perform in many laboratories, mainly due to the characteristics of the samples used^23,24.

Probe nick translation is a fundamental step for successful 3D multicolor DNA FISH; many differe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plates	Thermo Fisher Scientific	142475
6-well plates	Thermo Fisher Scientific	140675
Anti-Digoxigenin 488	DBA	DI7488
b-Mercaptoethanol	Sigma	M3148
bFGF	PeproTech	100-18B
Biotin 11 d-UTP	Thermo Fisher Scientific	R0081
BSA (bovine serum albumine)	Sigma	A7030
Coverlsips	Marienfeld	117500
CY3 d-UTP	GE Healthcare	PA53022
DAPI (4,6-diamidino-2-phenylindole)	Thermo Fisher Scientific	D21490
Deoxyribonucleic acids single strand from salmon testes	Sigma	D7656
Dextran sulfate (powder)	Santa Cruz	sc-203917A
Digoxigenin 11 d-UTP	Roche	11093088910
DMEM	Thermo Fisher Scientific	21969-035 500mL
DNA polymerase I	Thermo Fisher Scientific	18010-017
DNase I	Sigma	AMPD1
dNTPs (C-G-A-T)	Euroclone	BL0423A/C/G
EGF	Sigma	E9644.2MG
Ethanol	Sigma	02860-1L
FBS Hyclone	Thermo Fisher Scientific	SH30109
Formaldehyde solution	Sigma	F8775-25mL
Formamide	Sigma	F9037
Glutammine	Thermo Fisher Scientific	25030-024 100mL
Glycerol	Sigma	G5516-100mL
Glycogen	Thermo Fisher Scientific	AM9510
HCl	Sigma	30721
Human Cot-1 DNA	Thermo Fisher Scientific	15279-001
Insulin Human	Sigma	I9278-5 mL
MgCl2	Sigma	63069
NaAc (Sodium Acetate, pH 5.2, 3 M)	Sigma	S2889
NaCl	Sigma	S9888
Paraformaldehyde	Sigma	158127-25G
PBS (phosphate-buffered saline)	Sigma	P4417
Pennycillin/Streptavidin	Thermo Fisher Scientific	15070-063 100mL
Pepsin	Biorad	P6887
PhasePrep BAC DNA Kit	Sigma	NA0100-1KT
Poly-L-lysine solution	Sigma	P8920
ProLong Diamond Antifade Mountant	Thermo Fisher Scientific	P36970
PureLink Quick Gel Extraction & PCR Purification Combo Kit	Thermo Fisher Scientific	K220001
PureLink Quick Plasmid Miniprep Kit	Thermo Fisher Scientific	K210010
RNAse cocktail	Thermo Fisher Scientific	AM2288
Rubbercement	Bostik
Slides	VWR	631-0114
Streptavidina Alexa fluor 647	Thermo Fisher Scientific	S21374
Tri-Sodium Citrate	Sigma	1110379026
Tris-HCl	Sigma	T3253-500g
Triton X-100	Sigma	T8787-250mL
TWEEN 20	Sigma	P9416-100mL