In-Nucleus Hi-C in Drosophila Cells

Summary

The genome is organized in the nuclear space into different structures that can be revealed through chromosome conformation capture technologies. The in-nucleus Hi-C method provides a genome-wide collection of chromatin interactions in Drosophila cell lines, which generates contact maps that can be explored at megabase resolution at restriction fragment level.

Abstract

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, the mechanisms underlying TAD formation and boundaries are still under investigation. The application of the in-nucleus Hi-C method described here helped to dissect the function of architectural protein (AP)-binding sites at TAD boundaries isolating the Notch gene. Genetic modification of domain boundaries that cause loss of APs results in TAD fusion, transcriptional defects, and long-range topological alterations. These results provided evidence demonstrating the contribution of genetic elements to domain boundary formation and gene expression control in Drosophila. Here, the in-nucleus Hi-C method has been described in detail, which provides important checkpoints to assess the quality of the experiment along with the protocol. Also shown are the required numbers of sequencing reads and valid Hi-C pairs to analyze genomic interactions at different genomic scales. CRISPR/Cas9-mediated genetic editing of regulatory elements and high-resolution profiling of genomic interactions using this in-nucleus Hi-C protocol could be a powerful combination for the investigation of the structural function of genetic elements.

Introduction

In eukaryotes, the genome is partitioned into chromosomes that occupy specific territories in the nuclear space during interphase¹. The chromatin forming the chromosomes can be divided into two main states: one of accessible chromatin that is transcriptionally permissive, and the other of compact chromatin that is transcriptionally repressive. These chromatin states segregate and rarely mix in the nuclear space, forming two distinct compartments in the nucleus². At the sub-megabase scale, boundaries separate domains of high-frequency chromatin interactions, called TADs, that mark chromosomal organization

Protocol

1. Fixation

1. Start with 10 million Schneider's line 2 plus (S2R+) cells to prepare 17.5 mL of a cell suspension in Schneider medium containing 10% fetal bovine serum (FBS) at room temperature (RT).
2. Add methanol-free formaldehyde to obtain a final concentration of 2%. Mix and incubate for 10 min at RT, taking care to mix every minute.
   NOTE: Formaldehyde is a hazardous chemical. Follow the appropriate health and safety regulations, and work in the fume hood.
3. Quench the reaction by .......

Discussion

The in-nucleus Hi-C method presented here has allowed detailed exploration of Drosophila genome topology at high resolution, providing a view of genomic interactions at different genomic scales, from chromatin loops between regulatory elements such as promoters and enhancers to TADs and large compartment identification²⁵. The same technology has also been efficiently applied to mammalian tissues with some modifications³³. For example, when processing a tissue instead of a s.......

Materials

Name	Company	Catalog Number	Comments
16% (vol/vol) paraformaldehyde solution	Agar Scientific	R1026
Biotin-14-dATP	Invitrogen	CA1524-016
ClaI enzyme	NEB	R0197S
COVARIS Ultrasonicator	Covaris	LE220-M220
Cut Smart	NEB	B72002S
Dulbecco's Modified Eagle Medium (DMEM) 1x	Life Technologies	41965-039
Dynabeads MyOne Streptabidin C1	Invitrogen	65002
Fetal bovine serum (FBS) sterile filtered	Sigma	F9665
Klenow Dna PolI large fragment	NEB	M0210L
Klenow exo(-)	NEB	M0210S
Ligation Buffer	NEB	B020S
MboI enzyme	NEB	R0147M
NP40-Igepal	SIGMA	CA-420	Non-ionic surfactant for addition in lysis buffer
PE adapter 1.0	Illumina	5'-P-GATCGGAAGAGCGGTTCAGCAG GAATGCCGAG-3'
PE adapter 2.0	Illumina	5'-ACACTCTTTCCCTACACGACGCT CTTCCGATCT-3'
PE PCR primer 1.0	Illumina	5'-AATGATACGGCGACCACCGAGAT CTACACTCTTTCCCTACACGACG CTCTTCCGATCT-3'
PE PCR primer 2.0	Illumina	5'-CAAGCAGAAGACGGCATACGAG ATCGGTCTCGGCATTCCTGCTGA ACCGCTCTTCCGATCT-3'
Phenol: Chloroform:Isoamyl Alcohol 25:24:1	SIGMA	P2069
Primer 1 (known interaction, Figure 2A)	Sigma	5'-TCGCGGTAATTTTGCGTTTGA-3'
Primer 2 (known interactions, Figure 2A)	Sigma	5'-CCTCCCTGCCAAAACGTTTT-3'
Protease inhibitor cocktail tablet	Roche	4693132001
Proteinase K	Roche	3115879001
Qubit	ThermoFisher	Q33327
RNAse	Roche	10109142001
SPRI Beads	Beckman	B23318
T4 DNA ligase	Invitrogen	15224-025
T4 DNA polymerase	NEB	M0203S
T4 polynucleotide kinase (PNK)	NEB	M0201L
TaqPhusion	NEB	M0530S	DNA polymerase
Triton X-100			Non-ionic surfactant for quenching of SDS