Unbiased Interrogation of 3D Genome Topology Using Chromosome Conformation Capture Coupled to High-Throughput Sequencing (4C-Seq)

Abstract

The development and widespread implementation of chromosome conformation capture (3C) technology has allowed unprecedented new insight into how chromosomes are folded in three-dimensional (3D) space. 3C and its derivatives have contributed tremendously to the now widely accepted view that genome topology plays an important role in many major cellular processes, at a chromosome-wide scale, but certainly also at the level of individual genetic loci. A particularly popular application of 3C technology is to study transcriptional regulation, allowing researchers to draw maps of gene regulatory connections beyond the linear genome through addition of the third dimension. In this chapter, we provide a highly detailed protocol describing 3C coupled to high-throughput sequencing (referred to as 3C-Seq or more commonly 4C-Seq), allowing the unbiased interrogation of genome-wide chromatin interactions with specific genomic regions of interest. Interactions between spatially clustered DNA fragments are revealed by crosslinking the cells with formaldehyde, digesting the genome with a restriction endonuclease and performing a proximity ligation step to link interacting genomic fragments. Next, interactions with a selected DNA fragment are extracted from the 3C library through a second round of digestion and ligation followed by an inverse PCR. The generated products are immediately compatible with high-throughput sequencing, and amplicons from different PCR reactions can easily be multiplexed to dramatically increase throughput. Finally, we provide suggestions for data analysis and visualization.

AmpliSeq transcriptome analysis of human alveolar and monocyte-derived macrophages over time in response to Mycobacterium tuberculosis infection

“…TREM1 exemplifies a gene residing in a large cluster of homologues, covered with long LD bocks with frequent SNPs, an indicator of evolutionary selection pressures ( S1 Fig ). The gene cluster is decorated with numerous eQTLs (GTEx), many of which appear to interact with more than one gene in the cluster–possibly via chromatin looping among multiple enhancers and promoters [ 66 , 67 ]. We find numerous such multi-gene clusters among genes of the innate immune system that require combined analysis of the genetic influence of the entire cluster rather than that of single SNPs acting independently, as shown for a nicotinic receptor gene cluster [ 68 ].…”

Transcription factors orchestrate dynamic interplay between genome topology and gene regulation during cell reprogramming

“…4C-seq was performed as described previously72,73. Briefly, 0.5-1.0 million crosslinked nuclei were digested with Csp6I followed by ligation under dilute conditions.…”

The lncRNA Locus Handsdown Regulates Cardiac Gene Programs and Is Essential for Early Mouse Development

“…Circularized Chromosome Conformation Capture (4C) and Visualization 4C experiment was carried out as previously described (Brouwer et al, 2017;van de Werken et al, 2012). Briefly, 10 million mESCs and cardiomyocytes and 120 embryonic Hearts (E11.5) were crosslinked in 1% methanol-free formaldehyde in PBS for 10 minutes at room temperature.…”