Targeted Chromatin Capture (T2C): a novel high resolution high throughput method to detect genomic interactions and regulatory elements

Abstract

BackgroundSignificant efforts have recently been put into the investigation of the spatial organization and the chromatin-interaction networks of genomes. Chromosome conformation capture (3C) technology and its derivatives are important tools used in this effort. However, many of these have limitations, such as being limited to one viewpoint, expensive with moderate to low resolution, and/or requiring a large sequencing effort. Techniques like Hi-C provide a genome-wide analysis. However, it requires massive sequencing effort with considerable costs. Here we describe a new technique termed Targeted Chromatin Capture (T2C), to interrogate large selected regions of the genome. T2C provides an unbiased view of the spatial organization of selected loci at superior resolution (single restriction fragment resolution, from 2 to 6 kbp) at much lower costs than Hi-C due to the lower sequencing effort.ResultsWe applied T2C on well-known model regions, the mouse β-globin locus and the human H19/IGF2 locus. In both cases we identified all known chromatin interactions. Furthermore, we compared the human H19/IGF2 locus data obtained from different chromatin conformation capturing methods with T2C data. We observed the same compartmentalization of the locus, but at a much higher resolution (single restriction fragments vs. the common 40 kbp bins) and higher coverage. Moreover, we compared the β-globin locus in two different biological samples (mouse primary erythroid cells and mouse fetal brain), where it is either actively transcribed or not, to identify possible transcriptional dependent interactions. We identified the known interactions in the β-globin locus and the same topological domains in both mouse primary erythroid cells and in mouse fetal brain with the latter having fewer interactions probably due to the inactivity of the locus. Furthermore, we show that interactions due to the important chromatin proteins, Ldb1 and Ctcf, in both tissues can be analyzed easily to reveal their role on transcriptional interactions and genome folding.ConclusionsT2C is an efficient, easy, and affordable with high (restriction fragment) resolution tool to address both genome compartmentalization and chromatin-interaction networks for specific genomic regions at high resolution for both clinical and non-clinical research.

The second decade of 3C technologies: detailed insights into nuclear organization

“…This weakened the domain boundaries and thereby caused aberrant enhancer-promoter interactions with glioma oncogenes (Flavahan et al 2016). Depletion of cohesin or CTCF led to a decreased ratio of intra-TAD over inter-TAD contacts, indicative of boundary disruption (Seitan et al 2013;Sofueva et al 2013;Zuin et al 2014). However, this occurred to different degrees for each of the two proteins, perhaps because of differences in depletion efficiency.…”

“…One such strategy was termed targeted chromatin capture (T2C) (Kolovos et al 2014), which essentially offers an alternative to 5C technology. As depicted in Figure 2, the protocol basically follows standard 3C library preparation (using a six-cutter for the digest), but, instead of the sonication used in Hi-C protocols, T2C uses a second restriction digest with a four-cutter to fragment the library; sequencing adapters are then added via ligation.…”

Heat stress: A risk factor for skin carcinogenesis

“…TC-NER is initiated when an elongating RNA polymerase II is stalled as a result of DNA lesions, which disrupts transcription on the template strand of active genes (Katz et al, 2014;Kolovos et al, 2014). This is followed by the recruitment and modifications to Cockayne Syndrome B (CSB)…”

An Overview of Genome Organization and How We Got There: from FISH to Hi-C

“…Alternatively, selective genome capture may be performed with specific oligonucleotide to bind Hi-C libraries. The latter approach, considered a fourth-generation 3C technology, has already been successfully used by several groups to study cis-regulatory landscapes (capture-C [319]), to identify targets of breast cancer risk variants (capture-Hi-C [320]), and to obtain more detailed maps of the H19/Igf2 and ␤-globin networks (targeted chromatin capture [321]). …”