DNA interactions shape the genome to physically and functionally connect regulatory elements to their target genes. Studying these interactions is crucial to understanding the molecular mechanisms that regulate gene expression. In this... more
DNA interactions shape the genome to physically and functionally connect regulatory elements to their target genes. Studying these interactions is crucial to understanding the molecular mechanisms that regulate gene expression. In this chapter, we present a protocol for high-resolution Circular Chromosome Conformation Capture coupled to deep sequencing. This methodology allows to investigate short-range DNA interactions (<100 kbp) and to obtain high-resolution DNA interaction maps of loci. It is a powerful tool to explore how regulatory elements and genes are connected together.
3D chromatin organization is essential for many aspects of transcriptional regulation. Circular Chromosome Conformation Capture followed by Illumina sequencing (4C-seq) is among the most powerful techniques to determine 3D chromatin... more
3D chromatin organization is essential for many aspects of transcriptional regulation. Circular Chromosome Conformation Capture followed by Illumina sequencing (4C-seq) is among the most powerful techniques to determine 3D chromatin organization. 4C-seq, like other modifications of the original 3C technique, uses the principle of “proximity ligation” to identify and quantify ten thousands of genomic interactions at a kilobase scale in a single experiment for predefined loci in the genome.
In this chapter we focus on the experimental steps in the 4C-seq protocol, providing detailed descriptions on the preparation of cells, the construction of the circularized 3C library and the generation of the Illumina high throughput sequencing library. This protocol is particularly suited for the use of mammalian tissue samples, but can be used with minimal changes on circulating cells and cell lines from other sources as well. In the final section of this chapter, we provide a brief overview of data analysis approaches, accompanied by links to publicly available analysis tools.
Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes.... more
Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters cen- tered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics, and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra- and interchromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral lamina-associated domains. Interactions are inde- pendent of nearby H3K27me3-marked loci and determined by chro- mosomal distance and cell-type–specific scaling factors, thus induc- ing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Poly- comb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active, nuclear environments.
