Formation of new chromatin domains determines pathogenicity of genomic duplications

Abstract

Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.

Reciprocal insulation analysis of Hi-C data shows that TADs represent a functionally but not structurally privileged scale in the hierarchical folding of chromosomes

“…Irrespective of the mechanisms underlying their formation, genetic evidence suggests that TADs contribute to establish correct interaction patterns between enhancers and promoters (Symmons et al 2014;Lupiáñez et al 2015;Franke et al 2016). Consistent with this, transcriptional coregulation of neighboring genes is favored within TADs during differentiation (Nora et al 2012) and upon transcriptional responses to external stimuli (Le Dily et al 2014).…”

“…Several studies have provided evidence on the fundamental role of compartmentalization into TADs to instruct enhancer-promoter communication (Nora et al 2012;Symmons et al 2014;Lupiáñez et al 2015;Franke et al 2016), but they remain elusive on what makes TADs 'special' compared to other chromosomal folding layers, such as sub-TADs and notably contact domains or meta-TADs. In this study, we present a new domain-calling algorithm that is able to segment Hi-C interaction maps into nested sets of The number of sublevels in a domain correlates with the transcriptional activity within the domain (shown for domains at 65% RI in ESC).…”

Constrained release of lamina-associated enhancers and genes from the nuclear envelope during T-cell activation facilitates their association in chromosome compartments

“…As TADs and CDs likely represent orthogonal measures of the same structural units, we use the term TADs unless specifically referring to published data sets employing the CD approach. Topological constraints imposed by TADs favor looping interactions to cis-regulatory element:gene pairings within the same TAD (Lupianez et al 2015;Franke et al 2016;Hnisz et al 2016). However, a higher level of genome organization also occurs where multiple TADs many megabase pairs distal on one chromosome or on different chromosomes can be adjacent and share a functional state (Lieberman-Aiden et al 2009).…”

Enhancer-gene maps in the human and zebrafish genomes using evolutionary linkage conservation

“…Understanding how enhancers 20 regulate gene expression is therefore essential to understand how organisms develop and function throughout life. In fact, disruption of enhancer function has been shown to lead to abnormal gene expression and thus to diseases [4][5][6][7][8] . In addition, the majority of variants identified in Genome Wide Association Studies (GWAS) are found outside coding sequences 9,10 .…”