Background: The organization of chromatin in the nucleus plays an essential role in gene regulation. When considering the mammalian genome it is important to take into account that about half of the DNA is comprised of transposable elements. Given their repetitive nature, reads associated with these elements are generally discarded or randomly distributed among elements of the same type in genome-wide analyses. Thus, it is challenging to identify the activities and properties of individual transposons. As a result, we only have a partial understanding of how transposons contribute to chromatin folding and how they impact gene regulation.

word limit)Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that these associations act in isolation of other interacting partners within the genome. Indeed, the influence of multi-loci interactions in gene control remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these.Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4Cseq data sets that identify known pairwise interactions involving the Tcrb and Igk antigen receptor enhancers, in addition to novel tri-loci associations. We further show that enhancer deletions not only interfere with tri-loci interactions in which they participate, but they also disrupt pairwise interactions between other partner enhancers and this disruption is linked to a reduction in their transcriptional output. These findings underscore the functional importance of hubs and provide new insight into chromatin organization as a whole. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings. ! 3!