Identification of multi-loci hubs from 4C-seq demonstrates the functional importance of simultaneous interactions

Abstract

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 associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions 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 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEκ, 3′Eκ and Edκ, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings.

A Phase Separation Model for Transcriptional Control

“…This kinase is critical for initiation of transcription by RNA Polymerase II (RNAPII) and phosphorylates its repetitive C-terminal domain (CTD) (Larochelle et al, 2012). Furthermore, genetic deletion of constituent enhancers within SEs can compromise the activities of other constituents within the super-enhancer (Hnisz et al, 2015; Jiang et al, 2016; Proudhon et al, 2016; Shin et al, 2016), and can lead to the collapse of an entire super-enhancer (Mansour et al, 2014), although this interdependence of constituent enhancers is less apparent for some developmentally regulated super-enhancers (Hay et al, 2016). …”

Transcriptional Addiction in Cancer

“…Enhancer-associated proteins and RNAs, including TFs, co-factors such as Mediator, chromatin regulators, signaling factors, RNA polymerase II (RNAPII), enhancer-associated chromatin marks (H3K27Ac) and eRNAs, are all found at especially high density at the constituent enhancers of SEs. The constituent enhancers of SEs physically associate with one another (Figure 2C) (Dowen et al, 2014; Ji et al, 2016; Kieffer-Kwon et al, 2013) and can function as independent or interdependent components of these large transcription-regulating complexes to drive high-level expression of their associated genes (Hah et al, 2015; Hay et al, 2016; Hnisz et al, 2015; Jiang et al, 2016; Shin et al, 2016). …”

Multi-contact 3C data reveal that the human genome is largely unentangled

“…The method is based on conventional chromosome conformation capture (3C) 10 and the C-walk and multi-contact 4C approaches developed earlier [11][12][13] . 3C performed with the restriction enzyme DpnII produces long ligation products where multiple restriction fragments become ligated together to form DNA molecules that can be >6 kb long, and that are mostly linear (Supplemental Figure 1).…”

“…Although theoretical considerations and polymer simulations had suggested that chromosomes would display low levels of entanglement in cycling cells for kinetic reasons, it has proven difficult to experimentally assess the topological state of the genome in cells directly. Previous studies employed multi-contact data to explore the presence of sub-nuclear compartments 22 , and to test whether interactions between genes and regulatory elements occur in hubs or in pairs [11][12][13] . Here we find that the overall statistics of the composition and order of multi-contact data can also provide insights into the topological nature of the interfaces between chromosomes and chromosomal domains revealing a global lack of chromosomal entanglements.…”