ABSTRACT
Complex genomes show intricate organization in three-dimensional (3D) nuclear space. Current models posit that cohesin extrudes loops to form self-interacting domains delimited by the DNA binding protein CTCF. Here, we describe and quantitatively characterize cohesin-propelled, jet-like chromatin contacts as landmarks of loop extrusion in quiescent mammalian lymphocytes. Experimental observations and polymer simulations indicate that narrow origins of loop extrusion favor jet formation. Unless constrained by CTCF, jets propagate symmetrically for 1-2 Mb, providing an estimate for the range of in vivo loop extrusion. Asymmetric CTCF binding deflects the angle of jet propagation as experimental evidence that cohesin-mediated loop extrusion can switch from bi- to unidirectional and is controlled independently in both directions. These data offer new insights into the physiological behavior of in vivo cohesin-mediated loop extrusion and further our understanding of the principles that underlie genome organization.
Subject(s)
Chromatin , Chromosomal Proteins, Non-Histone , Animals , Chromatin/genetics , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Polymers/metabolism , Mammals/metabolism , CohesinsABSTRACT
Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure.
