ABSTRACT
CTCF is a critical regulator of genome architecture and gene expression that binds thousands of sites on chromatin. CTCF genomic localization is controlled by the recognition of a DNA sequence motif and regulated by DNA modifications. However, CTCF does not bind to all its potential sites in all cell types, raising the question of whether the underlying chromatin structure can regulate CTCF occupancy. Here, we report that R-loops facilitate CTCF binding through the formation of associated G-quadruplex (G4) structures. R-loops and G4s co-localize with CTCF at many genomic regions in mouse embryonic stem cells and promote CTCF binding to its cognate DNA motif in vitro. R-loop attenuation reduces CTCF binding in vivo. Deletion of a specific G4-forming motif in a gene reduces CTCF binding and alters gene expression. Conversely, chemical stabilization of G4s results in CTCF gains and accompanying alterations in chromatin organization, suggesting a pivotal role for G4 structures in reinforcing long-range genome interactions through CTCF.
Subject(s)
G-Quadruplexes , Animals , Mice , R-Loop Structures , CCCTC-Binding Factor/metabolism , Chromatin/genetics , Genomics , Binding SitesABSTRACT
Neuronal activation induces rapid transcription of immediate early genes (IEGs) and longer-term chromatin remodeling around secondary response genes (SRGs). Here, we use high-resolution chromosome-conformation-capture carbon-copy sequencing (5C-seq) to elucidate the extent to which long-range chromatin loops are altered during short- and long-term changes in neural activity. We find that more than 10% of loops surrounding select IEGs, SRGs, and synaptic genes are induced de novo during cortical neuron activation. IEGs Fos and Arc connect to activity-dependent enhancers via singular short-range loops that form within 20 min after stimulation, prior to peak messenger RNA levels. By contrast, the SRG Bdnf engages in both pre-existing and activity-inducible loops that form within 1-6 h. We also show that common single-nucleotide variants that are associated with autism and schizophrenia are colocalized with distinct classes of activity-dependent, looped enhancers. Our data link architectural complexity to transcriptional kinetics and reveal the rapid timescale by which higher-order chromatin architecture reconfigures during neuronal stimulation.
Subject(s)
Chromatin Assembly and Disassembly/physiology , Gene Expression/physiology , Genome/genetics , Neurons/physiology , Animals , Bicuculline/pharmacology , Brain-Derived Neurotrophic Factor/physiology , Chromatin Assembly and Disassembly/genetics , Cytoskeletal Proteins/physiology , Genome/drug effects , Humans , Mice , Nerve Tissue Proteins/physiology , Neurons/drug effects , Proto-Oncogene Proteins c-fos/physiology , Tetrodotoxin/pharmacology , Time FactorsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.