Long G4‐rich Regions in the Human Genome Primarily Function as Enhancers and Form Higher Order, Composite G4 Structures via a Novel Loop:Loop Kissing Interaction
Authors: Borchert Glen M. , Houserova Dominika , Langley Raymond J. , Gillespie Mark N. , Williams Jonathan D. 
Published: April 2020

Repetitive guanine‐rich (G‐rich) sequences have the propensity to form G‐quadruplex (G4) DNA, a stable non‐B form DNA structure. G4 sequences are typically found in association with regulatory elements and disproportionately occur at regions of instability linked with human disease. In the human genome, there are over 300,000 short, G‐rich sequences predicted to form minimal G4 structures. While the majority of these G4 sequences occur in relative isolation to one another, in stark contrast, mammalian antibody switch regions (~1,500 bp) are composed almost entirely of a series of closely neighboring G4‐capable sequences. Although numerous detailed structural analyses and genome‐wide examinations of roles in transcriptional regulation for minimal G4s have been reported, the occurrence, functional roles, and structural attributes of
Long
G4 capable regions (LG4s) ‐ like those occurring in antibody switch regions ‐ elsewhere in the genome are virtually unexplored. Using a novel computational approach searching for LG4 sequences similar to those found in switch regions, we recently identified 304 putative LG4 structures in the human genome and experimentally confirmed that 18 of these assume G4 structures in vitro. In addition, we find our LG4 loci are 5‐fold enriched in G4 ChIP datasets versus control loci. Using data from genome‐wide sequencing studies, we find that these regions frequently contain known promoter and enhancer elements, are prone to single nucleotide polymorphisms, insertions, deletions, and copy number variation, and are significantly associated with chromosomal rearrangements in malignancy. Notably, we find single stranded loops of neighboring minimal G4s in over 50% of our individual LG4 regions are frequently complementary to one another and experimentally confirm that neighboring G4 loops base pair with one another in vitro via a novel loop:loop kissing interaction. In addition to these complex local G4 structures, we also find evidence suggesting LG4 sequences form hybrid G4s with distal promoters with a LG4 and interacting promoter each contributing half the sequence necessary to form a composite G4. Strikingly, 217 of our LG4 sequences overlap with annotated enhancers, and we find the promoters of the inferred target genes for these enhancers to be markedly enriched in G4‐capable sequences suggesting G4s likely facilitate these promoter:enhancer interactions. That said, the average number of G triplets (GGG) potentially contributing to hybrid G4 formation within a single LG4 is ~74x the number of available G triplets found in the average inferred target gene promoter causing us to speculate that the high number of available G4 donor sequences allows LG4 enhancers to act as long “Velcro‐like” regions simultaneously interacting with a number of neighboring gene promoters to coordinate their expressions. In conclusion, our findings suggest LG4s adopt novel, higher order, composite G4 structures directly contributing to the inherent instability, regulatory capacity, and maintenance of these conspicuous genomic regions.

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