G-Quadruplex Structures Formed at the HOX11 Breakpoint Region Contribute to Its Fragility during t(10;14) Translocation in T-Cell Leukemia

Abstract

The t(10;14) translocation involving the HOX11 gene is found in several T-cell leukemia patients. Previous efforts to determine the causes of HOX11 fragility were not successful. The role of non-B DNA structures is increasingly becoming an important cause of genomic instability. In the present study, bioinformatics analysis revealed two G-quadruplex-forming motifs at the HOX11 breakpoint cluster. Gel shift assays showed formation of both intra-and intermolecular G-quadruplexes, the latter being more predominant. The structure formation was dependent on four stretches of guanines, as revealed by mutagenesis. Circular dichroism analysis identified parallel conformations for both quadruplexes. The non-B DNA structure could block polymerization during replication on a plasmid, resulting in consistent K ؉ -dependent pause sites, which were abolished upon mutation of G-motifs, thereby demonstrating the role of the stretches of guanines even on double-stranded DNA. Extrachromosomal assays showed that the G-quadruplex motifs could block transcription, leading to reduced expression of green fluorescent protein (GFP) within cells. More importantly, sodium bisulfite modification assay showed the single-stranded character at regions I and II of HOX11 in the genome. Thus, our findings suggest the occurrence of G-quadruplex structures at the HOX11 breakpoint region, which could explain its fragility during the t(10;14) translocation.

The role of fork stalling and DNA structures in causing chromosome fragility

“…Interspersed guanine runs—optimally four repeats of at least three Gs in a row separated by a spacer of one to seven nucleotides, can result in a G‐quadruplex structure‐forming on one DNA strand, leaving the partner DNA strand single‐stranded . G‐quadruplexes have been mapped to breakpoint regions of cancer‐causing translocations in humans, implicating them in causing fragility . G‐quadruplexes are involved in immunoglobulin class switching where they are stabilized by R‐loops and promote nicks via activation‐induced deaminase (AID) .…”

A Role for Non-B DNA Forming Sequences in Mediating Microlesions Causing Human Inherited Disease

“…However, the extent to which they are involved in mediating mutations on a genome‐wide scale has not been fully ascertained. Indeed, the association of non‐B DNA‐forming sequences with genomic instability has been best established in the area of triplet repeat expansion diseases [Iyer et al., ; Zhao and Usdin, ], and in gross chromosomal abnormalities, both in the germline [Cooper et al., ; Verdin et al., ; You et al., ; Wu et al., ; Javadekar and Raghavan, ] and in cancer [De and Michor, ; Nambiar et al., ; Jeitany et al., ; Lu et al., ; Williams et al., ]. Filling this knowledge gap is of particular importance in the field of medical genetics, given the widespread occurrence of non‐B DNA‐forming repeats in the human and other mammalian genomes [Du et al., ].…”

Snaps and mends: DNA breaks and chromosomal translocations

“…The correlation held true for almost 70% of the genes tested: E2A ( TCF3 ), BCR intron 14 and HOX11 in the case of lymphoid cancers, and TMPRSS2 in the case of prostate cancer . An analysis of the HOX11 breakpoint region involved in the t(10;14) translocation indicated the formation of two G‐quadruplex structures whose occurrence may be a reason for the fragility of HOX11 . Investigations are under way to further substantiate the correlation between G‐quadruplex structure formation and translocations.…”

“…In addition to sequence specificity, RAGs may also exhibit structure specificity by acting on non‐B‐DNA structures including heterologous loops, G‐quadruplexes and bubbles both in vitro and ex vivo (Fig. ).…”

“…Potential involvement of V(D)J recombination during these translocations is evident from analysis of translocation breakpoints [78]. However, based on recombination frequency and biochemical studies, it appears that the binding and cleavage efficiency of RAGs at these cryptic sites is several times weaker than that at the consensus RSSs In addition to sequence specificity, RAGs may also exhibit structure specificity by acting on non-B-DNA structures including heterologous loops, G-quadruplexes and bubbles both in vitro and ex vivo [6,49,51,56,[86][87][88][89][90][91][92][93][94][95][96] (Fig. 4).…”