| dc.description.abstract | Genome integrity is essential for normal cellular functions. Erroneous repair of DNA double-strand breaks (DSBs) could lead to chromosomal rearrangements, including chromosomal translocations that may result in altered protein expression or chimeric proteins. Cell-type specific translocations, as observed in human lymphomas, are important for oncogenic progression. One such instance is of t(14;18) translocation involving BCL2 (chromosome 18) and IgH (chromosome 14) loci, that causes follicular lymphoma (FL). In another illustrious example, diverse translocations involving a common partner, BCL6 (chromosome 3), have been reported in diffuse large B cell lymphoma (DLBL).
In the present study, impact of deviations in DNA structure in facilitating chromosomal fragility was investigated. In silico, in vitro and ex vivo approaches were employed to study the translocation breakpoint regions. Ex vivo assays were conducted to evaluate the effect of non-B DNA structures on physiological process such as transcription. Patient breakpoint analyses for BCL2 and BCL6 genes revealed breakpoint clusters in both the translocation cases. Bioinformatic studies suggest formation of G-quadruplex at BCL6 cluster III, while the nature of structure was unclear in case of BCL2 MBR. Gel shift assays showed faster mobility in case of both the translocation breakpoint sequences under study when compared to corresponding control sequences. Circular dichroism studies showed a characteristic G-quadruplex spectrum for BCL6. However, for BCL2, an uncharacteristic spectrum with a positive plateau pattern was observed, which was previously reported in literature for cruciform-like structures. Sodium bisulfite modification assay revealed single-strandedness at the fragile region when plasmid with BCL6 breakpoint was probed. These sequences had not been previously predicted by bioinformatic analysis; subsequent study of these revealed GNG motifs that aided in G4-structure formation. Additionally, gel shift assays, circular dichroism studies, and dimethyl sulphate probing supported the occurrence of G4-structure in these BCL6 sequences. However, no such protection at guanines was observed at BCL2, ruling out formation of G-quadruplex or triplex at this region. Further, it was observed that BCL2 peak III and BCL6 breakpoint region can block replication, unlike respective mutants, which allowed replication to proceed unhindered. Extrachromosomal episomal assay in pre-B cells, Nalm6 and Reh showed that BCL2 sequence can halt transcription. Similarly, BCL6 sequence could also halt transcription in the extrachromosomal episomal assay. Thus, our results suggest formation of G-quadruplex structures at BCL6 breakpoint region and a potential cruciform DNA structure at BCL2 MBR, with the capacity to affect replication and transcription. Furthermore, it is speculated that formation of such structures could impart fragility to breakpoint regions and eventually, lead to translocations, which needs to be investigated further.
Taking into account the impact of formation of non-B DNA structures on cellular processes, several techniques have been developed to help study non-B DNA structures in vitro. However, detecting their intracellular presence has been challenging. Potential solutions include developing small molecule probes and antibodies against these forms and along these lines; Recently, an antibody, BG4 was designed to study G4 structures in cells. In present study, BG4 antibody was further characterized by biochemical and biophysical methods. BG4 showed specific binding towards G-rich DNA derived from multiple genes, which formed G4-DNA, unlike its complementary C-rich sequence or oligomer containing random sequence. Competition assays further supported the antibody specificity. Further, BG4 bound the inter- and intra-molecular G4-DNA, of parallel orientation, in single-stranded DNA. Interestingly, gel shift assays for multiple sequences indicated that mere presence of G4-DNA motif on duplex DNA was insufficient for antibody binding. BioLayer Interferometry (BLI) revealed high affinity (Kd 17.4 nM) with a known G4 substrate. Dimethyl sulphate probing of BG4-bound G4 substrate indicated higher protection of guanine residues that were specifically involved in G4-formation. Importantly, BG4 bound the G4-DNA in telomere region within supercoiled DNA context, as demonstrated by mobility shift of supercoiled plasmid, unlike in control plasmid bearing random sequence. BG4 binding within cells indicated efficient foci formation in four cell lines tested, thus demonstrating presence of G4-DNA in cellular context. Importantly, number of BG4 foci, indicative of G4 structures, were shown to be modulated upon shRNA mediated knockdown of a G4-resolvase, WRN, and upon inducing nutrient deprivation in cells by serum starvation. It would be of interest to investigate how this antibody and others could be utilized to study influence of different cellular conditions on extent of formation of non-B DNA structures.
Taken together, our studies of the translocation breakpoint regions in BCL6 and BCL2 suggest a general mechanism associated with many common translocations seen in lymphoid cancers. Herein, formation of non-B DNA structures would generate partially single-stranded regions in the genome, which could render them susceptible to single-strand specific DNA nucleases or enzymes, thus promoting breakage and eventually, chromosomal translocations. In the other part of the study, further characterization has been conducted for an antibody known to detect one of such non-B DNA structures, G4-DNA, in in vitro and under two modified cellular conditions. Antibodies of such nature could be further utilized for determining G4 existence in certain genomic areas of interest, as probable diagnostic tools, and more ambitiously, as therapeutic agents. | en_US |
