Generation Of Translocations Research Articles

Are genomic translocations predictable? (Retrospective on DOI 10.1002/bies.201100122).

It is sometimes useful for biologists to be reminded that the rules governing the physical universe apply to biological systems. Thermodynamic principles stipulate that rates for chemical reactions are proportional to the concentration(s) of reactants, a basic rule that applies equally to inorganic chemical reactions as well as to complex genomic rearrangements such as translocation and recombination. Jerome Dejardin recently predicted that genomic translocation and recombination in pathologic circumstances are facilitated by alterations in chromatin that permit aberrant transcription factor binding [1]. Transcription factor binding is posited to result in co-localization of non-adjacent loci in three dimensional space within the nucleus. This transcription-factor driven colocalization increases local concentration of the reacting species for translocation or recombination, thus accelerating genomic rearrangement. This prediction, while simple in concept, is powerful in terms of predicting sites of aberrant genomic transactions: they are predicted to be concentrated in the vicinity of binding sites of transcription factors found in the cell type(s) of interest and to occur at loci with locally accessible chromatin features. Issues relevant to Dejardin’s predictions have recently been addressed experimentally by a pair of elegant studies. Roukos et al. [2] examined translocation in an engineered system utilizing ultrahigh-throughput imaging that permits real-time monitoring of DNA ends (the reactants) within nuclei. Double-strand breaks were induced at two distinct (and uniquely marked) genetic loci by a restriction enzyme. The resulting DNA ends sampled nuclear space, occasionally finding and pairing with a translocation partner. Importantly, the distance between the two induced double strand breaks prior to end joining was critical for the frequency of translocation: ends closer to each other prior to pairing were more likely to translocate. This study suggests that an important component of the prediction by Dejardin is likely to be true, i.e. increasing the proximity of DNA double-strand breaks to each other (increasing the local concentration of reactants) leads to a higher likelihood of translocation. While this study supports the ‘concentration’ component of Dejardin’s prediction, it does not speak to the nature of the chromatin surrounding such an event. Evidence speaking for the role of chromatin in the generation of translocations was recently published by Hakim and colleagues [3]. This study defined the nuclear interaction partners of enhancers of immunoglobulin heavy chain (the well characterized Igh enhancers Eμ and Eα) in activated B lymphocytes using chromosome conformation capture (4C) experiments. The interactome of these transcription factor binding sites was enriched in active chromatin marked by high levels of histone acetylation, RNA polymerase occupancy, and active transcription. Introduction of DSBs at Igh (using a restriction enzyme) resulted in productive translocation to regions with similar chromatin features. This study indicates that translocation events occur with higher frequency at regions that are in close proximity in three dimensional space and that bear local chromatin features consistent with increased accessibility – precisely as predicted. The predictions of Dejardin – that translocation and recombination may be driven by transcription factor binding, colocalization of non-adjacent loci, and alterations in chromatin features – appear to be in strong agreement with newly emerging experimental data. It remains to be determined whether all features of the prediction agree with experimental data. The field awaits the emergence of additional studies directed at understanding these genomic rearrangements that may shed new light on the ability of this model to predict their location.

Abstract 1776: Lymphomas associated with aberrant DNA rearrangements are suppressed by Mre11 mutation.

Abstract Many translocations associated with spontaneous human lymphoid malignancies are mediated by short regions of sequence homology (microhomology) at the breakpoints. The hallmark features of this error prone pathway are deletions of flanking sequence and short homologies of 1-25 bases at the junctions that serve to align the DNA ends prior to joining. In addition to its importance in the etiology of human cancers, microhomology-mediated joining or alternative NHEJ (aNHEJ) also appears to play a significant role in therapy-induced oncogenic translocations. Studies have provided compelling evidence that the DNA end-binding complex composed of Mre11 (nuclease), Rad50 (tetherer) and Nbs1 (signaler)–MRN–functions in the context of the aNHEJ pathway. In addition, in vivo and cell based studies have identified that deficiencies in components of the MRN complex reduce aNHEJ activity. However, it is not known whether the MRN complex participates in the generation of aNHEJ catalyzed translocations that can predispose to cancer. Therefore, our studies seek to better understand the contribution of Mre11/MRN to the aNHEJ pathway and thus, in generation of translocations. We have obtained mice harboring an Mre11 conditionally targeted null allele as well as an Mre11 knock-in mutation that abrogates nuclease activity. We have included mutant mice in this study that succumb to early onset Pro-B lymphomas associated with clonal translocations containing microhomologies of 2-8 nucleotides. Thus, the spontaneously arising tumors in this mutant background represent a valuable in vivo system to assess the involvement of Mre11/MRN in aNHEJ mediated translocations. We have determined that Mre11/MRN inactivation in the B-cell lineage suppresses Pro-B lymphoma in this mutant background. In future experiments we are investigating the impact of Mre11/MRN inactivation on various cancer phenotypes. Together, these studies will provide insights into the mechanisms underlying human lymphoma and may ultimately provide insights into targets for improved treatment of lymphoid malignancies. Citation Format: Cheryl J. Smith, Jeffrey Xie, David O. Ferguson, JoAnn M. Sekiguchi. Lymphomas associated with aberrant DNA rearrangements are suppressed by Mre11 mutation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1776. doi:10.1158/1538-7445.AM2013-1776

Regulation of telomere addition at DNA double-strand breaks

Telomeres constitute the ends of linear eukaryotic chromosomes. Due to the conventional mode of DNA replication, telomeric DNA erodes at each cell division. To counteract this, a specialized reverse transcriptase, telomerase, can elongate chromosome ends to maintain them at a constant average length. Because of their similarity to DNA double-strand breaks (DSBs), telomeres might be expected to induce a DNA damage response, which would lead to repair reactions and the generation of translocations or fusions. Many proteins present at telomeres prevent this by protecting (capping) the chromosome termini. Conversely, a DSB occurring in other regions of the genome, due, for instance, to a stalled replication fork or genotoxic agents, must be repaired by homologous recombination or end-joining to ensure genome stability. Interestingly, telomerase is able to generate a telomere de novo at an accidental DSB, with potentially lethal consequences in haploid cells and, at a minimum, loss of heterozygosity (LOH) in diploid cells. Recent data suggest that telomerase is systematically recruited to DSBs but is prevented from acting in the absence of a minimal stretch of flanking telomere-repeat sequences. In this review, we will focus on the mechanisms that regulate telomere addition to DSBs.

Potential G-quadruplex formation at breakpoint regions of chromosomal translocations in cancer may explain their fragility

Genetic alterations like point mutations, insertions, deletions, inversions and translocations are frequently found in cancers. Chromosomal translocations are one of the most common genomic aberrations associated with nearly all types of cancers especially leukemia and lymphoma. Recent studies have shown the role of non-B DNA structures in generation of translocations. In the present study, using various bioinformatic tools, we show the propensity of formation of different types of altered DNA structures near translocation breakpoint regions. In particular, we find close association between occurrence of G-quadruplex forming motifs and fragile regions in almost 70% of genes involved in rearrangements in lymphoid cancers. However, such an analysis did not provide any evidence for the occurrence of G-quadruplexes at the close vicinity of translocation breakpoint regions in nonlymphoid cancers. Overall, this study will help in the identification of novel non-B DNA targets that may be responsible for generation of chromosomal translocations in cancer.

Genomic Modifications Occur during Maturation in Myeloid Cells

Objective: A chromosomal translocation plays an important role on the leukemia-generation from the normal hematopoietic cells. Various kinds of translocation-patterns and -partners have been reported; however, a typical translocation has been reported like AML-1 and MTG8 in acute myelogenous leukemia (AML) (M2), and PML and RARα in AML (M3). This implies that some common mechanism exists on the chromosomal double strand breaks and the generation of translocations. We make a hypothesis that, when normal immature cells come into a maturation process, a specific event may occur on DNA level to induce irreversible changes toward cell-death. And a target region for the irreversible change may be the near site to chromosomal translocations. We compared the digested fragment-size for restriction-endonucleases using southern blotting among hematopoietic cell lines, normal bone marrow immature progenitor cells and normal blood mature neutrophils.Materials and Methods: Bone marrows and bloods were obtained from informed healthy persons, and mononuclear cells were prepared after Ficoll-Paque sedimentation method, and neutrophil-rich fractions were prepared with density gravity centrifugation and hypotonic RBC lyses method. Various kinds of human cell lines, cultured in DMEM with 10% FCS, were also prepared. High molecular weight DNA was extracted from these cells, and they were digested with several kinds of restriction endonucleases. After electrophoresis, the digested DNA was transferred to a nitrocellulose membrane, and southern blot-hybridization was examined. The probe used was AML-1, MTG-8, PML, and RARα, in which the 5′ and 3′ breakpoint regions observed in chromosomal translocations were selected, respectively. Results and Discussion: No changes on the blotted fragment-size were observed among in the cultured cell lines, bone marrow immature mononuclear cells, and blood mononuclear cells; however, when 5′-PML probe was hybridized after DNA was digested with HindIII, the observed blot size was changed between normal immature myeloid progenitor cells and normal blood mature neutrophilic granulocytes. This change was not due to the personal polymorphism. These observations indicate that when myeloid cells become mature to neutrophilic granulocytes, some change occurs at DNA level. PML works on the maintenance of nucleobody for cell division, and it is very important to determine whether PML gene is maintained in neutrophilic granulocytes. We now prepare a genomic library from blood neutrophils to isolate the target clone, and determine DNA sequence near at the HindIII site to identify the modification of DNA during maturation of myeloid cells.