Human Lymphoid Translocation Fragile Zones Are Hypomethylated and Have Accessible Chromatin

Abstract

Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones were found to be more accessible than the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more-refined models for why these zones are 100-to 1,000-fold more prone to undergo chromosomal translocation than the adjacent regions.C hromosomal translocations are seminal events in the development of many hematologic malignancies (1). Many hematopoietic malignancies are associated with recurrent reciprocal translocations and their resulting gene fusions or amplifications, which guide diagnosis, assessment of prognosis, and treatment. One example is the BCL2-IGH translocation, t(14;18)(q32;q21), observed in 85% of follicular lymphomas (FL) and 30% of diffuse large B cell lymphomas (DLBCL). A second example is the CCND1-IGH translocation, t(11;14)(q13;q32), observed in almost all mantle cell lymphomas (MCL). Oncogenes near sites of recurrent translocation often confer a selective growth advantage (2). For unknown reasons, human chromosomal translocation breakpoints near these oncogenes frequently cluster within tight DNA zones (often only 20 to 600 bp in length) known as fragile zones (see the green starbursts in the upper portion of Fig. 1A and 2A). These zones are 100-to 1,000-fold more sensitive to breakage than the adjacent DNA regions located, for example, only 20 bp to either side of the fragile zone (2-7). Hence, the DNA sequences surrounding each oncogene are not equally sensitive to breakage. The molecular basis for such an extreme breakage propensity within such a short zone of DNA is unknown, despite our progress on elucidation of other aspects of the mechanism described below (8).We have previously reported that breakpoints of human lymphoid translocation fragile zones are not randomly distributed (Fig. 1A and 2A). Pro-B/pre-B cell-stage translocations show a breakage propensity for the CG (commonly called CpG but designated CG in this paper) DNA sequence motif within the 20 to 600-bp fragile zones (3). We have proposed a model in which activation-induced deaminase (AID) initiates double-strand breaks (DSBs) by acting at the methylated form of these CG sites in single-stranded DNA (ssDNA) to deaminate the 5-methylcytosine to yield a T-G mismatch (3). The r...

Teneurins: An Integrative Molecular, Functional, and Biomedical Overview of Their Role in Cancer

“…We previously predicted the presence of several CpG-rich islands at the TENM2 and TENM4 gene regions (Graumann et al, 2017). Although we found no evidence of methylation-based transcriptional regulation of Teneurins in tumor cells, DNA hypomethylation might influence susceptibility to viral integration and translocation events by rendering DNA more accessible, as shown for some lymphoid malignancies (Cui et al, 2013; Lu et al, 2015). Finally, the occurrence of prevalent tandem rearrangements, defined by the involvement of genes that reside on the same strand and chromosome, has been related to an increased length of introns surrounding the fusion break points of both partner genes (Greger et al, 2014).…”

AID and Reactive Oxygen Species Can Induce DNA Breaks within Human Chromosomal Translocation Fragile Zones

“…A strong possibility is that the MBR and MTC are targeted by AID because of the transient ssDNA character they exhibit, evidenced by the fact that both sequences are sensitive to chemical probing with sodium bisulfite under native (non-denaturing) conditions (Tsai et al, 2009). While we have characterized aspects of the fragile zones in vitro and in human B cells (Cui et al, 2013; Lu et al, 2015; Tsai et al, 2009), transferring these DNA sequences to the genetically tractable S. cerevisiae system is a powerful approach to determine whether this transient ssDNA character is a source of fragility.…”

Microhomology-mediated end joining drives complex rearrangements and over-expression of MYC and PVT1 in multiple myeloma

“…The breakpoints at 8q24 surround an epigenetically active region, defined by the active chromatin marks H3K27Ac, H3K36me3 and, H3K4me1 as well as DNaseI hypersensitivity sites. It may be that epigenetically active, and therefore accessible, loci are preferred translocation partners 48,49 , and the nuclear localization of chromosomes may play a part too. 50 Each of these different rearrangements results in over-expression of MYC.…”