Loss of Bloom syndrome protein destabilizes human gene cluster architecture

Abstract

Bloom syndrome confers strong predisposition to malignancy in multiple tissue types. The Bloom syndrome patient (BLM) protein defective in the disease biochemically functions as a Holliday junction dissolvase and human cells lacking functional BLM show 10-fold elevated rates of sister chromatid exchange. Collectively, these phenomena suggest that dysregulated mitotic recombination drives the genomic instability underpinning the development of cancer in these individuals. Here we use physical analysis of the highly repeated, highly self-similar human ribosomal RNA gene clusters as sentinel biomarkers for dysregulated homologous recombination to demonstrate that loss of BLM protein function causes a striking increase in spontaneous molecular level genomic restructuring. Analysis of single-cell derived sub-clonal populations from wild-type human cell lines shows that gene cluster architecture is ordinarily very faithfully preserved under mitosis, but is so unstable in cell lines derived from BLMs as to make gene cluster architecture in different sub-clonal populations essentially unrecognizable one from another. Human cells defective in a different RecQ helicase, the WRN protein involved in the premature aging Werner syndrome, do not exhibit the gene cluster instability (GCI) phenotype, indicating that the BLM protein specifically, rather than RecQ helicases generally, holds back this recombination-mediated genomic instability. An ataxia-telangiectasia defective cell line also shows elevated rDNA GCI, although not to the extent of BLM defective cells. Genomic restructuring mediated by dysregulated recombination between the abundant low-copy repeats in the human genome may prove to be an important additional mechanism of genomic instability driving the initiation and progression of human cancer.

Human rRNA Gene Clusters Are Recombinational Hotspots in Cancer

“…Where possible, we compare the rDNA cluster lengths found in surgically excised nearby nontumor tissue to the gene cluster lengths found in the tumor itself, and to gene cluster lengths from the patients' peripheral blood as an additional control (lanes N, T, and B, respectively). Our prior experiences with both human blood and cell lines indicated that analysis of gene cluster lengths from 50 kb to ∼1 Mb is the most informative range for detecting recombination-mediated alterations (3,5), so this is the methodology we followed in this present work. Gene clusters larger than 1 Mb all run together unresolved at the top of the gel in the region (indicated by the ★) under these electrophoretic conditions, otherwise all unique gene clusters shorter than 1 Mb are resolved as individual bands.…”

“…On the other hand, if a tumor has lost control over recombination, the configuration of gene cluster lengths in the tumor will be different, relative to nontumor cells (5). If the rDNA cluster length restructuring occurred before clonal expansion of the prototumor cell, for example in the lungs of a heavy smoker, the altered length gene clusters will be present in the entirety of the subsequent tumor.…”

Neurodegeneration-associated instability of ribosomal DNA.

“…Moreover, this restructuring was also seen in fibroblasts from Ataxia Telangiectasia and Bloom syndromes, which are known to be caused by mutations in a DNA double strand break repair protein ATM and the BLM Rec Q helicase mentioned above [26,27]. Surprisingly, mutations in the Rec Q helicase WRN, which is implicated in Werner syndrome, did not alter the length of ribosomal DNA clusters [27]. Furthermore, as mentioned previously, the methylation status of the ribosomal DNA promoter and subsequent heterochromatin formation was directly linked to ribosomal DNA stability in a mammalian cell line [38].…”

“…For example, in human cancers approximately 50% of lung and colorectal solid tumors demonstrate ribosomal DNA restructuring [26]. Moreover, this restructuring was also seen in fibroblasts from Ataxia Telangiectasia and Bloom syndromes, which are known to be caused by mutations in a DNA double strand break repair protein ATM and the BLM Rec Q helicase mentioned above [26,27]. Surprisingly, mutations in the Rec Q helicase WRN, which is implicated in Werner syndrome, did not alter the length of ribosomal DNA clusters [27].…”

“…The tandem repeat arrangement of ribosomal DNA is well known for its relative instability compared to other regions of the genome [25][26][27]. The combination of this arrangement and the close physical proximity of all ten arrays inside the nucleolus potentially make them hot spots for homologous recombination.…”