Enhancement of microhomology-mediated genomic rearrangements by transient loss of mouse Bloom syndrome helicase

Abstract

Bloom syndrome, an autosomal recessive disorder of the BLM gene, confers predisposition to a broad spectrum of earlyonset cancers in multiple tissue types. Loss of genomic integrity is a primary hallmark of such human malignancies, but many studies using disease-affected specimens are limited in that they are retrospective and devoid of an appropriate experimental control. To overcome this, we devised an experimental system to recapitulate the early molecular events in genetically engineered mouse embryonic stem cells, in which cells undergoing loss of heterozygosity (LOH) can be enriched after inducible down-regulation of Blm expression, with or without site-directed DNA double-strand break (DSB) induction. Transient loss of BLM increased the rate of LOH, whose breakpoints were distributed along the chromosome. Combined with site-directed DSB induction, loss of BLM synergistically increased the rate of LOH and concentrated the breakpoints around the targeted chromosomal region. We characterized the LOH events using specifically tailored genomic tools, such as high-resolution array comparative genomic hybridization and high-density single nucleotide polymorphism genotyping, revealing that the combination of BLM suppression and DSB induction enhanced genomic rearrangements, including deletions and insertions, whose breakpoints were clustered in genomic inverted repeats and associated with junctional microhomologies. Our experimental approach successfully uncovered the detailed molecular mechanisms of as-yet-uncharacterized loss of heterozygosities and reveals the significant contribution of microhomologymediated genomic rearrangements, which could be widely applicable to the early steps of cancer formation in general.

Mutator Phenotype and DNA Double-Strand Break Repair in BLM Helicase-Deficient Human Cells

“…Yamanishi et al (28) developed an experimental system using mouse embryonic stem (ES) cells to detect LOH mutations in the adenine phosphoribosyltransferase gene. In this system, BLM expression and DSBs caused by I-SceI can be controlled, and the authors demonstrated that BLM-suppressed ES cells predominantly exhibited deletions after DSB repair.…”

“…The increase of SCEs in BS cells is thought to be due to a deficiency in BLM helicase during the resolution of HJs, thus underlining the importance of this enzyme. Indeed, a deficiency of BLM helicase is known to increase the LOH (26–28). It is possible to generate LOH via multiple mechanisms, and pro- and anti-HR functions of BLM helicase have been proposed (29, 30).…”

The Transient Inactivation of the Master Cell Cycle Phosphatase Cdc14 Causes Genomic Instability in Diploid Cells of Saccharomyces cerevisiae

“…The inactivation of caretaker genes is known to increase genetic instability and to play an important role in tumor development (Kinzler and Vogelstein 1997;Vogelstein and Kinzler 2004;van Heemst et al 2007;Yamanishi et al 2013). In this work, we have focused on the consequences for the genetic stability of S. cerevisiae of a transient temperature shift in diploid cells carrying the thermosensitive allele cdc14-1; CDC14 is an essential gene that encodes for the master cell cycle phosphatase required for exiting from mitosis.…”

“…Although caretakers are not directly responsible for the decision of a cell to divide, their loss leads cells to rapidly accumulate genomic aberrations and mutations that potentially result in disorganization of cell division and their subsequent transformation into cancer cells (Kinzler and Vogelstein 1997;van Heemst et al 2007). For example, mutations in genes involved in DNA repair pathways such as nonhomologous end joining, homologous recombination, mismatch repair, base excision repair, and nucleotide excision repair have been shown to lead to the accumulation of mutations within the genome that significantly increase the risk of carcinogenesis (Negrini et al 2010; Aguilera and García-Muse 2013 protein (BLM) activity leads to a significant increase in LOH events throughout the genome in mouse cell lines, pointing out a role of the BLM dysfunction in the early steps of tumorigenesis (Yamanishi et al 2013). In addition, it has been also shown that the transient inactivation of the BRCA2-and CDKN1A(p21)-interacting protein BCCIP is sufficient to promote tumorigenesis.…”

“…One study, however, reported that the transient loss of the helicase Bloom syndrome protein (BLM) activity leads to a significant increase in LOH events throughout the genome in mouse cell lines, pointing out a role of the BLM dysfunction in the early steps of tumorigenesis (Yamanishi et al 2013). In addition, it has been also shown that the transient inactivation of the BRCA2-and CDKN1A(p21)-interacting protein BCCIP is sufficient to promote tumorigenesis.…”