TREX2 exonuclease defective cells exhibit double-strand breaks and chromosomal fragments but not Robertsonian translocations

Dumitrache, Lavinia C.; Hu, Lingchuan; Hasty, Paul

doi:10.1016/j.mrfmmm.2008.11.012

Cited by 15 publications

(26 citation statements)

References 28 publications

(41 reference statements)

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“…Otherwise, TREX2 seems to be important for chromosomal stability. As such, TREX2 deletion in embryonic stem (ES) causes high levels of Robertsonian Translocations (27), but this effect is independent of its exonuclease activity and DNA binding domains (28). The biochemical activities and the mechanisms by which TREX2 prevents chromosomal instability and the consequences of TREX2 deficiency in the organism remain unknown.…”

Section: Introductionmentioning

confidence: 99%

Increased Susceptibility to Skin Carcinogenesis in TREX2 Knockout Mice

et al. 2009

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TREX2 is a proofreading 3 ¶-5 ¶ exonuclease that can be involved in genome maintenance; however, its biological role remains undefined. To better understand the function and physiologic relevance of TREX2, we generated mice deficient in TREX2 by targeted disruption of its unique coding exon. The knockout mice are viable and do not show relevant differences in growth, survival, lymphocyte development, or spontaneous tumor incidence compared with their wild-type counterparts over a period of up to 2 years. Also, we did not observe chromosomal instability or defects in cell proliferation and cell cycle upon loss of TREX2. We have observed that TREX2 expression is not ubiquitous, being expressed preferentially in tissues with stratified squamous epithelia, such as the skin or esophagus, and specifically in keratinocytes. Interestingly, TREX2-null mice are more susceptible to skin carcinogenesis induced by 7,12-dimethylbenz(a)anthracene (DMBA) compared with wild-type mice. This phenotype correlates with a reduction of DMBA-induced apoptosis in both the epidermis and keratinocytes of TREX2-null mice. Altogether, our results suggest a tumor suppressor role for TREX2 in skin carcinogenesis through which it contributes to keratinocyte apoptosis under conditions of genotoxic stress. [Cancer Res 2009;69(16):6676-84]

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Section: Introductionmentioning

confidence: 99%

Increased Susceptibility to Skin Carcinogenesis in TREX2 Knockout Mice

et al. 2009

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“…These breaks were almost exclusively in the pericentromere. In addition, the Trex2 H188A cells showed higher levels of spontaneous broken chromosomes than the trex2 null cells, suggesting a dominant negative-like effect (Dumitrache et al 2009). We looked at the level of broken chromosomes in cells after 5 hr exposure to camptothecin at 50 nM, 100 nM, or 200 nM followed by 4 hr exposure to colcemid.…”

Section: Trex2-deleted Cells Are Not Hypersensitive To Camptothecinmentioning

confidence: 99%

“…We made a null by deleting a single exon that contains all coding sequence (trex2 null and trex2 null-2 ) (Chen et al 2007a;Dumitrache et al 2009). In addition, by knock-in we introduced the wild-type human short isoform of TREX2 (Trex2 hTX2 ) and two variants with a single-amino-acid change adjacent to the Trex2 promoter (Chen et al 2007b;Dumitrache et al 2009). One variant is mutated in the catalytic domain (Trex2 H188A ) while the other variant is mutated in the DNA-binding domain (Trex2 R167A ).…”

Section: Substrains and Gene Targetingmentioning

confidence: 99%

“…Accordingly, the main insight from these experiments indicates that Trex2 deficiency does not affect the relative frequency of diverse DSB repair outcomes (HR vs. NHEJ and the types of NHEJ products). To explain the increase in DSB repair, we suggest DSB repair is elevated in trex2 null cells as a general compensatory response to increased spontaneous chromosomal breaks (Dumitrache et al 2009). In any case, these data do not support a role for endogenous Trex2 in promoting DSB repair.…”

Section: Trex2-deleted Cells Were Not Defective For Repairing I-scei-mentioning

confidence: 99%

“…We introduced into these trex2 null cells wild-type human TREX2 cDNA or human TREX2 cDNA with a single-amino-acid change in the catalytic domain (Trex2 H188A ) or DNA-binding domain (Trex2 R167A ); these cells were made by knock-in such that the human cDNA is expressed by the mouse Trex2 promoter in situ (Dumitrache et al 2009). The trex2 null and Trex2 H188A cells, but not the Trex2 hTX2 or Trex2 R167A cells, exhibited high levels of spontaneous broken chromosomes.…”

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Trex2 Enables Spontaneous Sister Chromatid Exchanges Without Facilitating DNA Double-Strand Break Repair

Dumitrache

Son

et al. 2011

Genetics

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Trex2 is a 39 / 59 exonuclease that removes 39-mismatched sequences in a biochemical assay; however, its biological function remains unclear. To address biology we previously generated trex2 null mouse embryonic stem (ES) cells and expressed in these cells wild-type human TREX2 cDNA (Trex2 hTX2 ) or cDNA with a single-amino-acid change in the catalytic domain (Trex2 H188A ) or in the DNA-binding domain (Trex2 R167A ). We found the trex2 null and Trex2 H188A cells exhibited spontaneous broken chromosomes and trex2 null cells exhibited spontaneous chromosomal rearrangements. We also found ectopically expressed human TREX2 was active at the 39 ends of I-SceI-induced chromosomal double-strand breaks (DSBs). Therefore, we hypothesized Trex2 participates in DNA DSB repair by modifying 39 ends. This may be especially important for ends with damaged nucleotides. Here we present data that are unexpected and prompt a new model. We found Trex2-altered cells (null, H188A, and R167A) were not hypersensitive to camptothecin, a type-1 topoisomerase inhibitor that induces DSBs at replication forks. In addition, Trex2-altered cells were not hypersensitive to g-radiation, an agent that causes DSBs throughout the cell cycle. This observation held true even in cells compromised for one of the two major DSB repair pathways: homology-directed repair (HDR) or nonhomologous end joining (NHEJ). Trex2 deletion also enhanced repair of an I-SceI-induced DSB by both HDR and NHEJ without affecting pathway choice. Interestingly, however, trex2 null cells exhibited reduced spontaneous sister chromatid exchanges (SCEs) but this was not due to a defect in HDR-mediated crossing over. Therefore, reduced spontaneous SCE could be a manifestation of the same defect that caused spontaneous broken chromosomes and spontaneous chromosomal rearrangements. These unexpected data suggest Trex2 does not enable DSB repair and prompt a new model that posits Trex2 suppresses the formation of broken chromosomes.

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One‐step knockin for inducible expression in mouse embryonic stem cells

Choi

Son

Hasty

2011

Genesis

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Transgenesis enables the elucidation of gene function; however, constant transgene expression is not always desired. The tetracycline responsive system was devised to turn on and off transgene expression at will. It has two components: a doxycycline controlled transactivator (TA) and an inducible expression cassette. Integration of these transgenes requires two transfection steps usually accomplished by sequential random integration. Unfortunately, random integration can be problematic due to chromatin position effects, integration of variable transgene units and mutation at the integration site. Therefore, targeted transgenesis and knockin were developed to target the TA and the inducible expression cassette to a specific location, but these approaches can be costly in time, labor and money. Here we describe a one-step Cre-mediated knockin system in mouse embryonic stem (ES) cells that positions the TA and inducible expression cassette to a single location. Using this system, we show doxycycline-dependent regulation of eGFP at the DNA topoisomerase 3β (Top3β) promoter. Since Cre-mediated recombination is used in lieu of gene targeting, this system is fast and efficient. Keywords knockin; gene targeting; inducible expression, Cre/loxP Constant transgene expression is not always desirable; therefore, the tetracycline responsive system was developed to regulate transgene expression. It is based on the Tn10-specific tetracycline-resistance operon found in Escherichia coli (Hillen and Berens, 1994) and has two basic components. One component is a tetracycline-controlled transactivator (TA) (Gossen et al., 1995) and the other component is a TA-inducible expression cassette with a nonfunctional promoter. This promoter contains a series of eight tet operator (tetO) sequences immediately upstream of a minimal CMV promoter, called tetO-mCMV. Transcription is induced after the TA binds to the tetO sequences. The investigator controls transcription with doxycycline (dox), a tetracycline derivative that binds the TA to regulate activity. Thus, the investigator controls transgene expression with dox.Both the TA and the inducible expression cassette must be integrated into cells by sequential rounds of random integration, targeted transgenesis or knockin. Random transgene integration can be problematic due to variable transgene copy number (Sandgren et al., 1991), epigenetic promoter silencing (al-Shawi et al., 1990), and mutagenesis (Covarrubias et al., 1986). To avoid these problems, targeted transgenesis (Jasin et al., 1996;Vivian et al., 1999) and knockin (Hanks et al., 1995;Shin et al., 1999) were developed. For targeted

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TREX2 exonuclease defective cells exhibit double-strand breaks and chromosomal fragments but not Robertsonian translocations

Cited by 15 publications

References 28 publications

Increased Susceptibility to Skin Carcinogenesis in TREX2 Knockout Mice

Increased Susceptibility to Skin Carcinogenesis in TREX2 Knockout Mice

Trex2 Enables Spontaneous Sister Chromatid Exchanges Without Facilitating DNA Double-Strand Break Repair

One‐step knockin for inducible expression in mouse embryonic stem cells

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