Postnatal growth failure, short life span, and early onset of cellular senescence and subsequent immortalization in mice lacking the xeroderma pigmentosum group G gene

Abstract

The xeroderma pigmentosum group G (XP-G) gene (XPG) encodes a structure-specific DNA endonuclease that functions in nucleotide excision repair (NER). XP-G patients show various symptoms, ranging from mild cutaneous abnormalities to severe dermatological impairments. In some cases, patients exhibit growth failure and life-shortening and neurological dysfunctions, which are characteristics of Cockayne syndrome (CS). The known XPG protein function as the 3' nuclease in NER, however, cannot explain the development of CS in certain XP-G patients. To gain an insight into the functions of the XPG protein, we have generated and examined mice lacking xpg (the mouse counterpart of the human XPG gene) alleles. The xpg-deficient mice exhibited postnatal growth failure and underwent premature death. Since XPA-deficient mice, which are totally defective in NER, do not show such symptoms, our data indicate that XPG performs an additional function(s) besides its role in NER. Our in vitro studies showed that primary embryonic fibroblasts isolated from the xpg-deficient mice underwent premature senescence and exhibited the early onset of immortalization and accumulation of p53.

FIG. 1

Gene targeting at the xpg locus. (A) Schematic representation of the insertional mutation at the mouse xpg locus. Two exons of the xpg gene, exons 3 and 4, are represented as black boxes. PCR primers are shown as arrows. The 3′ external probe used for Southern blot analysis is shown as a solid bar corresponding to the S-E fragment on the wild-type map on top of panel A, and the diagnostic fragments of 22.0 and 25.5 kb are shown as solid lines on the bottom of panel A. B, BamHI; X, XhoI; Bg, BglII; S, SphI; E, EcoRI. (B) PCR and Southern blot analyses of the targeted clone GG5. The predicted PCR products were PCR1 (with the neoS1 and TV2R4 primers) and PCR2 (with the TV2FD and TV2R4 primers) (shown in panel A). Southern blot analysis using the 3′ external probe also detected the predicted restriction fragments shown in panel A. M, size markers; ES, ES cells as a control. (C) PCR and Southern blot analyses of offspring from intercrosses between the chimeric males and C57BL/6J females. +/+, wild type; +/−, heterozygote; −/−, homozygous mutants. (D) Northern blot analysis of total RNA from newborn mice derived from a heterozygous intercross, using xpg cDNA as a probe. A β-actin cDNA probe was used as an internal control for estimation of total mRNA in each sample.

FIG. 2

Growth characteristics and life span of the xpg mutant mice. (A) Survival curve of mutant mice postpartum. (B) Average body weights of the xpg mutant mice (solid circles). The body weights of males and females from the normal group (i.e., wild-type and heterozygous mice) were combined (open circles). (C) Gross phenotypic appearance of an xpg mutant (−/−) (right), a heterozygous (+/−) littermate (middle), and a wild-type (+/+) littermate (left) at 16 days postpartum.

FIG. 3

Survival curves, removal kinetics of UV-induced DNA damage, and genetic instability tests for embryonic fibroblasts derived from xpg-deficient mice. UV survival curves (A), X-ray survival curves (B), and H₂O₂ survival curves (C) for cells derived from xpg-deficient mice are shown. Each point is an average of triplicate wells. (D and E) Removal kinetics of 6-4 photoproducts (D) and cyclobutane pyrimidine dimers (E). Ab, antibody. Each point represents an average of triplicate wells. (F) Growth properties of embryonic fibroblasts. Five independent experiments were carried out, and one of experimental data is shown in this figure. The timings of crisis and immortalization were somewhat different among experiments, but their tendencies were highly reproducible. (G) Accumulation of p53 in embryonic cells. All of the experiments were carried out with the embryonic fibroblasts from the wild type (open circles) and heterozygous (open squares) mice and from two homozygous xpg mutant mice (solid symbols) that originated from one litter at a low passage number (2 to 3).

FIG. 4

Histological and anatomical analyses of the xpg-deficient mice. Cross-sections from the small intestines of wild-type mice and mutant homozygotes were stained with hematoxylin and eosin at 0, 5, and 16 days postpartum. (A) Wild type (+/+) at day 0. (B) Wild type (+/+) at 5 days. (C) Wild type (+/+) at 16 days. (D) Mutant homozygote (−/−) at day 0. (E) Mutant homozygote (−/−) at 5 days. (F) Mutant homozygote (−/−) at 16 days. (A to F) Magnification, ×25. (G) Appearance of the stomach and intestines in a heterozygote (+/−) and a homozygote (−/−) at 21 days postpartum. Yellow arrows point to the stomachs. (H) Appearance of spleens from a heterozygote (+/−) and a homozygote (−/−) at 21 days postpartum. The spleens were very small in the −/− mice. (I) Sections from the livers of a heterozygote (+/−) and a homozygote (−/−) at 16 days postpartum stained with hematoxylin and eosin (magnification, ×150).