Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45

Abstract

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric nuclease complex critical for the induction of DNA fragmentation in vitro. To understand the in vivo role of DFF45 in programmed cell death, we generated DFF45 mutant mice. DNA fragmentation activity is completely abolished in cell extracts from DFF45 mutant tissues. In response to apoptotic stimuli, splenocytes, thymocytes, and granulocytes from DFF45 mutant mice are resistant to DNA fragmentation, and splenocytes and thymocytes are also resistant to chromatin condensation. Nevertheless, development of the immune system in the DFF45 mutant mice is normal. These results demonstrate that DFF45 is critical for the induction of DNA fragmentation and chromatin condensation in vivo, but is not required for normal immune system development.

Figure 1

Generation of DFF45 mutant mice. (A) Genomic DNA locus surrounding the first three exons (black boxes) of the DFF45 gene, the targeting vector, the mutant DFF45 gene locus, and the 5′ and 3′ hybridization probes. The exon probe contains 90, 170, and 40 bp from the first, second, and third exons of the DFF45 gene, respectively. (B and C) Identification of DFF45 mutant mice. Tail DNA samples from one litter of heterozygous intercross were digested with BglII, hybridized with a 5′ probe (B) or the exon probe (C). (D) Western blot analyses using spleen, thymus, heart, and liver extracts from wild-type (+/+) and DFF45 mutant mice (−/−) for the expression of DFF45 and caspase-3 (n = 4 mice each). Blots were probed with polyclonal antibodies against DFF45 or caspase-3, respectively.

Figure 2

Lack of DFF40 nuclease activity in DFF45 mutant mice. Cell extracts from spleen, thymus, and testes from both DFF45 mutant (−/−) and control (+/+) mice (n = 3 each) were isolated. DFF40 nuclease activity in these extracts was assayed by using hamster liver nuclei in the presence or absence of activated caspase-3. Genomic DNA samples were extracted, separated on 1.5% agarose gels, and visualized by ethidium bromide staining.

Figure 3

Resistance to DNA fragmentation in cells from DFF45 mutant mice. Genomic DNA samples were isolated from splenocytes (A) and thymocytes (B) from both mutant (−/−) and control (+/+) mice with or without actinomycin D, etoposide, and staurosporine treatment (n = 5 mice each), and from granulocytes with or without cycloheximide treatment (C, n = 3 mice each). All DNA samples were extracted, separated on 1.5% agarose gels, and visualized by ethidium bromide staining. N, no treatment; A, actinomycin D; E, etoposide; S, staurosporine; C, cycloheximide.

Figure 4

Resistance to chromatin condensation in cells from DFF45 mutant mice. Splenocytes and thymocytes from both DFF45 mutant (−/−) and wild-type control (+/+) mice (n = 3 each) were cultured in the absence or presence of actinomycin D, etoposide, and staurosporine. DAPI stainings of these cells were visualized under a fluorescence microscope, and 200 cells for each treatment condition were observed (A). Percentage of cells exhibiting chromatin condensation was counted and plotted (B). N, no treatment; A, actinomycin D; E, etoposide; S, staurosporine.

Figure 5

Normal immune system development in DFF45 mutant mice. Cells were harvested from lymphoid organs from wild-type (+/+) and DFF45 mutant (−/−) mice and analyzed for the expression of the indicated cell surface markers. Each plot shown is representative of results from three DFF45 mutant and three control mice, except for Peyer’s Patch, which is representative of results from two DFF45 mutant and two control mice. Numbers indicate the percentage of cells in each quadrant.