sir2α gene structure and targeted disruption.
Although there are seven genes in the human (16
) and mouse genomes that share a region homologous to the catalytic domain of the yeast SIR2
gene, one of these homologues, sir2
α, encodes a protein with a higher degree of sequence identity (40%) to the yeast Sir2p than the other six. The full-length cDNA sequence of sir2
α has been published (18
). We amplified a partial sir2
α cDNA encoding the C terminus of the SIR2α protein from reverse-transcribed mRNA isolated from the R1 line of ES cells (36
). This sir2
α cDNA was used to screen a bacteriophage genomic library derived from the DNA from strain 129/Sv mice (a gift from Douglas Gray). Two overlapping clones were isolated, subcloned into plasmid vectors, restriction mapped, and partially sequenced. The cloned genomic region consisted of the last seven exons of the sir2
α gene (Fig. ), a gene structure that is the same as that of its human homologue (accession number AL133551
We constructed two knockout vectors designed to delete exons 5 and 6 from the sir2
α gene. These two exons encode a region of the SIR2α protein that comprises a large portion of the catalytic domain, so we predicted that the result of homologous recombination would be a null sir2
α allele. Because the sir2
α gene is expressed in ES cells (McBurney et al., unpublished data), we created a knockout vector in which the selectable gene encoding hygromycin resistance would be driven by the sir2
α promoter following homologous integration into the sir2
α locus (35
) (see Fig. ). This vector was linearized and electroporated into R1 cells. Treated cells were selected in hygromycin, and drug-resistant colonies were expanded and screened by Southern blot hybridization with probes that identify both the 5′ and 3′ sides of the homologous recombination event.
A second knockout vector was created with the selectable gene encoding G418 resistance (35
) inserted into the same sir2
α targeting arms, and this linearized vector was electroporated into one of the R1 clones carrying the correctly targeted hygromycin resistance vector. Cells resistant to G418 were selected and expanded for analysis. Among the G418-resistant clones were those in which both wild-type alleles of sir2
α were replaced with the two knockout vectors (Fig. ). These sir2
α null cells grew well in culture and have been maintained for more than a year, indicating that the SIR2α protein is not essential for cell growth, viability, or immortality.
FIG. 2. Isolation of ES clones deleted for both sir2α genes. R1 ES cells (lane 1) that had homologously integrated the hygromycin resistance vector (lane 2) were electroporated with a second knockout construct carrying the lacZ-neomycin resistance (β-geo) (more ...)
We expected that the knockout alleles would be unable to produce SIR2α protein. To confirm this, we created a polyclonal rabbit antibody that recognizes the murine SIR2α protein (McBurney et al., unpublished data) and used this antibody to probe protein blots from the ES knockout cells (Fig. ). ES cells carrying one knockout allele had approximately half the SIR2α protein present in the diploid wild-type cells, and the double knockout had no detectable SIR2α protein.
FIG. 3. SIR2α protein is absent from sir2α−/− ES cells. Protein was isolated from the cells indicated and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred to membranes, (more ...)
The cells from the hygromycin-resistant clone isolated from the first electroporation experiment were aggregated with 2.5-day embryos derived from the CD1 mouse strain, and the mosaic blastocysts were transferred to the uteri of pseudopregnant foster mothers. Several of the male chimeras born from these manipulated embryos had their germ lines derived exclusively from the ES cells, and these were used to introduce the sir2α null allele into both the outbred CD1 line and the inbred 129/Sv strain. Animals carrying the sir2α null allele were identified by Southern blot hybridization, and these heterozygous animals were fertile and apparently normal.
sir2α null genotype is postnatal lethal.
Males and females heterozygous for the sir2α null allele were mated, and offspring were genotyped at weaning by Southern blotting (Fig. ). Animals carrying two sir2α null alleles were present among the viable offspring, but their proportion was about half what was expected, suggesting that there was prenatal or early postnatal loss of approximately half of the sir2α null offspring.
FIG. 4. Viable sir2α null offspring arise from crosses between heterozygous animals. Males and females that were genotyped as heterozygous for the mutated sir2α alleles were mated, and the DNA from their offspring was used for Southern blots to (more ...)
To investigate the possible loss of sir2α null embryos during gestation, we mated heterozygous animals and harvested mothers at various times after the appearance of the copulation plug. Each embryo was genotyped from DNA isolated from the fetal membranes, and each embryo was fixed for examination. sir2α null embryos were present at early and late stages of gestation in roughly the expected proportions, but some of these sir2α null embryos were abnormal (Fig. ) and would not have survived following birth. Thus, the reduced number of sir2 null animals at birth probably reflects the immediate postnatal loss of abnormal fetuses.
FIG. 5. sir2α null animals are not lost during fetal development but are smaller and occasionally have exencephaly. Animals heterozygous for the mutant sir2α allele were mated, and fetuses were harvested at daily intervals after embryonic day (more ...)
Most of the sir2α null embryos looked normal but were smaller than their wild-type or heterozygous littermates (Fig. ). Examination of some of the sir2α null fetuses indicated that they were retarded in certain developmental processes. For example, the mineralization of the digits in the sir2 null animals was delayed relative to their wild-type littermates (K. Reuhl et al., unpublished observations).
Following birth, the sir2α null mice were easily identified, as they were smaller than their littermates and their subsequent development was slower than that of their littermates. The most obvious developmental delay was eyelid opening. In the sir2α null animals, eyelids remained closed forever or for several months after birth.
The postnatal development of the sir2α null animals was dependent on their genetic background. On the inbred 129/Sv genetic background, the sir2α null animals invariably died before reaching 1 month of age. On the outbred genetic background from a mix of the CD1 and 129/Sv strains, the sir2α null mice were much more likely to thrive, although their stature was smaller than that of their littermates (Fig. ) and they all had the characteristic eyelid defect. Many of these sir2α null animals had a short or deviated snout as well but otherwise appeared normal. During the early postnatal period, these sir2α null animals were inevitably smaller than their littermates, but most thrived and eventually approached the size of the wild-type animals (Fig. ).
FIG. 6. Postnatal sir2α null animals are developmentally abnormal. sir2α null animals on the outbred background often survived past weaning but were characterized by a number of common developmental defects. Null animals were smaller than their (more ...)
FIG. 7. Some sir2α null animals thrive on the outbred genetic background. sir2α null animals (•) and their wild-type littermates (○) were weighed at weekly intervals. During the early postnatal development period, sir2α (more ...)
Examination of the internal tissues of the sir2 null animals indicated that many organs were smaller than those of wild-type animals, but the histology of most appeared normal. In some animals, the eyes, although normal in size, had abnormalities of the cornea, lens, and retina, but these may have been a secondary consequence of the fact that the eyelids of sir2α null mice do not open in a timely manner. Two organs consistently affected in the mutants were the lung and pancreas. Neutrophil infiltration of the lungs suggested chronic pulmonary infection that led to pneumonitis, pulmonary edema, and right ventricular hypertrophy. The pancreas showed patchy atrophy of the exocrine epithelium. These lung infections and pancreatic defects are probably responsible for the early postnatal lethality of the sir2α null animals. An examination of the lymphoid system in sir2α null animals indicated that the thymus contained normal numbers and proportions of T cells and that the spleen complement of B cells was normal. The only consistent abnormality appeared to be a reduced proportion of CD8-positive T cells in the spleens of sir2α null animals.
sir2α null animals do not reexpress a silenced transgene or prematurely erode their telomeres.
The yeast Sir2p is essential for maintaining the transcriptional inactivity of genes in this organism. The fact that the sir2α null mice develop relatively normally and that a very large proportion of the genes in a mammalian tissue remain silent suggest that the mammalian SIR2α may not be required to maintain gene inactivity in the mammalian genome. We probed Northern blots of RNA isolated from sir2α null and wild-type day-12.5 embryos and adult tissues. These blots yielded no evidence for ectopic expression of any of the endogenous genes that we assayed (IGF-II, IGF-IIR, endogenous retrovirus, gtl-2, SNRPN, Peg-3, H19, Lo-1, Zfp127, necdin, UBE3, and β2-microglobulin). Microarray analysis of RNA isolated from day-12.5 embryos also indicated that the vast majority of genes are normally expressed in the sir2α null animals.
Nevertheless, we set out to directly test whether the inactivation of a silent transgene could be modulated in sir2
α null animals. For this experiment, we used a previously described transgenic strain of mice that carry eight copies of a transgene consisting of the regulatory sequences of the mouse Pgk
gene driving the Escherichia coli lacZ
reporter sequence (32
). This Pgk-1,2-lacZ
transgene is expressed in all tissues investigated and encodes a fusion protein with β-galactosidase activity.
transgene remains active provided it is inherited through the male germ line; however, if this transgene is inherited through the female germ line, its expression is irreversibly abolished (26
). We interbred female mice carrying the sir2
α null allele with male animals carrying the active Pgk-1,2-lacZ
transgene. Animals heterozygous for the sir2
α null allele were mated with each other so that the Pgk-1,2-lacZ
gene was inherited from either the male or the female parent. Embryos were harvested at 9.5 days of gestation, each embryo was fixed and stained for β-galactosidase expression, and the fetal membranes of each embryo were used to isolate DNA, from which the genotype of each embryo was determined.
Embryos who inherited their Pgk-1,2-lacZ transgene from the male parent expressed β-galactosidase as determined by the 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) staining of these embryos (Fig. ). Embryos that inherited their Pgk-1,2-lacZ transgene from the female parent are expected to inherit this transgene in an inactive form. It is evident that the embryos shown in Fig. , numbers 1, 8, and 9, have inherited the Pgk-1,2-lacZ transgene along with at least one wild-type copy of sir2α. As expected, these embryos failed to express the transgene, as indicated by the absence of X-Gal staining. The embryos labeled 4 and 7 also inherited the Pgk-1,2-lacZ transgene, but these two embryos were also sir2α null. Because neither embryo 4 nor 7 was stained with X-Gal, we conclude that the absence of SIR2α did not result in compromised repression of the silent Pgk-1,2-lacZ transgene.
FIG. 8. sir2α null embryos do not reactivate expression of silent transgenes. Female mice carrying the sir2α null mutation were intercrossed with male mice carrying the Pgk-1,2-lacZ transgene, and animals heterozygous for the sir2α null (more ...)
Some embryos from these matings were allowed to develop to term, and the pups were allowed to mature. The internal tissues from these animals were analyzed for β-galactosidase activity. The results were similar to those seen in the embryos; sir2α null animals that had inherited the Pgk-1,2-lacZ transgene from their mothers did not express it, while the sir2α null animals that inherited the Pgk-1,2-lacZ transgene from their fathers did show staining with X-Gal in their internal tissues. Thus, the sir2α null animals did not express the silenced transgenes or modulate the expression of active transgenes.
The yeast Sir2p also plays a role in determining the rate at which yeast mother cells undergo senescence (20
). Work with Caenorhabditis elegans
also suggests that the sir2
homologue in worms may also play a similar role in regulating aging (46
). Because many of the sir2
α null animals died before weaning, one possibility is that they are aging prematurely. To investigate this possibility, we compared the lengths of the telomeres in both B and T lymphocytes from wild-type and sir2
α null littermates with animals of 4 months of age on the outbred CD1 background. The mean telomere lengths in these cells were identical (Fig. ). In addition, the chromosomes from the cells appeared normal, consistent with the absence of telomere erosion that might be predicted to occur if the sir2
α null animals underwent premature aging. Our oldest sir2
α null animals are now 17 months old.
FIG. 9. Telomeres do not erode prematurely in sir2α null mice. Splenocytes from sir2α null (panels A and B) and wild-type (panels C and D) mice were stimulated with concanavalin A (panels A and C) or lipopolysaccharide (panels B and D) to activate (more ...) sir2α expression is widespread, but the protein is associated with euchromatin.
To attempt to understand the phenotype of the sir2α null mice, we investigated the normal expression pattern of the sir2α gene by probing Northern blots of RNA isolated from various tissues. The sir2α transcript was readily detected in all tissues investigated (Fig. ) and was particularly evident in the testis and ovary.
FIG. 10. SIR2α expression is widespread throughout adult tissues. RNA was isolated from a variety of tissues from wild-type mice and subjected to electrophoresis, blotting, and hybridization to cDNA probes specific for sir2α (upper panel) and glyceraldehyde-3-phosphate (more ...)
The polyclonal rabbit antibody that recognizes the murine SIR2α protein (McBurney et al., unpublished data) was used for immunoblots of protein isolated from various murine tissues and to carry out immunofluorescence studies on these tissues. Despite the prevalence of the sir2α transcript in adult mouse tissues, we were able to detect protein only in the testis and ovary (see below).
We had noted previously that the SIR2α protein is expressed in abundance in embryonal carcinoma and embryonic stem cells (McBurney et al., unpublished data), so we carried out immunofluorescence experiments in early embryonic cells. The SIR2α protein was present and easily detected in zygotes, two-cell embryos (Fig. ), and cells of the blastocyst. At all stages, the distribution of the SIR2α protein was nuclear, but it was not concentrated in those regions of the nucleus thought to contain heterochromatin and identified by intense staining with DNA intercalating dyes such as Hoechst 33258. SIR2α also appeared to be excluded from the nucleolus. There was no staining with this antibody in cells from embryos that were sir2α null (data not shown), consistent with the prediction that no stable protein could be made from the knockout allele.
FIG. 11. SIR2α protein is expressed at high level in two-cell embryos. Wild-type two-cell embryos were recovered from the oviducts 24 h following mating. The zona pellucida was removed, and the embryos were fixed and stained with antibody to SIR2α (more ...)
In the ovary, the SIR2α protein was evident in large oocytes as well as in proliferating granulosa cells in larger follicles (Fig. ). Within both cell types, the SIR2 protein was nuclear and the intranuclear distribution was similar to that seen in early embryonic cells; the protein was nuclear but appeared to be associated with the euchromatin rather than the heterochromatin.
FIG. 12. SIR2α is present at high level in proliferating granulosa cells. Ovaries from wild-type mice were sectioned and stained with antibody to SIR2α (panel A) and with Hoechst 33258 (panel B). The merged image is shown in panel C. The box in (more ...)
In the testis, the SIR2α protein was present in the nuclei of spermatogenic cells only (Fig. ). In the developmental sequence of germ cells, a weak, finely speckled nuclear staining was observed in type A spermatogonia, while type B and intermediate spermatogonia displayed a slightly more intense nuclear staining with coarser positive spots. Spermatocytes and round spermatids were intensely positive with diffusely speckled nuclear staining. Soon after the spermatids began to elongate (step 9), the nuclear staining decreased to only a few spots and by step 11 was lost altogether. This staining pattern identifies the nuclear localization of the SIR2 protein in late spermatogonia, spermatocytes, and round spermatids, with the main expression in pachytene spermatocytes. There was no staining of Sertoli cells or of peritubular cells. Due to inherent autofluorescence, the staining of Leydig cells could not be assessed.
FIG. 13. SIR2α is expressed at high level during spermatogenesis. Frozen sections of testis from wild-type mice were stained with polyclonal antibody to SIR2α (panel A) and with Hoechst 33258 to stain DNA (panel B). In the two seminiferous tubules (more ...) sir2α knockout mice are sterile.
Although many sir2α null mice died before reaching maturity, some of those on the outbred background did survive, and these mature animals were caged with wild-type animals of the opposite sex. Both male and female sir2α null animals were sterile. Eleven sir2α null male animals were caged with wild-type females for up to 6 months (mean, 4 months), and none has sired a single litter despite evidence of copulation plugs in a number of the female mates. Seven sir2α null female animals were caged with wild-type males for up to 7 months (mean, 3.5 months). Only one of these females was fertile. She produced three litters but failed to suckle her offspring. However, some of these pups were fostered to another mother, with whom they were able to suckle and thrive and develop into fertile males and females.
The female reproductive tract in sir2α null animals was characterized by small ovaries in which the corpora lutea were conspicuously absent and the uterus was thin walled. Microscopic examination indicated that early, intermediate, and late stages of follicle development were present, but the absence of the corpus luteum indicated that ovulation did not occur. We examined the vaginal washes from two sir2α null females and two wild-type females maintained for 18 days in cages previously occupied by males. The wild-type females cycled through estrus every 4 to 6 days, but the sir2α null animals appeared to be arrested in diestrus. To determine if the absence of an estrous cycle was the cause of the infertility, we injected two sir2α null and two wild-type animals with hormones (5 IU of pregnant mare serum gonadotropin followed 46 h later with 5 IU of human choriogonadotropin) to induce ovulation. The next day, ovulated eggs were present in the oviducts of both sir2α null animals (16 and 19 eggs) as well as in both wild-type animals (29 and 35 eggs). This is consistent with the idea that female sterility in the sir2α null animals is due to hormonal inadequacy.
Analysis of spermatozoa from the cauda epididymis of wild-type and sir2α null mice showed striking differences in number, appearance, and motility. Wild-type mice had sperm counts of 48.8 × 106 ± 12.4 × 106, whereas the sir2α null animals had dramatically reduced numbers of mature sperm, with average counts of 2.6 × 106 ± 3.6 × 106 (data from three animals of each genotype). Virtually none of the sperm from the sir2α null mice were motile. Wild-type spermatozoa cell bodies had a consistent shape with a characteristic hook (Fig. ). In contrast, the majority of the sperm of the sir2α null mice were abnormal, with small, rounded, or smudged cell bodies and blunted or absent hooks (Fig. ). In addition, many of the sperm from sir2α null mice had retained cytoplasm around the nucleus, and many of the flagella appeared very fine and had no cell body attached.
FIG. 14. sir2α null mice have abnormal sperm morphology. Spermatozoa from the cauda epididymis were spread on a slide and stained with Dif-Quick stain. (a) Wild-type spermatozoa have a consistent nuclear shape with a characteristic hook. In contrast, the (more ...)
The male reproductive tract of sir2 null animals was characterized by organs significantly smaller than those of the wild-type littermates. All testes from sir2α null mice were subnormal in weight (wild type, 0.087 ± 0.013 g, n = 3; sir2 null, 0.056 ± 0.011 g, n = 3). Flow cytometry of testicular cells stained for DNA content indicated that all stages of spermatogenesis were present (Fig. ), but a histological inspection indicated that the spermatogenesis occurring in the testes of sir2α null mice was abnormal. In the least affected testis, only subtle evidence of abnormal spermatogenesis was apparent, manifested as an increased incidence of spermatocyte apoptosis, occasional multinucleated germ cells, decreased numbers of mature elongated spermatids, and increased numbers of retained elongated spermatids.
FIG. 15. DNA content distribution of testes from sir2α null mice indicates that all stages of spermatogenesis are present. Single-cell suspensions were created from the testes of 7-month-old wild-type (upper panel) and sir2α null (lower panel) (more ...)
In the most affected testis, many seminiferous tubules showed an obvious depletion of the normal complement of spermatocytes, round spermatids, and elongated spermatids, while the spermatogonia and the supportive cells, Sertoli cells, Leydig cells, and peritubular cells, were present in normal numbers (Fig. ). In addition to the obvious depletion of differentiating germ cells, abnormalities included an increased number and size of vacuoles within the seminiferous epithelium, an increased incidence of apoptotic spermatocytes and spermatids, retention of highly condensed spermatids, and morphologically abnormal round spermatids (Fig. , c, d, and e). The round spermatid abnormalities consisted of the sharing of acrosomes (Fig. ) and larger-than-normal cells in the stages right after meiosis (Fig. ).
FIG. 16. Spermatogenesis is abnormal in sir2α null mice. Testes from both wild-type (a) and sir2 null (b to e) mice were examined for histopathological abnormalities. At low power, the seminiferous tubules in the wild-type mouse appeared almost uniform (more ...)
It has been proposed that p53 is a substrate for the mammalian SIR2α deacetylase activity, that p53 activity is dependent on acetylation, and that p53 function is normally modulated by SIR2α deacetylation (25
). Apoptosis in the testis is dependent on p53 (4
), and therefore the elevated frequency of apoptotic cells might be a consequence of the absence of SIR2α in the testes of sir2
α null animals. We also predicted that the testes of the sir2
α null mice might be hypersensitive to radiation. Wild-type and sir2
α null mice were irradiated with 3 Gy and sacrificed 12 h later, and seminiferous tubules were examined for their incidence of apoptosis. X-irradiation increased the frequency of apoptotic cells in the testes of both wild-type and sir2
α null mice (Fig. ). However, testes from the the sir2
α null mice did not appear to be hypersensitive to the radiation.
FIG. 17. Increased numbers of apoptotic germ cells are present in the seminiferous tubules of sir2α null mice. Testis cross sections from wild-type (a) and sir2α null (b) mice were analyzed by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin (more ...)