Molecular Cancer Research Targeting the PI3-Kinase Pathway in Cancer Chemical and Biological Aspects of Inflammation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McBurney, M. W.
Right arrow Articles by Lemieux, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McBurney, M. W.
Right arrow Articles by Lemieux, M.
Molecular Cancer Research 1:402-409 (2003)
© 2003 American Association for Cancer Research

Signaling and Regulation

The Absence of SIR2{alpha} Protein Has No Effect on Global Gene Silencing in Mouse Embryonic Stem Cells1

Michael W. McBurney1,2, Xiaofeng Yang1,2, Karen Jardine1,2, Melissa Bieman1,2, John Th'ng3 and Madeleine Lemieux1,2

1 Ottawa Regional Cancer Centre and 2 University of Ottawa, Ottawa, Canada; and
3 Northwestern Regional Cancer Centre, Thunder Bay, Canada

Requests for reprints: Michael W. McBurney, Ottawa Regional Cancer Centre, 503 Smyth Road, Ottawa, K1H 1C4 Canada. Phone: (613) 737-7700x6887; Fax: (613) 247-3524. E-mail: michael.mcburney{at}orcc.on.ca


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The yeast sir2 gene plays a central role in mediating gene silencing and DNA repair in this organism. The mouse sir2{alpha} gene is closely related to its yeast homologue and encodes a nuclear protein expressed at particularly high levels in embryonic stem (ES) cells. We used homologous recombination to create ES cells null for sir2{alpha} and found that these cells did not have elevated levels of acetylated histones and did not ectopically express silent genes. Unlike yeast sir2 mutants, our sir2{alpha} null ES cells had normal sensitivity to insults such as ionizing radiation and heat shock, and they were able to silence invading retroviruses normally. These sir2{alpha} null cells were able to differentiate in culture normally. Our results failed to provide evidence that the mammalian SIR2{alpha} protein plays a role in gene silencing and suggest that the physiological substrate(s) for the SIR2{alpha} deacetylase may be nuclear proteins other than histones.

Key Words: targeted recombination • gene inactivation • retrovirus • embryonic stem cell


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Most of the genetic material in each mammalian cell remains transcriptionally silent. The mechanisms that establish and maintain the chromatin in the inactive configuration are thought to depend on modifications to the integral components of the chromatin such as DNA methylation and histone acetylation and methylation. The proteins that carry out these chromatin modifications and the means by which modified chromatin remains silent are emerging from studies carried out in simpler organisms such as yeast and Drosophila (1–3). Mammalian proteins homologous to those of these simpler organisms often share similar functions. For example, the mammalian homologues of Su(var)39 have been shown to catalyse methylation of core histones (4) and the mammalian homologues of HP1 are known to bind chromatin containing these methylated histones (5) and effect transcriptional inactivation of genes in the region of histone hypermethylation (reviewed in Ref. 6).

In yeast, a protein required for silencing genes in each of the well-investigated paradigms is that encoded by sir2 (reviewed in Refs. 2, 6, 7). The ysir2p has NAD+-dependent histone deacetylase (HDAC) activity (8–11) that is essential for its gene silencing function and ysir2p resides in the cell as part of a multi-protein complex that binds to and inactivates specific regions of the chromatin (2, 3). In addition to its role in mediating gene silencing, the ysir2p also plays roles in a variety of other cellular functions including cell cycle regulation, DNA repair, and cellular senescence (7).

At least seven mammalian genes share a region homologous to the catalytic domain of the ysir2p (12, 13). Given the prevalence and importance of gene silencing in the mammalian genome and the multitude of cellular processes requiring ysir2p, we undertook to investigate the role of one of the mammalian homologues of sir2 to determine if this protein plays a role in mammalian cells similar to that of ysir2p. We chose the murine homologue most similar to ysir2, sir2{alpha} (14). Our approach was to create an embryonic stem (ES) cell carrying two null alleles for the sir2{alpha} gene and determine if this cell was compromised in its ability to silence genes and carry out DNA repair. The sir2{alpha} null ES cells were indistinguishable from their parent.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
SIR2{alpha} Is Abundantly Expressed in ES Cells
We used a sir2{alpha} cDNA (14) to probe a Northern blot of RNA derived from a variety of cell lines and tissues. The mRNA encoding sir2{alpha} is abundant in ES cells as well as in embryonal carcinoma (EC) cells but is expressed at much lower levels by fibroblast cells and mouse tissues (Fig. 1).



View larger version (111K):
[in this window]
[in a new window]
  		FIGURE 1. The sir2{alpha} transcript is abundant in ES cells. Total RNA isolated from the cells and tissues indicated was separated by electrophoresis, blotted, and hybridized to probes specific for sir2{alpha}, enx1 (50), and CHD1 (51), three transcripts from genes thought to play roles in gene inactivation. The blot was also probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as a control for RNA loading. J1 and C52 (52) are ES cell lines; P19 (53) and C86 (54) are embryonal carcinoma cell lines; 3T3 and NR6 are fibroblast cell lines; and brain, kidney, and testis were RNAs isolated directly from adult mice. All cell lines are of murine origin.

 
To raise antibodies to the SIR2{alpha} protein, we created a his-tagged SIR2{alpha} fusion protein in bacteria, purified it on Ni columns, and used the purified protein as an immunogen to make a rabbit antiserum. The fusion protein comprised 505 amino acids from the COOH-terminal end of the SIR2{alpha} sequence. The crude antiserum was immunoadsorbed and subsequently eluted from the purified his-SIR2{alpha} protein. This purified antiserum was used to stain fixed ES cells. The SIR2{alpha} protein was nuclear and distributed in a fine punctate pattern. SIR2{alpha} appeared to be excluded from the nucleolus and those regions of the nucleus called nuclear bodies that stained strongly with DNA stains such as Hoechst 33258 (Fig. 2). Nuclear bodies normally house proteins with roles in gene silencing so the exclusion of SIR2{alpha} from these structures suggests that the function of the mammalian protein might be distinct from that of the ysir2p. The distribution of SIR2{alpha} changed during mitosis. Between prophase and telophase, the protein was distributed in the cytoplasm and appeared to be excluded from the region of the condensed chromosomes. SIR2{alpha} was also detected at much lower levels in mouse fibroblasts where it had similar intranuclear distribution (data not shown).



View larger version (72K):
[in this window]
[in a new window]
  		FIGURE 2. The SIR2{alpha} protein is nuclear and distributed in the euchromatin. R1 ES cells were fixed and stained with polyclonal antibody raised in rabbits that had been immunopurified and used for indirect immunofluorescence experiments (panels A and C). Preparations were counterstained with Hoechst 33258 for DNA (panels B, D, and F). In interphase cells, the SIR2{alpha} protein was located in the nucleus but is excluded from the nucleolus (A). In mitotic cells (C), the SIR2{alpha} protein was distributed throughout the cytoplasm and excluded from the region occupied by the condensed chromosomes. No staining occurred in preparations from which the primary antibody was omitted (E). Magnification bar in panel A = 10 µm.

 
On the basis of the full-length cDNA sequence, the SIR2{alpha} protein has a predicted molecular mass of 80,371 Da and a pI of 4.60. The SDS-PAGE mobility of the SIR2{alpha} protein suggests a molecular mass of about 120 kDa. This might indicate extensive post-translational modification to the protein, but in vitro transcription and translation of the sir2{alpha} cDNA resulted in the synthesis of protein that ran on SDS-PAGE with apparent molecular mass of 120 kDa, indicating that the protein migrates aberrantly during electrophoresis. Two-dimensional gels confirmed that the immunoreactive SIR2{alpha} protein had the expected pI of about 4.6. The SIR2{alpha} protein was eluted from isolated nuclei with relatively low salt (75 mM), suggesting that it is not a tightly bound chromatin protein and the native SIR2{alpha} protein was recovered from gel filtration columns in the region of 160 kDa. We interpret this to mean that the mouse SIR2{alpha} protein is not a component of a large stable complex of proteins as is the case for the ysir2p (15). Rather, the mammalian SIR2{alpha} protein resembles that from Drosophila (16) and may be present in the nucleoplasm as a dimer.

Characteristics of sir2{alpha} Null ES Cells
To investigate the function of SIR2{alpha}, we created an ES cell in which the sir2{alpha} alleles were sequentially targeted with knockout vectors designed to delete exons 5 and 6 (17). Because these exons encode important parts of the catalytic domain, we expected that the targeted sir2{alpha} genes would be converted to null alleles and immunoblots of proteins isolated from the single and double knockouts confirmed the absence of SIR2{alpha} protein from the sir2{alpha}-/- cell and the presence of half normal levels of SIR2{alpha} protein in sir2{alpha}+/- cells (Fig. 3).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 3. sir2{alpha} null cells differentiate in vitro. Wild-type R1 cells (sir2{alpha}+/+), cells carrying one mutant sir2{alpha} allele (sir2{alpha}+/-), and cells carrying two mutant sir2{alpha} mutant alleles (sir2{alpha}-/-) were aggregated in bacterial grade culture dishes in the absence of LIF, cultured as aggregates for 5 days, plated onto tissue culture grade dishes on day 5, and cultured for an additional 5 days. At intervals during the culture period, cells were harvested and protein extracted for SDS-PAGE. Following electrophoretic transfer to membranes, the blots were probed with antibody to SIR2{alpha} and for cytokeratin 8 recognized by the TROMA-1 monoclonal antibody. The cytokeratins reactive with TROMA-1 were distributed between 45 and 60 kDa perhaps because of proteolysis.

 
Because the SIR2{alpha} protein is nuclear and may play a role in the global regulation of gene expression, we set out to determine whether the sir2{alpha} null ES cells were able to differentiate in culture using the protocol normally employed for inducing differentiation. When aggregated and cultured as floating groups of cells, ES cells differentiate forming particularly large numbers of cells resembling extraembryonic endoderm characterized by the expression of cytokeratin 8 recognized by the TROMA-1 (18) antibody and the endoA cDNA (19, 20). Both the mRNA and protein are induced equally well in wild-type and sir2{alpha} null ES cells (Figs. 3 and 4). Similarly, another early differentiation marker, Brachyury, is also induced in the sir2{alpha} null cells. During the course of differentiation, the expression of the SIR2{alpha} protein and mRNA were down-regulated consistent with the observation that the levels of expression of sir2{alpha} are lower in differentiated tissues than in undifferentiated ES cells. In addition, the ES-specific Oct4 transcript was down-regulated in all cell lines during differentiation.



View larger version (93K):
[in this window]
[in a new window]
  		FIGURE 4. sir2{alpha} null cells differentiate in vitro. RNA was isolated from the differentiating ES cells and separated by electrophoresis on agarose gels before being transferred to membranes for hybridization to various radioactive probes. The probes used reacted with transcripts encoded by the genes indicated on the right. Oct4 is a marker for ES cells while Brachyury and endoA are markers for differentiating cells. The other transcripts (SNRPN, gtl2, lo1) were identified as amongst those absent from the double knockout cells in microarray experiments. GAPDH is the loading control.

 
The ysir2p is known to deacetylate histones and the mammalian SIR2{alpha} protein also has deacetylase activity on histone substrates (9–11, 14). To determine if the absence of the SIR2{alpha} protein in the null ES cells is accompanied by an increase in the level of histone acetylation, we analyzed the core histones on acid-urea gels looking for the extent of histone modification (Fig. 5). There was no apparent difference in global histone modification in the sir2{alpha} null ES cells while cells grown in the presence of an inhibitor of HDACs (trichostatin A) results in extensive histone hyperacetylation. Thus, it appears that the absence of SIR2{alpha} in ES cells has little effect on the overall level of histone acetylation. Yeast lacking the SIR2 protein have increased levels of acetylated histone H4 particularly in regions of the chromatin that are normally inactive (21). Mammalian cells have extensive regions of inactive chromatin so our result suggests that proteins other than histones may be the physiologically pertinent substrates for SIR2{alpha}.



View larger version (94K):
[in this window]
[in a new window]
  		FIGURE 5. The absence of SIR2{alpha} does not significantly affect the extent of histone acetylation. Histones from the cells indicated were isolated from cells growing on plastic or cultured as aggregates for 2 days to initiate differentiation. The histones were separated on acid-urea Triton gels (55) before being stained with coomassie blue. The control consists of histones isolated from HeLa cells treated for 24 h with the HDAC inhibitor trichostatin A and contains hyperacetylated histones. The labels on the left indicate the histone types and the labels on the right indicate the numbers of acetate groups on the histone H4 molecules (from none to 4). sir2{alpha}+/- and sir2{alpha}-/+ are two independent heterozygous clones carrying different homologously integrated knockout vectors.

 
In yeast, the sir2 protein is associated with the Ku dimer and plays a role in the repair of double-strand DNA breaks by the non-homologous end joining pathway (22). We compared the survival of the sir2{alpha} null ES cells following exposure to ionizing radiation and found that it was not different from that of wild-type ES cells in clonogenic survival assays (Fig. 6). ES cells null for the Ku70 protein were, as expected, radiation sensitive (23), so the lack of sensitivity of the sir2{alpha} null ES cells to radiation suggests that SIR2{alpha} may not be associated with the non-homologous end joining repair pathway. Yeast lacking sir2 are sensitive to other stresses such as heat shock (24) but we found that the sir2{alpha} null ES cells had normal levels of killing following hyperthermia at 43°C (data not shown).



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 6. sir2{alpha} null ES cells are not radiation sensitive. Exponentially growing cultures of wild-type R1 cells ({square}), sir2{alpha}-/- cells ({triangleup}), and Ku70-/- cells ({circ}) were irradiated with the indicated doses of X-rays and plated for colony formation on lethally irradiated STO feeder fibroblasts in the presence of LIF and ß-mercaptoethanol (56). Colonies that formed were fixed after 8 days and counted. The results shown are the averages from duplicate experiments.

 
Gene Silencing in sir2{alpha} Null Cells
We next undertook to look directly at the capacity of the sir2{alpha} null ES cells to initiate and maintain gene silencing using the paradigm of retrovirus infection. It has been well established that infection of undifferentiated ES cells with retroviruses results in the integration of provirus into the genome but these proviruses are transcriptionally silent (25, 26). We used a Moloney leukemia virus-based ecotropic virus consisting of the long terminal repeat (LTR) driving enhanced green fluorescent protein (GFP). Following infection by the recombinant retrovirus, cells were expanded and assessed for fluorescence and the presence of the provirus in the genomic DNA of the cell population (Fig. 7). 3T3 cells were the positive control for this experiment and these cells became strongly fluorescent following infection at a multiplicity of about 10. None of the infected ES cells, including the sir2{alpha} null cells, were fluorescent. Analysis of the genomic DNA from the cultures of infected cells indicated the presence of provirus DNA (approximately 7–15 copies per cell) in both the expressing 3T3 cells and the non-expressing ES cells.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 7. Retroviral silencing in ES cells does not depend on the SIR2{alpha} protein. The diagram at the bottom of the figure shows the structure of a Moloney leukemia virus-based retrovirus comprised of the LTR flanking the coding region for enhanced GFP. Virus stocks were made in {psi}2 cells (43) and used to infect cultures of the indicated cells at a multiplicity of infection of about 10. After the infected cultures were grown for 1 week, they were processed for flow cytometry to detect the GFP signal (right panels) and their DNA was isolated and digested with Kpn1 (panel A) or Kpn1 plus Not1 (panel B). Following electrophoretic separation and blotting to membranes, the DNA was hybridized to the GFP sequence. The intensity of the GFP signal indicated that the mean numbers of integrated virus per cell varied between 7 and 15. Not1 digestion proceeds only if the DNA recognition sequence is not methylated, so the distribution of GFP signal in panel B indicates that almost no methylation of the Not1 sequence occurred in proviruses in 3T3 cells while extensive de novo methylation occurred in the proviruses of all of the ES cells regardless of status of the sir2{alpha} genes. GFP fluorescence was monitored by flow cytometry and shows that infected 3T3 cells expressed high levels of GFP as evidenced by the strong fluorescent signals from these cells. However, all of the ES cells gave signals not different from uninfected cells. sir2{alpha}+/- and sir2{alpha}-/+ are two independent clones heterozygous at the sir2{alpha} locus.

 
The provirus contains a single Not1 site, a restriction enzyme that cuts DNA only if it is not methylated. The proviruses in 3T3 cells were virtually all unmethylated at the Not1 site, whereas the proviruses present in the ES cells were largely resistant to Not1 digestion, indicating that most of these Not1 sites were methylated. The methylation of provirus DNA is consistent with the presence of the de novo DNA methylase (DNMT3) in ES cells (27) and its absence from differentiated fibroblasts such as 3T3 cells.

The above evidence suggests that the sir2{alpha} null ES cells are capable of silencing infecting retroviral DNAs but leaves open the possibility that the function of SIR2{alpha} is to regulate a subset of cellular genes. Indeed, recent work suggests that the Drosophila sir2 homologue may serve to effect down-regulation of genes controlled by members of the HES family of DNA binding proteins (28). To investigate the idea that SIR2{alpha} regulates a smaller set of cellular genes, we isolated RNA from wild-type R1 cells and the sir2{alpha} null clone and used DNA microarrays to determine whether there are differences in the endogenous genes expressed by these two cells. The vast majority of the 36,000 gene and EST sequences on the Affymetrix murine genome array U74v2 were not differentially expressed in the two cells. Of those genes that were differentially abundant by more than 2.5-fold, only 2 were up-regulated while 104 were down-regulated in the sir2{alpha} null cells. Some of the down-regulated genes are normally imprinted (SNRPN, gtl2/meg3) while one of the others was isolated as a transcript preferentially abundant in two-cell embryos (lo1). A number of the differentially expressed genes have been verified by Northern blot analysis and semiquantitative reverse transcription-PCR. Fig. 4 shows that some of the differentially expressed genes that are expressed in the parental R1 cells are not expressed in the sir2{alpha} null ES cells.

Regulation of p53 Function
There have been a number of recent reports that indicate that the SIR2{alpha} deacetylase activity can use acetylated p53 as a substrate and that the p53 transactivation function is modulated by the SIR2{alpha} protein (29–31). Previous work with ES and EC cells established that p53 was present but irradiated cells did not arrest in G1, suggesting that p53 function was compromised in ES cells (32). If the idea is correct that SIR2{alpha} down-regulates p53 function, the high level of SIR2{alpha} in ES cells may explain why ES cells fail to arrest in G1 phase following irradiation. However, the studies implicating SIR2{alpha} in p53 regulation have all used assays derived from cells transfected with plasmids engineered to express SIR2{alpha}, p53, or both. Thus, the concentration of these proteins may be very high. To determine whether the SIR2{alpha} protein regulates p53 in ES cells expressing these proteins from their endogenous genes, we assessed the cellular response of wild-type and sir2{alpha} null ES cells to ionizing radiation.

Wild-type and sir2{alpha} null ES cells were irradiated with 10 Gy of X-rays and 12 h later, the cells were harvested to determine their distribution in the cell cycle and the amount of p53 protein. p53 protein was elevated in the irradiated cultures to the same extent in both wild-type and sir2{alpha} null cells (Fig. 8). In addition, in both cell types, the irradiated cells arrested in G2 phase despite the elevated level of p53. Thus, the absence of p53-mediated G1 arrest in ES cells is not due to the high level of SIR2{alpha} expression in these cells.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 8. sir2{alpha} null cells do not undergo p53-mediated arrest in G1 phase of the cell cycle following ionizing irradiation. Wild-type (sir2{alpha}+/+) and sir2{alpha} null (sir2{alpha}-/-) cells were irradiated with 10 Gy of X-rays and cultured for 12 h before being harvested, fixed, and processed for DNA determination by flow cytometry (left panels) or for SDS-PAGE (right panels). The flow cytometry profiles shown are for unirradiated (panels A and C) and irradiated (panels B and D) cells and show that in both cases, the irradiated cells arrest primarily in G2 phase of the cell cycle.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Despite the strong sequence similarity between the proteins encoded by the yeast sir2 gene and that of the mouse sir2{alpha} gene, mammalian ES cells lacking the SIR2{alpha} protein do not appear compromised in their ability to initiate or maintain genes in the inactive state. This conclusion is drawn from direct tests for ectopic expression of silent genes in sir2{alpha} null ES cells and from the ability of these cells to inactivate genes carried by recombinant retroviruses. sir2{alpha} null mice have been created (17) and their relatively mild phenotype is also consistent with the conclusion that SIR2{alpha} is not required for the cells to maintain genes in the transcriptionally inert state.

The distribution of SIR2{alpha} in the nucleus is consistent with its being associated with euchromatin. The Drosophila homologue of sir2 is also associated with actively transcribed regions of the genome (16, 33) although the protein may play a role in the down-regulation of genes mediated by certain transcription factors (28). A recent report (31) demonstrated association between the SIR2{alpha} protein and PML. PML is normally concentrated in the nuclear bodies and overexpression of PML in fibroblast cells resulted in the accumulation of SIR2{alpha} in the nuclear bodies (31). Our immunolocalization studies reported here and elsewhere (17) indicated that in ES cells, SIR2{alpha} is excluded from the nuclear bodies and this distribution did not change following X-irradiation or heat shock. This difference in distribution may be due to the fact that ES cells contain much higher levels of SIR2{alpha} than fibroblasts and that the amount of PML in ES cells is insufficient to result in visible accumulation of SIR2{alpha} in the nuclear bodies.

The absence of a phenotype affecting gene silencing in the sir2{alpha} null cells suggests either that the function of SIR2{alpha} in mammals is different from that of sir2 in yeast or that mammalian cells have redundant activities that mask the function of SIR2{alpha}. There are at least seven mammalian genes that share the conserved catalytic domain with sir2{alpha}; however, many of these homologues encode proteins that reside in the cytoplasm or mitochondria (34–36), suggesting that at least some of these sir2 homologues are unlikely candidates for redundant activities. Mammalian cells also contain large numbers of HDACs that do reside in the nucleus (12). Two of these HDACs have been knocked out in mice (37, 38) creating phenotypes very different from the sir2{alpha} knockout (17), suggesting that there may be rather distinct functions for each HDAC.

Many nuclear proteins are acetylated (39) and the SIR2{alpha} enzyme has been shown to be able to deacetylate at least some of these (29–31, 40). Particularly important amongst the possible substrates for SIR2{alpha} is p53. Acetylation of p53 is thought to be activating so the SIR2{alpha} activity would down-regulate the p53 transactivating function. This notion is attractive because the SIR2{alpha} protein is particularly abundant in ES cells and because ES cells do not display many of the p53-mediated functions despite the presence of normal p53 protein (32). However, sir2{alpha} null ES cells still do not respond to elevated p53 by G1 arrest, suggesting that there are other modulators of p53 activity in ES cells in addition to the SIR2{alpha} deacetylase.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture
Cells of the R1 line of ES cells (41) were maintained in cell culture on lethally irradiated STO feeder fibroblasts in DMEM, 10% FCS, and LIF essentially as described (42). The knockout vectors used to create null alleles of sir2{alpha} in R1 cells have been described (17).

Ecotropic retroviruses were created by co-transfecting {psi}2 cells (43) with the LTR-GFP construct shown in Fig. 7 along with Pgk-puro (44). Colonies were selected in 2 µg/ml puromycin and a number were picked and expanded. Culture supernatants from these clones were tested for virus titre and one clone selected for further expansion. Culture medium from this clone of transformed {psi}2 cells was harvested, filtered through a 0.22-µm filter, and used to infect cells in 2 ml cultures containing 104 cells at a m.o.i. of about 10. Cultures were expanded for 7 days following infection and harvested for DNA isolation and flow cytometry to quantitate GFP expression in these cells. Flow cytometry was performed on dispersed cultures of live cells using fluorescene filter sets.

Cell irradiation was done using a 250-kV X-ray source (45). Survival was assessed by colony formation in cultures containing lethally irradiated (100 Gy) STO feeder fibroblasts in medium supplemented with 10-4 M ß-mercaptoethanol. DNA content of cells was determined by propidium iodide staining as described (32).

Immunodetection
Immunofluorescence experiments were carried out on ES cells grown on coverslips essentially as described (46). Western blots were carried out also as described (47).

The rabbit antibody to the SIR2{alpha} protein was raised against bacterially synthesized and purified recombinant his-tagged SIR2{alpha} by Research Genetics, Inc. (Huntsville, AL). One of the two rabbits immunized had a significant titre of antiserum that was immunopurified before use by absorption and elution from nitrocellulose strips containing purified his-tagged SIR2{alpha} protein.

Nucleic Acid Procedures
Standard manipulation of plasmid DNAs was carried out according to the usual protocols (47). DNA was isolated from cells and tissues as described (48) and was digested with restriction enzymes under conditions recommended by the manufacturer before being subject to electrophoresis and blotting. Isolated DNA fragments were radiolabeled with 32P-dCTP by oligonucleotide priming and these were used to probe the blots (47). RNA was isolated using TRIzol (Invitrogen Life Technologies, Inc., Carlsbad, CA) and electrophoresed, blotted, and probed as described (49).


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
The authors are indebted to Dr. Douglas Gray for the gift of a phage genomic library from 129/Sv mouse DNA, to Drs. Brad Wouters and Roland Chiu for the GFP-containing retrovirus construct, and to Dr. Andras Nagy for the gift of the R1 cells.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 National Cancer Institute of Canada and the Canadian Institutes of Health Research. Back

Received September 26, 2002; revised January 24, 2003; accepted January 31, 2003.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

  1. Courey, A. J. and Jia, S. Transcriptional repression: the long and the short of it. Genes Dev., 15: 2786–2796, 2001.[Free Full Text]
  2. Moazed, D. Common themes in mechanisms of gene silencing. Mol. Cell, 8: 489–498, 2001.[Medline]
  3. Hediger, F. and Gasser, S. M. Nuclear organization and silencing: putting things in their place. Nat. Cell Biol., 4: E53–E55, 2002.[Medline]
  4. Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B. D., Sun, Z. W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C. P., Allis, C. D., and Jenuwein, T. Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature, 406: 593–599, 2000.[Medline]
  5. Aagaard, L., Laible, G., Selenko, P., Schmid, M., Dorn, R., Schotta, G., Kuhfittig, S., Wolf, A., Lebersorger, A., Singh, P. B., Reuter, G., and Jenuwein, T. Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31. EMBO J., 18: 1923–1938, 1999.[Medline]
  6. Richards, E. J. and Elgin, S. C. Epigenetic codes for heterochromatin formation and silencing. Rounding up the usual suspects. Cell, 108: 489–500, 2002.[Medline]
  7. Guarente, L. Sir2 links chromatin silencing, metabolism, and aging. Genes Dev., 14: 1021–1026, 2000.[Free Full Text]
  8. Bochar, D. A., Savard, J., Wang, W., Lafleur, D. W., Moore, P., Cote, J., and Shiekhattar, R. A family of chromatin remodeling factors related to Williams syndrome transcription factor. Proc. Natl. Acad. Sci. USA, 97: 1038–1043, 2000.[Abstract/Free Full Text]
  9. Smith, J. S., Brachmann, C. B., Celic, I., Kenna, M. A., Muhammad, S., Starai, V. J., Avalos, J. L., Escalante-Semerena, J. C., Grubmeyer, C., Wolberger, C., and Boeke, J. D. A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family. Proc. Natl. Acad. Sci. USA, 97: 6658–6663, 2000.[Abstract/Free Full Text]
  10. Landry, J., Sutton, A., Tafrov, S. T., Heller, R. C., Stebbins, J., Pillus, L., and Sternglanz, R. The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases. Proc. Natl. Acad. Sci. USA, 97: 5807–5811, 2000.[Abstract/Free Full Text]
  11. Tanny, J. C. and Moazed, D. Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: evidence for acetyl transfer from substrate to an NAD breakdown product. Proc. Natl. Acad. Sci. USA, 98: 415–420, 2001.[Abstract/Free Full Text]
  12. Gray, S. G. and Ekstrom, T. J. The human histone deacetylase family. Exp. Cell Res., 262: 75–83, 2001.[Medline]
  13. de Nigris, F., Cerutti, J., Morelli, C., Califano, D., Chiariotti, L., Viglietto, G., Santelli, G., and Fusco, A. Isolation of a SIR-like gene, SIR-T8, that is overexpressed in thyroid carcinoma cell lines and tissues. Br. J. Cancer, 86: 917–923, 2002.[Medline]
  14. Imai, S., Armstrong, C. M., Kaeberlein, M., and Guarente, L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature, 403: 795–800, 2000.[Medline]
  15. Ghidelli, S., Donze, D., Dhillon, N., and Kamakaka, R. T. Sir2p exists in two nucleosome-binding complexes with distinct deacetylase activities. EMBO J., 20: 4522–4535, 2001.[Medline]
  16. Barlow, A. L., van Drunen, C. M., Johnson, C. A., Tweedie, S., Bird, A., and Turner, B. M. dSIR2 and dHDAC6: two novel, inhibitor-resistant deacetylases in Drosophila melanogaster. Exp. Cell Res., 265: 90–103, 2001.[Medline]
  17. McBurney, M. W., Yang, X., Jardine, K., Hixon, M., Boekelheide, K., Webb, J. R., Lansdorp, P. M., and Lemieux, M. The mammalian SIR2{alpha} protein has a role in embryogenesis and gametogenesis. Mol. Cell Biol., 23: 38–54, 2003.[Abstract/Free Full Text]
  18. Kemler, R., Brulet, P., Schnebelen, M. T., Gaillard, J., and Jacob, F. Reactivity of monoclonal antibodies against intermediate filament proteins during embryonic development. J. Embryol. Exp. Morphol., 64: 45–60, 1981.[Medline]
  19. Ouellet, T., Levac, P., and Royal, A. Complete sequence of the mouse type-II keratin EndoA: its amino-terminal region resembles mitochondrial signal peptides. Gene, 70: 75–84, 1988.[Medline]
  20. Morita, T., Tondella, M. L., Takemoto, Y., Hashido, K., Ichinose, Y., Nozaki, M., and Matsushiro, A. Nucleotide sequence of mouse EndoA cytokeratin cDNA reveals polypeptide characteristics of the type-II keratin subfamily. Gene, 68: 109–117, 1988.[Medline]
  21. Braunstein, M., Rose, A. B., Holmes, S. G., Allis, C. D., and Broach, J. R. Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev., 7: 592–604, 1993.[Abstract/Free Full Text]
  22. Hasty, P. The impact energy metabolism and genome maintenance have on longevity and senescence: lessons from yeast to mammals. Mech. Ageing Dev., 122: 1651–1662, 2001.[Medline]
  23. Gu, Y., Jin, S., Gao, Y., Weaver, D. T., and Alt, F. W. Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination. Proc. Natl. Acad. Sci. USA, 94: 8076–8081, 1997.[Abstract/Free Full Text]
  24. Anderson, R. M., Bitterman, K. J., Wood, J. G., Medvedik, O., Cohen, H., Lin, S. S., Manchester, J. K., Gordon, J. I., and Sinclair, D. A. Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels. J. Biol. Chem., 277: 18881–18890, 2002.[Abstract/Free Full Text]
  25. Teich, N., Weiss, R. A., Martin, G. M., and Lowry, D. R. Virus infection of murine teratocarcinoma stem cell lines. Cell, 12: 973, 1977.[Medline]
  26. Gautsch, J. W. Embryonal carcinoma stem cells lack a function required for virus replication. Nature, 285: 110, 1980.[Medline]
  27. Okano, M., Bell, D. W., Haber, D. A., and Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 99: 247–257, 1999.[Medline]
  28. Rosenberg, M. I. and Parkhurst, S. M. Drosophila Sir2 is required for heterochromatic silencing and by euchromatic hairy/E(Spl) bHLH repressors in segmentation and sex determination. Cell, 109: 447–458, 2002.[Medline]
  29. Luo, J., Nikolaev, A. Y., Imai, S., Chen, D., Su, F., Shiloh, A., Guarente, L., and Gu, W. Negative control of p53 by sir2{alpha} promotes cell survival under stress. Cell, 107: 137–148, 2001.[Medline]
  30. Vaziri, H., Dessain, S. K., Eaton, E. N., Imai, S. I., Frye, R. A., Pandita, T. K., Guarente, L., and Weinberg, R. A. hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell, 107: 149–159, 2001.[Medline]
  31. Langley, E., Pearson, M., Faretta, M., Bauer, U. M., Frye, R. A., Minucci, S., Pelicci, P. G., and Kouzarides, T. Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence. EMBO J., 21: 2383–2396, 2002.[Medline]
  32. Schmidt-Kastner, P. K., Jardine, K., Cormier, M., and McBurney, M. W. Absence of p53-dependent cell cycle regulation in pluripotent mouse cell lines. Oncogene, 16: 3003–3011, 1998.[Medline]
  33. Van Steensel, B., Delrow, J., and Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat. Genet., 27: 304–308, 2001.[Medline]
  34. Afshar, G. and Murnane, J. P. Characterization of a human gene with sequence homology to Saccharomyces cerevisiae SIR2. Gene, 234: 161–168, 1999.[Medline]
  35. Yang, Y. H., Chen, Y. H., Zhang, C. Y., Nimmakayalu, M. A., Ward, D. C., and Weissman, S. Cloning and characterization of two mouse genes with homology to the yeast sir2 gene. Genomics, 69: 355–369, 2000.[Medline]
  36. Onyango, P., Celic, I., McCaffery, J. M., Boeke, J. D., and Feinberg, A. P. SIRT3, a human SIR2 homologue, is an NAD-dependent deacetylase localized to mitochondria. Proc. Natl. Acad. Sci. USA, 99: 13653–13658, 2002.[Abstract/Free Full Text]
  37. Lagger, G., O'Carroll, D., Rembold, M., Khier, H., Tischler, J., Weitzer, G., Schuettengruber, B., Hauser, C., Brunmeir, R., Jenuwein, T., and Seiser, C. Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO J., 21: 2672–2681, 2002.[Medline]
  38. Zhang, C. L., McKinsey, T. A., Chang, S., Antos, C. L., Hill, J. A., and Olson, E. N. Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy. Cell, 110: 479–488, 2002.[Medline]
  39. Sterner, D. E. and Berger, S. L. Acetylation of histones and transcription-related factors. Microbiol. Mol. Biol. Rev., 64: 435–459, 2000.[Abstract/Free Full Text]
  40. Hornbruch, A. and Wolpert, L. Position signalling by Hensen's node when grafted to the chick limb bud. J. Embryol. Exp. Morphol., 94: 257–265, 1986.[Medline]
  41. Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., and Roder, J. C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA, 90: 8424–8428, 1993.[Abstract/Free Full Text]
  42. Pirity, M., Hadjantonakis, A. K., and Nagy, A. Embryonic stem cells, creating transgenic animals. Methods Cell Biol., 57: 279–293, 1998.[Medline]
  43. Miller, D. G., Adam, M. A., and Miller, A. D. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol. Cell. Biol., 10: 4239–4242, 1990.[Abstract/Free Full Text]
  44. Schmidt-Kastner, P. K., Jardine, K., Cormier, M., and McBurney, M. W. Genes transfected into embryonal carcinoma stem cells are both lost and inactivated at high frequency. Somatic Cell Mol. Genet., 22: 383–392, 1996.[Medline]
  45. Myint, W. K., Ng, C., and Raaphorst, G. P. Examining the non-homologous repair process following cisplatin and radiation treatments. Int. J. Radiat. Biol., 78: 417–424, 2002.[Medline]
  46. Rudnicki, M. A. and McBurney, M. W. Cell culture methods and induction of differentiation of embryonal carcinoma cell lines. In: E. J. Robertson (ed.), Teratocarcinomas and embryonic stem cells, a practical approach, pp. 19–49. Oxford, United Kingdom: IRL Press, 1987.
  47. Maniatis, T., Fritsch, T., and Sambrook, J. Molecular cloning: a laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1982.
  48. Laird, P. W., Zidjerveld, A., Linders, K., Rudnicki, M. A., Jaenisch, R., and Berns, A. Simplified mammalian DNA isolation procedure. Nucleic Acids Res., 19: 4293–4293, 1991.[Free Full Text]
  49. Lau, S., Jardine, K., and McBurney, M. W. DNA methylation pattern of a tandemly repeated LacZ transgene indicates that most copies are silent. Dev. Dyn., 215: 126–138, 1999.[Medline]
  50. Hobert, O., Sures, I., Ciossek, T., Fuchs, M., and Ullrich, A. Isolation and developmental expression analysis of Enx-1, a novel mouse Polycomb group gene. Mech. Dev., 55: 171–184, 1996.[Medline]
  51. Delmas, V., Stokes, D. G., and Perry, R. P. A mammalian DNA-binding protein that contains a chromodomain and an SNF2/SWI2-like helicase domain. Proc. Natl. Acad. Sci. USA, 90: 2414–2418, 1993.[Abstract/Free Full Text]
  52. Li, E., Bestor, T. H., and Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell, 69: 915–926, 1992.[Medline]
  53. McBurney, M. W., Jones-Villeneuve, E. M. V., Edwards, M., and Anderson, P. Controlled differentiation of teratocarcinoma cells in culture. Nature, 299: 165–167, 1982.[Medline]
  54. McBurney, M. W. and Adamson, E. D. Studies on the activity of the X chromosome in female teratocarcinoma cells in culture. Cell, 9: 57–90, 1976.[Medline]
  55. Delcuve, G. P. and Davie, J. R. Western blotting and immunochemical detection of histones electrophoretically resolved on acid-urea-triton- and sodium dodecyl sulfate-polyacrylamide gels. Anal. Biochem., 200: 339–341, 1992.[Medline]
  56. Oshima, R. Stimulation of the clonal growth and differentiation of feeder layer dependent mouse embryonal carcinoma cells by ß-mercaptoethanol. Differentiation, 11: 149–155, 1978.[Medline]



This article has been cited by other articles:


Home page
 CarcinogenesisHome page
S. A. Gatz and L. Wiesmuller
Take a break--resveratrol in action on DNA
Carcinogenesis, February 1, 2008; 29(2): 321 - 332.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
J. M. Solomon, R. Pasupuleti, L. Xu, T. McDonagh, R. Curtis, P. S. DiStefano, and L. J. Huber
Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage
Mol. Cell. Biol., January 1, 2006; 26(1): 28 - 38.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
E. Michishita, J. Y. Park, J. M. Burneskis, J. C. Barrett, and I. Horikawa
Evolutionarily Conserved and Nonconserved Cellular Localizations and Functions of Human SIRT Proteins
Mol. Biol. Cell, October 1, 2005; 16(10): 4623 - 4635.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
N. Matsushita, Y. Takami, M. Kimura, S. Tachiiri, M. Ishiai, T. Nakayama, and M. Takata
Role of NAD-dependent deacetylases SIRT1 and SIRT2 in radiation and cisplatin-induced cell death in vertebrate cells
Genes Cells, April 1, 2005; 10(4): 321 - 332.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
D. Sereno, A. M. Alegre, R. Silvestre, B. Vergnes, and A. Ouaissi
In Vitro Antileishmanial Activity of Nicotinamide
Antimicrob. Agents Chemother., February 1, 2005; 49(2): 808 - 812.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H. Daitoku, M. Hatta, H. Matsuzaki, S. Aratani, T. Ohshima, M. Miyagishi, T. Nakajima, and A. Fukamizu
Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity
PNAS, July 6, 2004; 101(27): 10042 - 10047.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
S. W. Buck, C. M. Gallo, and J. S. Smith
Diversity in the Sir2 family of protein deacetylases
J. Leukoc. Biol., June 1, 2004; 75(6): 939 - 950.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H.-L. Cheng, R. Mostoslavsky, S.'i. Saito, J. P. Manis, Y. Gu, P. Patel, R. Bronson, E. Appella, F. W. Alt, and K. F. Chua
Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice
PNAS, September 16, 2003; 100(19): 10794 - 10799.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McBurney, M. W.
Right arrow Articles by Lemieux, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McBurney, M. W.
Right arrow Articles by Lemieux, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Revie