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First published online September 6, 2007
Stem Cells Vol. 25 No. 12 December 2007, pp. 3085 -3092
doi:10.1634/stemcells.2007-0478; www.StemCells.com
© 2007 AlphaMed Press

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STEM CELL GENETICS AND GENOMICS

Telomeric Transgenes Are Silenced in Adult Mouse Tissues and Embryo Fibroblasts but Are Expressed in Embryonic Stem Cells

Qing Gaoa, Gloria E. Reynoldsa, Lindsay Innesb, Mehrdad Pedramc, Ella Jonesd, Mustafa Junabid, Dong-wei Gaod, Michelle Ricoule, Laure Sabatiere, Henry Van Brocklind, Benjamin L. Francd, John P. Murnanea

aDepartments of Radiation Oncology, University of California, San Francisco, California;
bColumbia Medical School, New York, New York, USA;
cDepartment of Medical Genetics, Medical Sciences, University of Tehran, Tehran, Iran;
dDepartment of Nuclear Medicine, University of California, San Francisco, California, USA;
eLaboratoire de Radiobiologie et Oncologie, Commissariat à l'Energie Atomique, Fontenay-aux Roses, France

Key Words. Telomere • Telomere-position effect • Silencing • DNA methylation • Embryonic stem cell • Embryo fibroblast

Correspondence: John P. Murnane, Ph.D., Department of Radiation Oncology, University of California, San Francisco, 1855 Folsom Street, MCB 200, San Francisco, California 94103, USA. Telephone: 415-476-9083; Fax: 415-476-9069; e-mail: jmurnane{at}radonc.ucsf.edu

Received on June 18, 2007; accepted for publication on August 27, 2007.

First published online in STEM CELLS EXPRESS  September 6, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
In addition to their role in protecting the ends of chromosomes, telomeres also influence the expression of adjacent genes, a process called telomere-position effect. We previously reported that the neo and HSV-tk transgenes located adjacent to telomeres in mouse embryonic stem cells are initially expressed at low levels and then become gradually silenced upon passage in culture through a process involving DNA methylation. We also reported extensive DNA methylation in these telomeric transgenes in three different tissues isolated from mice generated from one of these embryonic stem cell clones. In the present study, we demonstrate that embryo fibroblasts isolated from two different mouse strains show extensive DNA methylation and silencing of the telomeric transgenes. Consistent with this observation, we also demonstrate little or no detectable expression of the HSV-tk telomeric transgene in somatic tissues using whole body imaging. In contrast, both telomeric transgenes are expressed at low levels and have little DNA methylation in embryonic stem cell lines isolated from these same mouse strains. Our results demonstrate that telomere-position effect in mammalian cells can be observed either as a low level of expression in embryonic stem cells in the preimplantation embryo or as complete silencing and DNA methylation in differentiated cells and somatic tissues. This pattern of expression of the telomeric transgenes demonstrates that subtelomeric regions, like much of the genome, are epigenetically reprogrammed in the preimplantation embryo, a process that has been proposed to be important in early embryonic development.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The caps on the ends of eukaryotic chromosomes, called telomeres, play an important role in protecting the ends of chromosomes and preventing chromosome fusion [1]. In addition to their role in protecting chromosome ends, telomeres can also influence the expression of nearby genes. The reversible silencing of genes near telomeres, termed telomere-position effect (TPE), has been extensively studied in yeast [2], where it has been found to involve changes in chromatin conformation, to be dependent upon both the distance from the telomere and telomere length, and to be mediated through Sir2p, a type III histone deacetylase [2–4]. Many of these studies were conducted using transgenes integrated near telomeres on truncated chromosome ends missing their original subtelomeric sequences [5–8]. Subsequent studies of either transgenes integrated at yeast native telomeres or endogenous yeast genes, however, revealed that some genes can escape TPE [9–11]. This variability in response to TPE results from the presence of DNA elements that function as insulators within subtelomeric regions of some yeast native telomeres [10, 11].

TPE has been implicated in the loss of expression of genes relocated near telomeres in a variety of human syndromes [12–16]. As in yeast, transgenes located near telomeres have been used to study TPE in mammalian cells. Two of these studies, Koering et al. [17] and Baur et al. [18], demonstrated TPE in human cancer cell lines. Both groups found that TPE is inhibited by trichostatin A, an inhibitor of type I and II histone deacetylases [19]. This observation led to the proposal that deacetylation of histones is involved in TPE, consistent with studies showing that histone deacetylation is important for chromatin condensation [20]. Furthermore, Koering et al. [17] found that 5-azacytidine (5-AzaC), an inhibitor of DNA methyltransferases [21], had no effect. As a result, they concluded that DNA methylation, a modification commonly associated with complete silencing, was not associated with TPE in human cells [17].

We previously investigated TPE in mouse embryonic stem (ES) cells, mouse embryo fibroblasts (MEFs), and transgenic mice, using selectable marker genes located adjacent to telomeres [22]. We found that silencing in normal mouse cells, unlike that in human tumor cells [17], was accompanied by DNA methylation. The role of DNA methylation in silencing in mouse ES cells was further demonstrated by the fact that treatment with 5-AzaC restored G418-resistance and expression of the telomeric transgenes. Tissues of mice that were generated from one of these ES cell clones also demonstrated extensive DNA methylation of the telomeric transgenes, suggesting that telomeric transgenes were silenced in adult tissues in vivo [22].

To provide additional insights into the biology and function of TPE, we have now expanded our studies by investigating the pattern of expression of telomeric transgenes in MEFs and ES cell lines isolated from transgenic mice, as well as in vivo in transgenic mice. These studies involve the analysis of the level of expression and the extent of DNA methylation of the neomycin-resistance (neo), puromycin-resistance (puro), and Herpes simplex virus thymidine kinase (HSV-tk) telomeric transgenes in MEFs and ES cell lines from two different transgenic mice. In addition, we have also analyzed the distribution of HSV-tk in individual tissues and organs, and using whole body imaging, using established protocols for the uptake of a radioactive substrate of HSV-tk. The results demonstrate that the silencing of subtelomeric genes follows a pattern typical of facultative heterochromatin, similar to that proposed for genes involved in embryonic development and cell differentiation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Generation of ES Cell Clones and Transgenic Mice Containing Telomeric Transgenes
Generation and propagation of ES cell clones containing telomeric transgenes was performed as previously described [22]. The isolation and analysis of ES cell clone A405 has been reported previously [23]. The analysis of ES cell clone 10P was performed using similar methods (Fig. 1). Briefly, the analysis of telomeric integration sites included fluorescence in situ hybridization (FISH) using both a plasmid probe and chromosome-specific probes to identify the location of the integrated plasmid [24], restriction mapping, BAL31 nuclease digestion to confirm that the new telomere created by the plasmid represents a terminal restriction fragment [23, 25], and rescue of the integrated plasmid and adjacent cellular DNA to determine the exact location of the integration site [23].


Figure 1
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  		Figure 1. A new telomere in embryonic stem (ES) cell clone 10P was seeded when a single copy of the pPPT2-tel plasmid integrated 1.4 megabases from the original end of chromosome 11. (A): The structure of integrated pPPT2-tel plasmid that was linearized with NotI prior to transfection to place telomeric repeat sequences on one end. The location of the vector sequences (amp/ori), puro gene, HSV-tk gene, tel repeats, and cellular DNA (chromosome 11) are shown. Also shown are the restriction sites used for analysis: B, E, H, Hp, N, P, and S. (B): Characterization of the integrated plasmid sequences by Southern blot analysis of 10P genomic DNA digested with a variety of restriction enzymes. The number and size of the restriction fragments and rescue of integrated plasmid DNA following hybridization with the pNPT{Delta} plasmid probe (without tel repeats) demonstrate that a single intact copy of pPPT2-tel is present. The presence of some bands at the LOR is consistent with the large size of terminal restriction fragments due to the long telomeres in mice. The additional light bands in some lanes are due to hybridization of the pgk promoter sequence in the plasmid probe with endogenous pgk sequences. (C): BAL31 nuclease digestion of genomic DNA from clone 10P demonstrates that the plasmid is located at a telomere. Following digestion for 0, 30, or 120 minutes with BAL31 nuclease, DNA was digested with HpaI and separated using pulsed-field gel electrophoresis (PFGE), and Southern blot analysis was performed using the pNPT{Delta} plasmid as a probe. The selective digestion of the large band demonstrated that it consists of a terminal restriction fragment containing a telomere. (D): Fluorescence in situ hybridization analysis of the integrated plasmid sequences demonstrated that the plasmid is located at the end of chromosome 11. Sequential hybridization was performed on the same metaphase spreads, first using the pNPT{Delta} plasmid probe (yellow) counterstained with propidium iodide (red), followed by multiplex fluorescence in situ hybridization (M-FISH) to identify the individual mouse chromosomes. Insets show the enlarged chromosome 11 containing the integrated plasmid sequences. The trisomy of chromosome 11 present in the late passage 10P ES cell culture used for the chromosome analysis shown here was not present in the early passage 10P ES cell clone used to generate the 10P mice or the ES and mouse embryo fibroblast cultures established from the 10P mice. Abbreviations: B, BamHI; E, EcoRI; H, HindIII; Hp, HpaI; Kb, kilobases; LOR, limit of resolution; N, NcoI; P, PvuII; S, SspI; tel repeats, telomeric repeat sequences.

 
The A405 and 10P transgenic mouse strains were established from ES cell clones A405 and 10P, respectively, in conjunction with the University of California San Francisco (UCSF) Cancer Center Transgenic Core Facility. Briefly, ES cells were injected into 50 blastocysts from C57BL/6J mice, resulting in the generation of chimeric mice. Breeding these mice with C57BL/6J mice generated agouti-colored mice derived from the ES cell clones. Southern blot analysis of tail DNAs using the pNPT{Delta} plasmid as a probe demonstrated that approximately half of these mice contained the telomeric plasmid sequences.

Isolation of Cell Lines from Mice Containing Telomeric Transgenes
MEF cultures were isolated from 13-day-old embryos of A405 and 10P mice by first mincing the embryos, digesting in 0.05% trypsin with 0.2 g/l EDTA (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) for 2 hours at 0°C, pelleting the tissue, and resuspending in growth medium consisting of Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum (HyClone, Logan, UT, http://www.hyclone.com) and gentamicin (UCSF Cell Culture Facility). Genomic DNA from the MEF cultures was then analyzed by polymerase chain reaction (PCR) to determine the presence of the HSV-tk transgene and confirmed by Southern blot analysis as previously described [23]. Isolation of mRNA and genomic DNA for gene expression and DNA methylation studies was performed on early-passage cultures (before passage 5) prior to senescence. To obtain MEF cultures expressing the telomeric transgenes, late-passage spontaneously immortalized MEF cultures were treated for 2 days with 10 mM 5-AzaC, followed by selection with 400 µg/ml G418 (data not shown). Individual clones selected from this G418-resistant MEF population either had resilenced the telomeric transgenes (G5) or did not resilence the telomeric transgenes (G9) upon growth without selection.

Isolation of ES cell lines from the A405 and 10P transgenic mice was performed as previously described [26]. Briefly, blastocysts were collected by flushing out the uterine horns of 3.5 days post coitum mice with M2 medium (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). These blastocysts were then transferred individually into 10-mm tissue culture wells containing a layer of primary mouse embryo fibroblast (PMEF) feeder cells (Specialty Media, Phillipsburg, NJ, http://www.specialtymedia.com) in ES medium and were cultured at 37°C in a 5% CO2 humidified incubator for approximately 10 days. The inner cell mass-derived outgrowths were then removed using a finely drawn micropipette, trypsinized, and transferred into new tissue culture wells containing PMEF feeder cells. After 4 days in culture, the ES-cell-like clumps were selected, expanded, and frozen for future use. Isolation of mRNA and genomic DNA for gene expression and DNA methylation studies was performed on ES cell lines after growth in culture for approximately 35 days.

Selection for both MEF cultures and ES cell lines expressing the neo, puro, and HSV-tk genes were performed in 400 µg/ml G418 (Invitrogen), 2 µg/ml puromycin, or 2 µM ganciclovir (Sigma-Aldrich), respectively, as previously described [22].

DNA Methylation and Gene Expression Assays
Genomic DNA was prepared as previously described [27]. The methylation status of the DNA at the plasmid integration sites was determined by digesting genomic DNA with either MspI or HpaII, as previously described [22].

Total mRNA was isolated using an RNeasy kit (Qiagen, Valencia, CA, http://www.qiagen.com). Analysis of transgene expression was performed in triplicate by quantitative real-time reverse transcription polymerase chain reaction (q-RT-PCR) in conjunction with the UCSF Cancer Center Genome Analysis core facility using TaqMan dual-labeled probes (PerkinElmer Life and Analytical Sciences, Norwalk, CN, http://www.perkinelmer.com) as previously described [22]. Expression of the mouse GAPDH gene was used as an internal control for normalization of the target genes.

Radiopharmaceutical Synthesis
125I-Radiolabeled 2'-fluoro-2'-deoxy-5-iodo-1-β-D-arabinofuranosyluracil (125I-FIAU) was prepared from the tin precursor, 5-trimethylstannyl-1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)uracil (FTAU; ABX Advanced Biochemical Compounds, Radeberg, Germany, http://www.abx.de/), as previously described in the literature [28]. Briefly, 2.3 mCi of 125I-sodium iodide (125I-NaI, 10 µl in 0.1 N NaOH) was added to a 4-ml flat-bottomed vial containing a stir bar and a chloroform solution of FTAU (25 µg in 100 µl). Five microliters of the peroxide solution (3:1 [vol/vol] acetic acid:30% hydrogen peroxide) was added, and the mixture was capped and sonicated for 1 minute using a Branson 2510 sonicator (Branson, Danbury, CT, http://www.bransonultrasonics.com). The desired compound was subsequently purified using gradient high-performance liquid chromatography (HPLC) at 1 ml/minute with a Phenomenex C12 proteo column (4 µm, 4.6 x 255 mm) (Phenomenex, Torrance, CA, http://www.phenomenex.com/). The collected fraction was dried at room temperature using a stream of nitrogen. The residue was reconstituted in phosphate-buffered saline (PBS) and used for the cell studies. HPLC purification following 125I-FIAU synthesis demonstrated that 95% of the collected activity was attributable to 125I-FIAU product. Purification by HPLC demonstrated 58%–62% 125I-FIAU labeling efficiency.

A procedure similar to that described above was used to obtain 123I-FIAU for imaging studies. In this case, four different reactions were run to minimize the amount of water in the reaction mixture. Approximately 22 µl (3.2–3.4 mCi) of the 123I-NaI stock solution was added to the vial containing FTAU (25 µg in 100 µl of acetonitrile). The peroxide solution (6 µl) was then added, and each mixture was sonicated for 1.5 minutes. Following HPLC, the fractions were grouped together, dried, and reconstituted before being administered to the mice.

FIAU-Uptake Assay with MEFs
The G5 and G9 MEF clones were grown in 3.0 g/l glucose-Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.12 mM gentamicin. G5 and G9 cells were plated at 0.5 x 106 cells per well of a six-well plate. After 24 hours, the medium was removed from all wells, and 700 µl of fresh medium containing 1 µCi of 125I-FIAU was added to each well. The cells were incubated at 37°C, 5% CO2 for a specified amount of time (20, 40, 60, or 120 minutes; n = 3 for each time point). Following incubation, the cells and medium were separated. Briefly, medium containing 125I-FIAU was pipetted from each well, and each well was then rinsed with 500 µl of PBS. Medium and rinse were combined, and the activity of this mixture was counted using a standard gamma well counter (Wizard; PerkinElmer Waltham, MA, http://www.perkinelmer.com). Cells were removed from the plate by adding 500 µl of trypsin to each well and incubating at 37°C, 5% CO2 for five minutes. The trypsinized cells were collected, the wells were rinsed with 500 µl of PBS, and the rinse and cells were combined for counting by a gamma well counter.

Biodistribution Study
Three 6-month-old male A405 transgenic mice and three 6-month-old male control mice (without the telomeric transgenes) were each administered 170–250 µCi of 123I-FIAU by tail-vein injection. To ensure identical genetic backgrounds, the A405 mice used in this study had been backcrossed six times with C57BL/6J mice, whereas the control group consisted of C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org). At 2 hours postinjection, mice were anesthetized with isoflurane and imaged using single-photon emission computed tomography and computed tomography (SPECT-CT) (Gamma Medica-Ideas, Northridge, CA, http://www.gm-ideas.com). An initial CT scan of the whole body was followed by pinhole SPECT of the thorax and abdomen. SPECT-CT was repeated at 24 hours. Following the imaging procedure (24 hours postinjection), animals were euthanized, their organs were dissected, and the 125I-FIAU activity was counted using a gamma well counter.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Development of Mouse Cells Containing Telomeric Transgenes
This study involves the analysis of TPE in mouse cells isolated from two different mouse strains, A405 and 10P, that contain transgenes located adjacent to a telomere. The A405 transgenic mouse used in these studies was established from ES cell clone A405, which contains a telomere that was seeded following integration of the pNPT2-tel plasmid 4 megabases (Mb) from the original end of chromosome 15 [22, 23]. The pNPT2-tel plasmid contains a neo gene with an HSV-tk promoter for positive selection in G418, an HSV-tk gene with a mouse phosphoglycerate kinase (pgk) promoter for negative selection in ganciclovir, and 0.8 kilobase (kb) of telomeric repeats for seeding the formation of new telomeres upon integration. The 10P mouse used in these studies was established from the 10P ES cell clone, which contains a new telomere generated by integration of the pPPT2-tel plasmid. The pPPT2-tel plasmid is similar to the pNPT2-tel plasmid except that it contains a puro gene instead of the neo gene for positive selection (Fig. 1A). Restriction mapping (Fig. 1B), BAL31 nuclease digestion (Fig. 1C), and FISH analysis (Fig. 1D) demonstrated that ES cell clone 10P has a single copy of pPPT2-tel integrated on the end of the long arm of chromosome 11. Rescue of the integrated plasmid sequences and adjacent genomic DNA (GenBank accession no. EF503725) demonstrated that the new telomere created by the integration of the pPPT2-tel plasmid in ES cell clone 10P was located 1.4 Mb from the original end of the chromosome.

Silencing of Telomeric Transgenes in MEFs
We previously reported that three different tissues taken from adult A405 mice showed extensive DNA methylation of telomeric transgenes, suggesting the presence of a strong TPE in somatic cells. A subsequent study by Gonzalo et al. [29] also reported that DNA methylation of subtelomeric DNA has an important role in telomere function. To investigate TPE in differentiated cells, we determined the expression levels of the neo and HSV-tk telomeric transgenes in primary MEFs. Primary MEF cultures isolated from both A405 and 10P mouse strains failed to form any colonies in medium containing G418 or puromycin, respectively, and grew well in the presence of ganciclovir, indicating that the neo, puro, and HSV-tk transgenes are silenced in all of the cells in these cultures. Consistent with this observation, using the GAPDH gene as an internal control, q-RT-PCR showed no expression of the puro or HSV-tk genes in two primary MEF cultures established from the 10P mouse strain and no significant expression of the neo and HSV-tk genes in four primary MEF cultures from the A405 mouse strain (Fig. 2).


Figure 2
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  		Figure 2. Telomeric transgenes are silenced in primary MEFs but expressed in ES cell lines. Total RNA isolated from primary 10P MEF cultures J402C and J402D and 10P ES cell lines JS647F and JS836C was analyzed by quantitative real-time reverse transcription polymerase chain reaction for the level of expression of the puro and HSV-tk genes. Total RNA from primary A405 MEF cultures E231C, E231D, M1-B, and M1-D and A405 ES cell lines N275F and N586I was analyzed for the level of expression of the neo and HSV-tk genes. Expression levels were determined in triplicate relative to an internal control for the expression of the GAPDH gene. Abbreviations: ES, embryonic stem; MEF, mouse embryo fibroblasts.

 
Expression of Telomeric Transgenes in ES Cell Lines
ES cells are pluripotent cells isolated from the preimplantation embryo [30]. Previous studies have proposed that passive and active demethylation of DNA in the preimplantation embryo is associated with the regulation of genes that are involved in development [31]. We previously reported that two ES cell lines isolated from the A405 mouse showed expression of the neo and HSV-tk genes [22]. To further investigate the expression of the telomeric transgenes in ES cell lines, we have now analyzed the level of expression and extent of DNA methylation of the telomeric transgenes in two additional ES cell clones from the A405 mouse strain and two ES cell lines from the 10P mouse strain. The ES cell lines from both the A405 and 10P mouse strains grew well in medium containing G418 and formed few colonies in ganciclovir, indicating the expression of the neo and HSV-tk genes. Consistent with this observation, the telomeric transgenes were expressed in all four of the A405 and 10P ES cell lines (Fig. 2). The levels of expression were very low, similar to the ES cell clones initially transfected with the pNPT2-tel or pPPT-tel plasmids [22]. Also similar to the original ES cell clones, the low level of expression of the neo and HSV-tk genes in the two A405 ES cell clones was maintained under selection with G418 but decreased upon passage in culture in the absence of G418 selection (data not shown).

DNA Methylation in Telomeric Transgenes in MEFs
We previously reported that DNA methylation is involved in the silencing of telomeric transgenes in mouse ES cells [22]. A role for DNA methylation in silencing of the telomeric transgenes in MEFs was indicated by the fact that treatment of an A405 MEF culture with 5-AzaC resulted in many cells acquiring resistance to G418 and expression of the telomeric transgenes (data not shown). The extent of DNA methylation of the telomeric transgenes in the MEF cultures was determined by Southern blot analysis following digestion with either HpaII or MspI (Fig. 3A). HpaII and MspI are isoschizomers that recognize the same 4-base pair restriction site; however, HpaII is inhibited by DNA methylation of the restriction site and MspI is not. As a result, methylated DNA is digested with MspI but not HpaII, whereas both enzymes digest unmethylated DNA. The genomic DNA was also digested with SacI, either alone or in combination with HpaII or MspI, to provide transgene-specific bands of a defined length to make the extent of digestion easier to observe. Because of the large number of sites recognized by HpaII and MspI in the transfected DNA sequences, the fragments generated by SacI/MspI digestion of the MEF DNAs are very small and run at the bottom of the gel. In contrast, the plasmid-specific bands in the SacI/HpaII-digested genomic DNAs from all of the MEF cultures are relatively large in size, demonstrating that the transgenes are extensively methylated. In some MEF cultures, the SacI/HpaII bands are somewhat smaller in size than the bands generated with SacI alone, demonstrating that some CpG methylation sites remain unmethylated in some cells. Regardless, it is clear from these results that the great majority of CpG sites in the telomeric transgenes are methylated in MEFs.


Figure 3
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  		Figure 3. Telomeric transgenes have extensive DNA methylation in primary mouse embryo fibroblasts (MEFs), but minimal DNA methylation in mouse embryonic stem (ES) cell lines. (A, B): Genomic DNA from primary 10P MEF cultures J402C and J402D and primary A405 cultures E231C, E231D, M1-B, and M1-D (A) or from 10P ES cell lines JS647F and JS836C and A405 ES cell lines N275F and N586I (B) was analyzed for DNA methylation. The DNA was digested with s, m, or h, and hybridization was performed using the pNPT{Delta} probe that does not contain tel repeats. MspI and HpaII recognize the same restriction site; however, MspI is not affected by methylation of the restriction site, whereas HpaII is inhibited by methylation of the restriction site. Digestion with SacI was included to provide transgene-specific bands of a defined length to make the extent of digestion easier to determine. Because of the large number of HpaII/MspI sites in the plasmid sequences, complete digestion resulted in small DNA fragments at the bottom of the gel (horizontal bar). The presence of large DNA fragments demonstrated a high degree of DNA methylation. Abbreviations: h, SacI plus HpaII; m, SacI plus MspI; s, SacI alone.

 
Minimal DNA Methylation in Telomeric Transgenes in ES Cell Lines
Consistent with the expression of the transgenes in ES cells, the telomeric plasmid sequences in all four ES cell lines contain relatively little DNA methylation, as is evident from the small size of the DNA digested with SacI/HpaII, resulting in small fragments at the bottom of the gel (Fig. 3B). However, although the level of DNA methylation was low compared with the MEFs, some DNA methylation was evident. It is not known whether this DNA methylation was present in the early embryo, or whether it was acquired during propagation in culture, as was observed in the original ES clones transfected with the plasmid [22].

Absence of Detectable Expression of the Telomeric HSV-tk Gene in Tissues of Adult Mice
To further investigate the extent of silencing in adult tissues, we took advantage of a well-established method to detect the HSV-tk protein in vivo. This approach uses the compound FIAU, which is taken up by tissues expressing the HSV-tk gene [32, 33]. To validate the system, uptake of 125I-FIAU was first analyzed in MEF clones that either do or do not express the telomeric HSV-tk protein. Although MEF cultures initially do not express the telomeric transgenes, clones in which they are expressed were generated using treatment with 5-AzaC (described in Materials and Methods). q-RT-PCR showed that similar to ES cells, clone G9 expresses the HSV-tk gene at low levels (1.5% of GAPDH), whereas clone G5 showed no detectable expression of the HSV-tk gene. As expected, clone G9 demonstrated a steady, linear increase in 125I-FIAU uptake over 120 minutes, whereas clone G5 showed no uptake of 125I-FIAU (Fig. 4A). The linear increase in uptake of 125I-FIAU in HSV-tk-expressing G9 cells and lack of uptake in silenced G5 cells over time supported the use of radiolabeled FIAU as a measure of HSV-tk transgene expression and TPE.


Figure 4
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  		Figure 4. Ex vivo quantitative evaluation of expression of the telomeric HSV-tk gene in tissues of adult mice. (A): Validation of differential 125I-radiolabeled 2'-fluoro-2'-deoxy-5-iodo-1-β-D-arabinofuranosyluracil (125I-FIAU) uptake by cells expressing HSV-tk using mouse embryo fibroblast clone G5, which does not express the HSV-tk gene, and clone G9, which does express the HSV-tk gene. (B): 123I-FIAU distribution measured in tissues of three A405 transgenic mice versus tissues of three nontransgenic control mice. Results are given as cpm/mg. No significant differences in FIAU uptake were observed except in the thyroid. Abbreviations: L. Int., large intestine; mins, minutes; Pancr., pancreas; S. Int., small intestine; Stom., stomach.

 
We next determined the extent of uptake of 123I-FIAU in vivo in A405 mice that contained the telomeric HSV-tk gene compared with control mice that did not contain the HSV-tk gene. This in vivo analysis of the biodistribution of 123I-FIAU was performed using two separate approaches, dissection and counting of 123I-FIAU in individual organs, and whole body imaging using combined SPECT-CT. Organ counting demonstrated that a significant whole-organ increase in 123I-FIAU uptake in the A405 transgenic mice compared with the control mice was limited to the thyroid (p ≤ .01) (Fig. 4B). The uptakes in all other organs in the A405 transgenic and control mice were not significantly different (p ≥ .01), although blood and stomach/small bowel uptake were generally greater in the A405 transgenic mice. SPECT-CT images confirmed the general lack of differences in whole body biodistribution between the A405 transgenic and control mice, with detectable uptake limited to the bladder and small foci of activity within the bowel in both (Fig. 5). The combined in vivo imaging and ex vivo assays at 24 hours after 123I-FIAU administration therefore failed to show significant differences in organ uptake within A405 transgenic versus control mice, with the exception of the thyroid. The significance of the difference in uptake in the thyroid in the A405 transgenic and control mice is not clear, but it may be explained by a greater fraction of free iodine within the A405 transgenic mice. Regardless, it is clear from these studies that most cells in adult tissues do not express the telomeric HSV-tk gene. However, because of the limited sensitivity of analysis of 123I-FIAU uptake, it cannot be completely ruled out that some cells in the tissues of adult mice express the HSV-tk gene.


Figure 5
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  		Figure 5. In vivo qualitative evaluation of expression of the telomeric HSV-tk gene in adult mice. 125I-Radiolabeled 2'-fluoro-2'-deoxy-5-iodo-1-β-D-arabinofuranosyluracil (125I-FIAU) distribution after 2 hours in a control mouse demonstrated excretion into the gallbladder, duodenum (presumed to be from biliary excretion), and urinary bladder. 123I-FIAU distribution after 24 hours in a control mouse and an A405 transgenic mouse demonstrated excretion in the urine, but no other foci of radiopharmaceutical uptake were detectable. Abbreviation: hr, hour.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
The results presented here and in our previous study [22] demonstrate that mammals have TPE. TPE in the mouse can be observed as either a low level of expression or complete silencing of telomeric transgenes. The first type of TPE, involving a low level of expression, is observed with telomeric transgenes in ES cells. Transgenes located near telomeres in ES cell lines are expressed at much lower levels than the same transgenes located at many interstitial sites, and the level of expression increases following rearrangements that result in the loss of the adjacent telomere [22]. The second type of TPE, involving complete silencing and DNA methylation, is observed with telomeric transgenes in mouse ES cell lines upon passage in culture [22]. Complete silencing of telomeric transgenes is also seen in mouse tissues and somatic cells. We previously reported extensive DNA methylation in the telomeric transgenes in three different tissues from the A405 mouse strain [22]. In the present study, we demonstrated that telomeric transgenes are silenced in most somatic cells and tissues and that they are silenced with extensive DNA methylation in MEF cultures isolated from the A405 and 10P mice. Consistent with our results, the analysis of subtelomeric regions in MEFs demonstrated extensive DNA methylation and histone modifications typical of heterochromatin [29, 34, 35].

A previous study with human tumor cells also found that TPE resulted in a low level of expression of a telomeric transgene [17]. However, complete silencing involving DNA methylation was not observed. We have also found an absence of complete silencing and DNA methylation of telomeric transgenes in human tumor cells (unpublished observations). Why TPE in human tumor cells should function differently from TPE in mouse ES cells is not clear. One possibility is that although ES cells are capable of de novo DNA methylation of integrated transgenes, this capability is diminished or nonexistent in human tumor cells. In fact, de novo DNA methylation is largely suppressed in differentiated somatic cells [36], and enzymes responsible for de novo DNA methylation are very low in somatic mouse tissues and human tumor cell lines compared with ES cells [37]. Thus, somatic cells and tumor cells are likely to be deficient in de novo DNA methylation, although they are capable of maintaining DNA methylation that is already present.

Although it is clear that TPE occurs in mammalian cells, its function remains to be determined. One possibility is that TPE is simply a consequence of the chromatin structure necessary for proper telomere function and that suppression of gene expression is an unintended consequence [10]. In this case, the introduction of telomeric transgenes may generate an indirect local reprogramming of the chromatin structure, which results in a low level of expression that is confined to ES cells because of their reduced level of DNA methylation. A second possibility that has been proposed is that TPE has a role in replicative cell senescence, in which telomere shortening in somatic cells that do not maintain telomere length eventually results in altered expression of subtelomeric genes involved in cell cycle arrest [38]. In fact, replicative senescence in human cells has been shown to result in alterations in expression of genes located near telomeres [39] and has been reported to result from changes in telomere structure but not length [40]. Consistent with these observations, changes in telomere length have been demonstrated to influence the structure of subtelomeric heterochromatin [41]. However, other studies have demonstrated that replicative cell senescence is associated with shortened telomeres being recognized as chromosome breaks, seemingly eliminating a role for TPE in cell senescence [42, 43].

A third possible function of TPE is the regulation of genes involved in embryonic development. This possibility is suggested by the fact that telomeric transgenes in differentiated cells are silenced and extensively methylated, whereas they are expressed with minimal DNA methylation in ES cells within the preimplantation embryo. Subtelomeric regions are therefore distinct from many heterochromatic regions, including centromeres [36] and imprinted genes [44, 45], which retain DNA methylation in the preimplantation embryo. In addition, intracisternal A-particle (IAP) transposons are also not demethylated in the preimplantation embryo [46]. Based on these differences, subtelomeric heterochromatin does not appear to serve solely a structural function or as a protective mechanism against foreign sequences [47, 48]. Therefore, because it has been proposed that the extensive demethylation of DNA in the preimplantation embryo is involved in the regulation of genes during embryonic development [49, 50], TPE may function as a part of this process. In fact, many genes are located close to telomeres in mouse, including the mBICD1 gene, which has been demonstrated in Drosophila to be involved in polarization of mRNA during oogenesis and embryogenesis [51]. Additional studies will be required to determine whether the progressive silencing and DNA methylation associated with TPE in ES cells is involved in the regulation of genes in early development.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
The work in the J.P.M. laboratory was supported by National Institute of Environmental Health Science Grant R01-ES008427. The work in the L.S. laboratory was supported by RISC-RAD contract FI6R-CT2003-508842.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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