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Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
Received 24 November 2004/ Returned for modification 15 December 2004/ Accepted 25 February 2005
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ABSTRACT |
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INTRODUCTION |
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The chromatin boundary activity at the H19/Igf2 locus requires the zinc-finger binding protein CCTC binding factor (CTCF) that binds, in a methylation-sensitive manner, to four sites within the ICR (3, 24). Mutations in the four CTCF sites eliminate boundary function and result in the accumulation of methylation on the maternal chromosome after fertilization, suggesting that CTCF is one of the factors that maintain the maternal ICR in an unmethylated state (40, 43). However, the CTCF sites are not required to establish the maternal allele in an unmethylated state during oogenesis, implying that other factors may be involved (40, 43). Using a different approach, Fedoriw et al. (16) have recently shown that inhibiting CTCF with a transgene expressing CTCF-specific RNA interference in oocytes leads to the acquisition of methylation on the ICR. One unlikely possibility to explain the contradiction between the two findings is that the mutations that eliminate binding in vitro are less deleterious in vivo. A more likely alternative is that CTCF binds elsewhere in the region or has an indirect effect on the ICR that does not require binding to DNA.
On the paternal ICR, mutations in the CTCF sites have no effect on the accumulation of DNA methylation in testes or somatic tissues (40, 43). Although the simplest explanation for the accumulation of methylation during spermatogenesis is the absence of CTCF to prevent it, the protein has been detected by immunofluorescence in spermatogonia that are undergoing methylation, suggesting that additional regulation is required (33). This is consistent with the finding that when two CTCF sites from the chicken ß-globin locus were inserted in place of the ICR at the H19 locus, paternal methylation did not occur and chromatin boundaries were formed on both alleles (46). More recently, Engel et al. (15) depleted the number of CpG residues in the ICR without disrupting the ability of CTCF to bind and showed that this had no effect on the establishment of paternal methylation in the germ line but resulted in a loss of maintenance methylation in the soma. Thus, sequences outside the CTCF sites themselves must play a role in methylation maintenance.
Taken together, these experiments suggest that there may be one or more proteins other than CTCF that play a role in the establishment and maintenance of parental-specific epigenetic differences at the ICR, and in vivo footprinting of the ICR has reinforced this conclusion (44, 45). Recently, two groups identified a series of sites within the ICR that bind specifically and exclusively to fetal and neonatal testes extracts derived from the stages of development when paternal methylation is being assembled on the ICR (5, 45). These sites contain a core that is similar to the consensus sequence for nuclear receptor extended half sites, and mutations in the half site eliminate binding. However, when Bowman et al. (5) mutated three of these sites in vivo, no effect on imprint establishment or maintenance was observed.
This report extends the search for additional proteins by using extracts from liver, a somatic tissue. We show that the ICR contains two sites that are very strong pause sites in vitro for the Ku70/80 heterodimer that has been implicated in nonhomologous recombination. Using chromatin immunoprecipitation (ChIP) assays and genetic mutations in mice, we conclude that these sites are not involved in the regulation of imprinting at the H19/Igf2 locus.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Electrophoretic mobility shift assays (EMSAs). DNA restriction fragments were radiolabeled with the Klenow fragment of DNA polymerase, while oligonucleotides were labeled with T4 polynucleotide kinase and annealed. The sequences of the oligonucleotide probes are shown in Table 1. All labeled probes were purified in 5% acrylamide gels. Mobility shift reactions were carried out in 40 µl at room temperature for 30 min in 20 mM HEPES-KCl (pH 7.9), 150 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 40,000 cpm radiolabeled DNA, 0.05 µg/µl poly(dI:dC) (0.015 µg/µl for recombinant human Ku70/80), 5% glycerol, 0.5% Triton X-100, and 1 to 5 µl protein extract (20 to 40 mg/ml). The reaction mixtures were analyzed without loading dye on 1.5-mm-thick 5% acrylamide gels.
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Chromatin immunoprecipitation. Adult liver (0.3 g) was sliced in 10 ml of phosphate-buffered saline with 1% formaldehyde and protease inhibitors (1 tablet/50 ml) at 25°C, homogenized for 10 strokes in a type B glass Dounce homogenizer, and incubated at 25°C for 10 min. The cross-linking was terminated with the addition of 500 µl of 2.5 M glycine. The samples were centrifuged at 4°C for 2 min at 2,000 rpm and resuspended three times in 10 ml of ice-cold phosphate-buffered saline. The final pellet was resuspended in 400 µl of lysis buffer (Upstate Biotechnology ChIP kit) and incubated on ice for 10 min, followed by the addition of 1.6 ml of ChIP dilution buffer (Upstate Biotechnology ChIP kit). The samples were sonicated three times with 10 pulses at 50% output, power 10, with a Branson Sonicator 2000 and centrifuged at 13,000 rpm at 4°C for 10 min. The immunoprecipitation was carried out on the supernatant using the Upstate Biotechnology ChIP kit and 10 µl of each antibody (CTCF polyclonal from Upstate [06-917], Ku70 polyclonal from Santa Cruz [sc-1487], Ku70 monoclonal from Neomarkers [Ab-4]). Quantitative PCR was performed on the precipitated DNA with a Bio-Rad iCycler. The A primers were F, 5'-TCTGCTGAATCAGTTGT, and R, 5'-GGGCTCTTTAGGTTTGG, and the B primers were F, 5'-AATCAACAAGGTCGGCTTACTC, and R, 5'-CTGCGATGTACGAGACTTCAC. The copy number was reported relative to that of CTCF, which was arbitrarily set at 100. In addition, all values were normalized to the copy number of each sample before immunoprecipitation.
Generation of H19Ku site– mice. Mutations in the two ATCGAT binding sites were introduced into a 4.7-kb XbaI genomic DNA fragment (from –5.5 to –0.8 kb relative to the H19 transcription start site) by site-directed mutagenesis (using the QuikChange site-directed mutagenesis kit from Stratagene) and integrated into the targeting construct of Schoenherr et al. (43). The vector was introduced into AB2.2 embryonic stem (ES) cells, and colonies were selected in the presence of G418 as described previously. Homologous recombination at the H19 locus was tested by Southern blot analysis with both external and internal probes. Detection of recombination between loxP sites to remove the neomycin selection cassette was achieved with the 3' external probe and a SpeI genomic DNA digest. The presence of the mutant binding sites was confirmed by PCR followed by digestion with ClaI to verify elimination of the ATCGAT ClaI sites. The primers that were used for HS site 1 were F, 5'-CTACATTCACACGAGCATCCAGG, and R, 5'-GATGTACGAGACTTCACTGCCGC. The primers that were used for HS site 2 were F, 5'-GGCAAACTCATGGGTCACTCAAGGC, and R, 5'-GGTACTCAAGCTTTGTCACAGCGG.
Analysis of RNA. RNase protection assays were performed using the RPAIII RNase protection assay kit from Ambion. The H19 and Igf2 probes were prepared as previously described (26) and incubated overnight with 10 µg of total neonatal liver RNA at 42°C. RNase digestion was carried out at 55°C for 1 h, and the protected fragments were separated on 5% acrylamide gels containing 8 M urea.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein (AFP) promoter (18) to reduce binding in a 100-fold excess (Fig. 1B). Furthermore, the two DNA binding sites were effective competitors against one another, suggesting that the same protein or protein complex bound each site (Fig. 1B). When the sequences of the sites were compared, a common ATCGAT motif was found. Base substitutions of each base pair within the ATCGAT motif severely inhibited the ability of HS site 2 to compete with itself for binding, while mutations of single base pairs outside of the ATCGAT motif had no effect (Fig. 1C). A search of the equivalent region in the human H19 gene revealed a single copy of the ATCGAT motif (data not shown). To assess the distribution of the binding activity, protein extracts were prepared from a variety of tissues at different times during development. The activity was found in whole embryo extracts prepared at embryonic day 10.5 and embryonic day 16.5 and in adult liver, oocytes, and neonatal testes, implying that the activity is ubiquitously expressed in mice (data not shown).
Purification of ATCGAT binding activity. To identify the ATCGAT binding activity, we prepared whole-cell extracts from 83 adult mouse livers, yielding 4.8 g of protein. The binding activity, which was followed with EMSA, separated on an S300 gel filtration column with an apparent molecular mass of 150 kDa (Fig. 2). The eluant was applied to a sequence-specific DNA affinity column containing multimerized copies of the ATCGAT binding site. The active fractions that eluted with 1 M KCl were pooled and applied to a Mono Q cation-exchange column. Two prominent bands of approximately 70 kDa and 80 kDa were visualized by silver staining in active fractions of the eluant (Fig. 2). These bands were sequenced by mass spectrometry at the Harvard Microchemistry Facility.
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DNA binding specificity of the Ku70/80 complex. Ku70/80 is thought to bind with high affinity to duplex DNA ends, but in a sequence-nonspecific manner. As such, its purification as the ATCGAT binding activity was unexpected. To verify that the ATCGAT binding activity is composed of Ku70 and Ku80, commercially available human recombinant Ku70 and Ku80 proteins were incubated in the presence of radiolabeled HS site 2. A retarded mobility complex indistinguishable from that obtained with the neonatal liver extract was observed (Fig. 3A). Furthermore, the recombinant Ku-DNA complex was competed specifically with site 2, but not with the AFP-nonspecific competitor. Finally, the human Ku heterodimer exhibited the same sequence specificity as the ATCGAT activity when challenged to bind the oligonucleotides containing base substitutions in site 2 (Fig. 1C and data not shown).
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Relative specificity of DNA binding by Ku70/80. A number of other sequence-specific Ku70/80 binding sites have been reported in the literature (14, 19, 42). Some of these have been shown to require free DNA ends and thus are thought to be strong internal pause sites, while others have been proposed to be sites of direct binding, based on the ability of Ku70/80 to bind to the site on circular DNA (19, 20). To compare the relative affinity of Ku70/80 for the ATCGAT binding site versus that of other sequences, we monitored the ability of sequences in both of these classes to compete for binding with the ATCGAT-containing probe (HS2). The best-characterized example of the direct binding class is the NRE1 element of the mouse mammary tumor virus long terminal repeat (20), a tandem repeat of GAAAGA that bears no sequence similarity to the ATCGAT sequence. As shown in Fig. 3C, lanes 3 and 4, an oligonucleotide containing the NRE1 sequence binds to Ku70/80, but the binding is effectively competed by HS site 2. Likewise, ATCGAT competes very effectively for pause sites, as illustrated in lanes 13 and 14 by the binding of Ku70/80 to an element in the U1 snRNA promoter. Indeed, of the 10 sequences tested, all but one bound Ku70/80 and was effectively competed with HS site 2. In contrast, none of the sequences competed with HS site 2 for binding, even at 500-fold excess (Fig. 3D). Thus, under the conditions of our assay, HS site 2 displays the strongest affinity for Ku70/80 of the sequence-specific binding sites identified to date.
Genome distribution of Ku binding sites. Given the observation that ATCGAT is a high-affinity site for Ku70/80, we used BLAST (1) to determine the distribution of this 6-mer in the mouse genome. We found that the sequence is found 104,497 times, which is very close to the expectation of 109,997 times based on the dinucleotide composition of the genome. The LINE-1 retrotransposon, which represents approximately 19% of the mouse genome, accounts for 26% of the ATCGAT hexamers in the genome. In this respect, the LINE-1 element is unique, as other classes of mouse repeats, with the exception of the IAPLTR1a I MM subfamily of IAP retrotransposons, did not contain this site (Table 2) (27). Within the 6.4-kb sequence of the consensus LINE-1 retrotransposon, ATCGAT is found twice, 1,233 bp apart.
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Two correctly targeted ES cell lines were identified and confirmed by Southern analysis with DNA probes that lie outside the vector (Fig. 5A). In addition, since the mutations eliminate ClaI recognition sites, the presence of the mutations could be verified by performing PCR followed by digestion with ClaI (data not shown). The two correctly targeted ES cell lines were used to generate mice, and the neomycin selection cassette was excised upon transmission from the male chimeras due to the expression of cre recombinase in testes (Fig. 5A). The resulting mice were designated H19Ku site–.
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To determine whether imprinting was affected by the mutations, we examined the expression of both H19 and Igf2 in reciprocal crosses between Mus domesticus mice carrying the H19Ku site– mutation and Mus castaneus mice. Figure 5C illustrates that the M. domesticus-derived H19 mRNA was transcribed from the mutant maternally inherited chromosome at levels comparable to a wild-type chromosome (lanes 1 and 2) and was appropriately silenced on the paternal chromosome (lanes 3 and 4). Likewise, the expression of the M. domesticus Igf2 gene displayed normal levels of expression on the paternal mutant chromosome (lanes 7 and 8) and no expression from the maternally inherited mutant chromosome (lanes 5 and 6). Thus, we could find no evidence for a role for the Ku70/80 binding sites in the establishment or maintenance of imprinting at the H19/Igf2 locus.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was initiated in order to identify additional factors that might play a role in the complex regulation of these imprinted genes. In vivo footprinting findings had suggested that such factors were present on the maternal ICR in the soma (44, 45). Using extracts prepared from neonatal liver as well as other tissues, we identified a ubiquitous protein complex that bound specifically to two identical binding sites in HS1 and HS2. Upon purification these proteins were identified as the heterodimer composed of Ku70 and Ku80. The two Ku70/80 sites in the H19 ICR do not appear to play an important role in either the establishment or maintenance of imprinting, based on our finding that mutations that eliminate in vitro binding have no physiological effect in vivo. This is consistent with chromatin immunoprecipitation results that suggested that Ku70/80 is not bound to the sites, at least in adult liver.
The binding site we have identified is a novel example of a high-affinity and sequence-specific Ku70/80 pause site. Ku70 and Ku80 are highly conserved proteins that play a central role in repairing double-strand breaks by the nonhomologous end-joining pathway and in V(D)J recombination (13, 21-23, 38, 47, 52). The complex has also been implicated in genome surveillance, the maintenance of telomeres, DNA replication, and the regulation of transcription (14). In some, but not all, of these processes Ku70/80 associates with the catalytic subunit of DNA-dependent protein kinase.
The role of Ku in nonhomologous recombination is thought to occur via its ability to bind with high affinity to free DNA ends and recruit DNA-dependent protein kinase and the rest of the complex required for end joining and repair (13, 21). Ku's affinity for free ends is rationalized by the crystal structure of the Ku complex, which predicts a preformed circle that can surround the DNA end and, in the presence of Mg2+, translocate along it (49). The C terminus of Ku70 contains a SAP DNA binding domain that packs against the base of Ku80 in the absence of DNA. However, when the crystal structure was solved with DNA, the SAP domain was disordered and in close apposition to the DNA (49). This suggests that during translocation, the SAP domain may be in a position to make contacts with individual base pairs. Our finding that the ATCGAT binding activity is dependent upon Mg2+ supports the view that the ATCGAT binding site is a high-affinity pause site.
Given the observation that ATCGAT is a high-affinity site for Ku70/80, we used BLAST to determine the distribution of this site in the mouse genome. We found that the ATCGAT sites are frequently found in LINE-1 retrotransposons. A role for Ku70/80 in transposition has been proposed for the P transposable element (P element) in Drosophila melanogaster (2, 37). P elements transpose through a cut-and-paste mechanism whereby the P element leaves its mobilization site and inserts itself into the new position as a double-stranded DNA molecule (30). Transposition requires the presence of 31-bp inverted repeats that flank the mobilized DNA and are also left behind at the site of mobilization (37). These inverted repeats contain sequence-specific binding sites for the Drosophila homolog of Ku70/80 (2). The repair of double-strand breaks after P element excision was severely affected in a strain with a mutation in the Ku70 gene (2). Thus, it is likely that Ku70 plays a role in NHEJ at the site of mobilization as well as the site of insertion.
In contrast, LINE-1 transposition in the mouse occurs through a copy-and-paste model where a single-stranded RNA copy of the element is transcribed at the mobilization site (34). This single-stranded RNA encodes an endonuclease-reverse transcriptase that creates a nick at the insertion site and uses the exposed 3' end to prime the synthesis of cDNA (17, 34, 36). Following the conversion of the cDNA to double-stranded DNA, a double-strand break is generated at the insertion site and the ends are joined, likely through the NHEJ pathway (34). It is at this step that the Ku70/80 binding sites in the LINE-1 element might play a role by sequestering the complex on the double-stranded DNA before end joining.
Once ligation has occurred, the Ku70/80 circular complex is trapped on the DNA (39). This complex, which would only occur after nonhomologous recombination, could serve as a signal to the cell that a new transposition event has occurred. Retrotransposons threaten the host genome by increasing the frequency of rearrangements via homologous recombination between nonallelic repeats, production of chimeric transcripts that combine retrotransposon and cellular sequences, and transcriptional interference from transcripts originating from retrotransposon promoters (12). In response, the mammalian genome attempts to shut down the process by methylating retrotransposon sequences (4, 50). In this cycle, it is crucial that the genome be able to detect new retrotranspositions so that they can be inactivated by methylation. It is possible that the binding of Ku70/80 to internal ATCGAT pause sites serves as such a mark.
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ACKNOWLEDGMENTS |
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S.M.T. was an investigator of Howard Hughes Medical Institute. This work was supported by a grant from the National Institute for General Medical Sciences.
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. Beall, E. L., A. Admon, and D. C. Rio. 1994. A Drosophila protein homologous to the human p70 Ku autoimmune antigen interacts with the P transposable element inverted repeats. Proc. Natl. Acad. Sci. USA 91:12681-12685.
3. Bell, A. C., and G. Felsenfeld. 2000. Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405:482-485.[CrossRef][Medline]
4. Bestor, T. H. 2003. Cytosine methylation mediates sexual conflict. Trends Genet. 19:185-190.[CrossRef][Medline]
5. Bowman, A. B., J. M. Levorse, R. S. Ingram, and S. M. Tilghman. 2003. Functional characterization of a testis-specific DNA binding activity at the H19/Igf2 imprinting control region. Mol. Cell. Biol. 23:8345-8351.
6. Davis, T. L., J. M. Trasler, S. B. Moss, G. J. Yang, and M. S. Bartolomei. 1999. Acquisition of the H19 methylation imprint occurs differentially on the parental alleles during spermatogenesis. Genomics 58:18-28.[CrossRef][Medline]
7. Davis, T. L., G. J. Yang, J. R. McCarrey, and M. S. Bartolomei. 2000. The H19 methylation imprint is erased and re-established differentially on the parental alleles during male germ cell development. Hum. Mol. Genet. 9:2885-2894.
8. DeChiara, T. M., A. Efstratiadis, and E. J. Robertson. 1990. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 345:78-80.[CrossRef][Medline]
9. de Vries, E., W. Vandriel, W. G. Bergsma, A. C. Amberg, and P. C. Vandervliet. 1989. HeLa nuclear-protein recognizing DNA termini and translocating on DNA forming a regular DNA mulitmeric complex. J. Mol. Biol 345:65-78.
10. Difilippantonio, M. J., J. Zhu, H. T. Chen, E. Meffre, M. C. Nussenzweig, E. E. Max, T. Ried, and A. Nussenzweig. 2000. DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation. Nature 404:510-514.[CrossRef][Medline]
11. Drewell, R. A., C. J. Goddard, J. O. Thomas, and M. A. Surani. 2002. Methylation-dependent silencing at the H19 imprinting control region by MeCP2. Nucleic Acids Res. 30:1139-1144.
12. Druker, R., and E. Whitelaw. 2004. Retrotransposon-derived elements in the mammalian genome: a potential source of disease. J. Inherit. Metab. Dis. 27:319-330.[Medline]
13. Dvir, A., S. R. Peterson, M. W. Knuth, H. Lu, and W. S. Dynan. 1992. Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II. Proc. Natl. Acad. Sci. USA 89:11920-11924.
14. Dynan, W. S., and S. Yoo. 1998. Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids. Nucleic Acids Res. 26:1551-1559.
15. Engel, N., A. G. West, G. Felsenfeld, and M. S. Bartolomei. 2004. Antagonism between DNA hypermethylation and enhancer-blocking activity at the H19 DMD is uncovered by CpG mutations. Nat. Genet. 36:883-888.[CrossRef][Medline]
16. Fedoriw, A. M., P. Stein, P. Svoboda, R. M. Schultz, and M. S. Bartolomei. 2004. Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting. Science 303:238-240.
17. Feng, Q., J. V. Moran, H. H. Kazazian, Jr., and J. D. Boeke. 1996. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell 87:905-916.[CrossRef][Medline]
18. Feuerman, M. H., R. Godbout, R. S. Ingram, and S. M. Tilghman. 1989. Tissue-specific transcription of the mouse alpha-fetoprotein gene promoter is dependent on HNF-1. Mol. Cell. Biol. 9:4204-4212.
19. Giffin, W., J. Kwast-Welfeld, D. J. Rodda, G. G. Prefontaine, M. Traykova-Andonova, Y. Zhang, N. L. Weigel, Y. A. Lefebvre, and R. J. Hache. 1997. Sequence-specific DNA binding and transcription factor phosphorylation by Ku autoantigen/DNA-dependent protein kinase. Phosphorylation of Ser-527 of the rat glucocorticoid receptor. J. Biol. Chem. 272:5647-5658.
20. Giffin, W., H. Torrance, D. J. Rodda, G. G. Prefontaine, L. Pope, and R. J. Hache. 1996. Sequence-specific DNA binding by Ku autoantigen and its effects on transcription. Nature 380:265-268.[CrossRef][Medline]
21. Gottlieb, T. M., and S. P. Jackson. 1993. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 72:131-142.[CrossRef][Medline]
22. Gu, Y., S. Jin, Y. Gao, D. T. Weaver, and F. W. Alt. 1997. 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.
23. Gu, Y., K. J. Seidl, G. A. Rathbun, C. Zhu, J. P. Manis, N. van der Stoep, L. Davidson, H. L. Cheng, J. M. Sekiguchi, K. Frank, P. Stanhope-Baker, M. S. Schlissel, D. B. Roth, and F. W. Alt. 1997. Growth retardation and leaky SCID phenotype of Ku70-deficient mice. Immunity 7:653-665.[CrossRef][Medline]
24. Hark, A. T., C. J. Schoenherr, D. J. Katz, R. S. Ingram, J. M. Levorse, and S. M. Tilghman. 2000. CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405:486-489.[CrossRef][Medline]
25. Hark, A. T., and S. M. Tilghman. 1998. Chromatin conformation of the H19 epigenetic mark. Hum. Mol. Genet. 7:1979-1985.
26. Jones, B. K., J. Levorse, and S. M. Tilghman. 1998. Igf2 imprinting does not require its own DNA methylation or H19 RNA. Genes Dev. 12:2200-2207.
27. Jurka, J. 2000. Repbase update: a database and an electronic journal of repetitive elements. Trends Genet. 16:418-420.[CrossRef][Medline]
28. Kaffer, C. R., M. Srivastava, K. Y. Park, E. Ives, S. Hsieh, J. Batlle, A. Grinberg, S. P. Huang, and K. Pfeifer. 2000. A transcriptional insulator at the imprinted H19/Igf2 locus. Genes Dev. 14:1908-1919.
30. Kaufman, P. D., and D. C. Rio. 1992. P element transposition in vitro proceeds by a cut-and-paste mechanism and uses GTP as a cofactor. Cell 69:27-39.[CrossRef][Medline]
31. Khosla, S., A. Aitchison, R. Gregory, N. D. Allen, and R. Feil. 1999. Parental allele-specific chromatin configuration in a boundary-imprinting-control element upstream of the mouse H19 gene. Mol. Cell. Biol. 19:2556-2566.
32. Leighton, P. A., R. S. Ingram, J. Eggenschwiler, A. Efstratiadis, and S. M. Tilghman. 1995. Disruption of imprinting caused by deletion of the H19 gene region in mice. Nature 375:34-39.[CrossRef][Medline]
33. Loukinov, D. I., E. Pugacheva, S. Vatolin, S. D. Pack, H. Moon, I. Chernukhin, P. Mannan, E. Larsson, C. Kanduri, A. A. Vostrov, H. Cui, E. L. Niemitz, J. E. Rasko, F. M. Docquier, M. Kistler, J. J. Breen, Z. Zhuang, W. W. Quitschke, R. Renkawitz, E. M. Klenova, A. P. Feinberg, R. Ohlsson, H. C. Morse III, and V. V. Lobanenkov. 2002. BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma. Proc. Natl. Acad. Sci. USA 99:6806-6811.
34. Luan, D. D., M. H. Korman, J. L. Jakubczak, and T. H. Eickbush. 1993. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72:595-605.[CrossRef][Medline]
35. Marshak, D. R. 1996. Strategies for protein purification and characterization: a laboratory course manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
36. Mathias, S. L., A. F. Scott, H. H. Kazazian, Jr., J. D. Boeke, and A. Gabriel. 1991. Reverse transcriptase encoded by a human transposable element. Science 254:1808-1810.
37. Mullins, M. C., D. C. Rio, and G. M. Rubin. 1989. cis-acting DNA sequence requirements for P-element transposition. Genes Dev. 3:729-738.
38. Nussenzweig, A., C. Chen, V. da Costa Soares, M. Sanchez, K. Sokol, M. C. Nussenzweig, and G. C. Li. 1996. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 382:551-555.[CrossRef][Medline]
39. Paillard, S., and F. Strauss. 1991. Analysis of the mechanism of interaction of simian Ku protein with DNA. Nucleic Acids Res. 19:5619-5624.
40. Pant, V., S. Kurukuti, E. Pugacheva, S. Shamsuddin, P. Mariano, R. Renkawitz, E. Klenova, V. Lobanenkov, and R. Ohlsson. 2004. Mutation of a single CTCF target site within the H19 imprinting control region leads to loss of Igf2 imprinting and complex patterns of de novo methylation upon maternal inheritance. Mol. Cell. Biol. 24:3497-3504.
41. Park, K. Y., E. A. Sellars, A. Grinberg, S. P. Huang, and K. Pfeifer. 2004. The H19 differentially methylated region marks the parental origin of a heterologous locus without gametic DNA methylation. Mol. Cell. Biol. 24:3588-3595.
42. Schild-Poulter, C., D. Matheos, O. Novac, B. Cui, W. Giffin, M. T. Ruiz, G. B. Price, M. Zannis-Hadjopoulos, and R. J. Hache. 2003. Differential DNA binding of Ku antigen determines its involvement in DNA replication. DNA Cell Biol. 22:65-78.[CrossRef][Medline]
43. Schoenherr, C. J., J. M. Levorse, and S. M. Tilghman. 2003. CTCF maintains differential methylation at the Igf2/H19 locus. Nat. Genet. 33:66-69.[CrossRef][Medline]
44. Szabo, P., S. H. Tang, A. Rentsendorj, G. P. Pfeifer, and J. R. Mann. 2000. Maternal-specific footprints at putative CTCF sites in the H19 imprinting control region give evidence for insulator function. Curr. Biol. 10:607-610.[CrossRef][Medline]
45. Szabo, P. E., G. P. Pfeifer, and J. R. Mann. 2004. Parent-of-origin-specific binding of nuclear hormone receptor complexes in the H19-Igf2 imprinting control region. Mol. Cell. Biol. 24:4858-4868.
46. Szabo, P. E., S. H. Tang, M. R. Reed, F. J. Silva, W. M. Tsark, and J. R. Mann. 2002. The chicken beta-globin insulator element conveys chromatin boundary activity but not imprinting at the mouse Igf2/H19 domain. Development 129:897-904.
47. Taccioli, G. E., T. M. Gottlieb, T. Blunt, A. Priestley, J. Demengeot, R. Mizuta, A. R. Lehmann, F. W. Alt, S. P. Jackson, and P. A. Jeggo. 1994. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science 265:1442-1445.
48. Thorvaldson, J. L., K. L. Duran, and M. S. Bartolomei. 1998. Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2. Genes Dev. 12:3693-3702.
49. Walker, J. R., R. A. Corpina, and J. Goldberg. 2001. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412:607-614.[CrossRef][Medline]
50. Walsh, C. P., and T. H. Bestor. 1999. Cytosine methylation and mammalian development. Genes Dev. 13:26-34.
51. Zhang, W. W., and M. Yaneva. 1992. On the mechanisms of Ku protein binding to DNA. Biochem. Biophys. Res. Commun. 186:574-579.[CrossRef][Medline]
52. Zhu, C., M. A. Bogue, D. S. Lim, P. Hasty, and D. B. Roth. 1996. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell 86:379-389.[CrossRef][Medline]
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