©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Specific Alu Binding Protein from Human Sperm Chromatin Prevents DNA Methylation (*)

(Received for publication, April 4, 1995; and in revised form, June 5, 1995)

Igor N. Chesnokov (1) Carl W. Schmid (1) (2)(§)

From the  (1)Section of Molecular and Cellular Biology and (2)Department of Chemistry, University of California, Davis, California 95616

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A protein from human sperm nuclei that specifically binds to Alu DNA repeats has been purified. The specific DNA binding site of this protein within the Alu sequence has been mapped by methylation interference and electrophoretic mobility shift assays. This sperm Alu binding protein selectively protects Alu elements from methylation in vitro and may be responsible for the unmethylated state of Alu sequences in the male germ line resulting in a parent-specific differential inheritance of Alu methylation.


INTRODUCTION

A significant fraction of human DNA consists of roughly one million Alu sequences that are broadly distributed throughout the genome and unusually rich in CpG dinucleotides(1, 2) . The CpG dinucleotide is the target for the mammalian DNA methyltransferase, and in vertebrates, CpG dinucleotides are indeed frequently methylated at position 5 of cytosine. The pattern of methylation of Alu in human sperm DNA is remarkably different from that in somatic tissue DNA. Despite their almost complete methylation in somatic tissues and oocytes, Alu repeats are hypomethylated in the male germ line(3, 4, 5, 6) . Differential methylation of Alu repeats may be associated with genomic imprinting in germ line cells or reflect differences in Alu transcriptional activity between these cell types.

However, CpG islands in housekeeping genes remain unmethylated(7) . One model to explain the selective methylation of DNA sequences proposes that steric hindrance by protein factors excludes DNA methyltransferase from CpG islands(7) . In agreement with this model, Sp1 sites located within the mouse or hamster adenine phosphoribosyltransferase gene promoter are required to protect a flanking CpG island from de novo methylation(8, 9) .

We hypothesize that a similar mechanism might explain undermethylation of Alu repeats in the male germ line. Here we describe a specific Alu binding protein (later called SABP (^1)for sperm Alu binding protein) isolated from human sperm chromatin that selectively protects Alu elements from methylation in vitro and may be responsible therefore for the unmethylated state of Alu sequences in the male germ line.


EXPERIMENTAL PROCEDURES

Purification of SABP

Sperm nuclei were isolated as described(10) . Proteins were extracted by 0.7 M NaCl in buffer containing 20 mM Tris-HCl (pH 7.5), 10% (v/v) glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM DTT (dithiothreitol), 2 mM MgCl(2). To purify SABP, sperm nuclear extract was applied to a Superose 12 (FPLC) HR 10/30 column (Pharmacia Biotech Inc.) at 0.5 ml/min in buffer containing 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 5% glycerol, 1 mM MgCl(2), 1 mM DTT, and 0.5-ml fractions were collected. In this and in subsequent purification procedures, fractions containing SABP activity were detected by electrophoretic mobility shift assay (EMSA), pooled, and concentrated using a Centricon 30 filter (Amicon). The activity from the Superose 12 column was applied to a Mono S (FPLC) HR 5/5 column (Pharmacia) in buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5% glycerol, 1 mM MgCl(2), 1 mM DTT. Bound proteins were eluted using a linear NaCl gradient (0.1-1.1 M, 25 ml) at 1.0 ml/min flow, and 1.0-ml fractions were collected. Sequence-specific affinity chromatography was performed essentially as described(11) . Complementary oligonucleotides 5` GATCTGTAATCCCA and 5` GATCTGGGATTACA were annealed, phosphorylated, and ligated(11) . The DNA oligomers were covalently attached to CNBr-activated Sepharose 4B according to the manufacturer's recommendations (Pharmacia). The concentration of covalently bound DNA in the affinity resin was 15-20 µg/ml of resin. Protein was applied to this column by gravity flow in wash buffer, which contains 0.1 M NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM MgCl(2), 1 mM DTT, and 10% (v/v) glycerol. Bound proteins were eluted with wash buffer adjusted to 1 M NaCl. Proteins were stored at -20 °C (for a short period of time) in wash buffer or at -70 °C in wash buffer possessing 50% glycerol.

Methylation Protection Assay

The methylation protection assay was based on a described procedure(12) . A PCR II plasmid (Invitrogene) with an Alu insertion (GenBank accession number U21664) (20-30 ng) called clone p280t9 was preincubated with SABP in the amounts indicated for 15 min at room temperature in the presence of 100-200 ng of DNA in buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl(2), 1 mM DTT, and 5% glycerol. Then 10 units of SssI methyltransferase were added together with S-adenosylmethionine (160 mM final concentration), and incubations were continued for 30 min at 30 °C. DNA was extracted with phenol/chloroform and chloroform and precipitated with ethanol. DNA (200 ng maximum) was digested with 10 units of the indicated restriction enzymes according to the manufacturer's recommended conditions. Restriction fragments were separated on a 1.2% agarose gel, transferred to a nylon membrane (Hybond N+, Amersham Corp.), and blotted with specific Alu or vector probes(5) . An oligonucleotide overlapping sequence of positions 81-103 within the Alu consensus sequence was used as the Alu-specific probe, and hybridization was performed as described(5) . The vector-specific probe was primer 1233 (New England Biolabs).

UV Cross-linking

UV cross-linking was performed essentially as described(13) . In brief, the gel-purified Alu insert from clone p280t9 was uniformly labeled and incubated with SABP in the presence of 1 µg of nonspecific DNA for 15 min at room temperature in buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM MgCl(2), and 5% (v/v) glycerol in a volume of 30 µl. Samples were subjected to UV irradiation at 254 nm using a Mineralight lamp (UVG-11) (Ultra-Violet Products, Inc.) for 20 min at room temperature. Then 70 µl of 5 mM CaCl(2) containing 10 mg/ml DNase I was added, and the samples were incubated for an additional 20 min at 37 °C. Proteins were precipitated with trichloroacetic acid analyzed by SDS-polyacrylamide gel electrophoresis, followed by autoradiography.

Other Methods

HeLa nuclear extracts were prepared as described(14) . EMSA was performed as described previously(15) . Unless indicated differently, 0.5 µg of DNA was included as a nonspecific competitor in all EMSA experiments. Oligonucleotides matching positions 1-23, 17-38, 35-55, 54-76, 76-98, and 100-120 in the Alu consensus sequence (1) were used in EMSA competition assay. For brevity, the oligonucleotide matching positions 17-38 (i.e. TCACGCCTGTAATCCCAGCAC) in the Alu consensus sequence is called oligonucleotide 17-38. The Alu insert from clone p280t9 was used for the methylation interference assay(16) , DNase I footprinting (16) , and Southwestern blotting analysis(17) , which were performed as described. Bio-Rad high range SDS-polyacrylamide gel electrophoresis molecular weight standards were used as markers.


RESULTS

Purification and Sequence Specificity of SABP

As indicated by a diffuse band (``complex 1'') in an EMSA experiment, crude protein extract from sperm nuclei contains a barely detectable DNA binding activity that may be Alu-specific (Fig. 1, lanes2 and 3). Using EMSA as the assay, this activity was partially purified by sequential FPLC chromatography on a Superose 12 column (Fig. 2, A and B; Table 1) followed by chromatography on a Mono S column (Fig. 3, A and B; Table 1). The increase in binding activity observed after the first chromatographic step suggests that a binding inhibitor present in the crude extract is removed by partial purification.


Figure 1: EMSA identification of SABP using a labeled Alu probe. Lane1 is a control with no added protein. Protein from the crude sperm nuclear extract (3 µg) was used for lanes2 and 3, and partially purified protein (0.3 µg) from the Mono S fraction was used for lanes4 and 5 and 10-12. HeLa nuclear extract (5 µg) was used for lanes 6-9. DNA was added as a nonspecific competitor for lanes 2, 4, and 6 and 8-12 (0.5 µg) and lanes 3, 5, and 7 (1 µg). Double-stranded oligonucleotide 17-38 was used as a specific competitor for lanes8 and 11 (20 ng) and lanes9 and 12 (40 ng). C1 and C2 indicate the positions of Alu-SABP complexes 1 and 2, and F indicates the position of the free Alu probe.




Figure 2: Superose 12 (FPLC) chromatography of sperm nuclear extract. A shows the protein elution profile from Superose 12. SABP activity was assayed by EMSA using 10 µl from the resulting fractions (B). Fractions 17-22 were pooled for further purification (Table 1). C1 and C2 indicate the positions of Alu-SABP complexes 1 and 2, and F indicates the position of the free Alu probe.






Figure 3: Mono S (FPLC) chromatography of partially purified SABP. A shows the protein elution profile from the Mono S column. SABP activity was assayed by EMSA using 10 µl from the resulting fractions (B). Fractions 19-21 were pooled for further purification (Table 1). C1 and C2 indicate the positions of Alu-SABP complexes 1 and 2, and F indicates the position of the free Alu probe.



Following partial purification, complex 1 forms a sharper EMSA band, and a second, diffuse band at higher molecular weights (``complex 2'') becomes more apparent (Fig. 1, lanes4 and 5). Competition experiments using double-stranded oligonucleotides overlapping the entire left monomer of the dimeric Alu consensus sequence (sequence positions 1-120) were used to define the region within the Alu repeat responsible for complex formation (see ``Experimental Procedures''). Only one, oligonucleotide 17-38, corresponding to the region immediately downstream from the A box competes for binding (Fig. 1, lanes10-12, and data not shown). The DNA binding activity initially observed in crude extracts is sequence-specific, and we refer to it as SABP.

SABP activity is not detected in total and partially purified extracts from HeLa cells (Fig. 1, lanes 6-9, and data not shown). The binding pattern of HeLa proteins with an Alu sequence(18, 19) differs from that of SABP binding (Fig. 1, lanes 6 and 7). Also, the binding of HeLa proteins to the Alu repeat is not disrupted by the double-stranded oligonucleotide 17-38 that competes for SABP binding (Fig. 1, lanes8 and 9).

Affinity-purified SABP forms sharper EMSA bands for the two Alu-DNA complexes than those formed with crude extracts and partially purified samples (Fig. 4A). As shown by a methylation interference assay (Fig. 4B) and DNase 1 footprinting (data not shown), SABP protects a region downstream from the A box promoter element for RNA polymerase III. The region identified by the methylation interference assays maps within Alu sequence positions 25-33 (1) in excellent agreement with the binding site deduced from the EMSA competition studies described above. This sequence is present twice within the dimeric structure of human Alu elements(1) , potentially accounting for SABP complexes 1 and 2 reported above. In conclusion, the affinity-purified sample is enriched for the original SABP binding activity (Table 1, Fig. 4A).


Figure 4: A, for EMSA, affinity-purified SABP (30 ng) was incubated with an end-labeled Alu fragment (5 ng) in the presence of increasing amounts (lane 1, 200 ng; lane2, 500 ng; lane3, 1000 ng; lane4, 2000 ng; lane5, 4000 ng; lane6, 6000 ng) of DNA. Lane7 shows a control without added SABP. C1 and C2 indicate positions of Alu-SABP complexes 1 and 2, and F indicates the position of the free Alu probe. B, for the methylation interference assay, fragments overlapping region 1-76 of the Alu repeat were subjected to G-specific chemical modification. Lane1 shows G-specific chemical cleavage of fragments that did not bind to affinity-purified SABP, and lane2 shows G-specific sequencing of fragments that did bind to SABP. The sequence responsible for binding, as indicated, corresponds to positions 25-33 within the Alu consensus sequence. The position of the RNA polymerase III A box promoter element is also indicated.



A Southwestern blotting experiment using a radioactive Alu sequence probe was performed to determine the molecular weight of SABP (Fig. 5A). A strong band at a 60 kDa position in sperm extracts is not apparent in extracts prepared from HeLa cells (Fig. 5A). These data are confirmed by a UV cross-linking experiment showing the molecular mass of SABP to be 60 kDa (Fig. 5B). Double-stranded oligonucleotide 17-38 competes for SABP in the UV cross-linking experiment (Fig. 5B). In agreement with the results from the Southwestern blot and UV cross-linking experiments, purified SABP consists of a 60-kDa polypeptide on a silver stained gel (Fig. 5C). A weak band of 50 kDa is also barely detectable on the same gel. The 60-kDa band is detectable at a 15-fold dilution (data not shown) suggesting limits on the abundance of other contaminating proteins.


Figure 5: A, for Southwestern blot analysis, 20 µg of protein from HeLa (HeLaex.) and sperm nuclear extracts (SNE) was added to each lane, and an end-labeled full-length Alu repeat was used as the radioactive probe. Positions of protein molecular mass markers are indicated, and the arrow indicates SABP activity. The probe also forms complexes at the top of the gel with sperm nuclear extract proteins trapped in the well. B, for UV cross-linking analysis, a uniformly labeled full-length Alu probe (20 ng) was cross-linked with 50 ng of affinity-purified SABP and digested with DNase I. Double-stranded oligonucleotide 17-38 (50 ng) was used as a specific competitor for lane2. C, 2 µg of sperm nuclear extract (lane1) and 300 ng of affinity-purified SABP (lane2) were examined by silver staining.



Purified SABP was digested with chymotrypsin, and the resulting peptides were separated and sequenced. The sequences obtained, GVFEPIGDEPRPD, QFGHQPGGN, FIQCVAETMK, and GLVFVL, do not show any significant matches with other proteins.

SABP Specifically Blocks Alu Methylation in Vitro

An in vitro methylation assay was employed (12) to test whether SABP can specifically protect a cloned Alu element from methylation without affecting the methylation of flanking vector sequences (Fig. 6). SABP was preincubated with the plasmid clone p280t9, which was then incubated with SssI (CpG) methyltransferase. Methylation of the clone was tested with methyl-sensitive restriction enzymes (BstUI and HhaI) using Southern blot analysis for the Alu insert (Fig. 6A) and for surrounding PCR II plasmid sequences (Fig. 6B). Cleavage of the single BstUI site within the Alu repeat (Fig. 6A, lane2) is, as expected, inhibited by in vitro methylation (Fig. 6A, lane1). However, increasing concentrations of SABP inhibit methylation of the Alu BstUI site (Fig. 6A, lanes3 and 4). As demonstrated by methylation of BstUI sites (12 sites) located in the vector sequence, this inhibition is Alu sequence-specific (Fig. 6B). SABP does not interfere with the nearly complete methylation of vector BstUI sites (Fig. 6B, lanes3 and 4), as compared with fully methylated (Fig. 6B, lane1) and fully unmethylated (Fig. 6B, lane2) vector controls. The differential methylation of Alu and vector CpGs provides an internal control for any nonspecific effect of SABP on methylation in vitro.


Figure 6: SABP selectively protects an Alu repeat from methylation in vitro. Southern blots of HindIII-XbaI-BstUI (lanes 1-4) or EcoRI-HhaI (lanes 5-8) digests of a cloned Alu repeat were probed with either an Alu-specific oligonucleotide (A) or a vector-specific oligonucleotide (B). In each case the predicted positions of BstUI uncut/cut and HhaI uncut/cut Alu and plasmid fragments are indicated. DNAs in lanes2 and 6 are unmethylated showing complete cleavage of the BstUI and HhaI sites, respectively. DNAs in lanes1 and 5 are methylated by SssI methyltransferase showing protection of the BstUI and HhaI sites, respectively. DNA samples in lanes3 and 7 were incubated with 50 ng of SABP, and samples in lanes4 and 8 were incubated with 80 ng of SABP.



These findings are confirmed by investigation of HhaI sites (Fig. 6, lanes 5-8). SABP binding fully protects the two Alu HhaI sites from methylation (Fig. 6A, lanes7 and 8) as compared with the unmethylated (Fig. 6A, lane6) and methylated (Fig. 6A, lane5) controls. Again this protection is highly specific to the cloned Alu insert and does not extend to the 31 vector HhaI sites (Fig. 6B). SABP does not protect vector sequences from methylation in vitro (Fig. 6B, lanes 7 and 8) as compared with the unmethylated (Fig. 6B, lane 6) and methylated (Fig. 6B, lane 5) vector controls. We conclude that SABP binding is sufficiently selective to protect an Alu element from in vitro methylation without significantly altering the methylation of adjacent CpGs.


DISCUSSION

The CpG content of human DNA is 1%, but the CpG content of Alu sequences approaches 9%. Alu repeats, which account for one-third of the 5`-methylcytosine residues in spleen DNA(3) , are also hypermethylated in many other somatic human cells (4, 5) and monkey oocytes(6) . In contrast, Alus are largely unmethylated in sperm DNA, whereas most potential methylation sites in sperm DNA are highly methylated(4, 5) .

Undermethylation of male germ line Alus might either cause or be caused by several male germ line specific requirements. 1) Methylation inhibits Alu transcriptional expression(4, 20, 21) , and interestingly the single documented example of Alu retrotransposition occurred in the developing male germ line(22) . Perhaps demethylation of Alus is required for their transcription during male gametogenesis. In agreement with a previous report(4) , we find RNA polymerase III-directed Alu transcripts in sperm cells using primer extension assay (data not shown). 2) Sperm chromatin, largely composed of nucleoprotamine as well as highly modified histones, is entirely different from chromatin in any other cell type(23, 24) . The peculiarities of these unusual chromatin structures might require a correspondingly unusual DNA methylation pattern. 3) Male and female gametes fulfill distinct roles in the early embryo as their expression is subject to genomic imprinting(25, 26, 27) . Differential germ line methylation of Alus might signal differences in gametic expression. Undoubtedly, other differences between male and female germ cells might cause or require differences in Alu methylation.

Since all DNA sequences that have been analyzed are unmethylated in primordial germ cells(28, 29) , we suspect that the patterns of Alu methylation in sperm and oocyte DNAs are also established during gametogenesis. The existence of SABP provides a simple explanation for how this pattern might be imposed. As assayed in vitro, SABP binding is sufficiently specific to selectively protect Alu CpGs from methylation. Whereas other CpGs in sperm DNA are hypermethylated, SABP binding might cause Alu hypomethylation. This model also provides an explanation for the presence of fully methylated and fully unmethylated Alus in sperm DNA(4, 5, 6) . Sperm DNA is segregated in a sequence-specific manner between nucleoprotamine and nucleohistone components(23) . The presence of SABP in one of these components and its absence in the other could result in the observed heterogeneity of Alu methylation. In analogy to this model, Sp1 binding has recently been implicated in protecting DNA sequences from methylation, thereby establishing a de novo methylation pattern(8, 9) . The activity reported here is evidently both tissue-specific and sequence-specific and therefore potentially responsible for generating a major difference in the methylation patterns inherited from male and female gametes.


FOOTNOTES

*
This research was supported by United States Public Health Service Grant GM 21346 and the Agricultural Experiment Station, University of California at Davis. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 916-752-9029; Fax: 916-752-3085.

^1
The abbreviations used are: SABP, sperm Alu binding protein; DTT, dithiothreitol; FPLC, fast protein liquid chromatography; EMSA, electrophoretic mobility shift assay.


REFERENCES

  1. Schmid, C. W., and Shen, C-K. J. (1985) in Molecular Evolutionary Genetics (MacIntyre, R. J., ed) pp. 323-358, Plenum Publishing Corp., New York
  2. Schmid, C. W., and Maraia, R.(1992)Curr. Opin. Genet. & Dev. 2,874-882 [Medline] [Order article via Infotrieve]
  3. Schmid, C. W. (1991)Nucleic Acids Res.19,5613-5617 [Abstract]
  4. Kochanek, S., Renz, D., and Doerfler, W.(1993)EMBO J.12,1141-1151 [Abstract]
  5. Hellmann-Blumberg, U., McCarthy Hintz, M. F., Gatewood, J. M., and Schmid, C. W.(1993) Mol. Cell. Biol.13,4523-4530 [Abstract]
  6. Rubin, C. M., VandeVoort, C. A., Teplitz, R. L., and Schmid, C. W.(1994) Nucleic Acids Res.22,5121-5127 [Abstract]
  7. Bird, A. P.(1986) Nature321,209-213 [Medline] [Order article via Infotrieve]
  8. MacLeod, D., Charlton, J., Mullins, J., and Bird, A. P.(1994)Genes & Dev.8,2282-2292
  9. Brandeis, M., Frank, D., Keshet, I., Siegfried, Z., Mendelsohn, M., Nemes, A., Temper, V., Razin, A., and Cedar, H.(1994)Nature371,435-438 [CrossRef][Medline] [Order article via Infotrieve]
  10. Zalensky, A. O., Yau, P., Breneman, J. W., and Bradbury, E. M.(1993)Mol. Reprod. Dev.36,164-173 [Medline] [Order article via Infotrieve]
  11. Kadonaga, J. T., and Tjian, R.(1986)Proc. Natl. Acad. Sci. U. S. A. 83,5889-5893 [Abstract]
  12. Kochanek, S., Renz, D., and Doerfler, W.(1993)Nucleic Acids Res. 21,2339-2342 [Abstract]
  13. Chodosh, L. A., Carthew, R. W., and Sharp, P. A.(1986)Mol. Cell. Biol. 6,4723-4733 [Medline] [Order article via Infotrieve]
  14. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G.(1983)Nucleic Acids Res. 11,1475-1489 [Abstract]
  15. Strauss, F., and Varshavsky, A.(1984)Cell37,889-901 [Medline] [Order article via Infotrieve]
  16. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, S., and Struhl, K. (eds) (1994) Current Protocols in Molecular Biology, pp. 12.3.1-12.3.3, John Wiley & Sons, New York _
  17. Lewis, J. D., Meehan, R. R., Henzel, W. L., Maurer-Fogy, I., Jeppesen, P., Klein, F., and Bird, A.(1992)Cell69,905-914 [Medline] [Order article via Infotrieve]
  18. Perelygina, L. M., Tomilin, N. V., and Podgornaya, O. I.(1987)Mol. Biol. Rep.12,111-116 [Medline] [Order article via Infotrieve]
  19. Chesnokov, I., Bozhkov, V., Popov, B., and Tomilin, N.(1991)Biochem. Biophys. Res. Commun.178,613-619 [Medline] [Order article via Infotrieve]
  20. Liu, W-M., and Schmid, C. W.(1993)Nucleic Acids Res.21,1351-1359 [Abstract]
  21. Liu, W-M., Maraia, R. J., Rubin, C. M., and Schmid, C. W.(1994)Nucleic Acids Res.22,1087-1095 [Abstract]
  22. Wallace, M. R., Andersen, L. B., Saulino, A. M., Gregory, P. E., Glover, T. W., and Collins, F. S.(1991)Nature353,864-866 [CrossRef][Medline] [Order article via Infotrieve]
  23. Gatewood, J. M., Cook, G. R., Balhorn, R., Bradbury, E. M., and Schmid, C. W.(1987) Science236,962-964 [Medline] [Order article via Infotrieve]
  24. Gatewood, J. M., Cook, G. R., Balhorn, R., Schmid, C. W., and Bradbury, E. M.(1990) J. Biol. Chem.256,20662-20666
  25. Surani, M. A. (1993)Nature366,302-303 [Medline] [Order article via Infotrieve]
  26. Razin, A., and Cedar, H. (1994)Cell77,473-476 [Medline] [Order article via Infotrieve]
  27. Barlow, D. P. (1994)Trends Genet.10,194-199 [CrossRef][Medline] [Order article via Infotrieve]
  28. Kafri, T., Ariel, M., Brandeis, M., Shemer, R., Urven, L., McCarrey, J., Cedar, H., and Razin, A.(1992)Genes & Dev.6,705-714
  29. Razin, A., and Cedar, H. (1993) DNA Methylation: Molecular Biology and Biological Significance (Jost, J. P., and Saluz, H. P., eds) pp. 343-357, Birkhauser Verlag, Switzerland

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.