(Received for publication, April 4, 1995; and in revised form, June 5, 1995)
From the
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.
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 (
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.
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
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
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.
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. 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.
)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.
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. 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
, 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
, 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
, 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
, 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
, 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
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.
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.
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.
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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.