Article |
2 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Address correspondence to Katherine L. Wilson, Dept. of Cell Biology, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: (410) 955-1801. Fax: (410) 955-4129. E-mail: klwilson{at}jhmi.edu
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Abstract |
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Key Words: HIV; retroviral preintegration complex; nucleus; emerin; nuclear envelope
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Introduction |
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In a two-hybrid screen (Furukawa, 1999), BAF was found to interact with lamin associated polypeptide (LAP)2ß, a nuclear inner membrane protein (Foisner and Gerace, 1993). Furthermore, BAF appeared to localize predominantly on chromatin in the nucleus (Furukawa, 1999), suggesting that BAF might function in the nucleus, despite its original purification from NIH3T3-cell cytosol (Lee and Craigie, 1998). LAP2ß is an abundant nuclear membrane protein with an 40-residue motif known as the LEM (LAP2, emerin, MAN1) domain. The LEM domain defines a growing family of nuclear membrane proteins (Lin et al., 2000), whose members include multiple splicing isoforms of LAP2 (Berger et al., 1996; Gant et al., 1999), plus emerin, MAN1 (Lin et al., 2000), Lem-3 (Lee et al., 2000), and otefin (Goldberg et al., 1998). Site-directed mutagenesis studies of LAP2 (Shumaker et al., 2001) and emerin (Lee et al., 2001) show that the LEM motif is essential for binding to BAF and to BAFDNA complexes. LEM domain structure, solved by NMR (Cai et al., 2001; Wolff et al., 2001), complements both the shape and hydrophobicity of a surface on the BAF dimer (Umland et al., 2000). Importantly, BAF can bind simultaneously to both LAP2 and DNA in vitro (Shumaker et al., 2001), suggesting that BAF might play a key role in attaching chromatin to the inner nuclear membrane. In addition, both LAP2ß and emerin interact with nuclear intermediate filament proteins named lamins (Foisner and Gerace, 1993; Clements et al., 2000; Vaughan et al., 2001), which comprise a key structural element of the nucleus and play critical roles in nuclear assembly (for review see Stuurman et al., 1998; Cohen et al., 2001). Collectively, these results suggest an important hypothesis: by binding to LEM proteins, BAF might link chromatin directly to the inner nuclear membrane and indirectly to lamin filaments. This model predicts that BAF may be essential for nuclear assembly, or for the structural integrity of interphase nuclei, or both.
In this study, we used site-directed mutagenesis to define and map functionally important surfaces on the BAF dimer. Through biochemical assays, we identified residues required for BAF to bind to DNA or emerin, and residues important for BAF dimer interactions. We then tested wild-type human BAF protein and our 25 point mutants for their effects on nuclear assembly in Xenopus egg extracts (Lohka and Masui, 1983; Wilson and Newport, 1988). The wild-type and mutant BAF proteins fell into four classes with respect to their nuclear assembly phenotypes, and were interpreted based on the biochemical activities of each mutant. Our results support the hypothesis that BAF has fundamental roles during nuclear assembly, and that BAF interactions with both DNA and LEM proteins are critical for chromatin decondensation and nuclear envelope growth.
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Results |
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We did one further assay, to anticipate the behavior of each BAF mutant when added to Xenopus extracts containing wild-type Xenopus BAF. Selected His-tagged BAF mutants were incubated with equal amounts of 35S-labeled wild-type BAF and then immunoprecipitated using anti-His antibodies (Fig. 4, A and B). This subunit exchange assay tested the ability of wild-type 35S-BAF dimers (and/or oligomers) to exchange or oligomerize with BAF mutants. Compared with wild-type BAF, we expected that mutants with weaker dimer interactions would exchange more frequently with 35S-labeled wild-type BAF. The wild-type control showed relatively low (ratio 0.1) levels of subunit exchange or oligomerization with wild-type 35S-BAF (Fig. 4 C, left). The same low signal was seen for mutants 25E and 41E (Fig. 4 C, left) and 14A (Fig. 4 C, right), relative to input His-tagged wild-type BAF. Note that a low signal was consistent with either (a) stable dimerization with no exchange of subunits, or (b) complete failure to dimerize or oligomerize. Slightly higher signals were seen for mutants 18A, 53E, 54E, and 75E, but this difference may not be significant. However, wild-type 35S-BAF exchanged at abnormally high levels with mutants 47E and 51E (Fig. 4 C). We concluded that mutants 47E and 51E had high potential to exchange subunits (forming heterodimers or heterooligomers) with wild-type BAF, and that other mutants (18A, 53E, 54E, and 75E) had a slightly increased potential. The surprising result was that mutants 51E and 47E, which map to
-helix 3 and were predicted not to dimerize (Umland et al., 2000), can form homodimers and weak heterodimers/heterooligomers with wild-type BAF. However, another surface of BAF, distinct from helix 3, and not affected by mutations 47E and 51E, might independently mediate dimer or oligomer interactions.
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To determine if exogenous BAF also disrupted intact (preassembled) nuclei, we assembled nuclei in Xenopus extracts for 1 h, and then added 0.5 or 5 µM BAF dimers. Exogenous BAF had no obvious effect on chromatin or nuclear growth, relative to buffer-treated controls (unpublished data). However, this does not rule out roles for BAF in interphase nuclear structure or function, because we do not know if exogenous BAF can disrupt preassembled endogenous BAF complexes.
Transmission electron microscopy analysis of BAF-arrested nuclei
When imaged by transmission electron microscopy (TEM) after 2 h of assembly, the control (no addition) nuclei had typical nuclear membranes with nuclear pore complexes (NPCs) (Fig. 7 A, NPCs marked by asterisks). In contrast, nuclei arrested by 5 µM added BAF had patches of double membranes separated by gaps (Fig. 7 B, arrow), and areas devoid of membrane (Fig. 7 B). However, the most prominent abnormalities in these condensed nuclei involved chromatin. There was a compressed outer shell of electron-dense chromatin (Fig. 7 B, paired arrowheads) that appeared to encapsulate the interior chromatin. Indirect immunofluorescence with a lamin-specific antibody showed that this outer shell did not include lamins (Fig. S1 D, available at http://www.jcb.org/cgi/content/full/jcb.200202019/DC1). BAF was detected on these nuclei by immunofluorescence, concentrated at the periphery (Fig. S1 B, available at http://www.jcb.org/cgi/content/full/jcb.200202019/DC1). Furthermore, the interior chromatin had a novel lattice-like morphology (Fig. 7 B). This lattice morphology was not seen in the sperm chromatin template (Fig. 7 C), or in chromatin of GTPS-arrested nuclei (Boman et al., 1992). These dominant effects of BAF on chromatin structure during nuclear assembly are novel and exciting. We hypothesized that the outer shell of condensed chromatin and interior lattice were due to unregulated DNA-bridging activity of excess BAF.
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Given that Xenopus extracts contain about 12.5 µM endogenous BAF dimers, we reinterpreted Figs. 6 and 7 as follows. Increasing the total BAF concentration in Xenopus extracts by only 4% (adding 0.5 µM xBAF dimers) enhanced chromatin decondensation and nuclear growth, suggesting a positive role for BAF in nuclear assembly. However, increasing BAF by 20% (adding 2.5 µM xBAF) compacted chromatin and blocked nuclear growth. The strong dominant effects of 20% extra BAF suggested that BAF is normally regulated by binding partners that are stoichiometrically limiting, such as LEM proteins (or novel partners), but not DNA (see Discussion).
Phenotypic analysis of mutant BAFs in nuclear assembly reactions
Human and Xenopus BAF are nearly identical (Fig. 5 A), and had the same effects on nuclear assembly (Fig. 6). Therefore, we tested all 25 mutant human BAFs (hBAFs) in Xenopus nuclear assembly extracts. Each mutant was added to nuclear assembly reactions at concentrations of 0.5, 2.5, or 5 µM, incubated for 2 h, and imaged by light microscopy. BAF mutants fell into four phenotypic classes termed wild-type (decondensed-to-condensed), inactive, always condensed, and inactive-to-condensed (Fig. 8).
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The three always condensed mutants (14A, 18A, and 47E) caused chromatin to condense at all concentrations tested (Fig. 8 C). All three had normal binding to DNA (Table I), but then split into two subclasses: mutants 14A and 18A can bind emerin, but 47E cannot (Table I). We concluded that DNA (but not emerin) binding activity is required for the always condensed phenotype. When seen by TEM, nuclei assembled with low (0.5 µM) or high (5 µM) amounts of mutant 14A had thin (Fig. 9, A and F) or thick (Fig. 9, B and G) shells of condensed chromatin, respectively, and patches of double membranes. Residues 14 and 18 define the top surface of the BAF dimer (see below). Because these mutants bind normally to both DNA and emerin, yet cause the condensed phenotype, we conclude that the top surface of BAF has a novel function.
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The inactive-to-condensed class (mutants 9A, 41E, 53E, and 64E) had no effect at low concentration, but still condensed chromatin at 5 µM (Fig. 8 D). This class comprised at least two subclasses when examined by TEM, exemplified by mutants 41E (binds to DNA and emerin) and 53E (binds DNA, but not emerin). At 5 µM, mutant 41E compressed chromatin like wild-type BAF (unpublished data). The same was true for mutant 53E, except that 53E also completely abolished membrane binding to chromatin (Fig. 9, E and I). Mutant 53E, which binds DNA but not emerin, suggests that BAF binding to LEM proteins may be crucial for membrane attachment to chromatin.
Mapping phenotypes to structure
We then mapped our results on the surface structure of the BAF dimer. Fig. 10 shows corresponding ribbon (A) and surface (B) structures of the front view of the human BAF dimer (Umland et al., 2000). In this orientation, dsDNA molecules would bind to the left and right ends, and the LEM-binding domain faces the reader. At right, the dimer is rotated down 90°C to show its top surface (Fig. 10 B).
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Discussion |
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Our current model is that LEM proteins bind centrally on the BAF dimer (as seen in Fig. 10 B, front view), whereas DNA binds to the left and right sides (Fig. 10 C). We propose that residues Pro-14 and Lys-18, on top of the BAF dimer, define a novel functional surface, because mutations at these sites had no effects on dimerization or binding to emerin or DNA, yet caused a distinct nuclear arrest phenotype (always condensed). We propose that this surface of BAF mediates either the oligomerization of BAF dimers (Zheng et al., 2000), or a novel function. Selected aspects of BAF function are discussed below.
DNA-binding activity
We identified only four mutants (6E, 25E, 27E, and 46E) that failed to bind 2006,000 bp dsDNA, and six mutants (9A, 25Q, 27Q, 54E, and 75E) with reduced DNA binding activity. The remaining 15 mutants bound to DNA as well as wild-type BAF in our assay, in contrast to previous reports that mutants 8E, 32E, 33E, 53E, 60E, and 64E do not bind short (21-bp) DNA fragments (Cai et al., 1998; Umland et al., 2000). We attribute this difference to DNA length. In previous studies, short DNA fragments were chosen deliberately to avoid BAF-mediated DNA-bridging activity (Lee and Craigie, 1998; Zheng et al., 2000). Thus, short-DNA assays may fail to detect BAF mutants that can bind longer DNA. Our conclusions are supported by evidence that mutations at residues Lys-6 and Lys-18 do not abolish BAF's barrier-to-autointegration activity (Harris and Engelman, 2000), which requires BAF binding to retroviral DNA. We suggest that our assay identified physiologically-relevant residues required to bind DNA. Our findings point to residues 6, 25, 27, and 46 (Fig. 10 C) as critical for BAF binding to DNA. These residues map predominantly to the left and right sides of the BAF dimer (Fig. 10 C), consistent with DNA binding sites predicted from the BAF crystal structure (Umland et al., 2000).
Model: BAF dimers have two conformational states
The three residues (51, 53, and 54) essential for binding to emerin clustered as shown in light blue (Fig. 10 B), and defined a LEM-domain binding site on the dimer. This experimentally determined LEM-binding valley is consistent with the shape and hydrophobicity of the LEM domain of LAP2, and with chemical shift mapping results (Cai et al., 2001). One major finding from our work was that all residues required to bind DNA were also important for emerin binding (see Table I). This finding supports two conformational states for BAF: a DNA-bound conformation (which enhances affinity for LEM proteins), and a DNA-free conformation. A DNA-induced conformational change in BAF was independently deduced from our previous mutational analysis of the LEM domain of LAP2; we found that a LEM domain carrying the m13 mutation could not bind BAF, but did bind BAF-DNA complexes, implying that the LEM domain sees a different BAF structure in the presence of DNA (Shumaker et al., 2001).
BAF residues essential for binding to emerin
We identified two mutants with wild-type DNA-binding that were completely defective for emerin binding: mutants 47E and 53E (Table I). When added at 5 µM, both mutants blocked membrane attachment to chromatin, suggesting that interactions between BAF and LEM proteins may be critical for nuclear membrane recruitment during nuclear assembly. Native residue Gly-47 is buried, and our data suggest that introducing a negative charge at this site caused BAF to form weak dimers. Given that the LEM-binding site spans the dimer interface, we conclude that BAF dimerization may be essential for binding to emerin. At low concentrations, mutant 47E blocked nuclear growth even though by TEM the chromatin and enclosing nuclear membranes appeared normal, except for a possible lack of NPCs. Growth arrest by 47E could be explained by lack of nucleocytoplasmic transport. However, we do not understand how BAF might affect pore formation. Native residue Lys-53 is more easily interpretable because it is surface exposed at the predicted LEM-binding site, and the 53E mutation cleanly disrupted binding to emerin without harming protein folding, dimerization, or DNA binding. Nuclei assembled in a high concentration of either mutant 53E or 47E lacked attached membranes. These mutants, which do not bind emerin, support a model in which DNA-bound BAF must interact with LEM proteins to recruit membranes and promote chromatin decondensation and nuclear envelope growth. Note that BAF was tested for binding to only one LEM protein, emerin, in our studies. It will be interesting to determine if our BAF mutants behave identically towards LAP2 and MAN1.
Inactive mutant G25E: nonfunctional in vitro, yet toxic in HeLa cells
Residues Gly-25 and Gly-27 are proposed to make crucial backbone contacts to DNA (Umland et al., 2000). DNA binding activity was abolished when Gly-25 was changed to a negatively charged Glu, consistent with charge repulsion of the DNA. As discussed above, mutant G25E was defective for all BAF functions assayed. Nonetheless, when mutant G25E is expressed in living HeLa cells, it has no apparent effect until mitosis, when it lethally disrupts nuclear assembly (Haraguchi et al., 2001). As cells progress into telophase, mutant G25E prevents wild-type BAF and emerin from localizing at the core region of telophase chromosomes, and blocks the assembly of emerin, LAP2ß and lamin A (but not lamin B) at the nuclear envelope (Haraguchi et al., 2001). We speculate that living cells can somehow rescue the G25E mutant. Alternatively, the G25E mutant might retain a function for which we did not assay, such as DNA-induced oligomerization (Zheng et al., 2000), or a novel function.
BAF localization in interphase cells
In cultured Xenopus cells, BAF localizes diffusely within the nucleus and concentrates near the nuclear envelope, consistent with its affinity for LEM proteins, most of which are anchored at the nuclear inner membrane. Nuclear envelopeenriched localization was not previously reported for BAF (Furukawa, 1999), but is seen in C. elegans embryos (unpublished data) and HeLa cells (Haraguchi et al., 2001). In telophase HeLa cells, the colocalization of BAF and emerin at specific regions on telophase chromosomes is critical for emerin to subsequently assemble at the nuclear envelope (Haraguchi et al., 2001). During interphase, BAF is also found in the nuclear interior and the cytosol. Inside the nucleus, we hypothesize that BAF interacts with LAP2, an abundant soluble (not membrane anchored) LEM protein that binds lamin A (Dechat et al., 1998, 2000a, 2000b). However, LAP2
fragments that lack the LEM domain localize normally in HeLa cells (Dechat et al., 1998), so the in vivo role of BAF with respect to LAP2
is not known. We also found BAF in the cytoplasm of cultured cells, consistent with the purification of BAF activity from the cytosol of NIH-3T3 cells (Lee and Craigie, 1998). Although BAF is small, it seems unlikely that it would diffuse out of the nucleus, unless its DNA- and LEM-binding activities were inhibited. Our results neither address nor explain the potential roles of cytoplasmic BAF.
Enhanced chromatin decondensation and nuclear growth: regulated BAF activity?
We are intrigued by the enhanced chromatin decondensation and nuclear growth caused by adding 4% extra BAF to assembling nuclei. We propose that these positive effects are due to regulated interactions with endogenous LEM proteins. We hypothesize that properly regulated BAF has positive roles during nuclear assembly, in promoting membrane attachment, chromatin decondensation, and nuclear growth. We further propose that adding 20% extra BAF to assembling nuclei might saturate endogenous LEM proteins, or novel regulatory partner(s), and lead to unregulated (inherently compressive?) BAF interactions with DNA. Other models are also possible. Our results support the idea that a fundamental function of BAF is to bind LEM proteins during nuclear assembly, and thereby attach chromatin to the nuclear inner membrane. In the nuclear interior, BAF interactions with soluble lamin-binding LEM proteins (such as LAP2) might link chromatin to intranuclear lamins. Our structurefunction analysis of BAF, and the subset of biochemically-distinct BAF mutants (14A, 18A, 25E, 47E, and 53E) identified here, will provide a useful foundation for understanding this highly-conserved chromatin protein.
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Materials and methods |
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Site-directed mutagenesis of human BAF
The cDNA encoding human BAF in the pET15b vector (Novagen, Inc.; Lee and Craigie, 1998) was mutagenized using the Quickchange site-directed mutagenesis kit (Stratagene, Inc.). For alanine-substitution mutagenesis, the entire plasmid was replicated by PCR using a pair of complementary mutagenic oligonucleotides as primers to replace the indicated residue with alanine (Fig. 1). Each primer included 624 nucleotides of perfect homology flanking the region to be mutated. The original DNA strands were destroyed by digestion with DpnI (Stratagene, Inc.), and the mutated DNA was transformed into Escherichia coli XL-1-blue cells. All mutations were verified by full-length dsDNA sequencing (unpublished data). Charge substitution mutations in BAF were described previously by Cai et al. (1998).
Recombinant BAF proteins
His-tagged BAF proteins were expressed in E. coli and purified essentially as described (Lee and Craigie, 1998), but were not dimer-purified and therefore also contained oligomeric BAF complexes (Zheng et al., 2000). For a detailed protocol for BAF purification, contact the corresponding author.
Synthesis of 35S-Cys/Met-labeled proteins
BAF proteins lacking the six-His tag were produced synthetically using coupled transcription and translation reactions (Promega Corp.). Template DNA was first amplified by PCR from plasmid DNA, using a 5' primer that contained a T7 promoter site and Kozak consensus sequence to drive protein expression in vitro, plus the first 20 nucleotides of the BAF open reading frame (5'-GATCCTAATACGACTCACTATAGGGAACAGCCACCATGACAACCTCCCAAAAGCA-3'). Our 3'primer comprised a poly (A)30 tail and the last 20 nucleotides of the cDNA sequence encoding BAF (5'-T[30]TCACAAGAAGGCGTCGCACC-3'). Proteins with a six-His tag were made using the plasmid DNA template (pET15b vector), which has a T7 promoter. A typical TNT reaction contained 40 µl reaction mix, 5 µl PCR-generated template DNA or 1 µg plasmid DNA, 2 µl 35S Ready Pro-Mix (Amersham Biosciences), and nuclease-free water for a final volume of 50 µl.
BAF binding to emerin
A cDNA encoding wild-type emerin residues 1222 in the pET11c vector (Novagen Inc.; Lee et al., 2001) was transformed into E. coli strain BL21 (DE3). Cells with this plasmid were grown to an OD600 of 0.6, and emerin expression was induced by 0.4 mM IPTG for 4 h. Cells were pelleted 5 min at 14,000 g, and resuspended in 2 x SDS sample buffer. Proteins from unfractionated bacterial lysates were separated on 10% SDS-PAGE gels, transferred to Immobilon-P PVDF membrane (Millipore Corp.), and blocked for 1 h in PBST containing 5% nonfat dry milk. Blots were washed twice in BRB (Blot Rinse Buffer; 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% Tween-20) for 5 min at 2224°C, and incubated with 5 µl 35S-cysteine/methionine-labeled probe protein (wild-type or mutant BAF) diluted 1:200 into BRB containing 0.1% fetal calf serum (final volume, 1 ml). Blots were incubated at 4°C overnight with the 35S-labeled in vitrotranscribed/translated probe protein, washed four times in BRB, dried and exposed to Hyperfilm MP (Amersham Biosciences).
Subunit exchange with wild-type BAF
35S-labeled wild-type BAF was mixed with each His-tagged mutant BAF, and interactions were assayed by coimmunoprecipitation. Specifically, 35S-labeled wild-type BAF protein (10 µl) was incubated with 500 ng unlabeled recombinant (wild-type or mutant) BAF protein for 30 min at 2224°C. We then added 300 µl of cold immunoprecipitation buffer (20 mM Hepes, pH 7.9, 150 mM NaCl, 10 mM EDTA, 2 mM EGTA, 0.1% Nonidet P-40, 10% glycerol, 1 mM DTT, 1 mM PMSF, and 20 ug/ml each aprotinin and leupeptin), plus 5 µl of anti-His rabbit polyclonal antibody (sc-803; Santa Cruz Biotechnology), and incubated at 4°C with constant mixing for 1 h. We then added 50 µl washed protein A Sepharose beads (Amersham Biosciences), incubated overnight at 4°C, centrifuged at 5,000 g for 5 min to pellet the beads, and washed pellets five times with ice-cold IP buffer. Bound proteins were removed from beads by boiling in 40 µl 2 x SDS sample buffer, subjected to SDS-PAGE on 412% gradient gels (Invitrogen Corp.), dried and exposed to Hyperfilm MP (Amersham Biosciences).
BAF binding to native DNA cellulose beads
DNA binding assays were performed as described (Kasof et al., 1999), with the following modifications. The 35S-labeled wild-type or mutant BAF protein (9 µl of a 50 µl TNT reaction; see above) was first incubated with 200 µl of NETN buffer (20 mM Tris, pH 8, 100 mM NaCl, 1 mM EDTA, 0.2% Nonidet P-40) and 50 µl native DNA cellulose beads (Amersham Biosciences) for either 2 h (or overnight) at 4°C. Samples were then washed four times in NETN, resolved by SDS-PAGE, dried and visualized by autoradiography.
Circular dichroism spectroscopy
We used a Jasco J720 spectrometer (Jasco) at 2224°C to measure spectra in the far UV region (200260 nm) at a protein concentration of 0.75 mg/ml in a quartz cuvette with a 100-nm path length.
Size exclusion chromatography
We used a Superdex 25 column (HR 3.2/30; Amersham Biosciences) on an Amersham Pharmacia Smart System. The column was equilibrated and run with 20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM DTT, 0.1 mM EDTA and 10% (wt/vol) glycerol. Protein (40 ml) was loaded at a concentration of 0.2 mg/ml and eluted at a flow rate of 50 ml/min.
Cloning of Xenopus BAF
A BLAST search with the human BAF cDNA yielded an EST with high homology in Xenopus laevis. This clone (GenBamk/EMBL/DDBJ accession no. AW641186) was obtained from Research Genetics, Inc. A full-length Xenopus BAF (xBAF) ORF was amplified by PCR from this clone using a 28-base 5' primer with an NdeI restriction site and a 30-base 3' primer with a BamHI site, and cloned into the NdeI and BamHI sites of the pET15b vector (Novagen Inc.). The insert was verified by double-stranded DNA sequencing (unpublished data).
BAF antibodies, immunoblotting, and indirect immunofluorescence
A peptide comprising residues 19-35 of xBAF (KSVQCLAGIGEALGHRL) was synthesized by Boston Biomolecules, and rabbit polyclonal antiserum was produced by Covance, Inc., in rabbit 3710, using as antigen the KLH-conjugated peptide. BAF antiserum 3710 was affinity purified by binding the BAF peptide (Reduce-Imm Reducing Kit and Sulfolink Kit, Pierce Chemical Co.), and used at dilutions of 1:100 for blots and 1:10 for indirect immunofluorescence (below).
For immunoblots, proteins were loaded onto 4-12% NuPAGE gels (Invitrogen Corp.), electrophoresed, and transferred onto Immobilon-P PVDF membranes for 30 min. Blots were blocked for 1 h at 25°C in PBS containing 0.1% Tween 20 (PBST) and 5% nonfat dry milk. Subsequent incubations in primary and secondary antibodies were done in PBST. xBAF was detected on blots using a 1:2,000 dilution of crude rabbit polyclonal serum 3710, or 1:100 dilution of affinity-purified serum 3710. Blots were incubated with primary antibody for 1 h at 25°C (or overnight at 4°C), washed in PBST for 20 min three times, followed by horseradish peroxidase-conjugated goat antirabbit antibodies (1:50,000 dilution; Pierce Chemical Co.), then washed in PBST for 20 min three times. Proteins were visualized by enhanced chemiluminescence and exposure to Hyperfilm MP (Amersham Biosciences).
For indirect immunofluorescence of cultured Xenopus epithelial cells (A6 and XLK-WG lines), cells were fixed in 4% paraformaldehyde and washed in PBS containing 0.1% Tween 20 and 1% bovine serum albumin. The Xenopus kidney epithelial cells (XLK-WG line; pseudodiploid) were a gift from Dr. Joe Gall (Carnegie Institute, Baltimore, MD).
Immunofluorescence of in vitro assembled nuclei
Aliquots (5 µl) of 12 µl nuclear assembly reactions were placed on a microscope slide coated with 0.1% polylysine, coverslipped, and flash frozen in liquid nitrogen. Samples were then fixed at 20°C in methanol then acetone (20 min each). Slides were placed in 0.1% PBST and 5% milk for 10 min, and then rinsed quickly in PBST. Slides were incubated for 30 min at 2224°C with 50 µl primary antibody (peptide-purified serum 3710 against xBAF, or mAb S49 against Xenopus lamin B3, a gift of R. Stick [Bremen University, Bremen, Germany]) diluted to 1:25 and 1:100 in PBS, respectively. Slides were then washed with PBS three times (10 min each), incubated 30 min with a 1:250 dilution of the fluorochrome-labeled secondary antibody, washed in PBS three times (10 min each), and mounted with 10 µg/ml Hoechst 33342 dye in 3.7% formaldehyde.
Quantitation of endogenous BAF in Xenopus egg cytosol
Various amounts (1, 2, 4, and 8 µl) of a 1:10 dilution of Xenopus egg cytosol were compared with purified recombinant xBAF (5, 10, 20, 40, and 80 ng) on western blots. Samples were immunoblotted as described above, probed with affinity-purified 3710 antibodies and alkaline phosphatase-conjugated donkey antirabbit antibody (1:1,000 dilution; Jackson ImmunoResearch Laboratories), and visualized by a colorimetric reaction (AP conjugate substrate kit; BioRad Laboratories). Blots were quantitated using FluorChem v.2 software (Alpha Innotech, Corp.). Intensities for known amounts of recombinant BAF were plotted as a standard curve using the Sigma Plot software (Jandel Scientific). The intensity of BAF in 1 µl diluted cytosol fell within the linear range of detection, and the amount of endogenous BAF was calculated from the standard curve. The Xenopus egg cytosol fraction contains 25 µM monomeric BAF, or 12.5 µM BAF dimers.
Structural modeling of BAF
Residues were mapped using coordinates for the crystal structure of BAF (PDB Id: 1CI4; Umland et al., 2000). BAF was manipulated using the GRASP program (A. Nicholls, Department of Biochemistry and Molecular Biophysics, Columbia University, New York).
Transmission EM
Xenopus nuclear assembly reactions (100 µl) were fixed by mixing with 700 µl membrane wash buffer (MWB: 250 mM sucrose, 50 mM KCl, 2.5 mM MgCl2, 50 mM Hepes, pH 8, 1 mM ATP) plus 700 µl 2x fix buffer (250 mM sucrose, 100 mM Hepes, pH 8, 1.5 mM MgCl2, 1.5 mM CaCl2, 1% glutaraldehyde, 0.5% paraformaldehyde), and incubating 20 min at 2225°C. Samples were then pelleted in a horizontal microcentrifuge (Beckman Microfuge ETM) for 1 min at full speed (14,000 rpm), and washed three times (5 min each) in cacodylate buffer (0.2 M cacodylate, pH 7.4, 1.5 mM MgCl2, 1.5 mM CaCl2). Samples were then fixed 1 h in cacodylate buffer containing 4% reduced osmium (OsO4), washed twice (5 min each) in distilled water, and stained 20 min in 1% uranyl acetate in distilled water. Samples were dehydrated by a graded series of 5 min incubations in 50, 70, and 90% ethanol, followed by three 3-min incubations in 100% ethanol. Samples were then embedded in Spurr's medium, sectioned (80120 nm thick), and visualized using a Philips CM120 transmission electron microscope.
Supplemental materials available
Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200202019/DC1) shows immunofluorescence staining for BAF and lamins in nuclei assembled in 5 µM added exogenous wild-type BAF.
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Footnotes |
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* Abbreviations used in this paper: BAF, barrier-to-autointegration factor; CD, circular dichroism; dsDNA, double-stranded DNA; hBAF, human BAF; LAP, lamin-associated polypeptide; LEM, LAP2, emerin, MAN1; NPC, nuclear pore complex; TEM, transmission electron microscopy.
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Acknowledgments |
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This work was funded by a National Institutes of Health grant (RO1 GM48646) to K.L. Wilson.
Submitted: 6 February 2002
Revised: 28 May 2002
Accepted: 11 June 2002
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References |
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