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Originally published In Press as doi:10.1074/jbc.M509917200 on October 3, 2005

J. Biol. Chem., Vol. 280, Issue 51, 42252-42262, December 23, 2005
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Binding of Barrier to Autointegration Factor (BAF) to Histone H3 and Selected Linker Histones Including H1.1*

Rocío Montes de Oca, Kenneth K. Lee1, and Katherine L. Wilson2

From the Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, September 8, 2005 , and in revised form, September 21, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Barrier to autointegration factor (BAF) is an essential conserved double-stranded DNA-binding protein in metazoans. BAF binds directly to LEM domain nuclear proteins (e.g. LAP2, Emerin, and MAN1), lamin A, homeodomain transcription factors, and human immunodeficiency virus type 1-encoded proteins. BAF influences higher order chromatin structure and is required to assemble nuclei. BAF also facilitates retroviral preintegration complex insertion into target DNA in vitro, through unknown mechanisms. We report that BAF binds directly and selectively to linker histone H1.1 (among three subtypes tested) and core histone H3 with affinities of ~700 nM and ~100-200 nM, respectively, in vitro and in vivo. Mutations at the bottom and top surfaces of the BAF dimer disrupted or enhanced, respectively, this binding and affected H1 and H3 similarly. Biochemical studies showed that C-terminal residues 108-215 of histone H1.1 and the N-terminal tail plus helix {alpha}N in the core of histone H3.1 were each necessary and sufficient to bind BAF. Based on its interactions with histones and DNA, we propose BAF might bind nucleosomes in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Barrier to autointegration factor (BAF)3 is a conserved metazoan protein (reviewed by Segura-Totten and Wilson (1)). BAF was originally identified because of its ability to restore integration competence to salt-extracted retroviral preintegration complexes (PICs) isolated from the cytoplasm of cells infected with either Moloney murine leukemia virus (2, 3) or human immunodeficiency virus type 1 (HIV-1) (4). BAF is a host component of HIV-1 virions (5) and retroviral PICs (4, 6). BAF is proposed to associate with the PIC through direct binding to retroviral DNA (2), matrix protein (5), and LAP2{alpha}, a host-encoded LEM domain protein (see below) (7). Human BAF is a 10.3-kDa (89-residue) protein that exists as a dimer in solution (8, 9) and can also form higher order oligomers in the presence of DNA (2, 10). BAF binds in a non-sequence-specific manner to double-stranded DNA in vitro (10). When incubated with 21-bp double-stranded DNA fragments, BAF forms nucleoprotein complexes consisting of six dimers (10), the structure and implications of which are unknown. BAF has been proposed to change conformation in the presence of DNA, since a human autoantigen identified as BAF is only recognized by autoantibodies when bound to DNA (11). In cultured vertebrate cells and Caenorhabditis elegans embryos, BAF is present in both the cytoplasm and nucleus with significant enrichment near the nuclear envelope (12-14). However, in Drosophila embryos, BAF is primarily chromatin-associated (15). Thus, BAF localization is proposed to be regulated by post-translational modifications as well as by direct binding to its multiple partners (see Ref. 1).

BAF binds directly to a family of nuclear proteins bearing the conserved LEM domain motif (13, 16-21). Most LEM domain proteins (e.g. LAP2{beta}, Emerin, MAN1) are lamin-binding proteins anchored at the nuclear inner membrane (22, 23). Another LEM domain protein, LAP2{alpha}, has no transmembrane domain and colocalizes with A-type lamins in the nuclear interior (24). Lamins form filaments essential for nuclear structure and function. Mutations in A-type lamins cause at least 10 tissue-specific diseases, including Emery-Dreifuss muscular dystrophy and progeria (accelerated "aging") syndromes (25). Interestingly, BAF also binds lamin A in vitro (26).

The roles of BAF in the nucleus have been enigmatic. BAF can repress Crx-dependent gene transcription in vivo (27) but also has potent large scale effects on chromatin architecture during nuclear assembly (12). When small amounts of exogenous BAF (4% above endogenous) are added to cell-free nuclear assembly reactions, the resulting nuclei are larger than normal and have more evenly dispersed chromatin (12). Higher levels of added BAF (20-40% above endogenous) produce the opposite effect, blocking nuclear assembly and profoundly compacting chromatin (12). These results showed that BAF influences chromatin structure and gene expression but did not reveal its mechanism. To explain these various results, we hypothesized that BAF might interact with histones, which organize chromatin and modulate gene activity by affecting chromatin compaction.

Core histones (H2A, H2B, H3, and H4) form nucleosomes, the fundamental unit of chromatin structure (28). Other proteins, including linker histones (H1), high mobility group (HMG) proteins (29), and regulatory proteins such as MeCP2 and Sir3 (30, 31), further organize nucleosomes into compacted higher order structures (32). Transcriptionally active and silenced chromatin are distinguished by at least two mechanisms: post-translational modifications of core histones (33-35) and enrichment for different subtypes of histone H1 (36, 37).

Mammals have at least seven genes encoding subtypes of histone H1 (38, 39). Linker histones range in mass from ~21-23 kDa and have a conserved tripartite domain organization (40). Their short N-terminal tail ("N-tail") is proposed to bind DNA (41) and is post-translationally modified (42-44). The central globular domain includes a DNA-binding motif (45, 46) and is thought to contact the nucleosome at the site of DNA entry/exit crossover (47). The ~100-residue C-terminal tail ("C-tail") is highly basic, essential for DNA condensation (48, 49), and independently capable of condensing DNA (50, 51). Histone H1 subtypes differ most in their C-tail domains, which are proposed to confer unique properties to each subtype (52-54). We report here that BAF binds selectively and with physiologically relevant affinities to linker histones and core histone H3, in vitro and in vivo. We propose that BAF might influence higher order chromatin structure by binding to specific histones and, potentially, nucleosomes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Purified Proteins and [35S]Met-Labeled Probes—Total avian erythrocyte histones (catalog number 13-107) and purified calf thymus histone H1 (catalog number 14-155) were obtained from Upstate USA, Inc. (Charlottesville, VA). Recombinant yeast core histones were purified from bacteria (55, 56). 35S-Labeled BAF and other probes were synthesized in eukaryotic coupled transcription/translation reticulocyte lysates (TNT®; Promega, Madison, WI) as described (12) in the presence of [35S]methionine (RedivuePRO-MIX; Amersham Biosciences), using the T7 promoter of pET plasmids encoding human wild type or mutant BAF (12), human histone H3.1 (a kind gift of T. Owen-Hughes and J. Workman), human histone H1.1 or H1.2 (see below), or wild type emerin (20). Bacterially expressed emerin and BAF proteins were purified as described (12, 20). BAF dimers were isolated by size exclusion chromatography (Superdex 200 16/60 column; Amersham Biosciences) after histidine tag cleavage with thrombin as described (10). Bacterially expressed GST-fused histones were purified as described below. BSA was purchased from Sigma (catalog number A7906-500G).

Human Histone H1 Constructs for in Vitro Transcription/Translation—Recombinant human histones H1.1 and H1.2 were PCR-amplified from a HeLa cDNA library. For full-length human H1.1, we used a 5' primer with an NdeI site (5'-GTATTCATATGTCTGAAACAGTGCCTCCCGCC-3') and 3' primer with a BamHI site (5'-GTTATTGGATCCTTACTTTTTCTTGGGTGCCGCTTTC-3') and cloned into the NdeI and BamHI sites of pET15b vector (Novagen, Inc., Madison, WI). Full-length H1.2 was amplified using a 5' primer with an NdeI site (5'-GTTATTCATATGTCCGAGACTGCTCCTGCCGCTC-3') and 3' primer with a BamHI site (5'-GTTATTGGATCCCTATTTCTTCTTGGGCGCCGCCTTC-3') and cloned into pGEM®-T Easy vector (Promega Corp., Madison, WI). Inserts were verified by double-stranded DNA sequencing (data not shown). The mouse H1o cDNA in vector pET11d was a kind gift from X. Lu and J. Hansen (Colorado State University).

Proteolysis Assays, SDS-PAGE, and Blot Overlay Assays—Purified calf thymus linker histones were dissolved in buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 2 mM MgCl2) to a final concentration of 2 mg/ml. Chymotrypsin (Sigma) was added to a final concentration of 100 ng/ml. Samples were digested at 10 °C as described (57), and aliquots were removed at the times indicated, quenched by the addition of SDS-sample buffer, resolved by SDS-PAGE (NuPAGETM 4-12% bis-Tris gels; Invitrogen), and transferred to Protran nitrocellulose membranes (Schleicher and Schuell). Membranes were blocked and probed as described (20) with 35S-labeled proteins synthesized in eukaryotic TNT® reactions and then washed, dried, and exposed to x-ray film (Denville Scientific Inc., Metuchen, NJ).

Microtiter Binding Assays—Microtiter assays were done essentially as described (26), but using PCR-amplified DNA as the substrate to produce each 35S-labeled protein in vitro. For linker histones, the initiating methionine contained the radiolabel. DNA fragments encoding portions of H1.1 were PCR-amplified from the full-length H1.1 plasmid, using 5' primers that contained a T7 promoter site and Kozak consensus sequence to drive protein expression in vitro plus the following nucleotides to specify fragments A and B (5'-TCTGAAACAGTGCCTCCCGCC-3'), fragment C (5'-GCTAGCAAAAAGAGCGTCAAG-3'), fragment D (5'-TCCTCCGTGGAAACCAAGCCC), fragments E and H (5'-TTCAAGCTCAACAAGAAGGCG-3'), fragment F (5'-TCGGGTTCCTTCAAGCTCAAC-3'), and fragment G (5'-GGAACGTTGGTGCAGACAAAG-3'). The 3' primer comprised a poly(A)30 tail (shown as [30]) plus 21 or 27 nucleotides to specify fragment A (5'-T[30]CCCCGTGGCCTTTTTGAGCTT-3'), fragment B (5'-T[30]AGTTTTGGGTTTTTTTGGATTCTTGGA-3'), fragment H (5'-T[30]TTACTTTTTCTTGGGTGCCGC-3'), and fragments C-G (5'-T[30]TTACTTTTTCTTGGGTGCCGC-3'). Full-length H1.1, H1.2, and H1o proteins and truncated H1.1 polypeptides were synthesized in eukaryotic TNT® reactions, as described above. To prepare microtiter wells, purified recombinant substrate protein (e.g. BAF) was diluted to 100 µl in transport buffer (20 mM HEPES, pH 7.4, 110 mM KOAc, 2 mM MgOAc, 1 mM EGTA) and incubated overnight at 4 °C in Immulon®4 HBX polystyrene 96-well microtiter plates (Thermo Labsystems, Franklin, MA) or HisGrabTM HBC 8-well strip plates (Pierce), at the indicated concentrations, in triplicate. Each solution was then gently aspirated, replaced with 200 µl of transport buffer containing 30 mg/ml BSA, and incubated for 3 h at 22-24 °C to block nonspecific binding sites. Wells were then washed and incubated overnight at 4 °C with known amounts of each 35S-labeled probe in a final volume of 100 µl of transport buffer per well. Wells were rinsed five times with transport buffer and eluted with 50 µl of 5% SDS, and bound proteins were quantified by scintillation counter. Molar amounts of total, and bound 35S-labeled proteins were calculated as described in Refs. 26 and 58.

GST Pull-down Assays—Full-length H3.1 or H3.1 residues 1-64 (counting the initiating methionine as residue number 1) were cloned into the pETGEXCT vector (a gift from S. Buratowski, Harvard Medical School) to fuse GST to the C terminus of each. A plasmid encoding GST fused to the N terminus of the H3.1 tail (residues 1-47; "GST-tail") was obtained from J. Workman (Stowers Institute for Medical Research). Recombinant GST-fused histones were expressed in E. coli (BL21) and purified as follows. Cells were lysed in phosphate-buffered saline (10 mM phosphate-buffered saline, 138 mM NaCl, pH 7.4) containing 350 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitors.

Lysates were clarified by centrifugation (10 min, 20,000 rpm in an Avanti J-20XP centrifuge, 4 °C), and the supernatants were loaded onto a GST fast flow column (Amersham Biosciences). Bound proteins were eluted in phosphate-buffered saline, pH 8, plus 350 mM NaCl and 40 mM glutathione. Eluted proteins were dialyzed overnight at 4 °C into 100 volumes of buffer (20 mM Tris-HCl, pH 7.6, 300 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol), purified by ion exchange chromatography (Mono S HR 10/10; Amersham Biosciences), washed with 15 column volumes of 20 mM Tris-HCl, pH 7.6, plus 300 mM NaCl, and eluted using a linear 0.3-1.0 M NaCl gradient over 15 column volumes. Equal amounts (~10 µg) of each fusion protein or GST alone were allowed to bind 20 µl of glutathione-agarose beads (Sigma) for 3 h at 4 °C. After washing with immunoprecipitation (IP) buffer (see below), beads were incubated at 4 °C overnight with gentle rotation with ~20 µg of purified untagged BAF dimers in a final reaction volume of 200 µl. Beads were then washed three times with IP buffer, and the bound proteins were eluted with 2x SDS sample buffer, resolved by SDS-PAGE (4-12% NuPAGETM gels) and detected by Coomassie Blue stain.

Transfections and Immunoprecipitations—Full-length human H1.1 and H1.2 were cloned into the pcDNA3.1 Myc-His expression vector (Invitrogen) to generate C-terminally Myc-His-tagged proteins. Full-length H3.1 was cloned into the pcDNA3.1 FLAG-HA expression vector, a kind gift from C. Lerin (Johns Hopkins University), to generate an N-terminally FLAG-HA-tagged protein. The pEGFP-C1 expression vector (CLONTECH) was used to express green fluorescent protein (GFP). Each construct and its corresponding empty vector were transiently transfected into HeLa cells using LT1 (Mirus, Madison, WI) as per the manufacturer's instructions and allowed to express for 24 h. Transfected cells were lysed in IP buffer (20 mM HEPES, pH 8, 150 mM NaCl, 0.1% Nonidet P-40, 10 mM EDTA, 2 mM EGTA, 2 mM dithiothreitol, protease inhibitors) and treated with benzonase (300 units/µl; EMD Chemicals Inc., Gibbstown, NJ) for 30 min on ice to degrade DNA. Lysates were then sonicated four times (15 s each) and centrifuged (10 min, 14,000 rpm in an Eppendorf 5415C microcentrifuge, 4 °C), and the supernatants were precleared with Protein A-Sepharose (Amersham Biosciences) for 1 h at 4 °Cand then recentrifuged to obtain cleared cell lysates. For each immunoprecipitation, 300 µl of precleared lysate was incubated overnight at 4 °C with 7.5 µl of rabbit anti-BAF serum 3273 (59) plus 7.5 µl of rabbit anti-BAF serum 5045 (described below) or no serum, as a control. We then added 15 µl of Protein A-Sepharose, incubated for 2 h at 4 °C, pelleted (1,500 rpm in an Eppendorf 5415C microcentrifuge, 4 °C), and washed four times with IP buffer. Bound proteins were eluted using 100 mM glycine, pH 3, and neutralized by adding 4x sample buffer containing 60 mM Tris base to a final concentration of 1x sample buffer and 15 mM Tris base. Samples were resolved by SDS-PAGE (4-12% NuPAGETM gels), and proteins were detected using monoclonal antibodies against Myc (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), HA (1:1500 dilution; Covance, Inc., Berkeley, CA), GFP (1:500 dilution; Santa Cruz Biotechnology), or rabbit serum 3273 against BAF (59) at 1:40,000 dilution.



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  		FIGURE 1.
BAF binds linker histones and core histone H3. Purified recombinant yeast core histone octamer (yeast; 1.5 µg), 1.5 µg of total avian histones purified from erythrocytes (bird), or 1 µg of linker histones purified from calf thymus (calf) were resolved by 15% SDS-PAGE (Tris-glycine buffer) and either stained with Coomassie Blue (A) or transferred to membranes and probed with [35S]BAF (B) or [35S]emerin (residues 1-222; C) produced in coupled transcription/translation extracts. The autoradiograms are shown in B and C. The brackets indicate linker histones. Core histones (H2A, H2B, H3, and H4) and the avian erythrocyte-specific linker histone H5 are indicated. Due to high charge/mass ratios, histones migrate slower than predicted by mass. Please note that the seven linker subtypes resolve incompletely in this gel system, forming only three bands. The data shown are representative of at least three independent experiments.

 
Anti-BAF Serum 5045—To facilitate immunoprecipitation of BAF, rabbit serum 5045 was raised against full length (His-tagged) purified recombinant human BAF by Covance and used in combination with serum 3273, which recognizes human BAF residues 4-20 (59).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We first used [35S]BAF to probe blot-immobilized histones from three sources. Purified bacterially expressed yeast core histones, total histones purified from avian erythrocytes, and linker histones purified from calf thymus were resolved on duplicate SDS-polyacrylamide gels. One gel was stained with Coomassie, revealing the expected migration pattern for histones (Fig. 1A). The other gel was transferred to nitrocellulose and probed with either [35S]BAF (Fig. 1B) or [35S]emerin as a negative control (Fig. 1C). No binding was observed between [35S]emerin and histones. However, BAF consistently recognized at least three linker histone bands in the avian and calf samples and core histone H3 in the yeast and avian samples (Fig. 1B). Very weak binding to histones H4 and H2B was seen on longer exposures (data not shown). We concluded that BAF binds core histone H3 and at least three of the seven linker histones that comigrate in SDS-polyacrylamide gels. Since the yeast core histones were expressed in bacteria, we further concluded that eukaryotic post-translational modifications of H3 were not required to bind BAF.

BAF Binds Recombinant Histone H1.1 with Submicromolar Affinity but Not H1.2 or H1o—To independently confirm and quantify BAF binding to linker histones, we used a microtiter binding assay in which linker histones were immobilized in wells. BSA served as a negative control. [35S]BAF bound a mixture of native (potentially post-translationally modified) purified calf thymus linker histones with an apparent equilibrium affinity of ~1 µM (n = 3 triplicate sets; data not shown). This result confirmed the interaction but did not reveal whether BAF discriminated between histone H1 subtypes. We therefore tested three purified recombinant subtypes (human H1.1 and H1.2, mouse histone H1o) for which cDNAs were available. These subtypes are encoded by three different genes (39) and have distinct properties in living cells (36, 37, 60, 61). Mouse and human H1o proteins are 94.3% identical (62), and we assume that the mouse protein behaves like human H1o. All three subtypes have nearly identical globular domains (Fig. 2A, GD) but differing N- and C-tail domains (Fig. 2A). Histones H1.1, H1.2, and H1o were each expressed and [35S]Met-labeled in eukaryotic transcription/translation reactions (see "Materials and Methods") and incubated with recombinant purified BAF immobilized in microtiter wells. BAF bound significantly to H1.1, with only weak binding to H1.2 or H1o even at high input levels of [35S]H1.2 or [35S]H1o (Fig. 2B). BAF bound [35S]H1.1 with an equilibrium affinity of 702 nM (range 519-915 nM, n = 3 triplicates) in the microtiter assay (Fig. 2C). These findings independently confirmed the blot overlay results and showed that, of the three subtypes tested, BAF binds selectively to H1.1 with physiologically relevant affinity (see "Discussion"). We concluded that BAF discriminates in vitro between three related linker histones. The other four H1 subtypes remain to be tested.

BAF Binds the C-tail of Histone H1.1 plus Crucial Residues in the Globular Domain in Vitro—We used two approaches to map the H1.1 domain recognized by BAF. We first digested the mixture of purified calf thymus linker histones with chymotrypsin, which cleaves preferentially at a unique conserved phenylalanine (Phe-108 in H1.1) in the globular domain near the C-tail (see Fig. 2A, asterisks) (63). Next, aliquots were removed from the chymotrypsin reaction at different times and quenched by adding SDS-PAGE loading buffer. All samples were resolved by SDS-PAGE, transferred to nitrocellulose, stained with Ponceau S to visualize the extent of cleavage (Fig. 3A, left), washed, and then probed with wild type [35S]BAF (Fig. 3A, right). The mixture of linker histones migrated as a doublet band under these gel conditions; similarly, chymotrypsin cleavage yielded two major C-tail bands as expected, due to different length C-tails in this mixture of linker subtypes (57), plus one N-tail band. Total avian histones were used as size standards (Fig. 3A, S), since the C-terminal cleavage products are known to migrate between H1 and the core histones (64). [35S]BAF bound full-length linker histones and C-tail fragments (Fig. 3A, right, double asterisk) but not the faster migrating N-tail fragment (Fig. 3A, right, single asterisk). Thus, BAF appeared to recognize the C-tail domain of histone H1.1.



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  		FIGURE 2.
BAF binds linker histone H1.1 with submicromolar affinity but does not bind H1.2 or H1o in vitro. A, amino acid sequences of human H1.1 and H1.2, mouse H1o, and rat H1d. Rat H1d is included for reference, because its "HMG-box-like fold" (boxed) contains characterized conserved (S/T)PKK motifs (overlined within box) (72, 73). Identical residues are shaded black; similar residues are shaded gray. The three domains characteristic of linker histones (N-tail, globular domain (GD), and C-tail) are marked by dotted, solid, and dashed lines, respectively. The conserved chymotrypsin cleavage site (Phe-108 in H1.1) is marked by asterisks, and residues Gly-94 and Ser-105 (relevant to Fig. 3) are marked by filled circles. B, microtiter assay results. Increasing concentrations of [35S]H1.1, [35S]H1.2, or [35S]H1o were incubated with constant amounts of BAF (or negative control BSA) immobilized in microtiter wells; moles bound were plotted after subtracting the BSA background (n = 3 triplicates). C, the equilibrium affinity of H1.1 for BAF determined in microtiter assays was 702 nM (range 519-915 nM, n = 3 triplicates). Increasing concentrations of [35S]H1.1 were added to constant amounts (2 µg) of recombinant His-tagged BAF immobilized in wells. Double reciprocal plots (not shown) were used to determine the affinity constant as described (26, 58). The bars in B and C indicate S.D.

 
To identify a minimal binding site for BAF, we synthesized several 35S-labeled fragments of human histone H1.1 (Fig. 3B). Each radiolabeled fragment was incubated with purified recombinant BAF immobilized in microtiter wells, with BSA-containing wells as negative controls. Full-length [35S]H1.1 bound BAF (Fig. 3D, WT), as expected. Large C-terminal deletions (Fig. 3B, fragments A and B) or N-terminal deletions (Fig. 3B, fragment C) greatly reduced or disrupted binding (data not shown), suggesting that the entire C-tail was essential for binding. However, the C-tail alone (Fig. 3B, fragment D) was insufficient for binding (data not shown), unless we included seven residues of the globular domain to recreate the C-terminal chymotrypsin fragment (Fig. 3, B and D; fragment E). Extending this polypeptide by either 3 or 14 residues to reconstitute half or all (respectively) of a {beta}-hairpin structure (45) in the globular domain (Fig. 3C) inhibited binding to BAF (Fig. 3, B and D; fragments F and G). We speculate that these additional residues (105-107 and 94-107 in H1.1) are normally anchored to the globular domain in the full-length histone, and without this domain they can flexibly disrupt binding to BAF. Interestingly, binding to BAF was also lost when five residues were removed from the C terminus of fragment E to generate fragment H (Fig. 3B, data not shown). The equilibrium affinity of fragment E for BAF was 559 nM (range 440-661 nM, n = 3 triplicates; Fig. 3E). This affinity was comparable with that of full-length H1.1 (range 519-915 nM; Fig. 2C). We concluded that the last seven residues of the globular domain (Phe-108 to Ala-114) plus the entire C-tail of H1.1, totaling 108 residues, were necessary and sufficient to directly bind BAF in vitro.

BAF Binds Core Histone H3 and Human Histone Variant H3.1 in Vitro—To investigate whether BAF discriminated between different forms of histone H3, we used bacterially expressed Xenopus H3 (xH3; 98.5% identical to human H3) and in vitro transcribed/translated human histone H3.1 (hH3.1) (65). Both H3 and H3.1 are expressed and incorporated into chromatin during S-phase (replication-coupled), and their synthesis decreases during cell differentiation (65). The binding affinity of [35S]hH3.1 for recombinant purified human BAF was 106 nM (range 87-108 nM, n = 3 triplicates; Fig. 4A). Similar results were found with purified N-terminally His-tagged BAF immobilized on a nickel matrix (HisGrabTM plates; data not shown; see "Materials and Methods"). The equilibrium affinity of [35S]BAF for xH3 was 190 nM (range 128-262 nM, n = 3 triplicates; Fig. 4B), almost 2-fold weaker than hH3.1 but still significant. These findings independently confirmed the blot overlay results (Fig. 1) and showed that BAF binds replication-coupled isoforms of histone H3 with physiologically relevant affinities of ~100-200 nM in vitro. From the blot overlay results (Fig. 1B), BAF also binds yeast histone H3, which corresponds to the human replication-independent histone variant H3.3; this form of H3 is used to remodel chromatin and replaces H3 and H3.1 in actively expressed genes (66). Thus, BAF is a high affinity partner that binds all major isoforms of histone H3 in vitro.



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  		FIGURE 3.
The C-tail plus seven globular domain residues of H1.1 are necessary and sufficient to bind BAF. A, linker histones purified from calf thymus were digested with chymotrypsin for 0-30 s or 1-90 min, as indicated, and resolved by SDS-PAGE (see "Materials and Methods"). Gels were transferred to nitrocellulose, stained with Ponceau S solution, and scanned (left) and then washed and probed with [35S]BAF (autoradiogram at right). Chymotrypsin preferentially cleaves linker histones at a unique phenylalanine residue (Phe-108; see Fig. 2A), yielding two fragments that migrate as shown (63, 64). N, GD, and C indicate the N-tail (dark gray), globular domain (medium gray), and C-tail (light gray), respectively. The double asterisks indicate [35S]BAF bound to the C-terminal fragment; the single asterisk indicates the position of the N-terminal fragment. B, schematic representation of wild type (WT) H1.1 (residues 1-215) and fragments thereof (A-H), each of which was synthesized and [35S]Met-labeled in eukaryotic transcription/translation reactions and tested for binding to BAF in microtiter assays. Results are summarized at the right (BAF binding) and shown for fragments E, F, and G, in D. C, atomic structure of the globular domain of histone H5 (45), showing the conserved positions of Phe-108 (F108) and the N-terminal residues of fragment F (Ser-105) and fragment G (Gly-94) from H1.1 (indicated by black dots in Fig. 2A). D, BAF binds wild type (WT) H1.1 and fragment E (black circles), but not fragment F or G (black triangles and crosses, respectively), in the microtiter assay. Increasing concentrations of each [35S]H1.1 polypeptide were incubated with constant amounts of BAF immobilized in wells. Signals for 35S-labeled H1.1 fragments A-D and H were all indistinguishable from BSA background (not shown). The filled and open symbols indicate histone binding to BAF- or BSA-containing wells, respectively. The data shown are typical of at least three independent experiments. E, equilibrium affinity curve of histone H1.1 fragment E for BAF. Increasing concentrations of [35S]H1.1 fragment E were incubated with constant amounts of recombinant BAF immobilized in wells. Double reciprocal plots (not shown) were used to determine the affinity constant, 559 nM (range 440-661 nM; n = 3 triplicates). The bars in D and E indicate S.D.

 
BAF Binds the Globular Domain of H3.1 in Vitro—To determine which domains in H3 were recognized by BAF, we generated full-length H3.1 and fragments thereof as GST fusion proteins (diagrammed in Fig. 4C). Most of the N-terminal tail (residues 1-41 counting the initiating methionine as number 1) and portions of the first {alpha}-helix of the core region (residues 46-57; {alpha}N) are surface-exposed in the context of the nucleosome (67) and therefore potentially accessible to BAF. Bacterially expressed, purified GST fusion proteins were immobilized on glutathione-agarose, incubated with purified recombinant BAF dimers (10.6 µM), resolved by SDS-PAGE, and stained with Coomassie Blue (Fig. 4D). BAF bound directly and equally (densitometry data not shown) to full-length H3.1-GST (Fig. 4D, lane 6) and H3.1 residues 1-64 (Fig. 4D, tail+{alpha}N-GST, lane 8). BAF did not bind detectably to the N-terminal tail (Fig. 4D, GST-tail, lane 10). These interactions were specific, since BAF did not bind GST-beads or beads alone (Fig. 4D, lanes 4 and 2, respectively). We concluded that direct interaction requires helix {alpha}N residues in the core region of histone H3.1. The first 47 residues of H3, comprising the N-tail (residues 1-40) and the "short linker" (residues 41-45), were not sufficient to bind BAF in vitro but might contribute to binding. We concluded that BAF binds directly to core region {alpha}N and potentially also the H3 tail, which are exposed and available in the context of the nucleosome. BAF bound substoichiometrically to full-length H3 in both the microtiter (Fig. 4, A and B; 2-20% bound) and GST pull-down assays (Fig. 4D, ~3% of input BAF bound). Thus, our measured affinities are conservative estimates; histone monomers are probably suboptimal ligands for BAF, since histones associate as tetramers (e.g. H3-H4) or octamers (e.g. in the nucleosome) in vivo.

BAF Associates with H1 and H3 in Vivo—To investigate whether BAF associates with histones in vivo, we used antibodies against BAF to immunoprecipitate lysates from HeLa cells transiently transfected to express either H1.1-Myc, H1.2-Myc, HA-H3.1, or, as controls, the corresponding empty vector plus GFP (see "Materials and Methods"). Immunoblots of input, pellet, and supernatant fractions (I, P, and S, respectively) were probed with antibodies against either Myc, HA, GFP, or BAF (Fig. 4E). Confirming our in vitro results, endogenous BAF co-immunoprecipitated with both Myc-tagged H1.1 and HA-tagged H3.1 (Fig. 4E, lanes 5 and 14, respectively). The association was specific, since BAF did not pellet in the absence of antibody (Fig. 4E, lane 17), and control GFP protein did not pellet with BAF (Fig. 4E, lanes 2 and 11). Interestingly, endogenous BAF also co-immunoprecipitated with Myc-tagged H1.2 (Fig. 4E, lane 8), to which BAF did not bind directly in vitro; this result suggested either indirect association (via nucleosomes or a third protein) or positive regulation of binding (e.g. by posttranslational modification of one or both proteins) in vivo (see "Discussion"). These results show that BAF associates with linker histones and H3.1 in vivo.



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  		FIGURE 4.
BAF binds core histone H3 with nanomolar affinity in vitro and associates with H3 and H1 in vivo. A and B, BAF binds core histone H3 with nanomolar affinity in microtiter assays. Increasing concentrations of [35S]Met-labeled hH3.1 were added to wells containing immobilized recombinant BAF (A). In a reciprocal experiment (B), increasing amounts of [35S]BAF were added to wells containing constant amounts of purified bacterially expressed wild type Xenopus histone H3 (xH3; 1 µg/well). The affinity constants, determined using double reciprocal plots (not shown), were 106 nM (range 87-180 nM; n = 3) for hH3.1 and 190 nM (range 128-262 nM; n = 3) for xH3. The bars indicate S.D. C, schematic representation of GST-fused human histone H3.1 and polypeptides thereof, each of which was purified as a recombinant protein and tested for binding to BAF in D. D, pull-down assays using purified recombinant histones and purified recombinant BAF. H3.1-GST, tail+{alpha}N-GST (residues 1-64 of H3.1), GST-tail (residues 1-47 of H3.1), or GST alone were bound to glutathione-agarose beads and incubated with isolated untagged BAF dimers. The precipitates were resolved by SDS-PAGE gels and stained with Coomassie Blue. Amounts loaded were 0.5% of the input and supernatant (I and S, respectively) and 30% of the pellet (P). The asterisks indicate GST fusion proteins, and the arrowhead indicates untagged BAF dimers. E, Western blot analysis of immunoprecipitates (IP) from HeLa cells transiently transfected with either Myc-tagged H1.1 (H1.1-Myc), Myc-tagged H1.2 (H1.2-Myc), HA-tagged H3.1 (HA-3.1), H1.1-Myc plus HA-H3.1, or each empty vector (Myc-v or HA-v) plus GFP as negative controls. Samples were immunoprecipitated using anti-BAF sera 3273 and 5045 (IP {alpha}BAF, lanes 1-15)orno antibody as the negative control (IP-No Ab, lanes 16-18). The input and supernatant fractions (0.25% of H1.2-Myc lysates, 0.5% other lysates) and corresponding pellets (35% of H1.2-Myc lysates, 70% others) were immunoblotted using antibodies against Myc ({alpha}Myc), HA ({alpha}HA), GFP ({alpha}GFP), or BAF ({alpha}BAF). ns, nonspecific band detected by anti-Myc antibody.

 
Mutations in BAF Surface Residues That Disrupt or Enhance Histone Binding—To map BAF residues involved in binding to histones, we used blot overlay assays. Core and linker histones purified from avian erythrocytes were resolved by SDS-PAGE, transferred to nitrocellulose, cut into strips, and probed with wild type [35S]BAF or each of 18 different [35S]BAF missense mutants synthesized in eukaryotic transcription/translation reactions (Fig. 5A). As a control, 25% of each probe was resolved by SDS-PAGE and visualized by autoradiography (Fig. 5A, bottom) to verify similar levels of each probe and quantify binding of each mutant relative to wild type BAF (data not shown). A second set of eight alanine substitution mutations in BAF (68) were also tested (Fig. 5B). In general, mutations that either disrupted or enhanced BAF binding to histones affected H1 and H3 similarly. This was unexpected, since the BAF-binding regions of H1 and H3, mapped above, are dissimilar in sequence and structure. Several BAF mutants showed slightly reduced (mutants D9A, I26A, and K41E) or severely reduced (K6A, G25E, L46E, G47E, and V51E) binding to histones, relative to the amount of each input probe. Other mutations slightly enhanced (K18A, K41A, K54A, R60E, R75E, C80A, and R82E) or greatly enhanced (R8A, P14A, K53E/A, W62A, and K64E) binding to histones (Fig. 5, A and B). The remaining BAF mutants (V29A, K32E, K33E, R37A, and L50A) gave signals similar to wild type BAF. BAF mutants were also tested for binding to blot-immobilized purified calf thymus linker histones (rather than avian histones) with similar results except for two mutations; V29A abolished binding to calf histones, and K53E was indistinguishable from wild type (data not shown). All results are compared in TABLE ONE with previously tested binding partners emerin, DNA, and the C-terminal domain of MAN1 (MAN1-C) (12, 68, 69).


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  		TABLE ONE
Mutations in BAF surface residues that disrupt or enhance histone binding

 



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  		FIGURE 5.
BAF residues implicated in binding to histones. A and B, blot overlay assays for [35S]BAF binding to blot-immobilized histones. Total histones purified from avian erythrocytes (24 µg) were resolved by SDS-PAGE (15% acrylamide, Tris-glycine buffer (A) or 4-12% NuPAGETM (B)), transferred to nitrocellulose, and either cut into strips (A) or resolved in individual lanes (B) prior to cutting into strips. wt, wild type. Each strip was incubated with 200,000 cpm of 35S-labeled wild type BAF or BAF missense mutants. BAF mutants tested in A and B were previously characterized in Refs. 12 and 68, respectively. Below each panel is an autoradiograph showing 25% of each input [35S]BAF probe. The asterisks indicate residues switched to glutamic acid; all others are alanine substitutions. C, modeling of wild type BAF residues involved in binding to histones. The two BAF monomers are colored white and gray. The PyMol program (available on the World Wide Web at pymol.sourceforge.net; Mac OS/10 version) with BAF crystal structure coordinates (Protein Data Bank accession code 1CI4) (9) downloaded from the Protein Data Bank (on the World Wide Web at www.pdb.org) was used to locate and color wild type residues in which mutations greatly enhanced (dark pink), slightly enhanced (light pink), slightly reduced (light blue), or abolished (dark blue) binding to histones. The front view was rotated 90° toward or away from the reader to obtain the top and bottom views, respectively. To obtain the side view (DNA-binding surface), the front view was rotated 90° to the right.

 
PyMol software was used to color on the BAF dimer structure the positions of wild type residues in which mutations either partially reduced or enhanced (light blue or light pink respectively) or severely reduced or enhanced (dark blue or dark pink, respectively) BAF binding to histones (Fig. 5C). Two "strongly enhancing" mutations localized to the center front and center back (Fig. 5C, front view, K53E/A), whereas other strongly enhancing mutations mapped to the top (R8A and P14A) or bottom (W62A and K64E; Fig. 5C) of the BAF dimer. Several "enhancing" mutations (K53E, R60E, K64E, R75E, and R82E) switched a positively charged to a negatively charged residue; since histones are basic, enhanced binding might be due to charge effects, with the exception of residue Lys-53, since the alanine substitution mutant also showed an "enhanced" binding phenotype. Other "enhancing" mutations that cannot be explained by charge effects were R8A, P14A, and K18A, located on the top surface of the BAF dimer, suggesting that the top surface has an interesting role with respect to histones.

Wild type residues Val-51 (on the bottom surface) plus Lys-6 and Gly-25 (located at the proposed DNA binding surface; Fig. 5C, side view; see Ref. 9) were the only surface-exposed residues tested in which a mutation significantly reduced binding to histones (Fig. 5C, bottom and side views). However, this analysis did not reveal any "histone-specific" mutations. The G25E mutant was a negative control, because it does not dimerize, has no binding activity, and is inactive when added to nuclear assembly reactions (TABLE ONE; see also Ref. 69). Mutation of residue Val-51, which defines a hydrophobic groove on the bottom surface of BAF, also disrupts binding to emerin, MAN1-C, and DNA (TABLE ONE). Similarly, the Lys-6 mutation also abolishes binding to DNA and MAN1-C (TABLE ONE; other partners have not yet been tested). We conclude that the BAF-histone interaction involves several surfaces of BAF, including the hydrophobic groove defined by Val-51, and potentially also the DNA-binding surfaces.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We show that BAF binds linker histone H1.1 and core histone H3 with affinities of ~500-900 and ~100-200 nM in vitro, respectively. These affinities are biologically relevant, since BAF has an estimated concentration of ~9 µM near the nuclear inner membrane (26) and 1.9 µM within the nucleus.4 BAF binds emerin more tightly than histone H1.1 in vitro (Kd of 200 nM versus 700 nM), consistent with the enriched localization of endogenous BAF at the nuclear envelope of cultured Xenopus and HeLa cells (12, 59) and the reduced mobility of emerin-bound GFP-BAF in living HeLa cells (14). Since chromatin is abundant near the nuclear envelope, BAFs high affinity in vitro for H3 (~100-200 nM) and H1.1 (~700 nM) may also contribute to its enrichment at the nuclear periphery. However, given these relatively high affinities in vitro, why would GFP-BAF have such rapid (ms) recovery half-times in vivo (14)? One possibility is that GFP perturbs BAF binding to histones. Alternatively, BAF might have dynamic roles in chromatin structure, similar to HMG proteins and linker histones, which are also mobile in vivo (70). If BAF-histone interactions are dynamic, they might be regulated by post-translational modifications in vivo. Indeed, BAF is phosphorylated on Ser-4 and other residues at multiple stages of the cell cycle in vivo.5 Endogenous BAF can also form stable (immobile) complexes with LAP2{alpha} and telomeric chromatin at early stages of nuclear assembly (21). Furthermore, BAF, H1, and H3 co-purify from HeLa cell nuclei as stable components of a 1-MDa emerin-containing native complex,6 supporting their in vivo interactions. Thus, whereas most BAF appears rapidly mobile, subpopulations of BAF can be relatively immobile in vivo, suggesting both positive and negative regulation of BAF interactions in the nuclei of living cells. Our results, which demonstrate direct binding of BAF to core histone H3 and at least one linker histone (H1.1) in vivo and in vitro, are a major step toward understanding how BAF interacts with chromatin in cells.

BAF Residues Involved in Binding Histones—Our analysis of BAF missense mutants suggests that histones might contact the bottom groove of the BAF dimer and potentially also the side (where DNA binds) and top surfaces. Further experiments are needed to determine whether BAF binds histones and DNA cooperatively, competitively, or noncompetitively.

Four Ala substitution mutations in BAF (R8A, P14A, K18A, and W62A) and the K64E mutation each enhanced binding to histones but exhibited wild type binding to emerin and/or DNA (12, 68). Thus, these wild type residues and particularly the "top" surface of the BAF dimer might normally restrict or inhibit binding to histones. Interestingly, two of these mutants have dominant effects on chromatin when added to nuclear assembly reactions (TABLE ONE; see Ref. 12); even small amounts of mutant proteins P14A or K18A dominantly compress chromatin (TABLE ONE, C-C phenotype), supporting the notion that the top surface of BAF is physiologically relevant to histone binding and higher-order chromatin structure. The other "enhancing" mutations map on the bottom edge (W62A and K64E) or top edge (R8A) of the dimer, and current information is insufficient to explain their phenotypes. Nevertheless, the dominant chromatin compression phenotype caused by mutants P14A and K18A is consistent with and possibly explained by their enhanced binding to histones.

Two residues in which mutations significantly reduced binding to histones (L46E and G47E) are buried in the dimer interface; these mutant proteins purify as dimers by gel filtration chromatography (data not shown), but the dimers are weak, as shown by increased rates of exchange with wild type BAF (12, 68). The L46E mutation completely inactivates BAF both in vitro and in nuclear assembly reactions (12, 68). In contrast, mutation G47E abolishes binding to emerin, MAN1-C, and histones, but not DNA (12, 69) (see TABLE ONE). Mutant G47E dominantly compresses chromatin (TABLE ONE, C-C phenotype) even when added at low levels (4% over wild type) to Xenopus nuclear assembly reactions (12). Thus, we speculate that this mutant is dominant, because it can still cross-bridge DNA but fails in its "positive" role (chromatin decondensation) due to lack of binding to histones or LEM domain proteins. It is worth emphasizing that BAF is known to have positive roles in vivo; BAF is required to decondense chromatin during nuclear assembly, and this activity requires interactions with at least two types of partners: LEM domain proteins and lamins (13, 71). Our current findings add two new interactors, histones H3 and H1, to this picture.

BAF Interacts with the Tail of Histone H1.1—The last seven residues of the globular domain plus the C-tail of histone H1.1 were necessary and sufficient to bind BAF. Very few residues distinguish the BAF-binding region of H1.1 from H1.2, which did not bind BAF directly in vitro. Indeed, the seven critical residues (FKLNKKA) in the globular domain are identical in H1.1 and H1.2, suggesting that they are required but that specificity is determined exclusively by the C-tail. Of the three H1 subtypes we tested, only one (H1.1) bound directly to BAF in vitro. Four other subtypes are currently being tested; at least two (in addition to avian-specific histone H5) are expected to bind BAF based on the number of bands visualized in the blot overlay experiments (Fig. 1B).

Unexpectedly, BAF associated with both H1.1 and H1.2 in vivo (Fig. 4E). We hypothesize that H1.2 either binds BAF indirectly, via other protein(s) or by association with nucleosomes, or directly after post-translational modification(s) of one or both proteins. In this regard, BAF as well as linker histones are known to be post-translationally modified. BAF is phosphorylated in Ser-4 and other residues at many stages of the cell cycle.5 In mammalian cells, linker histones are heavily modified by acetylation, methylation, and phosphorylation (43); interestingly, cyclin-dependent kinases phosphorylate linker histones at the consensus motif S/T-P-X-Z (where X represents any amino acid, and Z is a basic amino acid) (42). These motifs are thought to mediate DNA condensation and are located in the "HMG-box-like fold" domain in the linker histone tail (72, 73). H1.1 has two (S/T)PXK motifs, whereas H1.2 has at least four (S/T)PXK motifs (74). Of all linker histones, H1.1 and H1.2 have the shortest C-tails, and both bind and dissociate from chromatin rapidly (recovery times of ~100 s in FRAP experiments) (74). Interestingly, both subtypes associate preferentially with open/active chromatin (37), consistent with our co-immunoprecipitation of both subtypes from HeLa cells.

Histone H1.2 is highly abundant and is expressed in most tissues examined (37). In contrast, H1.1 expression is restricted to thymus, spleen, neurons, testis, and lymphocytes (60, 75, 76). Four of these tissues (thymus, spleen, neurons, and lymphocytes) are notably susceptible to infection by HIV-1 (see below). However, general conclusions about the properties of BAF-binding linker histones must await further studies of the remaining four subtypes. We speculate that BAF binds directly to at least one ubiquitous subtype, since BAF itself is expressed nearly ubiquitously.

BAF Interacts with Helix {alpha}N of H3—Histone H3 has three {alpha}-helices ({alpha}1, {alpha}2, and {alpha}3) and two intervening loops (L1 and L2) that comprise its "histone fold domain" plus an {alpha}-helical extension named {alpha}N (residues 46-57; with initiating methionine as number 1), which connects to the N-tail (residues 1-41) via a short linker (residues 42-45) (67). Helix {alpha}N binds 13 bp of the DNA superhelix and contacts parts of core histones H4 and H2A but also has residues that are exposed on the nucleosome surface (67). We found that residues 1-64 were sufficient to bind BAF directly in vitro, whereas residues 1-47 (the tail and short linker) were insufficient. Thus, we deduce BAF binds directly to helix {alpha}N but cannot rule out additional interactions with the linker or N-tail. Interestingly, BAF binding to the H3 core ({alpha}N domain) has the potential for direct post-translational regulation. Other regions of the core are known to be modified in vivo (e.g. Lys-79 methylation during telomeric silencing) (77), and a recent peptide mass fingerprint analysis showed methylation of either Arg-52, Arg-53, or Lys-56 in helix {alpha}N in vivo (33). We speculate that these (and other) exposed residues in {alpha}N might directly contact BAF. Further regulation of BAF-H3 binding via post-translational modifications of the H3 tail is also possible.

Implications for BAF Function during Retroviral Preintegration—By binding histones (or nucleosomes), BAF has the potential to influence the mechanism or sites of HIV-1 integration into human chromosomes. HIV-1 integrates preferentially into regions containing active genes (78). This bias could be a consequence of more accessible chromatin structure; in this case, BAF might simply help position the incoming viral DNA on histones or nucleosomes. Alternatively, we speculate that BAF might facilitate retrovirus insertion in vivo via preferential association with histone H1.1 (and/or H1.2), which, as noted above, are enriched in active chromatin and highly expressed in cells and tissues that can be infected by HIV-1. A small number of BAF mutants were previously tested for their ability to reconstitute the DNA integration activity of salt-extracted HIV-1 PICs in vitro (68); only two mutants had reduced (mutant K6A) or undetectable (mutant I26A) PIC reconstitution activity, and both also had reduced binding to histones in our assays (TABLE ONE). Thus, BAF interactions with histones might be relevant to HIV-1 integration.

Model: BAF Interacts with Nucleosomes—We propose that a major physiological target for BAF is the nucleosome. This model is based on the direct binding of BAF to the globular domain of histone H3 and to selected linker histones, including H1.1, and its direct nonspecific binding to double-stranded DNA. This model is also consistent with the rapid (~260-ms) diffusional mobility of BAF in vivo (14), since HMG and linker histones, which regulate nucleosome compaction in vivo, are also mobile with diffusional half-times on the order of seconds (70). If BAF were to influence nucleosome compaction or decompaction, this would provide a mechanism for its observed effects on higher order chromatin structure and nuclear assembly (see Introduction) and potentially explain why BAF is essential in metazoans.

To date, chromatin immunoprecipitation experiments have yielded no evidence that BAF associates preferentially with either silenced (K9-methylated H3) or active (K4-methylated H3) chromatin.7 Indeed, the ability of BAF to influence both chromatin compaction and decondensation in cell-free Xenopus nuclear assembly extracts, which are inactive for gene expression, suggest that BAF might organize higher order chromatin structure irrespective of its transcriptional status. Testing the hypothesis that BAF regulates nucleosome organization will be a major focus of future work.


   FOOTNOTES
 
* This work was funded by National Institutes of Health Grant RO1 GM48646 (to K. L. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Stowers Institute for Medical Research, Kansas City, MO 64110. Back

2 To whom correspondence should be addressed: Dept. of Cell Biology, 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.

3 The abbreviations used are: BAF, barrier to autointegration factor; PICs, preintegration complexes; HIV-1, human immunodeficiency virus type 1; H1, linker histones; HMG, high mobility group; GST, glutathione S-transferase; IP, immunoprecipitation; GFP, green fluorescent protein; BSA, bovine serum albumin; xH3, Xenopus H3; hH3, human H3; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Back

4 J. M. Holaska and K. L. Wilson, unpublished estimate. Back

5 L. Bengtsson and K. L. Wilson, unpublished observations. Back

6 J. M. Holaska and K. L. Wilson, unpublished observations. Back

7 R. Montes de Oca and K. L. Wilson, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank K. Witwer for initiating this project, M. Eddins for purifying GST-histones, and K. Tifft for giving advice on the chymotrypsin assays. We thank the Wilson laboratory, especially M. Mansharamani and J. Holaska, for stimulating discussions and advice. We are grateful to J. Workman, M. Carrozza, and C. Kiesecker (Stowers Institute for Medical Research) for providing yeast histones; F. Gordon (University of Texas Health Science Center at San Antonio), X. Lu, and J. Hansen (Colorado State University) for mouse H1o and xH3 cDNAs and proteins; and T. Owen-Hughes (University of Dundee) for the human H3.1 construct. We thank J. Avalos for help with atomic modeling.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Segura-Totten, M., and Wilson, K. (2004) Trends Cell Biol. 14, 261-266[CrossRef][Medline] [Order article via Infotrieve]
  2. Lee, M. S., and Craigie, R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1528-1533[Abstract/Free Full Text]
  3. Lee, M. S., and Craigie, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9823-9827[Abstract/Free Full Text]
  4. Chen, H., and Engelman, A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15270-15274[Abstract/Free Full Text]
  5. Mansharamani, M., Graham, D. R., Monie, D., Lee, K. K., Hildreth, J. E., Siliciano, R. F., and Wilson, K. L. (2003) J. Virol. 77, 13084-13092[Abstract/Free Full Text]
  6. Suzuki, Y., and Craigie, R. (2002) J. Virol. 76, 12376-12380[Abstract/Free Full Text]
  7. Suzuki, Y., Yang, H., and Craigie, R. (2004) EMBO J. 23, 4670-4678[CrossRef][Medline] [Order article via Infotrieve]
  8. Cai, M., Huang, Y., Zheng, R., Wei, S. Q., Ghirlando, R., Lee, M. S., Craigie, R., Gronenborn, A. M., and Clore, G. M. (1998) Nat. Struct. Biol. 5, 903-909[CrossRef][Medline] [Order article via Infotrieve]
  9. Umland, T. C., Wei, S.-Q., Craigie, R., and Davies, D. R. (2000) Biochemistry 39, 9130-9138[CrossRef][Medline] [Order article via Infotrieve]
  10. Zheng, R., Ghirlando, R., Lee, M. S., Mizuuchi, K., Krause, M., and Craigie, R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8997-9002[Abstract/Free Full Text]
  11. Forne, I., Carrascal, M., Martinez-Lostao, L., Abian, J., Rodriguez-Sanchez, J. L., and Juarez, C. (2003) J. Biol. Chem. 278, 50641-50644[Abstract/Free Full Text]
  12. Segura-Totten, M., Kowalski, A. M., Craigie, R., and Wilson, K. L. (2002) J. Cell Biol. 158, 475-485[Abstract/Free Full Text]
  13. Liu, J., Lee, K. K., Segura-Totten, M., Neufeld, E., Wilson, K. L., and Gruenbaum, Y. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 4598-4603[Abstract/Free Full Text]
  14. Shimi, T., Koujin, T., Segura-Totten, M., Wilson, K. L., Haraguchi, T., and Hiraoka, Y. (2004) J. Struct. Biol. 147, 31-41[CrossRef][Medline] [Order article via Infotrieve]
  15. Furukawa, K., Sugiyama, S., Osouda, S., Goto, H., Inagaki, M., Horigome, T., Omata, S., McConnell, M., Fisher, P. A., and Nishida, Y. (2003) J. Cell Sci. 116, 3811-3823[Abstract/Free Full Text]
  16. Shumaker, D. K., Lee, K. K., Tanhehco, Y. C., Craigie, R., and Wilson, K. L. (2001) EMBO J. 20, 1754-1764[CrossRef][Medline] [Order article via Infotrieve]
  17. Cai, M., Huang, Y., Ghirlando, R., Wilson, K. L., Craigie, R., and Clore, G. M. (2001) EMBO J. 20, 4399-4407[CrossRef][Medline] [Order article via Infotrieve]
  18. Wolff, N., Gilquin, B., Courchay, K., Callebaut, I., Worman, H. J., and Zinn-Justin, S. (2001) FEBS Lett. 501, 171-176[CrossRef][Medline] [Order article via Infotrieve]
  19. Laguri, C., Gilquin, B., Wolff, N., Romi-Lebrun, R., Courchay, K., Callebaut, I., Worman, H. J., and Zinn-Justin, S. (2001) Structure (Camb.) 9, 503-511[Medline] [Order article via Infotrieve]
  20. Lee, K. K., Haraguchi, T., Lee, R. S., Koujin, T., Hiraoka, Y., and Wilson, K. L. (2001) J. Cell Sci. 114, 4567-4573[Medline] [Order article via Infotrieve]
  21. Dechat, T., Gajewski, A., Korbei, B., Gerlich, D., Daigle, N., Haraguchi, T., Furukawa, K., Ellenberg, J., and Foisner, R. (2004) J. Cell Sci. 117, 6117-6128[Abstract/Free Full Text]
  22. Burke, B., and Stewart, C. L. (2002) Nat. Rev. Mol. Cell. Biol. 3, 575-585[CrossRef][Medline] [Order article via Infotrieve]
  23. Bengtsson, L., and Wilson, K. L. (2004) Curr. Opin. Cell Biol. 16, 73-79[CrossRef][Medline] [Order article via Infotrieve]
  24. Vlcek, S., Just, H., Dechat, T., and Foisner, R. (1999) EMBO J. 18, 6370-6384[CrossRef][Medline] [Order article via Infotrieve]
  25. Mounkes, L. C., and Stewart, C. L. (2004) Curr. Opin. Cell Biol. 16, 322-327[CrossRef][Medline] [Order article via Infotrieve]
  26. Holaska, J., Lee, K., Kowalski, A., and Wilson, K. (2003) J. Biol. Chem. 278, 6969-6975[Abstract/Free Full Text]
  27. Wang, X., Xu, S., Rivolta, C., Li, L. Y., Peng, G., Swain, P. K., Sung, C., Swaroop, A., Berson, E. L., Dryja, T. P., and Chen, S. (2002) J. Biol. Chem. 277, 43288-43300[Abstract/Free Full Text]
  28. Richmond, T. J., and Davey, C. A. (2003) Nature 423, 145-150[CrossRef][Medline] [Order article via Infotrieve]
  29. Reeves, R. (2001) Gene (Amst.) 277, 63-81[CrossRef][Medline] [Order article via Infotrieve]
  30. Georgel, P. T., Palacios DeBeer, M. A., Pietz, G., Fox, C. A., and Hansen, J. C. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 8584-8589[Abstract/Free Full Text]
  31. Georgel, P. T., Horowitz-Scherer, R. A., Adkins, N., Woodcock, C. L., Wade, P. A., and Hansen, J. C. (2003) J. Biol. Chem. 278, 32181-32188[Abstract/Free Full Text]
  32. Ramakrishnan, V. (1997) Crit. Rev. Eukaryot. Gene Expr. 7, 215-230[Medline] [Order article via Infotrieve]
  33. Zhang, L., Eugeni, E. E., Parthun, M. R., and Freitas, M. A. (2003) Chromosoma 112, 77-86[CrossRef][Medline] [Order article via Infotrieve]
  34. Vermaak, D., Ahmad, K., and Henikoff, S. (2003) Curr. Opin. Cell Biol. 15, 266-274[CrossRef][Medline] [Order article via Infotrieve]
  35. Wang, Y., Fischle, W., Cheung, W., Jacobs, S., Khorasanizadeh, S., and Allis, C. D. (2004) Novartis Found. Symp. 259, 3-21, 163-169[Medline] [Order article via Infotrieve]
  36. Parseghian, M. H., Newcomb, R. L., and Hamkalo, B. A. (2001) J. Cell. Biochem. 83, 643-659[CrossRef][Medline] [Order article via Infotrieve]
  37. Parseghian, M. H., and Hamkalo, B. A. (2001) Biochem. Cell Biol. 79, 289-304[CrossRef][Medline] [Order article via Infotrieve]
  38. Kinkade, J. M., Jr., and Cole, R. D. (1966) J. Biol. Chem. 241, 5798-5805[Abstract/Free Full Text]
  39. Albig, W., Kioschis, P., Poustka, A., Meergans, K., and Doenecke, D. (1997) Genomics 40, 314-322[CrossRef][Medline] [Order article via Infotrieve]
  40. Wolffe, A. P. (1997) Int. J. Biochem. Cell Biol. 29, 1463-1466[CrossRef][Medline] [Order article via Infotrieve]
  41. Belikov, S., and Karpov, V. (1998) J. Biomol. Struct. Dyn. 16, 35-39[Medline] [Order article via Infotrieve]
  42. Swank, R. A., Th'ng, J. P., Guo, X. W., Valdez, J., Bradbury, E. M., and Gurley, L. R. (1997) Biochemistry 36, 13761-13768[CrossRef][Medline] [Order article via Infotrieve]
  43. Garcia, B. A., Busby, S. A., Barber, C. M., Shabanowitz, J., Allis, C. D., and Hunt, D. F. (2004) J. Proteome Res. 3, 1219-1227[CrossRef][Medline] [Order article via Infotrieve]
  44. Kuzmichev, A., Jenuwein, T., Tempst, P., and Reinberg, D. (2004) Mol. Cell 14, 183-193[CrossRef][Medline] [Order article via Infotrieve]
  45. Ramakrishnan, V., Finch, J. T., Graziano, V., Lee, P. L., and Sweet, R. M. (1993) Nature 362, 219-223[CrossRef][Medline] [Order article via Infotrieve]
  46. Goytisolo, F. A., Gerchman, S. E., Yu, X., Rees, C., Graziano, V., Ramakrishnan, V., and Thomas, J. O. (1996) EMBO J. 15, 3421-3429[Medline] [Order article via Infotrieve]
  47. Vignali, M., and Workman, J. L. (1998) Nat. Struct. Biol. 5, 1025-1028[CrossRef][Medline] [Order article via Infotrieve]
  48. Thoma, F., Losa, R., and Koller, T. (1983) J. Mol. Biol. 167, 619-640[Medline] [Order article via Infotrieve]
  49. Allan, J., Mitchell, T., Harborne, N., Bohm, L., and Crane-Robinson, C. (1986) J. Mol. Biol. 187, 591-601[CrossRef][Medline] [Order article via Infotrieve]
  50. Moran, F., Rodriguez, A. T., Suau, P., and Montero, F. (1989) Biophys. Chem. 33, 133-141[CrossRef][Medline] [Order article via Infotrieve]
  51. Rodriguez, A. T., Perez, L., Moran, F., Montero, F., and Suau, P. (1991) Biophys. Chem. 39, 145-152[CrossRef][Medline] [Order article via Infotrieve]
  52. De, S., Brown, D. T., Lu, Z. H., Leno, G. H., Wellman, S. E., and Sittman, D. B. (2002) Gene (Amst.) 292, 173-181[CrossRef][Medline] [Order article via Infotrieve]
  53. Clark, D. J., and Thomas, J. O. (1988) Eur. J. Biochem. 178, 225-233[Medline] [Order article via Infotrieve]
  54. Roth, S. Y., and Allis, C. D. (1992) Trends Biochem. Sci. 17, 93-98[CrossRef][Medline] [Order article via Infotrieve]
  55. Luger, K., Rechsteiner, T. J., and Richmond, T. J. (1999) Methods Mol. Biol. 119, 1-16[Medline] [Order article via Infotrieve]
  56. Gelbart, M. E., Rechsteiner, T., Richmond, T. J., and Tsukiyama, T. (2001) Mol. Cell. Biol. 21, 2098-2106[Abstract/Free Full Text]
  57. Bradbury, E. M., Cary, P. D., Chapman, G. E., Crane-Robinson, C., Danby, S. E., Rattle, H. W., Boublik, M., Palau, J., and Aviles, F. J. (1975) Eur. J. Biochem. 52, 605-613[Medline] [Order article via Infotrieve]
  58. Black, B. E., Levesque, L., Holaska, J. M., Wood, T. C., and Paschal, B. M. (1999) Mol. Cell. Biol. 19, 8616-8624[Abstract/Free Full Text]
  59. Haraguchi, T., Koujin, T., Segura-Totten, M., Lee, K. K., Matsuoka, Y., Yoneda, Y., Wilson, K. L., and Hiraoka, Y. (2001) J. Cell Sci. 114, 4575-4585[Medline] [Order article via Infotrieve]
  60. Lennox, R. W., and Cohen, L. H. (1984) Dev. Biol. 103, 80-84[CrossRef][Medline] [Order article via Infotrieve]
  61. Alami, R., Fan, Y., Pack, S., Sonbuchner, T. M., Besse, A., Lin, Q., Greally, J. M., Skoultchi, A. I., and Bouhassira, E. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 5920-5925[Abstract/Free Full Text]
  62. Albig, W., Drabent, B., Kunz, J., Kalff-Suske, M., Grzeschik, K. H., and Doenecke, D. (1993) Genomics 16, 649-654[CrossRef][Medline] [Order article via Infotrieve]
  63. Parseghian, M. H., Clark, R. F., Hauser, L. J., Dvorkin, N., Harris, D. A., and Hamkalo, B. A. (1993) Chromosome. Res. 1, 127-139[CrossRef][Medline] [Order article via Infotrieve]
  64. Losa, R., Thoma, F., and Koller, T. (1984) J. Mol. Biol. 175, 529-551[CrossRef][Medline] [Order article via Infotrieve]
  65. Tagami, H., Ray-Gallet, D., Almouzni, G., and Nakatani, Y. (2004) Cell 116, 51-61[CrossRef][Medline] [Order article via Infotrieve]
  66. Malik, H. S., and Henikoff, S. (2003) Nat. Struct. Biol. 10, 882-891[CrossRef][Medline] [Order article via Infotrieve]
  67. Luger, K., Möder, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Nature 389, 251-260[CrossRef][Medline] [Order article via Infotrieve]
  68. Harris, D., and Engelman, A. (2000) J. Biol. Chem. 275, 39671-39677[Abstract/Free Full Text]
  69. Mansharamani, M., and Wilson, K. L. (2005) J. Biol. Chem. 280, 13863-13870[Abstract/Free Full Text]
  70. Catez, F., Yang, H., Tracey, K. J., Reeves, R., Misteli, T., and Bustin, M. (2004) Mol. Cell. Biol. 24, 4321-4328[Abstract/Free Full Text]
  71. Margalit, A., Segura-Totten, M., Gruenbaum, Y., and Wilson, K. L. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 3290-3295[Abstract/Free Full Text]
  72. Bharath, M. M. S., Ramesh, S., Chandra, N. R., and Rao, M. R. S. (2002) Biochemistry 41, 7617-7627[CrossRef][Medline] [Order article via Infotrieve]
  73. Bharath, M. M. S., Chandra, N. R., and Rao, M. R. S. (2002) Proteins 49, 71-81[CrossRef][Medline] [Order article via Infotrieve]
  74. Th'ng, J. P., Sung, R., Ye, M., and Hendzel, M. J. (2005) J. Biol. Chem. 280, 27809-27814[Abstract/Free Full Text]
  75. Rasheed, B. K., Whisenant, E. C., Ghai, R. D., Papaioannou, V. E., and Bhatnagar, Y. M. (1989) J. Cell Sci. 94, 61-71[Abstract/Free Full Text]
  76. Franke, K., Drabent, B., and Doenecke, D. (1998) Biochim. Biophys. Acta 1398, 232-242[Medline] [Order article via Infotrieve]
  77. Ng, H. H., Feng, Q., Wang, H., Erdjument-Bromage, H., Tempst, P., Zhang, Y., and Struhl, K. (2002) Genes Dev. 16, 1518-1527[Abstract/Free Full Text]
  78. Schroder, A. R., Shinn, P., Chen, H., Berry, C., Ecker, J. R., and Bushman, F. (2002) Cell 4, 521-529

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