INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Structural change of chromatin in eukaryotic cells has an impact on biological processes such as gene expression, replication, and maintenance (57, 59). Disruption and reformation of nuclear architecture involve global remodeling of chromatin organization in mitotic and meiotic cell cycle (29, 47). Thus, chromatin remodeling has been one of the hot topics in molecular and cell biology in recent years (3, 12, 22, 52, 53, 55).

Chromatin consists of a repeating unit, nucleosome, in which about 200 bp of DNA wrap around a core histone octamer (H2A, H2B, H3, H4)₂. Mixing of histones with DNA at physiological ionic strength generally results in precipitation of histone-DNA aggregates. It is therefore thought that nucleosome assembly-remodeling factors are required to mediate nucleosome assembly under physiological conditions (22). These factors are classified into two classes at least. One includes so-called histone chaperones, proteins which recruit and/or deposit histones to DNA. Nucleoplasmin is the first identified histone chaperone; it was originally identified as an abundant nucleosome assembly factor in oocytes of Xenopus laevis (31). It is reported that in Xenopus egg extracts, core histones are complexed with nucleoplasmin and a pair of other histone chaperones, N1 and N2 (10, 28). From somatic cells, other nucleosome assembly factors, such as chromosome assembly factor I (CAF-I) and nucleosome assembly protein I (NAP-I), have been identified by using DNA replication-dependent or -independent nucleosome assembly systems (18, 50). Recent progress in our understanding of chromatin remodeling from considerable experimental efforts has identified the second class of nucleosome assembly-remodeling factors, i.e., chromatin remodeling factors dependent on ATP hydrolysis (22, 53, 55). One of these ATP-dependent chromatin remodeling factors, ACF, can act with NAP-I or CAF-I to mediate the formation of periodic nucleosome arrays (20).

Dynamic remodeling in chromatin structure takes place during early development, represented by sperm chromatin decondensation and pronuclei formation upon fertilization (47). During spermatogenesis in Xenopus, subtypes of somatic histones are replaced with sperm-specific basic proteins. Xenopus sperm chromatin contains all four histones, but the amounts of H2A and H2B are considerably less than those of H3 and H4 (11, 23, 45). Demembraned Xenopus sperm chromatin is decondensed by incubation with Xenopus egg extracts in cell-free systems (11, 12, 23, 34, 42, 46). In the past decade, demembraned Xenopus sperm chromatin has been used extensively to study functions of histone binding proteins (7, 21, 24, 42, 45, 46). Again first studied was nucleoplasmin, which is most likely the actual player to decondense sperm chromatin in Xenopus eggs (42, 46). Depletion of nucleoplasmin from egg extracts results in a much lower rate of chromatin decondensation than that in the mock-depleted control extracts (46). Recently, Drosophila embryo extracts used to study the nucleosome assembly and fractionation of the extracts led to the identification of factors that decondense Xenopus sperm chromatin (6, 7, 19-21, 24). Thus, it is thought that factors which had been found originally as nucleosome assembly factors can be involved in remodeling of chromatin structure.

In the course of study to establish a cell-free adenovirus DNA replication system with a viral DNA template complexed with viral basic proteins (adenovirus core), we identified and purified a host factor designated template activating factor I (TAF-I) (35). Subsequently, we showed that TAF-I is also able to stimulate transcription from the adenovirus core but not from the naked adenovirus DNA as a template, suggesting that TAF-I functions as a remodeling factor of the structure of the adenovirus core (36). TAF-I was purified as a fraction containing 41- and 39-kDa proteins from uninfected HeLa cell extracts (35). cDNA cloning of human TAF-I revealed that the 39-kDa protein TAF-I is encoded by a previously identified gene called SET, which is fused to the CAN/NUP214 gene in a case of acute undifferentiated leukemia (39, 56). The 41-kDa protein, TAF-I, differs from TAF-I only at its amino-terminal region. TAF-I and TAF-I are highly acidic proteins with their pIs of 4.23 and 4.12, respectively. TAF-I has structural homology with NAP-I, and it was shown that TAF-I facilitates assembly of nucleosomes in the supercoiling assay, in which TAF-I introduces negative supercoiling in circular DNA when incubated with histones and DNA topoisomerase I (25, 39). We previously showed that NAP-I is also able to stimulate DNA replication and transcription of adenovirus core, suggesting a functional similarity between TAF-I and NAP-I (25, 39). Importantly, it has been indicated that TAF-I remodels the structure of the chromatin reconstituted with a DNA fragment and purified histones and stimulates transcription from it (44).

In this paper, we studied the function of TAF-I in decondensation of Xenopus sperm chromatin. We found that recombinant human TAF-I mediates the decondensation of sperm chromatin by releasing sperm basic proteins. Immunoblotting revealed the existence of the Xenopus homologue of TAF-I in oocyte nuclei and in the eggs. We cloned its cDNA and found that Xenopus TAF-I is active in sperm chromatin decondensation. Domain analyses of TAF-I suggested that the acidic region and the region possibly required for the structural integrity are essential for chromatin decondensation. The results presented here extend our knowledge on the structure-function relationships of histone-binding chromatin remodeling proteins.

	MATERIALS AND METHODS

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Preparation of proteins. Recombinant human TAF-I (hTAF-I) and its deletion mutants with a six-histidine tag at their amino termini were prepared as described previously (39). To generate hTAF-IPME, DNA fragments were prepared by PCR-mediated amplification with a cDNA clone as a template and oligonucleotide primers. TAF-I cDNA was separated into two parts, parts 1 and 2, in the middle of the region to be mutated. Each DNA fragment corresponding to two parts was amplified by PCR with oligonucleotide primers as follows: 5'-GGCAGCCATATGTCGGCGCCGGCGGCCAAAGTC-3' and 5'-CATTAGATCTGTCTTCTTCATTTTGTTCTTCATCAATGTGTTC-3' as an amino-terminal primer and a carboxyl-terminal primer, respectively, for part 1 of PME; 5'-GACAGATCTAATGAACAAGAAAGTGAGGAGATTTTG-3' and 5'-CGCGGATCCTTAGTCATCTTCTCCTTCATC-3' as an amino-terminal primer and a carboxyl-terminal primer, respectively, for part 2 of PME. Amplified DNA fragments for part 1 was treated with Klenow fragment (Takara Shuzo, Kusatsu, Japan) to blunt the ends and with polynucleotide kinase (TOYOBO, Osaka, Japan) to phosphorylate the 5' ends and cloned into the EcoRV site of plasmid pBluescript (Stratagene). The resultant plasmid was designated pBluescript-TAF-IPME. Amplified DNA fragments for part 2 were digested with BamHI and BglII and cloned into BamHI-BglII sites of pBluescript-TAF-IPME. A DNA fragment generated by digestion of the plasmid with both NdeI and BamHI were cloned into NdeI-BamHI sites of plasmid pET14b (Novagen). To obtain recombinant TAF-I protein, Escherichia coli BL21 (DE3) was transformed with each plasmid. Recombinant TAF-I was overexpressed by the addition of isopropyl--D-thiogalactopyranoside to the bacterial culture (800 ml). Bacterial cells were sonicated in 30 ml of sonication buffer consisting of 20 mM Tris HCl (pH 8.0) and 100 mM NaCl. The cell lysate was centrifuged at 12,000 × g for 10 min, and the supernatant was applied to a Talon metal affinity column (Clontech). Six-histidine-tagged TAF-I proteins were eluted with sonication buffer containing 100 mM imidazole. hTAF-IN1 and hTAF-IC3 were further purified by the isolation from a polyacrylamide gel and subjected to the denature-renature protocol (36). For preparation of glutathione S-transferase (GST)-hTAF-I and GST-AR, DNA fragments were obtained by PCR using the TAF-I cDNA clone as a template with the following primers: 5'-GGCGCGGATCCATGTCGGCGCAGGCGGCCAAAGTC-3' for GST-hTAF-I and 5'-CGCGGATCCGATGATGAAGAAGGAGAAGGAG-3' for GST-AR as amino-terminal primers, and 5'-CCGGAATTCCTTAGTCATCTTCTCCTTCATC-3' as a common carboxyl-terminal primer. The PCR products were digested with BamHI and EcoRI and cloned into BamHI-EcoRI sites of plasmid pGEX2TK (Amersham Pharmacia Biotech) for GST-hTAF-I or pGEX4T-3 for GST-AR. GST fusion recombinant proteins were overexpressed in the E. coli BL21 (DE3) strain as described above. Bacterial cells were suspended in phosphate-buffered saline (PBS) and sonicated. The soluble fractions were applied to a glutathione-Sepharose 4B column (Amersham Pharmacia Biotech), and the GST fusion proteins were eluted with 10 mM reduced glutathione in 50 mM Tris HCl (pH 8.0) as described in the manufacturer's instructions.

Preparation of extracts. To prepare the oocyte lysates used for detection of Xenopus TAF-I, defolliculated oocytes were prepared by treating frog ovaries with collagenase, and healthy stage VI oocytes (300 µl) were homogenized in 1 ml of 90 mM HEPES (pH 7.5)-70 mM KCl-5% sucrose-1 mM dithiothreitol (DTT). The homogenate was centrifuged in a microtube at 12,000 × g rpm for 10 min at 4°C, and the supernatant was used as the oocyte lysate. To examine the localization of proteins, nuclei were isolated from oocytes manually. Nuclear or cytoplasmic fractions from 10 oocytes were pooled and analyzed by immunoblotting as described below.

Decondensation of Xenopus sperm chromatin. Demembraned Xenopus sperm chromatin was prepared as described previously (51). In a standard assay, the demembraned sperm chromatin was incubated at a final concentration of 5 × 10³ sperm/µl with TAF-I at room temperature in 10 µl of reaction mixture containing 8 mM HEPES (pH 7.5), 8 mM KCl, 2 mM MgCl₂, 200 mM sucrose, 3.3 mM ATP, 33 mM creatine phosphate, 0.33 mg of phosphocreatine kinase per ml, and 0.8 mM DTT. After incubation, a 2-µl aliquot of the reaction mixture was added to 5 µl of PBS containing 10 µg of Hoechst 33258 per ml, 50% glycerol, and 7.4% formaldehyde on a slide glass. The DNA stained with the dye was visualized under a fluorescent microscope (Olympus). To analyze the chromatin-bound and released proteins during chromatin decondensation by TAF-I, sperm chromatin (1.5 × 10⁶) was incubated with 150 µg of GST or 250 µg of GST-hTAF-I at room temperature for 60 min under the conditions for the decondensation assay. The mixture was centrifuged at 12,000 × g for 10 min to separate the chromatin from the released proteins. The resulting supernatants were incubated with 20 µl of glutathione-Sepharose 4B by gentle agitation at 4°C for 60 min. Protein complexes bound to the Sepharose beads were precipitated by centrifugation and washed extensively with buffer A (20 mM Tris-HCl [pH 7.5], 1 mM EDTA, 10% glycerol, 1 mM DTT) containing 50 mM NaCl and then with buffer A containing 200 mM NaCl. Proteins were eluted with buffer A containing 1 M NaCl and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The chromatin was resuspended in buffer A containing 50 mM NaCl, and then HCl was added at a final concentration of 0.5 N. The mixture was incubated on ice for 10 min, and insoluble proteins were removed by centrifugation for 10 min. The HCl-soluble proteins (basic proteins) were concentrated by precipitation with trichloroacetic acid and analyzed by SDS-PAGE.

Chemical cross-linking analysis. Dimer formation of TAF-I was examined by chemical cross-linking of TAF-I proteins. One hundred nanograms of TAF-I was cross-linked at room temperature for 30 min with 0.05% glutaraldehyde in a buffer containing 30 mM HEPES (pH 7.9), 0.5 mM EDTA, 50 mM NaCl, and 10% glycerol. The reaction was stopped by adding the sample buffer for SDS-PAGE. Samples were then heated at 98°C for 2 min and separated by SDS-7.5% PAGE.

Restriction enzyme sensitivity assay. Adenovirus core (30 ng) was incubated with TAF-I at 30°C for 30 min in a buffer containing 12.5 mM MgCl₂, 60 mM KCl, 20 mM NaCl, 12 mM HEPES (pH 7.9), 1 mg of BSA per ml, and 8% glycerol and then digested at 30°C for 2 min with 1 unit of PvuII. DNA was purified and separated in a 1% agarose gel. DNA was transferred to a nylon membrane and visualized by hybridization with radiolabelled DNA spanning nucleotide positions 455 to 628 of the adenovirus genome around the E1A promoter region (25, 44).

cDNA cloning of Xenopus TAF-I cDNAs. A Xenopus oocyte cDNA library was constructed in the ZIPLOX vector by using SuperScript Lambda System (Life Technologies Inc.). A degenerated oligonucleotide set of 5'-GARAARGARCARCARGARGC-3' (R = G or A) encoding amino acids EKEQQEA and 5'-CCYTCYTCRTCRTCCATRTC-3' (R = G or A, Y = T or C) encoding amino acids DMDDEEG was used for reverse transcriptase-mediated PCR using Xenopus oocyte mRNA. Nucleotide sequence analysis of amplified DNA cloned into a plasmid vector revealed that the amino acid sequence encoded by the cloned DNA showed high homology with human TAF-I. Using this DNA fragment, the Xenopus oocyte cDNA library was screened. Positive clones were plaque-purified twice, and the cDNA clones were recovered in plasmids by using in vivo excision as described in the manufacturer's protocol and sequenced. To construct Xenopus TAF-I1 and TAF-I2 expression plasmids, PCR was performed with the cDNA clones as templates with primer sets of 5'-GGCAGCCATATGTCGGCGCCGGCGGCCA-3' for xTAF-I1 or 5'-GGCAGCCATATGTCGGCGCCCAAAGTCAGTAAAAAG-3' for xTAF-I2 as amino-terminal primers and 5'-AGCCCGCTCGAGATCATCTTCACCTTCGTCTTCC-3' as a common carboxyl-terminal primer. The PCR products were digested with NdeI and XhoI and inserted into NdeI-XhoI sites of pET24b (Novagen).

Immunoblot analysis. Cell-free transcription-coupled translation in rabbit reticulocyte lysate was performed with 1 µg of expression plasmids for xTAF-I1 or xTAF-I2 (see above) in a 50-µl reaction mixture with the TNT T7 Quick Coupled Transcription/Translation System (Promega). The cell-free translation products, Xenopus extracts, and purified proteins were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The following procedures were performed at room temperature. The membrane was blocked with 10% BSA in PBS plus 0.2% Tween 20 for 1 h, followed by incubation with first antibodies in PBS containing 5% BSA and 0.2% Tween 20 for 1 h. After washing with three changes of PBS containing 0.3% Triton X-100, the membrane was incubated with a secondary antibody, goat anti-mouse IgG antibody conjugated to horseradish peroxidase (Promega), in PBS containing 1% BSA and 0.2% Triton X-100 for 1 h. The membrane was washed as described above, and the signals were detected with ECL Western blotting detection reagent (Amersham Pharmacia Biotech).

Nucleotide sequence accession number. The complete nucleotide sequences of xTAF-I1 and xTAF-I2 cDNA obtained in this study will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases under accession no. AB022691 and AB022692, respectively.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

TAF-I, NAP-I, and nucleoplasmin share the acidic region in their carboxyl-terminal regions (13, 15, 39). The acidic region of nucleoplasmin has been suggested to be important for its interaction with histones (13). As shown in Fig. 1A, TAF-I has an acidic region with glutamic and aspartic acid stretches located at its carboxyl-terminal region (also see Fig. 7 for amino acid sequence). The carboxyl-terminal third of NAP-I is also acidic, consisting of tripartite highly acidic regions (Fig. 1A). This structural similarity prompted us to examine whether TAF-I can act as a chromatin remodeling factor in a sperm chromatin decondensation assay. We then found that this is indeed the case.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Structure of TAF-I and NAP-I. (A) Schematic diagrams of TAF-I, TAF-I, and NAP-I. TAF-I- and TAF-I-specific regions are indicated by gray and hatched boxes, respectively. The TAF-I acidic region and the NAP-I tripartite acidic regions are shown by black boxes. Bar, 100 amino acids. (B) Recombinant proteins used in this study. Purified recombinant human TAF-I and TAF-I and yeast and mouse NAP-I proteins (yNAP-I and mNAP-I, respectively) were analyzed by SDS-10% PAGE and stained with Coomassie brilliant blue. The sizes of the molecular mass markers (Bio-Rad) are shown on the right.

TAF-I, but not TAF-I, efficiently decondenses Xenopus sperm chromatin. Demembraned Xenopus sperm chromatin was incubated with recombinant human TAF-I and TAF-I (Fig. 1B), and the chromosomal DNA was stained with Hoechst 33258 stain and visualized under a fluorescent microscope. After incubation with TAF-I for 60 min, sperm chromatin was fully decondensed (Fig. 2A). Although TAF-I shares all the amino acid sequence with TAF-I except its amino-terminal specific region (Fig. 1A), it showed a very weak decondensation activity. In samples taken after incubation for 10 and 30 min with TAF-I, we did not observe any decondensed sperm chromatin. At 60 min, we found about 1% of sperm chromatin decondensed; its size was slightly larger than that of the condensed chromatin but not as large as that of the decondensed chromatin with TAF-I (Fig. 2A and data not shown).

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Decondensation of Xenopus sperm chromatin by TAF-I and TAF-I. (A) Time course of decondensation by TAF-I and TAF-I. Sperm chromatin (5 × 104 sperm) was incubated without (top panels) or with 5 µg of recombinant hTAF-I (middle panels) or hTAF-I (lower panels) as described in Materials and Methods. At the indicated times, aliquots were mixed with fixation buffer containing Hoechst 33258 stain and the chromosomal DNA was visualized under a fluorescent microscope. For the sample from the 60-min incubation with hTAF-I, two panels are shown to represent a weak decondensation activity (see text for details). Bar, 10 µm. (B) Dose response of hTAF-I in chromatin decondensation. Sperm chromatin (5 × 104 sperm) was incubated with 0, 0.5, 1.5, and 5 µg of hTAF-I for 10 and 60 min.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Chromatin decondensation by NAP-I. Sperm chromatin (5 × 104 sperm) was incubated with 5 µg of hTAF-I (B), mouse NAP-I (C), and yeast NAP-I (D) for 10 min. The chromosomal DNA was visualized as described in Materials and Methods. Sperm chromatin that was not incubated with any proteins is shown in panel A.

Domains of TAF-I required for the chromatin decondensation. Having established that acidic proteins TAF-I and NAP-I decondense Xenopus sperm chromatin, we wished to determine the domain which is required for this activity by using recombinant human TAF-I derivatives. We compared the activities of TAF-I and TAF-I and found that the chromatin decondensation caused by TAF-I was to a very limited extent in contrast to the efficient decondensation by TAF-I (Fig. 2A). In the first of the domain analyses, shown in Fig. 4, we used deletion mutants of hTAF-I. hTAF-IN1, which lacks the amino-terminal -specific domain, represents the / common regions (Fig. 4E). hTAF-IN1 binds to histones as efficiently as full-length hTAF-I (44). Sperm chromatin incubated with this mutant TAF-I was decondensed as efficiently as that with full-length hTAF-I. Given that TAF-I differs from TAF-I only at its amino-terminal region, these results suggest that the -specific region has a negative effect on the chromatin decondensation activity. In marked contrast, hTAF-IC3 [previously termed TAF-I(1-225) in reference 39] lacking the carboxyl-terminal acidic domain did not show any decondensation activity (Fig. 4A).

View larger version (31K): [in this window] [in a new window]   FIG. 4.   Domains required for chromatin decondensation. (A to D) Sperm chromatin was incubated with 5 µg of hTAF-I and its derivatives for 60 min. The chromosomal DNA was stained with Hoechst 33258 stain and visualized under a fluorescent microscope. In panel B, the gel-isolated hTAF-IN1 and hTAF-IC3 were used (see Materials and Methods). (E) The results of domain analysis for sperm chromatin decondensation are summarized. The decondensation activity of each hTAF-I derivative is shown to the right of its schematic diagram. + and , active and inactive in the decondensation assay, respectively. hTAF-I has a very weak activity, indicated by (+). Point mutations in hTAF-IPME are shown by X in the schematic diagram.

View larger version (25K): [in this window] [in a new window]   FIG. 5.   Dimerization of TAF-I. (A) Chemical cross-linking of hTAF-I proteins. One hundred nanograms of hTAF-IPME (lanes 1 and 2) or hTAF-I (lanes 3 and 4) was cross-linked with (lanes 2 and 4) or without (lanes 1 and 3) 0.05% glutaraldehyde. Samples were analyzed by SDS-7.5% PAGE, and proteins were detected by silver staining. Lane 5 shows marker proteins. Monomer and dimer are indicated by an arrowhead and an arrow, respectively. The cross-linked product migrating faster than the monomer appears to be a compact form of TAF-I due to intramolecular cross-linking. (B) Restriction enzyme sensitivity assay. Adenovirus core (30 ng) was incubated at 30°C for 30 min without (lanes 1 and 2) or with 100 ng (lanes 3 and 6), 200 ng (lanes 4 and 7), or 400 ng (lanes 5 and 8) of hTAF-I (lanes 3 to 5) and hTAF-IPME (lanes 6 to 8) and then digested with PvuII. Lane 1 shows the undigested DNA. DNA was purified and separated by electrophoresis in a 1% agarose gel. DNA fragments around the E1A promoter region were detected by Southern blotting. One-hundred-seventy-three- and 628-bp-long fragments were shown by the probe after partial digestion with PvuII.

TAF-I in the Xenopus oocytes and eggs. So far we have used recombinant hTAF-I in the decondensation assay of Xenopus sperm chromatin. Next we looked for TAF-I in Xenopus oocytes and eggs. We have prepared monoclonal antibodies against either hTAF-I- or hTAF-I-specific peptides as well as a monoclonal antibody that recognizes the / common region (40). Immunoblotting was employed to detect the Xenopus protein(s) by using these antibodies (Fig. 6A). With / common and -specific antibodies, we found a Xenopus protein which comigrated with hTAF-I purified from HeLa cells. TAF-I-specific antibody detected no protein in the Xenopus oocyte and egg extracts. These results suggest the existence of the  form of TAF-I in Xenopus oocytes and eggs. We next examined the localization of the Xenopus TAF-I protein in the oocytes. Nuclei were isolated from the oocytes manually under a microscope, and the resultant nuclear and cytoplasmic fractions were used for immunoblotting with anti-TAF-I antibody (Fig. 6B). The signal detected with anti-TAF-I antibody was found only in the oocyte nucleus. Although TAF-I was originally purified from HeLa cytoplasmic fractions, immunocytochemical analysis indicated its accumulation in the nuclei of somatic cells (35, 40). Previously, NAP-I homologues were detected in Xenopus oocytes (15). We then performed an immunoblotting of the same oocyte fractions with anti-NAP-I monoclonal antibody (17) and found the cytoplasmic localization of Xenopus NAP-I (Fig. 6B).

View larger version (37K): [in this window] [in a new window]   FIG. 6.   Existence of TAF-I, but not TAF-I, in Xenopus oocytes and eggs. (A) Detection of TAF-I in Xenopus oocytes and eggs. Xenopus oocyte lysates (30 µg of protein; lanes 1, 5, and 9), egg extracts (30 µg of protein; lanes 2, 6, and 10), purified HeLa TAF-I (lanes 3, 7, and 11), and recombinant human TAF-I (40 ng; lanes 4, 8, and 12) were analyzed by SDS-10% PAGE and transferred to a polyvinylidene difluoride membrane. TAF-I was detected with monoclonal antibodies which recognize human TAF-I and TAF-I common region (clone KM1725 in reference 40) (lanes 1 to 4), TAF-I-specific region (clone KM1720) (lanes 5 to 8), or TAF-I-specific region (clone KM1712) (lanes 9 to 12) by immunoblotting (see Fig. 7 for the epitope of each antibody). It should be noted that recombinant hTAF-I has a six-histidine tag at its amino terminus, which might have resulted in its lower mobility than that of the native hTAF-I (compare a band in lane 4 and the lower band in lane 3). Molecular masses of marker proteins are shown on the left. (B) Localization of TAF-I and NAP-I in oocytes. A Xenopus oocyte was separated into nucleus and cytoplasm. Lysates of total oocyte (T), nuclear (N), and cytoplasmic fractions (C) were analyzed by immunoblotting with anti-TAF-I antibody (lanes 1 to 3). Immunoblotting with anti-NAP-I antibody of the same oocyte fractionation is also shown (lanes 4 to 6). The positions of TAF-I and NAP-I are indicated by arrows. (C) Chromatin decondensation activity of TAF-I in heat-labile fraction of egg extracts. Xenopus egg extracts were heated at 80°C for 10 min and separated into heat-stable and heat-labile fractions. The heat-labile fraction was subjected to the denature-renature protocol, and TAF-I was depleted from the renatured heat-labile fraction. Left panels show immunoblotting analysis using anti-TAF-I antibody of egg extracts (lane 1), heat-labile fraction solubilized in guanidine-containing buffer (lane 2), heat-stable fraction (lane 3), mock-depleted renatured heat-labile fraction (lane 4), TAF-I depleted renatured heat-labile fraction (lane 5), and sequential dilutions of renatured heat-labile fraction (lane 6, 100%; lane 7, 30%; lane 8, 10%; lane 9, 3%). Right panels show chromatin decondensation with these fractions. Sperm chromatin (104 sperm) was incubated for 10 min with egg extracts, mock-depleted renatured heat-labile fraction, and TAF-I-depleted renatured heat-labile fraction. The chromosomal DNA was stained with Hoechst 33258 stain and visualized under a fluorescent microscope. (D) Sperm chromatin decondensation by TAF-I immunopurified from oocyte and egg extracts. The oocyte and egg extracts and recombinant hTAF-I were subjected to immunoprecipitation with anti-TAF-I antibody or control mouse IgG as described in Materials and Methods. The immunoprecipitates of oocyte extracts (labeled "o") (lanes 1, 5, and 13), egg extracts (labeled "e") (lanes 2, 6, and 14), and recombinant hTAF-I (labeled "r") (lanes 3 and 7) with anti-TAF-I antibody or the immunoprecipitates of recombinant hTAF-I with control mouse IgG (lanes 4 and 8) were analyzed by SDS-PAGE followed by staining with Coomassie brilliant blue (lanes 1 to 4) and by immunoblotting with anti-TAF-I antibody (lanes 5 to 8) and with anti-nucleoplasmin monoclonal antibody (lanes 13 and 14). Two microliters (lanes 9 and 11) and 0.6 microliter (lanes 10 and 12) of oocyte (lanes 9 and 10) and egg (lanes 11 and 12) extracts were also analyzed by immunoblotting with antinucleoplasmin antibody. Positions of TAF-I, IgG heavy chain (H) and light chain (L), and nucleoplasmin (NP) are indicated. It is important to note that the egg nucleoplasmin has lower mobility than the oocyte nucleoplasmin due to the hyperphosphorylation (32, 43, 48). The proteins released at pH 11 from the protein G beads were assayed for mediating the decondensation of sperm chromatin (2 × 104) for 60 min. The panel labelled "control" shows the sperm chromatin incubated with no added protein. The panel labelled "IP control" shows the sperm chromatin incubated with the proteins released after the immunoprecipitation of recombinant hTAF-I with control mouse IgG.

cDNA cloning of xTAF-I and its characterization. To obtain direct evidence that the Xenopus TAF-I indeed acts as a chromatin decondensation factor, we decided to clone its cDNA. Using degenerated oligonucleotides encoding amino acid sequences of hTAF-I, a DNA fragment from Xenopus mRNA whose sequence showed high homology with hTAF-I was amplified. This DNA fragment was used to screen the Xenopus oocyte cDNA library. We have obtained several positive clones, and two of them, which are designated xTAF-I1 and xTAF-I2, were completely sequenced (Fig. 7). Alignment of amino acid sequence deduced from the cloned cDNAs shows 96% identity between xTAF-I1 and xTAF-I2 and 93 to 94% identity between xTAF-I and hTAF-I. Since there was one amino acid difference between the xTAF-I sequence and the peptide sequence which was used to raise the monoclonal antibody against hTAF-I (Fig. 7A), we performed cell-free translation (Fig. 8A). The products of the cell-free transcription-coupled translation using xTAF-I cDNAs were specifically recognized by our anti-TAF-I antibody. These results confirmed that we have cloned xTAF-I cDNAs which actually encode the protein(s) detected by the immunoblotting in oocyte and egg extracts (Fig. 6). We then purified recombinant xTAF-I and used it in the chromatin decondensation assay (Fig. 8B). In a 60-min incubation with xTAF-I1, sperm chromatin was decondensed as efficiently as with hTAF-I.

View larger version (70K): [in this window] [in a new window]   FIG. 7.   Sequence comparison of TAF-I. (A) The complete amino acid sequences of Xenopus TAF-I1 and TAF-I2, human TAF-I (accession no. M93651), and TAF-I (D45198) aligned by use of a CLUSTALW program. Amino acids identical in all four sequences are indicated by asterisks, conserved substitutions are indicated by colons, and semiconserved substitutions are indicated by periods at the bottom of the alignment. The four amino acids mutated in hTAF-IPME are highlighted with black shading. The peptide sequence to produce monoclonal antibodies against TAF-I ( and  common, EQQEAIEHIDEVQNE; -specific, RPPPALGPEETSASA; -specific, SKKELNSNHDGADET) are boxed. (B) Amino acid sequence alignment of amino-terminal regions of TAF-I/SET from different organisms. Sequences are aligned from human, rat (accession no. S68589 and S68987), Xenopus, puffer fish (fugu fish; AF007219), Drosophila (U30470), C. elegans (Z54236), and yeast (Z71522) TAF-I/SET. Only the amino-terminal portion of the alignment is shown.

View larger version (22K): [in this window] [in a new window]   FIG. 8.   Analysis of recombinant Xenopus TAF-I. (A) Immunoblotting of cell-free translation products. Transcription-coupled translation in rabbit reticulocyte lysate was performed with no DNA (lane 1), expression plasmids of xTAF-I1 (lane 2), and xTAF-I2 (lane 3). Aliquots were subjected to immunoblotting with anti-TAF-I antibody. (B) Decondensation activity of xTAF-I. Sperm chromatin (5 × 104 sperm) was incubated with 5 µg of hTAF-I or xTAF-I1 for 60 min.

TAF-I directly interacts with sperm basic proteins. To examine protein composition of the chromatin decondensed by TAF-I, we analyzed chromatin-bound basic proteins by SDS-PAGE (Fig. 9). Before incubation with TAF-I, sperm chromatin contains sperm-specific proteins SP2 to SP6 in addition to histones H3 and H4 and small amounts of H2A and H2B. SP3 to SP5 comigrate towards each other under this condition (23). Almost all SP3 to SP6 and some of SP2 were removed from the chromatin by incubation with GST-hTAF-I (Fig. 9, lane 3). These results indicate that TAF-I mediates the decondensation of Xenopus sperm chromatin by releasing sperm-specific basic proteins. We performed a GST pulldown assay to test whether TAF-I interacts directly with core histones and/or sperm proteins upon chromatin decondensation. Protein complexes formed in the decondensation reaction were precipitated with GST-hTAF-I. SP3 to SP5 and SP6 were coprecipitated with GST-hTAF-I (Fig. 9, lane 6). In this experiment, we detected faint bands corresponding to SP2 and histone H4. These results demonstrate that TAF-I decondenses the sperm chromatin by interacting directly with sperm-specific basic proteins and releasing them from the chromatin.

View larger version (40K): [in this window] [in a new window]   FIG. 9.   Interaction of TAF-I with chromatin basic proteins. Sperm chromatin was incubated with GST (lanes 2 and 5) or GST-hTAF-I (lanes 3 and 6) under the conditions for chromatin decondensation for 60 min. The chromatin was precipitated by centrifugation, and chromatin-bound basic proteins were analyzed by SDS-15% PAGE (lanes 2 and 3). Proteins released during chromatin decondensation were subjected to a GST pulldown assay. Proteins eluted from the glutathione-Sepharose beads at 1 M NaCl were analyzed (lanes 5 and 6). Total proteins of sperm chromatin were electrophoresed in parallel (lane 1 and 4). The gel was stained with Coomassie brilliant blue. The position of sperm-specific basic proteins (SP2 to SP6) and core histones (H3 and H4) are indicated. Arrowheads on the left of the gel show the positions of marker proteins of 21.5 and 14.4 kDa.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this paper, we have shown that histone-binding acidic protein TAF-I, which was originally identified in somatic cells, has an activity to mediate the decondensation of Xenopus sperm chromatin dependent on its acidic region. We found the  form of TAF-I in Xenopus egg and oocyte extracts. Although it remains to be examined whether xTAF-I indeed functions in sperm chromatin decondensation in eggs, we found that both xTAF-I in the oocyte and egg extracts and its recombinant form are active in the chromatin decondensation assay. The extent of chromatin decondensation by purified TAF-I is comparable to that performed by egg extracts or by purified nucleoplasmin, but the time course of decondensation by TAF-I was slow (Fig. 2) (45, 46). Philpott et al. (46) observed decondensation of sperm chromatin in their nucleoplasmin-depleted egg extracts that occurred more slowly than that in the mock-depleted extracts and suggested the existence of other decondensation factors. Our observation supports this notion in that TAF-I present in egg extracts could have a redundant function to decondense sperm chromatin at a lower rate than that of nucleoplasmin.

Molecular mechanism of chromatin decondensation by TAF-I. We have shown that the sperm chromatin incubated with TAF-I has lost most of its sperm-specific basic proteins upon chromatin decondensation. The similar change in the protein composition of chromatin occurs when sperm chromatin was incubated with egg extracts or purified nucleoplasmin (45). We also showed the direct interaction of TAF-I with those released basic proteins in solution. These results suggest that TAF-I decondenses the sperm chromatin by interacting with sperm-specific basic proteins on the chromatin and releasing them from the chromatin.

TAF-I and other histone chaperones. Xenopus oocytes contain a large stockpile of proteins and mRNAs that will be used upon oocyte maturation and fertilization to midblastula transition, when zygotic transcription is repressed (9). The stored proteins in the oocytes include core histones to package newly synthesized DNA in the embryos (58). There are two complexes in the oocytes which contain histones and specific histone binding proteins: one consists of histones H2A and H2B and nucleoplasmin, and the other complex contains H3 and H4 and N1 (10, 28). Nucleoplasmin was found to be increasingly associated with maternal mRNA upon oocyte maturation (37). It remains to be examined whether xTAF-I is bound to core histones in the oocytes and eggs. Nucleoplasmin and N1 and N2 are the most abundant acidic proteins in the oocyte nucleus (30, 38). A homology to a bipartite nuclear localization signal consisting of two runs of basic amino acids separated by a spacer region, which was originally identified in nucleoplasmin, is found in hTAF-I and xTAF-I sequences (KRSSQTQNKASRKR) (41). Indeed, TAF-I was found in nuclear fractions of somatic cells and Xenopus oocytes (references 1 and 40 and this study). Our preliminary estimation indicates that 40 to 60 ng of TAF-I is present in one egg (36a). Assuming that one Xenopus egg contains 250 ng of nucleoplasmin and about 50 ng of TAF-I, it is thought that the contribution of xTAF-I to sperm chromatin decondensation in the eggs would be small. Indeed, we showed that depletion of TAF-I after the heat fractionation of the egg extracts resulted in the significant decrease of chromatin decondensation activity. We also proved that xTAF-I has a function in chromatin decondensation by isolating its cDNA and using the recombinant protein in the decondensation assay.

Cellular functions of TAF-I. We have originally identified TAF-I as an acidic host factor for DNA replication and transcription of the adenovirus core and proposed its possible function as a chromatin disassembly factor (35). Recently it was shown that TAF-I stimulates transcription from nucleosomal templates (44). TAF-I was also found to facilitate nucleosome assembly in the supercoiling assay (25), suggesting that TAF-I can act as a chromatin assembly or remodeling factor. NAP-I can be involved in promoter-specific chromatin remodeling to activate transcription from nucleosomal templates in conjunction with ATP-utilizing chromatin assembly factor, ACF (20). ACF also functions in assembly of regularly spaced nucleosomes together with CAF-I as well as with NAP-I. Considering the structural and functional similarity between TAF-I and NAP-I, one can speculate that TAF-I cooperates as a nucleosome assembly or remodeling protein with ACF or other chromatin remodeling factors. In the cell-free transcription system, TAF-I activated transcription of the adenovirus core from the E1A promoter but not from the major late promoter (36). Taken together, TAF-I can induce a local change in chromatin or chromatin-like structure as well as global chromatin remodeling such as sperm chromatin decondensation.