WSTF-ISWI chromatin remodeling complex targets heterochromatic replication foci
2002; Springer Nature; Volume: 21; Issue: 9 Linguagem: Inglês
10.1093/emboj/21.9.2231
ISSN1460-2075
Autores Tópico(s)Williams Syndrome Research
ResumoArticle1 May 2002free access WSTF–ISWI chromatin remodeling complex targets heterochromatic replication foci Ludmila Bozhenok Ludmila Bozhenok Marie Curie Research Institute, Oxted, Surrey, RH8 0TL UK Search for more papers by this author Paul A. Wade Paul A. Wade Department of Pathology, Emory University, Atlanta, GA, USA Search for more papers by this author Patrick Varga-Weisz Corresponding Author Patrick Varga-Weisz Marie Curie Research Institute, Oxted, Surrey, RH8 0TL UK Search for more papers by this author Ludmila Bozhenok Ludmila Bozhenok Marie Curie Research Institute, Oxted, Surrey, RH8 0TL UK Search for more papers by this author Paul A. Wade Paul A. Wade Department of Pathology, Emory University, Atlanta, GA, USA Search for more papers by this author Patrick Varga-Weisz Corresponding Author Patrick Varga-Weisz Marie Curie Research Institute, Oxted, Surrey, RH8 0TL UK Search for more papers by this author Author Information Ludmila Bozhenok1, Paul A. Wade2 and Patrick Varga-Weisz 1 1Marie Curie Research Institute, Oxted, Surrey, RH8 0TL UK 2Department of Pathology, Emory University, Atlanta, GA, USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:2231-2241https://doi.org/10.1093/emboj/21.9.2231 Correction(s) for this article WSTF–ISWI chromatin remodeling complex targets heterochromatic replication foci17 June 2002 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Williams Syndrome Transcription Factor (WSTF), the product of the WBSCR9 gene, is invariably deleted in the haploinsufficiency Williams–Beuren Syndrome. Along with the nucleosome-dependent ATPase ISWI, WSTF forms a novel chromatin remodeling complex, WICH (WSTF–ISWI chromatin remodeling complex), which is conserved in vertebrates. The WICH complex was purified to homogeneity from Xenopus egg extract and was found to contain only WSTF and ISWI. In mouse cells, WSTF interacts with the SNF2H isoform of ISWI. WSTF accumulates in pericentric heterochromatin coincident with the replication of these structures, suggesting a role for WSTF in the replication of heterochromatin. Such a role is supported by the in vitro activity of both the mouse and frog WICH complexes: they are involved in the assembly of regular spaced nucleosomal arrays. In contrast to the related ISWI-interacting protein ACF1/WCRF180, WSTF binds stably to mitotic chromosomes. As dysfunction of other chromatin remodeling factors often has severe effects on development, haploinsufficiency of WSTF may explain some of the phenotypes associated with this disease. Introduction Williams–Beuren syndrome is a developmental disorder characterized by congenital vascular and heart disease, particular facial features, growth deficiency, mental retardation and characteristic behavior patterns (Lenhoff et al., 1997; Francke, 1999). This disorder is caused by haploinsufficiency of ∼1.6 Mb of chromosome 7 (Francke, 1999). The deletion is uniform in size because it arises by inter- or intrachromosomal crossover events within regions of high sequence identity flanking the typical deletion. More than 15 genes reside in the deleted area and several of those may contribute to the complex multi-system clinical phenotype (Francke, 1999). One of the genes that invariably maps to the deletion region is WBSCR9, which encodes an ∼170 kDa protein called Williams syndrome transcription factor (WSTF) (Lu et al., 1998; Peoples et al., 1998). WSTF is expressed in many tissues and is subject to alternative splicing (Lu et al., 1998; Peoples et al., 1998). WSTF belongs to a family of proteins with similar domain structure, the BAZ (Jones et al., 2000a), also referred to as the WAL family (Poot et al., 2000). WSTF is related in subdomain architecture to ACF1 and WCRF180, subunits of the chromatin remodeling factors ACF, WCRF and CHRAC (Ito et al., 1999; Bochar et al., 2000; Guschin et al., 2000; LeRoy et al., 2000; Poot et al., 2000; Eberharter et al., 2001). hACF1 (LeRoy et al., 2000; Poot et al., 2000), BAZ1A (Jones et al., 2000a) and WCRF180 (Bochar et al., 2000) are essentially identical (with only three amino acid differences between the sequences published by Poot et al. and Bochar et al., which may reflect polymorphism), and this protein is the human ortholog of the Drosophila ACF1 protein (Ito et al., 1999). hACF1 (WCRF180/BAZ1A) and WSTF share an N-terminal WAC (WSTF/ACF1/cbp146) domain (Ito et al., 1999), which is followed by a DDT domain (Doerks et al., 2001), BAZ motifs (Jones et al., 2000a), a WAKZ domain (Ito et al., 1999), a PHD finger and a C-terminal bromodomain (Haynes et al., 1992) (see Figure 1). The WAC domain, DDT domain, BAZ and WAKZ motifs had been identified by their conservation in various proteins; no functionality has been assigned to these domains and motifs. A difference between WSTF and ACF1/WCRF180 is highlighted by the fact that domain analysis tools (e.g. RPS-BLAST, http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) identify a FERM domain (Chishti et al., 1998) related sequence in WSTF (Figure 1), but not in ACF1, despite the fact that the significance of this domain in WSTF is unclear. Figure 1.WSTF is conserved in vertebrates. The domain structure of WSTF is shown above. Bars below show the matching positions of corresponding ESTs from X.laevis (1–4), zebrafish (D.rerio, 5–7) and S.tropicalis (8–9). Accession numbers: 1, BI447904; 2, BG264264; 3, BI447594; 4, BG345707; 5, AI794397; 6, AI436874; 7, AW466480; 8, BG514920; 9, BG515236. Download figure Download PowerPoint The dynamic nature of eukaryotic chromatin requires special mechanisms to alter histone–DNA contacts. ATP-dependent chromatin remodeling factors use the energy gained from ATP hydrolysis to remodel nucleosomes (Kingston and Narlikar, 1999; Varga-Weisz, 2001). Most of these enzymes are multi-subunit complexes and all contain a SNF2 superfamily ATPase (Eisen et al., 1995). Four classes of ATP-dependent chromatin remodeling factors can be distinguished that contain either the SNF2, CHD/Mi-2, INO80 or ISWI ATPases (Kingston and Narlikar, 1999; Varga-Weisz, 2001). Three ISWI-containing complexes, nucleosome remodeling factor (NURF), CHRAC and ACF (Tsukiyama et al., 1995; Ito et al., 1997; Varga-Weisz et al., 1997), have been purified from Drosophila extracts and either disrupt or enhance the periodic organization of nucleosome arrays. Related ISWI-containing complexes have subsequently been purified from human cell extracts, Xenopus and budding yeast (LeRoy et al., 1998; Tsukiyama et al., 1999; Bochar et al., 2000; Guschin et al., 2000; LeRoy et al., 2000; Poot et al., 2000). ISWI complexes mobilize nucleosomes, resulting in alterations in the translational position of the histone octamer without displacement from DNA (Hamiche et al., 1999; Längst et al., 1999). Genetic analysis implicates ISWI in transcriptional regulation and maintenance of chromosome structure (Deuring et al., 2000; Goldmark et al., 2000). Three BAZ/WAL proteins interact independently with ISWI to form chromatin remodeling factors: ACF1/WCRF180 in the ACF, WCRF and CHRAC complexes from fly, frog and human (Ito et al., 1999; Bochar et al., 2000; Guschin et al., 2000; LeRoy et al., 2000; Poot et al., 2000; Eberharter et al., 2001), TIP5 (also called BAZ2A and WALp3) in the NoRC complex (Strohner et al., 2001) and NURF301 in NURF (Xiao et al., 2001). Whereas ISWI alone has nucleosome remodeling activity, ACF1 enhances the ATP-dependent chromatin assembly activity of ISWI (Ito et al., 1999) and the efficiency of ISWI-catalyzed nucleosome sliding by an order of magnitude, and determines the directionality of this sliding (Guschin et al., 2000; Eberharter et al., 2001). The largest subunit of NURF, NURF301, shares structural motifs with ACF1 and also modulates the nucleosome mobilizing property of ISWI (Xiao et al., 2001). Here we show that WSTF forms a nucleosome remodeling factor containing ISWI and that this factor is conserved across vertebrate phyla. Mammalian WSTF is targeted to pericentromeric heterochromatin during its replication and is distinguished from the related ACF1 protein by its ability to interact with mitotic chromatin. We suggest that WSTF is important for the dynamics of chromosome structure in vertebrates, which may explain some aspects of Williams–Beuren syndrome. Results WSTF is a highly conserved protein in vertebrates Searches of expressed sequence tag (EST) databases using the BLAST algorithm (Altschul et al., 1997) revealed that WSTF is highly conserved in mammals, with multiple ESTs from several organisms showing near sequence identity. In addition, many ESTs from amphibia (Xenopus laevis and Silurana tropicalis) and zebrafish (Danio rerio) share considerable sequence identity with human WSTF (Figure 1). Frog and fish ESTs more closely resemble human WSTF than human ACF1/WCRF180, and can be assigned as WSTF. This sequence conservation suggests a specific function for WSTF in vertebrates. To study the localization and function of WSTF, we raised a rabbit polyclonal antiserum against an N-terminal peptide of human WSTF. The affinity-purified antibody detected a protein of ∼175 kDa in mouse NIH 3T3 (Figure 2A) and human (HeLa) nuclear extracts (Figures 2B and 3A). No cross-reactivity with mouse or human ACF1 (WCRF180) or with any protein in Drosophila embryo extract, which contains much ACF1, was observed (see Figure 2A and D). We concluded that this antiserum specifically recognizes the WSTF protein. Figure 2.WSTF and ISWI form a complex in mouse and human cells. (A) Western blot analysis of NIH 3T3 nuclear extract and Drosophila embryo extract using affinity-purified anti-WSTF antibody. (B) Co-immunoprecipitation of WSTF with affinity-purified anti-ISWI antibodies in HeLa cell nuclear extracts (left panel) and NIH 3T3 cell nuclear extracts (right panel). Input (12%); IP, immunoprecipitate (100%); sup, supernatant of immunoprecipitate. (C) Co-immunoprecipitation of ISWI with affinity-purified anti-WSTF antibodies. Input (4%); IP, immunoprecipitate (100%). Control immunoprecipitations in (B) and (C) were with the same amount of purified rabbit IgG. (D) Fractionation of WSTF, hACF1(WCRF180) and ISWI from crude HeLa nuclear extract by Superose-6 gel filtration chromatography. Upper panel: overlay of three separate western blots against WSTF, hACF1 (WCRF180) and hISWI of the fractions. Lower panel: fractionation of WSTF in HeLa nuclear extract immunodepleted with antibodies against hISWI (mock depletion was with pre-immune serum). Size standards were thyroglobulin (670 kDa) and catalase (232 kDa). Download figure Download PowerPoint Figure 3.WSTF interacts with the SNF2H isoform of ISWI. (A) Western blot of identical amounts of nuclear extract proteins from mouse ES cells, ES cells driven to differentiation (dES), NIH 3T3 and HeLa cells. (B) Western blot of affinity-purified mouse WSTF–ISWI complex: input, 10%; peptide eluate, 100%. (C) Immunoprecipitation of WSTF– and ACF1–ISWI complexes from ES cell nuclear extract. The complexes were eluted from the antibodies with the antigenic peptides and analyzed by western blots: input, 16%; peptide eluate, 100%. Download figure Download PowerPoint WSTF forms a complex with ISWI We used the anti-WSTF rabbit polyclonal antiserum in co-immunoprecipitation experiments to analyze the interaction of WSTF with human and mouse ISWI. An affinity-purified antiserum against human ISWI (α-hISWI), which recognizes both the SNF2H and SNF2L isoforms and co-immunoprecipitates hACF1 (Poot et al., 2000), efficiently depleted WSTF as well as hACF1 from nuclear extracts of HeLa and NIH 3T3 cells [Figure 2B, ‘input’ versus supernatant (‘sup’)]. Both WSTF and hACF1 were found in the ISWI immunoprecipitate (Figure 2B, ‘IP’). Conversely, the antiserum against WSTF (α-WSTF) precipitated both WSTF and ISWI (Figure 2C) from NIH 3T3 extracts. The WSTF–ISWI complex could be eluted from the immunocomplexes bound to protein A beads with the antigenic peptide (see below). We concluded that WSTF and ISWI form a complex in both human and mouse nuclei. The failure of the α-WSTF antiserum to co-immunoprecipitate hACF1 protein excluded the possibility of a ternary complex containing WSTF, hACF1 and ISWI (data not shown). We did not observe an interaction between WSTF and HuCHRAC-17, the histone-fold-containing subunit of HuCHRAC (Poot et al., 2000), or with topoisomerase II (data not shown). We fractionated crude HeLa nuclear extract by gel filtration with Superose-6 and probed the fractions for the presence of WSTF, hACF1/WCRF180 and ISWI by western blot analysis (Figure 2D). WSTF fractionated in a single peak at ∼700 kDa. hACF1/WCRF180 ran in a slightly larger complex, consistent with earlier findings (Bochar et al., 2000; Poot et al., 2000). Most ISWI ran in a broad peak that includes both the hACF1/WCRF180- and WSTF-containing fractions. However, the majority of ISWI is found in the hACF1/WCRF180-containing fractions. To test if all the WSTF is in a complex with ISWI, we depleted the ISWI from crude HeLa nuclear extract with the anti-hISWI antiserum (Poot et al., 2000) and fractionated this depleted extract over the gel filtration column. The ISWI depletion not only removed most of the WSTF from the extract, but also the high molecular weight WSTF complex, whereas a mock depletion of the extract with the pre-immune serum had no effect on the fractionation of WSTF and ISWI (Figure 2D, ‘-mock’). We could further purify the ∼700 kDa WSTF–ISWI complex from both HeLa and NIH 3T3 extracts over several ion-exchange columns such as MonoQ, where the complex runs in one peak (data not shown). Taken together, the data strongly suggest that most of the WSTF is associated with ISWI in NIH 3T3 and HeLa cell extracts (although we cannot exclude that there is a minor fraction of WSTF in a complex that does not fractionate over the Superose-6). We attribute the fact that not all WSTF can be depleted with the antisera against ISWI to a weaker association of ISWI with WSTF as compared with hACF1, which can be depleted more efficiently (Figure 2B), and, therefore, to the presence of ‘free’ WSTF under some conditions. We next examined WSTF, ACF1 and the two isoforms of ISWI (SNF2L and SNF2H) (Okabe et al., 1992; Aihara et al., 1998; Lazzaro and Picketts, 2001) in nuclear extracts of different cell lines (Figure 3A). All extracts exhibited similar levels of SNF2H protein. SNF2L was observed only in the embryonic stem (ES) cell and differentiated embryonic stem cell extracts; we were not able to detect SNF2L in nuclear extracts of NIH 3T3 cells, which represent a more differentiated, fibroblastic cell type, nor in HeLa nuclear extract. The NIH 3T3 cell extracts exhibited dramatically higher levels of WSTF than the ES cells or HeLa extracts. The levels of ACF1 paralleled those of WSTF. Interestingly, the differentiated embryonic cell nuclear extracts contained an ACF1 isoform with slightly slower electrophoretic migration. This may represent a cell-type-specific splice variant of ACF1 as the ES cells differentiate into a very heterogeneous cell mixture. Because the NIH 3T3 cell nuclear extract is devoid of detectable SNF2L, we conclude that both WSTF and ACF1 interact with the SNF2H isoform of mammalian ISWI, as demonstrated previously for ACF1 in HeLa cell extracts (LeRoy et al., 2000; Poot et al., 2000). Indeed, the antiserum specific to the H-isoform of ISWI detected the SNF2H in affinity-purified WSTF– ISWI complex (Figure 3B), whereas SNF2L was not detectable in the extract or the affinity-purified complex (not shown). Therefore, WSTF and ACF1 may compete for the same pool of SNF2H in the nucleus. We performed co-immunoprecipitation experiments with ES cell nuclear extract to find out whether WSTF and ACF1 interact with SNF2L in an extract that contains both ISWI isoforms (Figure 3C). The efficiency of ISWI co-precipitation with the antibodies against WSTF and ACF1 was low in this extract, probably because there is little WSTF and ACF1 in the ES extract to start with. We eluted the proteins from the antibody complexes bound to protein A with the antigenic peptides to reduce background. An antibody against mouse ACF1 precipitated SNF2H and some SNF2L together with the ACF1. However, we could detect only SNF2H in the immunoprecipitate of the antibody against WSTF. Although we cannot exclude the possibility that there is some interaction of SNF2L with WSTF, all experiments together indicate that SNF2H is the predominant isoform of ISWI with which WSTF interacts. A two-subunit WSTF–ISWI complex from Xenopus egg extract Previously, four discrete ISWI complexes, ISWI-A, -B, -C and -D, have been purified from Xenopus egg extracts, and ISWI-C was shown to contain the Xenopus homolog of ACF1 (Guschin et al., 2000). We examined the remaining ISWI complexes for WSTF with the WSTF antiserum. The antiserum strongly recognized the 200 kDa subunit of the ISWI-B complex (Figure 4A and B) and failed to react with any components of the ISWI-A and ISWI-D complexes (data not shown). The sequence [nine amino acids (aa)] of one tryptic peptide, derived from ‘p200’ obtained by Edman degradation, and five sequences obtained by mass spectrometry (13–16 aa) all matched sequences from WSTF ESTs. We therefore conclude that ‘p200’ is the Xenopus WSTF homolog. Furthermore, this WSTF–ISWI complex consists of only two subunits. A more careful analysis of the size of the WSTF from Xenopus indicated that it is actually smaller than 200 kDa, at ∼180 kDa. Figure 4.WSTF forms a complex with ISWI in Xenopus egg extracts. (A) Western blot of co-purification of Xenopus WSTF (anti-human WSTF antibodies) and Xenopus ISWI (anti-Xenopus ISWI antibodies) over MonoS chromatography during ISWI-B purification. This is the penultimate step in the purification scheme. (B) SDS–PAGE of the final purified complex showing the two polypeptides. Immunoblot shows the antibody reactivity with the respective antisera. Download figure Download PowerPoint The WSTF–ISWI complex is a chromatin remodeling factor We examined the WSTF–ISWI complexes for nucleosome remodeling activities. The mouse complex was tested following immuno-affinity purification and peptide elution. A property of several ISWI complexes is the ability to create regular nucleosomal arrays from irregular chromatin in vitro (Varga-Weisz et al., 1997; Tsukiyama et al., 1999; Guschin et al., 2000; LeRoy et al., 2000; Poot et al., 2000; Längst and Becker, 2001). To test whether the WSTF–ISWI complexes share this activity we assembled a chromatin-like structure on plasmid DNA with a Drosophila chromatin assembly extract in the absence of ATP, and stripped endogenous nucleosome spacing activities with a sarcosyl wash. This template is characterized by the absence of a regular nucleosome repeat structure when assayed by partial micrococcal nuclease digest (Varga-Weisz et al., 1997). Addition of mock eluate in the presence of ATP did not improve the regularity of the chromatin structure (Figure 5A, left panel, lanes 1–4). However, the mouse WSTF–ISWI complex reconfigures the chromatin to a regularly spaced nucleosome array in an ATP-dependent fashion (Figure 5A, left panel, lanes 5–8). The same activity was exhibited by the purified frog complex (Figure 5A, right panel). There is some residual spacing activity in the sarcosyl-stripped chromatin in the presence of ATP (Figure 5A, right panel, buffer control, lanes 1 and 2 versus 3 and 4); however, the frog complex significantly improved the regularity of the nucleosomal array in an ATP-dependent manner (lanes 5 and 6 versus 7 and 8). The frog complex also increased the internucleosomal spacing (repeat length): the DNA protected by three nucleosomes (trinucleosome) runs below the 491 bp marker in the control experiment (Figure 5A, right panel, lane 4, arrow), whereas it runs with this marker in the chromatin that was remodeled by the frog WSTF–ISWI complex (lanes 7 and 8, arrow). Figure 5.The WSTF–ISWI complex is a chromatin remodeling factor, WICH. (A) Left panel: the mouse WSTF–ISWI complex reconfigures irregular chromatin into a regular nucleosomal array. Eluates from the pre-immune serum (+control) or anti-WSTF antiserum (+WICH) were added to sarcosyl-stripped chromatin, assembled in Drosophila embryo extracts without ATP. Addition of 1 mM ATP was as indicated. Micrococcal nuclease digestion was for 30 s (lanes 1, 3, 5 and 7) or 60 s (lanes 2, 4, 6 and 8). Lanes labeled ‘m’ contain size marker DNA fragments: 0.49, 1.1 and 1.2 kbp. Arrows indicate mono-, di-, tri- and tetranucleosome DNA fragments (from the bottom upwards). Right panel: same experiment as in the left panel, but with the purified Xenopus WICH (300 ng MonoS fraction, purification buffer as control). Arrows indicate the position of the trinucleosome DNA fragment. (B) WICH mobilizes nucleosomes. Nucleosomes were assembled with polyglutamic acid as carrier on plasmid DNA. This chromatin was incubated with mock eluate (+control) or the immunopurified WSTF complex (+mWICH). Addition of 1 mM ATP was as indicated above the panels. Micrococcal nuclease digestion was for 30 s (lanes 1, 3, 5 and 7) or 60 s (2, 4, 6 and 8). Lanes labeled ‘m’ contain size marker DNA fragments: 0.49, 1.1 and 1.2 kbp. Arrows indicate a change of internucleosomal repeat length. Download figure Download PowerPoint To assess the ability of the WSTF–ISWI complex to mobilize nucleosomes, we assembled a chromatin-like structure on plasmid DNA with purified HeLa histones and polyglutamic acid as carrier. This type of artificial chromatin is characterized by a non-physiological ‘close-packed’ nucleosomal spacing. Purified ACF complex from Drosophila and Xenopus has been shown previously to reposition nucleosomes in a salt and ATP-dependent manner to a spacing with greater internucleosomal distances (Ito et al., 1997; Guschin et al., 2000). We found that addition of the affinity-purified mouse WSTF–ISWI complex also caused a repositioning of the nucleosomes to a greater spacing in an ATP-dependent manner (Figure 5B, compare lane 5 with 7, and 6 with 8). These findings indicate that the interaction of WSTF with ISWI results in a nucleosome remodeling factor that can mobilize and ‘space’ nucleosomes. We named this complex the WSTF–ISWI chromatin remodeling complex, or WICH. WSTF is targeted to pericentromeric heterochromatin during its replication We next determined the localization of WSTF in mouse cells using indirect immunofluorescence and confocal microscopy. The affinity-purified α-WSTF antibody predominantly stained the nuclei of NIH 3T3 cells (Figure 6). In addition, ∼40–50% of the nuclei of an asynchronous log-phase culture exhibited foci of staining over a more even background. These foci were similar in number, size and distribution to the pericentromeric heterochromatin foci. We determined whether the WSTF foci are localized to pericentromeric heterochromatin by staining the cells for both WSTF and M31 (also called HP1β), a marker for pericentromeric heterochromatin (Wreggett et al., 1994). Indeed, the WSTF foci co-localized with the M31 foci (Figure 6) and, therefore, WSTF targets pericentromeric heterochromatin in at least a subset of the cell population. Figure 6.WSTF co-localizes with M31 (mouse HP1β), a marker protein for mouse pericentromeric heterochromatin. NIH 3T3 cells were fixed and stained with affinity-purified anti-WSTF antibodies (top, FITC, green) and rat monoclonal antibodies against M31 (middle, Texas Red). Lower panel: merged image (yellow). Bar = 10 μm. Download figure Download PowerPoint To understand the restriction of pericentromeric heterochromatin localization of WSTF to a subset of cells, we analyzed this localization pattern through the replication phase in NIH 3T3 cells. Levels of WSTF were constant and its association with ISWI was maintained throughout S phase (data not shown). To demarcate cells passing through S phase, we used the nucleotide analog bromodeoxyuridine (BrdU), which is incorporated into newly synthesized DNA and can be visualized with anti-BrdU antibodies. Cells were arrested at the G1/S boundary using an inhibitor of DNA polymerase α, aphidicolin, and then released into S phase by washing with fresh medium. S phase in NIH 3T3 cells usually takes 7–8 h to complete and occurs in stages (early, middle, late and very late S phase), which are characterized by specific patterns of BrdU incorporation (Fox et al., 1991; O‘Keefe et al., 1992). Cells blocked at the G1/S boundary showed WSTF foci over a granular nuclear staining; however, the foci were rather weak compared with the general nuclear staining in these cells (Figure 7A, ’G1/S block‘). One hour after release from the G1/S block, overall WSTF staining became more intense and even, and it did not show pronounced foci but was excluded from the pericentromeric heterochromatin (see below; Figure 7B, ’1 h‘). There was no co-staining of WSTF with the BrdU incorporation sites. Two to 4 h after release, BrdU incorporation was seen in a granular pattern throughout the nucleus. At this time the WSTF staining became more uneven and there was an extensive overlap of the WSTF staining and the BrdU incorporation staining (Figure 7A, ’2–4 h‘). At ∼5 h into S phase the cells replicated the pericentromeric heterochromatin, which was visualized by the incorporation of the BrdU into large foci (Fox et al., 1991; O’Keefe et al., 1992). At this time, the WSTF staining was clearly concentrated into large foci, which co-localized with the BrdU foci (Figure 7A, ‘5 h’). At very late S stage, 6–7 h after release from the chemical block, BrdU incorporation again became more granular and was particularly apparent around the perimeter of the nucleus. At that point, WSTF staining persisted in the pericentromeric heterochromatin; however, the foci were not as intense as at the 5 h time point (Figure 7A, ‘6–7 h’). At this time, there was little co-localization of BrdU incorporation foci and WSTF staining. Figure 7.WSTF is targeted to pericentromeric heterochromatin during its replication. (A) NIH 3T3 cells were synchronized at the G1/S border, released into S phase, fixed at the indicated times after release and stained for WSTF (top, FITC, green) and BrdU (middle, Texas Red) incorporation. The merged image (yellow) is shown at the bottom. Arrows indicate cells with WSTF foci at late S phase. (B) WSTF (top, green) and M31 (HP1β, middle, red) staining through S phase. Bottom panel: merged image (yellow). Bar = 10 μm. Download figure Download PowerPoint The most intriguing result from this time course experiment was the accumulation of WSTF in the foci of replicating heterochromatin. To better understand the relationship between pericentromeric heterochromatin and WSTF through S phase, we stained the cells at different points through S phase with antibodies against the pericentromeric heterochromatin protein M31, also called HP1β (Wreggett et al., 1994), together with antibodies against WSTF. WSTF is somewhat enriched in these pericentromeric foci before the release of the G1/S block (Figure 7B,‘G1/S block’) and it is also found throughout the nucleus. One hour after release from the G1/S block, WSTF is distributed more evenly in the nucleus in a granular pattern. Co-localization analysis with M31 shows that WSTF is actually excluded from the pericentromeric heterochromatin at this point (Figure 7B, ‘1 h’). Five hours into S phase, when cells replicate the pericentromeric heterochromatin, WSTF is clearly enriched in the M31-stained foci. These data document a dynamic association between WSTF and pericentromeric heterochromatin. WSTF co-localizes with DNA replication foci at middle to late S phase (2–5 h after G1/S). Outside of this time, there is no close link between WSTF localization and DNA replication foci. These data indicate a close relationship between the replication of the pericentromeric heterochromatin and WSTF accumulation at these structures in S phase. ACF1 is targeted to replicating heterochromatin in NIH 3T3 cells too; however, unlike WSTF, it remained in these structures into G2 phase (N.Collins, C.García-Jiménez, G.Dellaire and P.Varga-Weisz, submitted). The N-terminal ∼350 amino acids of hACF1, including the WAC domain, have been shown to target a marker protein to pericentromeric heterochromatin in mouse cells (Tate et al., 1998), indicating a role for the WAC domain in heterochromatin targeting. However, we did not find targeting of the N-terminal 400 amino acids, including the WAC domain of WSTF fused to green fluorescent protein to heterochromatin, although the fusion protein is nuclear (I.Kukimoto and P.Varga-Weisz, unpublished data). Further studies are necessary to reveal the mechanism of heterochromatin targeting of WSTF. Stable association of WSTF with mitotic chromosomes distinguishes it from ACF1/WCRF180 Immunostaining of fixed cells and spread chromosomes indicated that WSTF is associated with metaphase chromosomes. In less condensed chromosomes, such as in the earlier stages of chromosome condensation, enrichment of WSTF at the pericentromeric heterochromatin is apparent (Figure 8A, compare left and right sets of chromosomes). We have not been able to detect ACF1 (WCRF180) on mitotic chromosomes in fixed cells with indirect immunofluorescence experiments using antisera against both the N- and C-terminal peptides of ACF1 (N.Collins and P.Varga-Weisz, unpublished observations). To verify this clear distin
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