Targeting of Krüppel-associated Box-containing Zinc Finger Proteins to Centromeric Heterochromatin

IMPLICATION FOR THE GENE SILENCING MECHANISMS*

Eishou MatsudaDagger, Yasutoshi AgataDagger, Manabu Sugai, Tomoya Katakai, Hiroyuki Gonda, and Akira Shimizu§

From the Center for Molecular Biology and Genetics, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

Received for publication, November 27, 2000, and in revised form, January 18, 2001




    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Krüppel-associated box-containing zinc finger proteins (KRAB-ZFPs) repress transcription via functional interaction with the corepressor KRAB-associated protein-1 (KAP-1). KAP-1 directly interacts with heterochromatin protein 1 (HP1), a dose-dependent regulator of heterochromatin-mediated silencing. Here we show that two KRAB-ZFPs that we previously identified, KRAZ1 and KRAZ2, are targeted to foci of centromeric heterochromatin containing HP1alpha through the interaction with KAP-1. Centromeric targeting potential of KRAZ1 and KAP-1 is strictly correlated with their silencing activities; a KRAB mutant of KRAZ1 that is unable to bind KAP-1 and KAP-1 deletions unable to bind HP1 cannot localize to centromeric foci nor repress transcription. We provide evidence that this correlation is likely to be functionally relevant. First, overexpression of the VP16 transactivation domain fused with the KAP-1 deletion that binds to KRAB but not to HP1 leads to dramatic redistribution of KRAZ1 from centromeric foci and simultaneously converts KRAZ1-mediated silencing into strong transcriptional activation. Second, a specific inhibitor of histone deacetylases, trichostatin A, effectively redistributes KRAZ1 and KAP-1 from centromeric foci and partially relieves their silencing activities. These data strongly suggest that KRAB-ZFPs/KAP-1 silence transcription by dynamic recruitment of the target locus to the specific gene silencing compartment, centromeric heterochromatin, in a histone deacetylase-dependent manner.




    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Krüppel-associated box (KRAB)1 was originally identified as an evolutionarily conserved motif that is presumably contained in about one-third of the several hundred Krüppel-like zinc finger proteins (ZFPs) (1). It is found almost exclusively in the N terminus of the Krüppel-like ZFPs that contain C2H2-type zinc finger domains in their C terminus and is subdivided into KRAB-A and -B domains (1). Several KRAB domains have been shown to repress transcription when heterologously tethered to the promoter (2-4). The KRAB-A domain present in all KRAB motifs is responsible for transcriptional silencing (2-4), and substitutions of conserved residues in this domain abrogate the repressor function (2, 3).

KRAB-associated protein-1 (KAP-1) (5), also identified as TIF1beta (6, 7) or KRIP-1 (8), was isolated as a protein that interacts with multiple KRAB domains but not with those containing KRAB-A mutations. KAP-1 is thought to function as a corepressor for the large class of KRAB-containing ZFPs, because 1) KAP-1 itself represses transcription when directly targeted to DNA; 2) overexpression of KAP-1 enhances KRAB-mediated silencing; and 3) KAP-1 functionally interacts with KRAB in mammalian cells and exerts a repressor function in the complex with the DNA-bound KRAB domains (5-9). In the N-terminal region, KAP-1 contains the RBCC domain (RING finger, B boxes, and coiled-coil region) that is essential for binding to KRAB and participates in multimerization of KAP-1 (5, 6, 9, 10). The central region of KAP-1 is a core domain essential but not sufficient for the full silencing activity (6, 9). The C-terminal region that has also been shown to be required for full silencing activity contains a plant homeodomain finger and a bromodomain (6, 9). The functions of these domains are still unclear but have been implicated in chromatin-mediated transcriptional regulation and interaction with the acetylated histone tail (11-13).

A clue toward elucidation of the mechanisms of KRAB/KAP-1-mediated silencing was provided by the finding that KAP-1 interacts with heterochromatin protein 1 (HP1) via its central repressor domain (14, 15). HP1 constitutes a subfamily of the chromatin organization modifier (chromo) superfamily and is a dose-dependent regulator of heterochromatin-mediated silencing known as position effect variegation, suggesting that HP1 is a functional and structural component of heterochromatin (for review see Ref. 16). Three HP1 members, HP1alpha , HP1beta (M31/MOD1), and HP1gamma (M32/MOD2), have been identified in both human (17-19) and mouse (7, 17). KAP-1 binds directly with all three HP1 proteins in vitro (14, 15). However, these three subtypes exhibit distinct subnuclear localization patterns depending on the species and cell type (20, 21). For example, in mouse NIH3T3 cells, HP1alpha was found almost exclusively in foci of centromeric heterochromatin (15). HP1beta localizes to centromeric foci, and additional nuclear speckles appear to represent euchromatin (14, 15). HP1gamma is predominantly distributed in euchromatic regions (14, 15). KAP-1 was shown to colocalize with HP1beta in pericentromeric heterochromatin and with HP1gamma in euchromatic regions (14), suggesting that KAP-1 may exert its silencing activity by a heterochromatin-mediated mechanism. However, there is no evidence indicating a correlation between subnuclear localization of KAP-1 or KRAB-ZFPs and their silencing function.

In this report, we demonstrate that two KRAB-ZFPs, KRAZ1 and KRAZ2, which we identified previously (9), colocalize with KAP-1 in foci of centromeric heterochromatin containing HP1alpha . Centromeric localization of KRAZ1/2 is dependent on the interaction with KAP-1, and in turn, binding to HP1 is essential for centromeric recruitment of KAP-1. We further provide data that strongly suggest that KRAB-ZFPs/KAP-1 repress transcription by locus targeting to centromeric heterochromatin in a histone deacetylase-dependent manner.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Plasmids-- Recombinant DNA techniques were performed according to standard protocols (22). The pGL3-G5SV and expression plasmids for GAL4-, Myc-, and VP16-fused KAP-1 deletions have been described previously (9). To express full-length KRAZ1 and KRAZ2, cDNAs encoding amino acids 42-652 of KRAZ1 and 1-606 of KRAZ2 (GenBankTM accession numbers AB024004 for KRAZ1 and AB024005 for KRAZ2) were inserted into the pEF-BOS-GAL4 or pEF-BOS-6xMyc-tagged vectors (9). To construct KRAB mutants of KRAZ1/2, the KRAB-containing regions of full-length KRAZ1/2 were replaced by the corresponding fragments containing the amino acid substitutions in KRAB-A domains (aspartic acid-valine to alanine-alanine; DV to AA) (9). A firefly luciferase reporter plasmid, pGL3-G5T7, was constructed by subcloning the fragment carrying five GAL4-binding sites excised from pGL3-G5SV (9) and the fragment carrying seven repeats of tetracycline operator and the cytomegalovirus promoter excised from pUHD10-3 (23) into pGL3-Basic vector (Promega). To express tetracycline-controlled transactivator (tTA), the fragment containing tTA excised from pRetro-Off (CLONTECH) was inserted into pEF-BOS bsr (9).

Transfection and Luciferase Assay-- Transient transfection of NIH3T3 cells using LipofectAMINETM (Life Technologies, Inc.) and luciferase assay using the Dual-Luciferase reporter assay system (Promega) were performed as described (9). Firefly luciferase activity from pGL3-G5SV or pGL3-G5T7 was normalized for transfection efficiency with sea-pansy luciferase activity from the internal control plasmid, pRL-SV40 (Promega). -Fold repression was calculated by comparing normalized luciferase activity of each GAL4 fusion with that of GAL4DBD alone, which represents the basal activity of the reporter used. Each transfection experiment was repeated at least three times in duplicate, and the results are presented as averages with standard deviations of duplicates in representative experiments. For TSA treatment, 16 h after transfection, cells were treated with 100 ng/ml TSA (Wako) or an equal volume of Me2SO as a vehicle control and incubated for an additional 32 h.

GST Pull-down Assay-- The expression plasmids for GST-HP1alpha and GST-HP1gamma (24) were kindly provided by Dr. H. J. Worman (Centre National de la Recherche Scientifique (CNRS), Paris, France). Protein purification and GST pull-down assay were performed as described (9). Briefly, 1-5 µg of GST or GST fusion proteins immobilized on glutathione-Sepharose 4B (Amersham Pharmacia Biotech) were incubated with 35S-labeled VP16-KAP-1 deletions that were in vitro-translated by using the T7 TNT system (Promega). After washing, bound proteins were separated by 10-12% SDS polyacrylamide gel electrophoresis and analyzed by a Bio-image analyzer (Fujix BAS 5000).

Antibodies-- The primary antibodies (Abs) used were as follows: (i) rabbit polyclonal Ab against human KAP-1 (nucleotides 1882-2673 that are 96% identical to mouse KAP-1) (25), which was kindly provided by Dr. S. C. Lee (National Taiwan University, Taiwan, Republic of China); (ii) mouse anti-mouse HP1alpha monoclonal antibody (mAb) 2HP-1H5 (15), which was kindly provided by Dr. P. Chambon (CNRS, Strasbourg, France); (iii) rabbit polyclonal Ab against human HP1alpha (amino acids 181-191 that are 91% identical to mouse HP1alpha ) (21), which was kindly provided by Dr. J. C. Courvalin (CNRS, Paris, France); (iv) mouse anti-GAL4 mAb (RK5C1; Santa Cruz Biotechnology); (v) rabbit anti-VP16 polyclonal Ab (CLONTECH); and (vi) unlabeled and Cy3-labeled mouse anti-Myc mAb (9E10; Santa Cruz Biotechnology). Labeling was performed by a Cy3 labeling kit (Amersham Pharmacia Biotech). All of the following secondary Abs were purchased from Jackson ImmunoResearch Laboratories, Inc.: donkey Texas Red-labeled anti-mouse or rabbit IgG and donkey FITC-labeled anti-mouse or rabbit IgG. Species specificities of the secondary antibodies were confirmed by control staining.

Immunoprecipitation and Western Blotting-- These were performed as described (9). Briefly, GAL4DBD, GAL4-KRAZ1, or GAL4-KRAZ1 mutant were transiently transfected into NIH3T3 cells, together with VP16-KAP-1 N835, and immunoprecipitated with rabbit polyclonal anti-GAL4 Ab (Upstate Biotechnology). Immunoprecipitates were analyzed by Western blotting using rabbit anti-VP16 Ab.

Immunofluorescent Microscopy-- For conventional and confocal microscopy analysis, NIH3T3 cells were plated 24 h before transfection on eight chamber slides (Nunc, Inc.) precoated with 0.1% gelatin and transfected using LipofectAMINETM as described (9). Cells were fixed 24-48 h after transfection with 3% paraformaldehyde in phosphate-buffered saline, followed by permeabilization with 1% Triton X-100. Cells were then incubated with primary antibodies for 60 min. After washing three times with 0.2% Tween 20 in phosphate-buffered saline, cells were incubated with secondary antibodies for 60 min. DNA was labeled with 0.1 µg/ml 4',6'-diamidino-2-phenylindole (DAPI; Sigma) for 5 min, and the slides were mounted with PermaFluorTM (Shandon/Lipshaw). All staining experiments were repeated more than twice, and at least 100 transfected cells were examined in each experiment with conventional microscopy (DIAPHOT-300; Nikon) or confocal laser microscopy (MRC-1024; Bio-Rad). The fluorescence signals from the two fluorochromes were recorded simultaneously in one scan and saved separately on two channels to be processed independently with the Adobe Photoshop 5.0 software (Adobe Systems Inc.).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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KRAZ1 and KRAZ2 Colocalize with KAP-1 and HP1alpha to Centromeric Foci-- We previously reported that N-terminal KRAB-containing regions of KRAZ1 and KRAZ2 repress transcription when heterologously targeted to the nucleus as fusions with GAL4DBD (9). We observed, however, that these regions alone did not localize to the nucleus (data not shown), whereas full-length KRAZ1 and KRAZ2 containing zinc fingers in the C terminus (15 and 9 repeats, respectively; see Fig. 1A) were nuclearly localized (Fig. 1B). We thus further investigated the subnuclear localization of KRAZ1/2 within this more physiological context of full-length proteins. Each Myc-tagged full-length protein was transiently expressed in mouse NIH3T3 cells and examined by immunofluorescent staining with an anti-Myc antibody (Fig. 1B). In ~20% of transfected cells, KRAZ1 exhibited a unique dot-like structure in interphase nuclei, whereas the remaining 80% presented a diffuse distribution (Fig. 1B, a and c). KRAZ2 also displayed similar nuclear dots though to a lesser extent than KRAZ1 (Fig. 1B, e and g), suggesting that these dot-like structures might be a common characteristic in many KRAB-containing ZFPs. Although the levels of KRAZ1/2 expression varied widely among individual transfected cells, there was no correlation between the presence of the dot-like structures and the expression level (data not shown), suggesting that dot formation is not likely because of artifacts caused by overexpression. Moreover, KRAZ1/2 nuclear dots coincide with DAPI-stained dots that correspond to foci containing centromeric heterochromatin (Fig. 1B, d and h) (21, 26), suggesting that KRAZ1/2 specifically localize to centromeric foci. We focused on KRAZ1 for further investigation, because the cell population showing nuclear dots was consistently larger for KRAZ1 than KRAZ2. However, basically similar data were obtained for KRAZ2.



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Fig. 1.   A fraction of KRAZ1 and KRAZ2 localize to the heterochromatic dot-like structures. A, a schematic diagram of the KRAZ1 and KRAZ2 proteins and amino acid sequences of their KRAB domains. The KRAB-A, KRAB-B, and the multiple zinc finger domains are shown. The consensus sequence of the KRAB domains and DV residues substituted to AA in the KRAB-A mutants are indicated. B, localization of full-length KRAZ1 and KRAZ2 in interphase nuclei. NIH3T3 cells were transiently transfected with Myc-tagged full-length KRAZ1 and KRAZ2 and stained with Cy3-labeled anti-Myc mAb. Conventional microscope images of representative cells exhibiting a diffuse or dot-like pattern are shown in left panels. Right panels represent counterstaining of the same nuclei with DAPI.

KRAZ1 functionally interacts with KAP-1 through its KRAB domain (9), and KAP-1 was shown to directly bind to all three HP1 proteins, HP1alpha , HP1beta , and HP1gamma in vitro (14, 15). Thus, we examined whether KRAZ1 colocalizes with KAP-1 and HP1s within the nucleus by using immunofluorescent confocal microscopy. Among the HP1 members, we focused on HP1alpha , because endogenous HP1alpha exclusively localized to centromeric foci as reported previously (see Refs. 15 and 21 and Fig. 2A, b). Consistent with the previous observation (14), endogenous KAP-1 was concentrated in dot-like structures in nearly 50% of the nuclei (Fig. 2A, a). KAP-1 dots colocalized exclusively with HP1alpha (Fig. 2A, c), indicating that KAP-1 localized to foci of centromeric heterochromatin. Overlaying of the corresponding images revealed that the nuclear dots of KRAZ1 coincided entirely with endogenous KAP-1 and HP1alpha in centromeric foci (Fig. 2A, d-i). Furthermore, like other proteins that are specifically targeted to centromeric foci such as HP1beta /MOD1, CAF1 p150, and Suv39 h1 (26, 27), dot-like structures by KRAZ1 were resistant to Triton X-100 extraction (data not shown), suggesting that KRAZ1 is specifically targeted to centromeric foci and tightly associated with heterochromatin. The colocalization of KRAZ1 dots with KAP-1 and HP1alpha were further confirmed by using a different combination of antibodies, anti-mouse KAP-1 (1Tb-1A9, which was kindly provided by Dr. P. Chambon; see Ref. 15), and anti-human HP1alpha antibodies (21) (data not shown).



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Fig. 2.   Centromeric localization of KRAZ1 is associated with silencing activity. A, localization of KRAZ1 but not its KRAB mutant, together with endogenous KAP-1 and HP1alpha . Confocal microscope images of single optical sections of representative cells are shown. NIH3T3 cells were transiently transfected with Myc-tagged full-length KRAZ1 or its KRAB mutant (KRAZ1 mut) and stained with Cy3-labeled anti-Myc Ab (red; d, g, j, and m). Endogenous KAP-1 was stained with anti-hKAP-1 Ab followed by Texas Red-labeled anti-rabbit IgG (red; a) or FITC-labeled anti-rabbit IgG Ab (green; e and k). Endogenous HP1alpha were stained with anti-mHP1alpha and FITC-labeled anti-mouse IgG Abs (green; b, h, and n). Right panels show the merged images of left and middle panels (yellow). B, transcriptional silencing by full-length KRAZ1 but not its KRAB mutant. NIH3T3 cells were transiently transfected with the reporter plasmid pGL3-G5SV (100 ng) carrying five GAL4-binding sites upstream of the SV40 promoter, together with increasing amounts (2, 10, and 50 ng) of the expression plasmids for GAL4DBD, GAL4-KRAZ1, or GAL4-KRAZ1 mut. Transcriptional silencing activity is shown as -fold repression relative to GAL4DBD alone. Luc, luciferase. C, in vivo interaction of KAP-1 with full-length KRAZ1 but not its KRAB mutant. NIH3T3 cells were transiently transfected with VP16-KAP-1 N835 and each GAL4 fusion as indicated. GAL4 fusions were immunoprecipitated with anti-GAL4 Ab. Immunoprecipitates and 10% of inputs were run by 10% SDS polyacrylamide gel electrophoresis and analyzed for the presence of KAP-1 by Western blotting with anti-VP16 Ab.

Centromeric Targeting of KRAZ1 Correlates with Silencing-- To determine whether centromeric localization of KRAZ1 is associated with KRAZ1-KAP-1 interaction and transcriptional silencing, we first assessed the silencing activity of full-length KRAZ1 fused with GAL4DBD (Fig. 2B). KRAZ1 significantly repressed transcription in a dose-dependent manner, whereas a mutation introduced in KRAB severely compromised the silencing activity. Immunoprecipitation analysis revealed that full-length KRAZ1 but not its KRAB mutant bound to KAP-1 (Fig. 2C). These results indicate that the silencing activity of KRAZ1 is correlated with the ability to interact with KAP-1. Next, we analyzed localization of the Myc-tagged KRAB mutant of full-length KRAZ1. Although in multiple experiments we examined hundreds of transfected cells that expressed the mutant at various levels, no centromeric localization was ever detected, only a diffuse pattern of staining (Fig. 2A, j-o). This is not attributed to instability of the KRAZ1 mutant protein, because comparable expression levels of expected-sized products were detected for both wild-type and mutant KRAZ1 by Western blotting (data not shown). Thus these data demonstrate that centromeric targeting of KRAZ1 is a specific event that strictly depends on the interaction with KAP-1 via KRAB and that the silencing activity of KRAZ1 is correlated with the ability to colocalize with KAP-1 to centromeric foci.

Association of KAP-1 Centromeric Localization with Silencing Activity-- Based on the finding that KAP-1 directly interacts with HP1 proteins through its core repressor domain (6, 9, 14, 15), we next addressed the possibility that centromeric localization of KAP-1 in turn depends on the interaction with HP1 and correlates with its silencing activity. To this end, we first assessed interaction of KAP-1 deletions (9) with HP1 proteins in vitro. GST pull-down assay revealed that KAP-1 deletions N562, N835, 396C, and 396-711 bound to both GST-HP1alpha and -gamma but not to GST alone (Fig. 3, A and C). In contrast, no binding was detected for KAP-1 N487 and 697C in this assay, indicating that amino acids 487-562 of KAP-1 are necessary for interaction with HP1 in vitro. These results are consistent with the reports showing that amino acids 396-562 constitute the core repressor domain (9) and that amino acids 483-510 constitute the HP1 binding domain (BD) (14, 15).



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Fig. 3.   Centromeric localization of KAP-1 is associated with its silencing activity. A, schematic diagram of KAP-1 deletion constructs and their silencing activities (taken from Ref. 9). Silencing activities are shown as -fold repression relative to GAL4DBD alone. Note that those of the top three constructs fused with GAL4DBD are shown in parentheses and that the corresponding images in B are of Myc-tagged constructs. Bromo, bromodomain; PHD, plant homeodomain. B, localization of KAP-1 deletions transiently expressed in NIH3T3 cells and endogenous HP1alpha in interphase nuclei. Confocal microscope images of representative cells are shown. In the upper six rows, Myc-KAP-1 deletions (red; left panels) were stained with Cy3-labeled anti-Myc Ab, and endogenous HP1alpha (green; middle panels) were stained with anti-mHP1alpha and FITC-labeled anti-mouse IgG Abs. In the lower three rows, GAL4-fused KAP-1 deletions that lack KRAB BD (red; left panels) were stained with anti-GAL4 and Texas Red-labeled anti-mouse IgG Abs, and endogenous HP1alpha (green; middle panels) were stained with anti-hHP1alpha and FITC-labeled anti-rabbit IgG Abs. Right panels show the merged images of left and middle panels (yellow). C, in vitro interaction between KAP-1 deletions and HP1 proteins. VP16-KAP-1 deletions shown in A were 35S-labeled in vitro and incubated with GST alone, GST-HP1alpha , or GST-HP1gamma . Bound proteins and 10% of the input were subjected to 10-12% SDS polyacrylamide gel electrophoresis and analyzed by a Bio-image analyzer (Fujix BAS 5000).

We next investigated subnuclear localization of the KAP-1 deletions tagged with Myc epitope and containing the nuclear localization sequence of the SV40 large T antigen (9) (Fig. 3, A and B). KAP-1 N562 and N835 that contain the RBCC domain and HP1BD colocalized with HP1alpha to centromeric foci, whereas KAP-1 N487 and 694C lacking HP1BD could not, indicating that HP1BD is essential for centromeric targeting. Surprisingly, however, KAP-1 396C and 396-711 that retain HP1BD but not RBCC could not localize to centromeric foci, indicating that HP1BD alone is not sufficient for centromeric targeting in vivo but that RBCC might also be required. Considering the fact that RBCC serves as the interface for KAP-1 homo-oligomerization upon binding to KRAB (10), the above result suggests that KAP-1 oligomerization is required for centromeric targeting. To address this possibility, we examined the localization of RBCC deletions of KAP-1 fused with GAL4DBD that can form a homodimer (28). Although GAL4-KAP-1 697C that lacks HP1BD still could not localize to centromeric foci, GAL4-KAP-1 396C and 396-711 that contain HP1BD became targeted to centromeric foci (Fig. 3B), indicating that the requirement for RBCC can be replaced with GAL4DBD. These results demonstrate that HP1BD and oligomerization through RBCC are required and sufficient for centromeric targeting of KAP-1.

To determine whether KAP-1 centromeric localization correlates with silencing activity, we next re-evaluated the silencing activities of GAL4-KAP-1 deletions that we previously reported (9) (Fig. 3A). Because GAL4DBD can replace RBCC for centromeric targeting of KAP-1 as shown above, all the KAP-1 constructs containing HP1BD could thus localize to centromeric foci, and all of them except 396-711 showed high silencing activities (N562, N835, and 396C). The 396-711 region contains the core repressor domain that is essential but not sufficient for full silencing activity; either the N- or C-terminal portion is also required for full repression (9). Nonetheless, KAP-1 constructs lacking HP1BD could not localize to centromeric foci and exhibited weak or almost no silencing activity (N487 and 697C). This is not attributed to instability of these deletions, as an expected-sized product was a major fraction detected for each KAP-1 deletion by Western blotting (data not shown). We thus conclude that the centromeric targeting ability of KAP-1 is associated with its silencing activity.

Centromeric Localization of KRAZ1 and KAP-1 Is Functionally Involved in Silencing-- To determine whether correlation of the centromeric targeting potential of KRAZ1, as well as KAP-1, with their silencing activities has functional significance, we used the mammalian two-hybrid assay by which we previously showed that silencing mediated by the KRAB-containing region of KRAZ1 fused to GAL4DBD could be converted to strong activation of transcription upon coexpression of KAP-1 deletions fused to the VP16 transactivation domain (VP16AD) (9). Like the KRAB-containing region, full-length KRAZ1 fused with GAL4DBD repressed transcription in comparison with GAL4DBD alone in the presence of VP16AD (Fig. 4A). When GAL4-KRAZ1 was coexpressed with a VP16-KAP-1 N487 construct that binds to KRAB of KRAZ1 but not HP1 (Fig. 3C), however, transcription was strongly activated (Fig. 4A), indicating that KAP-1 N487 functionally interacts with full-length KRAZ1. In contrast, cotransfection with VP16-KAP-1 N562 that binds to both HP1 and KRAB of KRAZ1 (Fig. 3C) inhibited the transcriptional activation to almost the basal level (Fig. 4A). This result suggests that although KAP-1 N562 interacts with KRAZ1 in vivo, the silencing activity of KAP-1, which is probably mediated via HP1 interaction, dominates over the VP16AD transactivation effect.



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Fig. 4.   A VP16AD-fused KAP1 deletion causes redistribution of KRAZ1 from centromeric foci and transcriptional activation. A, transcriptional activity by GAL4-KRAZ1 and VP16-KAP-1 in mammalian-two hybrid assay. NIH3T3 cells were transiently transfected with the reporter plasmid pGL3-G5SV (100 ng) and expression plasmids for GAL4DBD or GAL4 full-length KRAZ1 (50 ng) and for VP16 AD, VP16-KAP-1 deletions N487 or N562 (200 ng) were used. The schematic diagram of VP16-KAP-1 deletions and relative luciferase activities are shown. B, redistribution of full-length KRAZ1 from centromeric foci by VP16-KAP-1 N487 but not N562. Confocal microscope images of representative cells are shown. NIH3T3 cells were transiently transfected with Myc-tagged full-length KRAZ1, and VP16-KAP-1 deletions are indicated. In left panels, Myc-KRAZ1 expression was visualized with anti-Myc and FITC-labeled anti-mouse IgG Abs (green; a and g) or with Cy3-labeled anti-Myc Ab (red; d and j). In middle panels, VP16-KAP-1 deletions (red; b and h) were detected with anti-VP16 and Texas Red-labeled anti-rabbit IgG Abs, and endogenous HP1alpha (green; e and k) was detected with anti-mHP1alpha and FITC-labeled anti-mouse IgG Abs. Right panels show the merged images of left and middle panels (yellow).

We next examined localization of Myc-tagged full-length KRAZ1 coexpressed with VP16-KAP-1 deletions (Fig. 4B). When coexpressed with KAP-1 N562 that inhibits transactivation by VP16AD, KRAZ1 could colocalize with KAP-1 N562 and endogenous HP1alpha to centromeric foci (Fig. 4B, g-l). This result suggests that KRAZ1 interacts with KAP-1 N562 within centromeric foci wherein strong transcriptional activation by VP16AD is inhibited by KAP-1 silencing activity via HP1 interaction. In contrast, upon coexpression of KAP-1 N487 that leads to strong transcriptional activation, KRAZ1 was dramatically redistributed from centromeric foci, together with KAP-1 N487, to exhibit a diffuse nuclear staining, whereas HP1alpha remained in centromeric foci (Fig. 4B, a-f). This observation suggests that overexpressed KAP-1 N487 interacts with KRAZ1 and displaces KRAZ1 from centromeric foci in a dominant manner over endogenous KAP-1, resulting in activation of reporter gene transcription elsewhere in the nucleus away from foci.

Silencing Activity and Centromeric Localization of KRAZ1/KAP-1 Depend on Histone Deacetylation-- The recent observation that TSA, a specific inhibitor of histone deacetylases (HDACs), can relieve, at least in part, the silencing activities of KAP-1 and HP1 proteins (15) prompted us to test whether TSA can also relieve KRAZ1-caused silencing and centromeric localization of KRAZ1, as well as KAP-1. To test the effect of TSA on silencing, we used a reporter plasmid carrying five GAL4-binding sites and driven by the tetrocycline repressor and VP16AD chimera (tTA) (23) (Fig. 5A). Without TSA treatment, GAL4 fusions with KRAZ1, KAP-1 N562, and N835 that can localize to centromeric foci efficiently repressed transcription, whereas the KRAZ1 mutant that failed to localize in foci did not. TSA treatment, however, could partially relieve the silencing activities of KRAZ1, as well as KAP-1 N562 and N835, indicating that KRAZ1/KAP-1-mediated silencing is, at least in part, dependent on HDAC activity.



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Fig. 5.   TSA causes redistribution of KRAZ1/KAP-1 from centromeric foci and partial relief of their silencing activity. A, effect of TSA on KRAZ1- and KAP-1-mediated transcriptional silencing. NIH3T3 cells were transiently transfected with the reporter plasmid pGL3-G5T7 (100 ng) carrying five GAL4-binding sites upstream of seven repeats of the tetrocycline operator (Tet O) and the cytomegalovirus promoter, together with the expression plasmids for tTA (10 ng) and for GAL4DBD or GAL4-KRAZ1, KRAZ1 mutant, and KAP-1 deletions (125 ng). The relative luciferase (Luc) activities of GAL4 fusions are shown as percentages of GAL4DBD alone in the absence or presence of TSA (50 ng/ml). B, redistribution of KRAZ1 and KAP-1 from centromeric foci by TSA. Confocal microscope images of representative cells are shown. In the top panels, endogenous KAP-1 in NIH3T3 cells treated with (b) or without (a) TSA for 24 h were stained with anti-hKAP-1 and Texas Red-labeled anti-Rabbit IgG Abs. In the lower panels, NIH3T3 cells were transiently transfected with Myc-tagged, full-length KRAZ1 (c and f), KAP-1 N562 (i), or KAP-1 N835 (l) and treated with TSA from 24 to 36 h after transfection. Cells were stained with anti-Myc and FITC-labeled anti-mouse IgG Abs (green; c) or with Cy3-labeled anti-Myc Ab (red; f, i, and l). Endogenous KAP-1 (red; d) was detected with anti-hKAP-1 and Texas Red-labeled anti-rabbit IgG. Endogenous HP1alpha (green; g, j, and m) were stained with anti-mHP1alpha and FITC-labeled anti-mouse IgG Abs. Right panels show the merged images of left and middle panels (yellow).

We next investigated whether TSA influences localization of KRAZ1 and KAP-1. Surprisingly, culture with TSA caused redistribution of endogenous KAP-1 from centromeric foci throughout the nucleus in all cells (Fig. 5B, a-b). Furthermore, TSA also caused redistribution of Myc-tagged KRAZ1, KAP-1 N562, and N835 in all the transfected cells (Fig. 5B, c-n), accompanied by partial relief of their silencing activities. In contrast, HP1alpha remained in the foci with TSA, indicating that centromeric localization of HP1alpha does not depend on HDACs. Taken together, these data demonstrate that centromeric localization of KRAZ1 and KAP-1 requires TSA-sensitive HDAC activity and further suggest that their centromeric targeting ability is functionally relevant to the silencing activity.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we demonstrated that KRAB-ZFPs are targeted to foci of centromeric heterochromatin through the interaction with KAP-1 and HP1alpha and that the ability of KRAB/KAP-1 to localize to foci is strictly correlated with their silencing activities. This correlation is likely to be functionally relevant, because overexpression of a VP16AD-KAP-1 N487 that binds to KRAB but not to HP1 led to dramatic redistribution of KRAB-ZFPs from centromeric foci and simultaneously to reversion of KRAB-mediated silencing to strong transcriptional activation. In addition, HDAC inhibition by TSA also resulted in redistribution of KRAB-ZFPs, as well as KAP-1, from centromeric foci and in partial relief of their silencing activities. Furthermore, VP16AD-KAP-1 N562 that binds to both KRAB and HP1 could indeed colocalize with KRAB-ZFPs to centromeric foci; however, their interaction did not lead to transcriptional activation. This indicates that centromeric foci are indeed a transcriptionally inert compartment where even strong transactivation by VP16AD is severely suppressed. Therefore, these data strongly suggest that one of the mechanisms for KRAB/KAP-1-mediated silencing is sequestration of target gene loci to the transcriptionally inert compartment, centromeric foci, where HP1 proteins and HDACs are concentrated (29, 30), but the RPB1 subunit of RNA polymerase II is excluded (15). A similar mechanism has been implicated for the gene regulation by the lymphocyte-specific Ikaros protein (29, 31-34). However, transcriptional silencing when assessed even in a bulk cell population is quite efficient despite the fact that only a small fraction of cells exhibit centromeric localization of KRAB/KAP-1. This indicates that another mechanism must operate, at least in the cells that do not exhibit KRAB/KAP-1 dots. For example, KRAB-ZFPs may recruit KAP-1 and HP1 to the target gene loci, leading to local conversion of euchromatic regions into transcriptionally inactive, higher order heterochromatin-like structures (14, 15). Based on the following findings, we propose that both mechanisms might be functional within the individual cells and that active transition may occur between the two silencing states. First, KAP-1 localizes to both centromeric heterochromatin and euchromatic regions (14, 15). Although only HP1alpha exclusively localizes to centromeric foci, KAP-1 can bind to not only HP1alpha but also HP1beta and -gamma (14, 15), which are not exclusively associated with centromeric heterochromatin (14, 15, 20). HP1beta and -gamma might thus be the components of local heterochromatin-like structures that are formed on the KRAB/KAP-1-targeted loci and may be involved in the transition between centromeric and local euchromatic regions. Alternatively, it is conceivable that such local heterochromatic domains defined by HP1beta and HP1gamma might constitute fine local compartments for gene silencing. Second, in both the two-hybrid assay and the TSA experiment, localization of KRAB/KAP-1 and hence the presumable target locus itself is not static, but dynamically mobile in the nucleus, whereas the position of HP1alpha was relatively fixed. It would be intriguing to test whether the dynamic localization of KRAB-ZFPs might be also regulated by the cell cycle as previously postulated for KAP-1 (14).

TSA-caused redistribution of KRAB/KAP-1 from centromeric foci is quite rapid, because TSA treatment for only 6 h led to complete redistribution (data not shown), suggesting that inhibition of HDACs causes primarily dislodgment of KRAB/KAP-1 from foci rather than prevention of de novo targeting to foci. Therefore, stable anchorage or incorporation of the KRAB/KAP-1-bound target locus into heterochromatin in the foci may require hypoacetylated nucleosomes in the target locus. More importantly, TSA treatment simultaneously causes partial relief of silencing, indicating the involvement of HDAC activity in the silencing mechanism. This is reminiscent of the finding that TSA treatment in fission yeast relieves silencing of centromeric marker genes that require Swi6p, a yeast HP1 homolog (35). Thus, involvement of the HP1 family proteins and HDACs is evolutionarily conserved in silencing at centromeric heterochromatin. However, the finding that HDAC inhibition by TSA could not completely relieve silencing suggests that, unlike the direct HDAC recruitment to the localized target locus as previously shown for many repressors (36, 37), HDACs might be indirectly involved in KRAB/KAP-1-mediated silencing, possibly by stabilizing their centromeric localization as described above or because of fortuitous colocalization within centromeric foci where HDACs are concentrated (29, 30).

Nonetheless, it should be noted that TSA could cause complete redistribution of KRAB/KAP-1 from centromeric foci despite the incomplete relief of silencing. This suggests that redistribution is required, but by itself not sufficient, for transcriptional activation from the formerly silenced locus. This view is consistent with recent studies using beta -globin transgenes, which have shown that localization away from centromeric heterochromatin is required for general hyperacetylation and an open chromatin structure of the locus but not sufficient for fully active transcription (38) and that a functional enhancer suppresses silencing of a transgene and prevents its localization close to centromeric heterochromatin (39). Consistent with this idea, the presence of VP16AD is absolutely required for transcriptional activation in both our mammalian two-hybrid assay and the partial relief of silencing by TSA. The same KAP-1 deletion N487 tagged with only Myc epitope without VP16AD caused similar redistribution of KRAZ1 but did not activate transcription or relieve silencing even to the basal level (data not shown). We thus speculate that, in the absence of functional enhancers and activators, the silencing status might be persistent and dominant even after redistribution of KRAB/KAP-1 from centromeric foci. The molecular nature of the persistent silencing status might be the stable heterochromatin complex that is formed within centromeric foci and moves together with the target locus after redistribution. This idea would provide another explanation for the fact that silencing is quite efficient despite the small cell fraction showing KRAB/KAP-1 dots. Finally, identification of the target genes that are regulated by KRAB/KAP-1 will provide the opportunity, by using in situ hybridization, to directly test whether the target locus can be localized to centromeric foci in the repressed state and excluded from foci when activated.


    ACKNOWLEDGEMENTS

We are grateful to Drs. P. Chambon, J. C.  Courvalin, S. C. Lee, and H. J. Worman for materials, to Dr. H. Kurooka for valuable suggestions and discussion, and to Drs. K. Shibahara and R. Yu for critical reading of the manuscript. We thank S. Hirano for technical assistance.


    FOOTNOTES

* This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors equally contributed to this work.

§ To whom correspondence should be addressed. Tel.: 81-75-751-4189; Fax: 81-75-751-4190; E-mail: shimizu@virus.kyoto-u.ac.jp.

Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010663200


    ABBREVIATIONS

The abbreviations used are: KRAB, Krüppel-associated box; ZFP(s), zinc finger protein(s); DBD, DNA-binding domain; AD, activation domain; GST, glutathione S-transferase; BD, binding domain; HDAC(s), histone deacetylase(s); KAP-1, KRAB-associated protein-1; HP1, heterochromatin protein 1; tTA, tetracycline-controlled transactivator; TSA, trichostatin A; Ab(s), antibody(ies); mAB, monoclonal antibody; FITC, fluorescein isothiocyanate; DAPI, 4',6'-diamidino-2-phenylindole.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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