JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M403997200 on June 7, 2004

J. Biol. Chem., Vol. 279, Issue 32, 33799-33805, August 6, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/32/33799    most recent
M403997200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, A.
Right arrow Articles by Chen, J. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, A.
Right arrow Articles by Chen, J. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Identification of a Novel Family of Ankyrin Repeats Containing Cofactors for p160 Nuclear Receptor Coactivators*

Aihua Zhang, Percy Luk Yeung, Chia-Wei Li, Shih-Chieh Tsai, Gia Khanh Dinh, Xiaoyang Wu{ddagger}, Hui Li§, and J. Don Chen

From the Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635

Received for publication, April 9, 2004 , and in revised form, May 24, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Members of the p160 nuclear receptor coactivators interact with liganded nuclear receptors to enhance transcription of target genes. Here we identify a novel family of ankyrin repeats containing cofactors (ANCOs) that interact with the p160 coactivators. ANCO-1 binds to the conserved Per-Arnt-Sim (PAS) region of the p160 coactivators. It encodes a large nuclear protein with five ankyrin repeats, and parts of its sequences have been reported as nasopharyngeal carcinoma susceptibility protein and medulloblastoma antigen. Immunofluorescence staining reveals discrete nuclear foci of ANCO-1 that are distinct from known nuclear structures. Intriguingly, ANCO-1 also colocalizes and interacts with histone deacetylases. Transient reporter gene assay shows that ANCO-1 expression inhibits ligand-dependent transactivation by both steroid and nonsteroid nuclear receptors. Taken together, we have identified a novel family of ankyrin repeats containing cofactors that may recruit histone deacetylases to the p160 coactivators/nuclear receptor complex to inhibit ligand-dependent transactivation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear receptors (NRs)1 are DNA-binding transcription factors that control hormone-dependent gene expression in many biological processes (1, 2). NRs contain an N-terminal activation domain (AF-1), a central DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD) that also contains a ligand-dependent activation function (AF-2). Two classes of cofactors, known as coactivators and corepressors, mediate the activation and repression functions of NRs, respectively. Among the corepressors, silencing mediator of retinoid and thyroid hormone receptors and nuclear receptor corepressor are two highly related proteins that bind primarily to unliganded NRs (3–6). The mechanism of silencing mediator of retinoid and thyroid hormone receptors/nuclear receptor corepressor-mediated transcriptional repression is known to be mediated through histone deacetylases (HDACs) (7–9).

Contrary to the corepressors, the p160 NR coactivators bind to liganded NRs to mediate transcriptional activation through recruitment of histone acetyltransferases (10). The p160 coactivators include SRC-1 (11), GRIP1/TIF2 (12–14), and RAC3/ACTR/AIB1/pCIP/TRAM-1 (15–19). These coactivators bind to the hydrophobic cleft in LBD through LXXLL motifs (20, 21). These coactivators also interact with histone acetyltransferases such as CREB-binding protein/p300 and P/CAF (22, 23), tethering histone acetyltransferase activity to target promoters. Genetic studies suggest that the p160 coactivators are involved in regulating hormonal responses in mice (24–26). Among the p160 coactivators, RAC3 AIB1 is clinically important because it is amplified in breast cancers (17). Furthermore, RAC3 forms a stable complex with estrogen receptor {alpha} in breast cancer cells (27), suggesting that RAC3 may play an important role in the development of breast cancer.

The p160 coactivators share a common domain structure, including a highly conserved N-terminal basic-helix-loop-helix (bHLH) and Per-Arnt-Sim (PAS) domains (10). The PAS domain is separated into A and B regions, which are conserved among many PAS family proteins (28). The bHLH-PAS domain is implicated in mediating protein-protein interaction (see Refs. 34–36). We thought to search for new proteins that may regulate the function of RAC3 by interacting with the conserved bHLH-PAS domain. Previously, we have identified a human homologue of the yeast MMS19 as a RAC3-interacting protein, which also interacts with estrogen receptor {alpha} in a ligand-independent manner (29). Here, we report the identification and characterization of another RAC3-interacting protein, designated ANCO-1 for ankyrin repeats containing cofactor-1. We found that ANCO-1 and likely its related protein ANCO-2 may represent a novel class of nuclear receptor corepressors that may inhibit transcriptional activity of NRs through interfering with the coactivator function of p160 by recruiting HDACs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and Yeast Two-hybrid Screen—The bait plasmid pGBT-RAC3 bHLH-PAS (amino acids 1–408) used in the yeast two-hybrid screen and the other RAC3 constructs are as described previously (15, 30). A yeast two-hybrid screen of the human placenta cDNA library (Clontech) was conducted in Y190 cells with ~2 million transformants. Three RAC3-interacting cDNA clones, RS14, RS2, and RS13, were isolated, and they encode the C-terminal amino acids 2183–2663, 2279–2663, and 2369–2663 of ANCO-1, respectively. The RS2 and RS13 sequences match to a genomic clone on chromosome Xq27.1–Xq27.3; however, the RS14 N-terminal region differs from the genomic sequence. These clones were later found to match exactly to another genomic clone on chromosome 16q24.3 (RP11-781H20), and further analysis indicates that the sequence on Xq27.1 may be a pseudogene. Stepwise search and assembly of many overlapping expressed sequence tag clones in comparison with the genomic sequence predicted a full-length ANCO-1 of ~9.1 kb. The full-length ANCO-1 vector was constructed using DNA fragments from RS13, the genomic clone PR11-781H20, and the expressed sequence tag clone tf70e12. Constructions of other plasmids were achieved by standard methods, and details are available upon requests.

GST Pull-down Assay—GST fusion proteins were expressed in BL-21 cells and purified by standard glutathione agarose beads. The [35S]methionine-labeled proteins were synthesized in reticulocyte lysate (Promega). Five µg of GST fusion proteins conjugated to glutathione agarose beads were incubated with 5 µl of in vitro translated proteins at 4 °C overnight in a binding buffer as described before (31). Pellets were washed four times with binding buffer, and bound proteins were eluted in SDS sample buffer followed by SDS-PAGE and autoradiography.

Northern Blot—Multiple tissue and cancer cells Northern blots were purchased from Clontech and probed with 32P-labeled ANCO-1 cDNA fragments using the ExpressHyb solution. The blot was washed twice for 20 min in 2x SSC/0.1% SDS at room temperature and subject to autoradiography at -70 °C.

Immunofluorescence Microscopy—Cells were grown on cover glasses and then fixed with cold methanol/acetone (1:1) mixture for 2 min and processed for immunofluorescence staining as described previously (32). Thirty-six hours after transfection, cells were stained with primary antibodies followed by rhodamine- or fluorescein-conjugated secondary antibodies. Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole, and cover glasses were mounted with Pro-Long anti-fade reagents (Molecular Probes). The images were visualized with a Zeiss Axiovert 200 microscope and captured with the Axiocam and Axiovision software. The HA antibodies were purchased from MBL International Corp, and HDAC antibodies were from Affinity Bioreagents. The RNA splicing speckles were detected with SC35 monoclonal antibodies. The PML oncogenic domains and interphase centromeres were detected by anti-PML polyclonal antibodies and human autoimmune sera GS, respectively.

Co-immunoprecipitation Assay—The co-immunoprecipitation assay was conducted as described before (31). COS-7 cells were transfected with FLAG-HDAC3 and HA-ANCO-1C, and the total cell lysate was prepared 48 h after transfection. The HDAC3 and ANCO-1C proteins were immunoprecipitated with monoclonal anti-FLAG or polyclonal anti-HA antibodies, respectively. The agarose beads-conjugated FLAG and HA antibodies were purchased from Sigma. Western blot was conducted using ECL reagents from Amersham Biosciences.

Cell Culture and Transient Transfection Assays—Cells were grown at 37 °C with 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 5 µg/ml gentamycin (Invitrogen). One day prior to transfection, cells were seeded in 12-well plates at a density of 50,000 cells/well in phenol-red free Dulbecco's modified Eagle's medium supplemented with 10% charcoal resin-stripped fetal bovine serum. Transfection was performed using calcium/phosphate precipitate method as described (31). After 10 h, cells were washed with phosphate-buffered saline and re-fed with fresh medium containing indicated treatments. Cells were harvested 36–48 h after treatments for luciferase and {beta}-galactosidase assays as described previously (15).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of ANCO-1—ANCO-1 was identified from a yeast two-hybrid screen as a RAC3-interacting protein. Three ANCO-1 cDNA clones encoding overlapping C-terminal domains were isolated. To confirm the interaction and to map the interacting surface on RAC3, these cDNA clones were rescued and retransformed into yeast cells together with a series of RAC3 constructs. As illustrated in Fig. 1A, the RS13 clone encoding amino acids 2369–2663 of ANCO-1 (ANCO-1C) interacted with the PAS-B, PAS-A/B, and bHLH-PAS fragments of RAC3, whereas it had no interaction with the vector, bHLH, PAS-A, or the receptor-interacting domain of RAC3. Similar results were obtained with RS14 and RS2 clones (not shown). The interaction of ANCO-1C with RAC3 in vitro was then analyzed by GST pull-down assay (Fig. 1B), in which ANCO-1C bound specifically to the PAS-A, PAS-B, and PAS-A/B fragments. These data suggest that ANCO-1 interacts specifically and preferentially with the PAS region of RAC3.



View larger version (60K):
[in this window]
[in a new window]
  		FIG. 1.
ANCO-1 interacts with p160 coactivators. A, the yeast two-hybrid interaction between RAC3 and ANCO-1C. Yeast cells were cotransformed with indicated Gal4 DBD-RAC3 fusions and Gal4 activation domain (AD)-ANCO-1C. {beta}-galactosidase expressions were analyzed in three different colonies. All RAC3 clones and ANCO-1C alone did not activate {beta}-galactosidase expression. RID, receptor-interacting domains. B, a GST pull-down assay showing interactions of ANCO-1C with RAC3 PAS-B domain. C, a GST pull-down assay showing interactions of ANCO-1C and ANCO-1Ct fragments with 35S-labeled RAC3, TIF2, and SRC-1. D, a mammalian two-hybrid assay showing interactions between VP16-ANCO-1C and ANCO-1-(1802–2663) fragments with RAC3 PAS-A/B (Gal4-PAS-A/B) in COS-7 cells. Relative luciferase activities were determined from three independent experiments after normalization with cotransfected {beta}-galactosidase. E, the expression pattern of ANCO-1 in normal human tissues and cancer cell lines. Northern blots were hybridized with the 32P-labeled N-terminal untranslated region or C-terminal conserved region of ANCO-1 probes. The membranes were stripped and hybridized again with {beta}-actin probe for comparison.

 
To investigate whether ANCO-1 also interacts with full-length p160 coactivators, a GST pull-down assay was conducted using in vitro translated full-length RAC3, TIF2, and SRC-1. We found that both ANCO-1C and the ANCO-1Ct fragment (amino acids 2597–2663) bound strongly to the full-length RAC3, TIF2, and SRC-1 (Fig. 1C). These data suggest that ANCO-1C may interact with full-length p160 coactivators through the C-terminal 67 amino acids of ANCO-1. This interaction was further confirmed by mammalian two-hybrid assay in COS-7 cells (Fig. 1D), in which the RAC3 PAS-A/B fragment interacted strongly with ANCO-1C and a larger ANCO-1 fragment (amino acids 1802–2663).

Northern blot analysis revealed a large 10-kb transcript in many human tissues and cancer cells, which represents the full-length ANCO-1. The expression level of ANCO-1 varies modestly among different tissues and cancer cell lines, with the highest expression in skeletal muscle and chronic myeloid leukemia K562 cells. The amounts of actins are approximately equal in every lane, confirming equal loading of RNA. Intriguingly, the N-terminal probe also detected two smaller transcripts of ~3.5-kb, whereas the C-terminal probe detected an additional 7.5-kb transcript, suggesting the existence of possible alternative splicing variants of ANCO-1.

Amino Acid Sequences of ANCO-1 and ANCO-2—The full-length ANCO-1 contains 2663 amino acids with an estimated molecular size of 298 kDa (Fig. 2). The only known domain in ANCO-1 is the five 33-amino-acid-long ankyrin repeats (33). Interestingly, this ankyrin region is identical to a nasopharyngeal carcinoma susceptibility protein LZ16 (GenBankTM accession number AF121775 [GenBank] ), which has a cytosine deleted on codon Gly-326 leading to frameshift and early termination. Comparison of existing expressed sequence tag sequences did not find this deletion in other sequences, suggesting that this mutation might be unique in the nasopharyngeal carcinoma cells. A recent study also showed a partial medulloblastoma antigen sequence corresponding to amino acids 382–1120 of ANCO-1 but contains a 205-bp deletion after codon Lys-937 (34), causing a frameshift relative to ANCO-1. Beside ankyrin repeats, ANCO-1 does not contain other noticeable domains. Hydrophobicity analysis shows that the central region of ANCO-1 is highly charged and contains many putative nuclear localization signals. Furthermore, an ANCO-1-related protein of unknown function was found in GenBank (KIAA0874 and GAC-1, GenBank accession number NM_015208 [GenBank] and AF317425 [GenBank] ). We name this ANCO-1-related protein ANCO-2. The similarity between ANCO-1 and ANCO-2 is most striking at the N-terminal and C-terminal domains (67 and 81%, respectively). ANCO-2 also interacts with p160 coactivators in GST pull-down assay (not shown), suggesting that ANCO-1 and ANCO-2 may be functionally related.



View larger version (93K):
[in this window]
[in a new window]
  		FIG. 2.
Amino acid sequences and comparison between ANCO-1 and ANCO-2. The alignment was produced by ClustlalW algorithm in the MacVectorTM software. Identical or conserved amino acids are shown in shaded boxes. ANCO-1 and ANCO-2 proteins are most conserved at the N-terminal region that contains five ankyrin repeats (arrows) and the C-terminal ANCO-1C domain.

 
Localization of ANCO-1 at Nuclear Foci and Association with HDACs—The interaction of ANCO-1 with transcriptional coactivators suggests that ANCO-1 may be involved in gene regulation. To test this hypothesis, we examined anticipated nuclear localization of ANCO-1 by immunofluorescence staining. HeLa and 293T cells were transfected with HA-ANCO-1 expression vector and double-stained with anti-HA antibodies and antibodies that recognize known nuclear structures. Intriguingly, we found that not only was ANCO-1 indeed localized in the nucleus; it was also accumulated at 10–50 discrete foci (Fig. 3). These ANCO-1 nuclear foci are distinct from other known nuclear domains such as the SC35 RNA splicing speckles, PML oncogenic domains, and interphase centromeres. To determine whether ANCO-1 nuclear foci may be linked to transcriptional regulation, we analyzed colocalization of ANCO-1 with several transcriptional coregulators. Surprisingly, we found ANCO-1 nuclear foci associated closely with HDAC3, HDAC4, and HDAC5 (Fig. 3). These associations are specific because ANCO-1 did not colocalize with the nuclear HDAC1 or the cytoplasmic HDAC6.



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 3.
Colocalization studies of ANCO-1 with known nuclear structures and HDACs. The 293T cells stably transfected with HA-ANCO-1 were double-immunostained for HA and endogenous SC35, PML, or interphase centromeres (GS) as described under "Materials and Methods." For HDAC colocalization, HeLa cells were transiently transfected with HA-ANCO-1 and respective FLAG-HDACs and double-immunostained for HA and FLAG tags. DAPI, 4',6-diamidino-2-phenylindole.

 
To investigate the association of ANCO-1 with HDACs, we performed a GST pull-down assay to determine whether ANCO-1 interacts directly or indirectly with HDACs. The data show that the ANCO-1C domain binds to HDAC3 (Fig. 4A), as well as HDAC4 and HDAC5 (data not shown). The HDAC-interacting domain of ANCO-1 is separable from the coactivator-binding domain because the ANCO-1Ct fragment bound p160 coactivators (Fig. 1C) but not HDAC3 (Fig. 4A). Furthermore, a co-immunoprecipitation assay also demonstrated the formation of an ANCO-1/HDAC3 protein complex in vivo (Fig. 4B). These data strongly suggest that, in addition to the p160 coactivators, ANCO-1 may also interact with specific HDACs.



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 4.
Interaction of ANCO-1 with HDACs in vitro and in vivo. A, ANCO-1 interacts with HDAC3 in GST pull-down assay. The Coomassie panel shows the amounts of individual GST proteins used in the GST-pull-down experiment. The HDAC3 protein was produced in reticulocyte lysate using the TNT T7 in vitro transcription/translation system. The newly synthesized protein was labeled with [35S]methionine as described under "Materials and Methods." MS marker, molecular size marker. B, ANCO-1C co-immunoprecipitates with HDAC3. COS-7 cells were cotransfected with HA-ANCO-1C and FLAG-HDAC3. Cell lysates were prepared and immunoprecipitated with monoclonal anti-FLAG or anti-HA antibodies followed by Western blotting. Asterisks indicate the IgG heavy chain. IP, immunoprecipitation.

 
ANCO-1 Inhibits Transcriptional Activation by Nuclear Receptors—The interaction of ANCO-1 with HDACs suggests that ANCO-1 may be a negative cofactor on transcriptional regulation by NRs. To test this hypothesis, we transiently overexpressed ANCO-1 in COS-7 cells and analyzed its effect on ligand-dependent transcriptional activation by NRs. We found that ANCO-1 overexpression resulted in a concentration-dependent inhibition of ligand-induced transactivation by several NRs, including those for mineralocorticoid, androgen, progesterone (PR), and glucocorticoid (Fig. 5, A and B). In contrast, ANCO-1 overexpression had no effect on the basal activity of the MMTV-Luc reporter (Fig. 5C). ANCO-1 also had no effect on a Gal4-dependent reporter in the presence of Gal4 DBD or Gal4-VP16 fusion protein (Fig. 5D), suggesting that ANCO-1 may inhibit NR-dependent transcription selectively. Furthermore, ANCO-1 expression also inhibited retinoic acid-dependent transactivation of the Gal4-dependent reporter by Gal4-RAR{alpha} in a concentration-dependent manner (Fig. 5E). These data strongly suggest that ANCO-1 may function as a negative coregulator for NRs by inhibiting ligand-dependent transcriptional activation.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 5.
ANCO-1 inhibits ligand-dependent transcriptional activation. A, ANCO-1 inhibits aldosterone (Aldo.)-dependent mineralocorticoid (MR)-mediated transactivation on MMTV-Luc reporter. ANCO-1 also inhibits dihydroxytestosterone (DHT)-dependent androgen-mediated transactivation on MMTV-Luc reporter. The indicated amounts of full-length ANCO-1 expression plasmid (in µg) were cotransfected with 0.25 µg of receptor plasmid in COS-7 cells. B, ANCO-1 inhibits progesterone (Prog.)-dependent PR-mediated transactivation on MMTV-Luc reporter. ANCO-1 also inhibits dexamethasone (Dex)-dependent glucocorticoid (GR)-mediated transactivation on MMTV-Luc reporter. C, ANCO-1 has no effect on the basal MMTV promoter activity. D, ANCO-1 has no effect on the Gal4-dependent thymidine kinase promoter activity in the presence of Gal4 DBD or Gal4 DBD-VP16 activation domain fusion (Gal4-VP16). E, ANCO-1 inhibits all-trans retinoic acid (atRA, 100 nM)-dependent Gal4-RAR{alpha}-mediated transactivation. F, ANCO-1 overexpression inhibits TIF2 coactivation on PR-mediated transactivation. G, TIF2 overexpression reverts ANCO-1-mediated transcriptional inhibition on PR-mediated transactivation of MMTV-Luc reporter.

 
Finally, we determined the involvement of p160 coactivators on ANCO-1-mediated transcriptional inhibition. We tested whether the inhibition of PR-mediated transactivation by ANCO-1 involves competition with the coactivator TIF2. Consistent with previous experiments, expression of ANCO-1 inhibited PR/progesterone-mediated transactivation on the MMTV-Luc reporter, whereas expression of TIF2 enhanced the transactivation (Fig. 5F, lanes 3 and 4). Increasing concentrations of ANCO-1 progressively diminished the TIF2-mediated coactivation, suggesting that ANCO-1 may antagonize the coactivator function of TIF2. Conversely, increasing concentrations of TIF2 also overcame ANCO-1-mediated transcriptional repression on PR/progesterone-mediated transactivation in a dose-dependent manner (Fig. 5G). Consistently, a Gal4 DBD-ANCO-1 fusion protein was able to repress basal transcription of the Gal4-dependent luciferase reporter (not shown), suggesting that ANCO-1 contains intrinsic transcriptional repression activity. Taken together, these data indicate that the ability of ANCO-1 to inhibit ligand-dependent transcriptional activation by nuclear receptors may involve antagonizing the function of p160 coactivators by recruiting HDACs to the coactivator/NR complex.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have identified a novel family of ankyrin repeats containing coregulator proteins: ANCO-1 and ANCO-2. ANCO-1 was isolated in a yeast two-hybrid screen as a RAC3-interacting protein, whereas ANCO-2 was identified in GenBank as a protein related to ANCO-1. ANCO-1 interacts with p160 coactivators and HDAC corepressors, and it inhibits ligand-dependent transcriptional activation by NRs. We speculate that recruitment of HDACs by ANCO-1 is likely to neutralize the histone acetyltransferase activity present in the coactivator complex, leading to the attenuation of transcriptional activation. Thus, the ANCO proteins represent a novel class of nuclear receptor corepressors that may inhibit ligand-dependent transcription by antagonizing function of coactivators through recruitment of HDACs.

The bHLH-PAS domain of p160 coactivators has been shown to interact with several secondary coactivators such as hMMS19 (29), BAF57 (35), and CoCoA (36), as well as other transcription factors (37). Unlike most other proteins that require both PAS-A and PAS-B domains for efficient binding (29), ANCO-1 binds preferentially to the PAS-B domain in vivo, although binding to PAS-A domain also occurs in vitro. The interaction between ANCO-1 and the PAS domain has been confirmed by yeast two-hybrid, mammalian two-hybrid, and GST pull-down assays. Furthermore, we show that ANCO-1 is capable of interacting with full-length p160 coactivators; thus, their interactions are not limited to separate domains.

The N-terminal domain of the ANCO-1 was found in a nasopharyngeal carcinoma susceptibility protein, and the central region of ANCO-1 was recently isolated as a tumor antigen present in childhood medulloblastoma (34). Therefore, it is reasonable to speculate that ANCO-1 may serve as a molecular marker for the tumors. The ANCO-1 gene is located at 16q24.3, a region that is frequently deleted in cancer (34, 38). This finding supports the possibility that ANCO-1 may be a cancer-related gene. One striking feature of ANCO-1 is the presence of five ankyrin repeats at the N-terminal region. Ankyrin repeats are 33 amino acids long and involved in protein-protein interactions (33). Indeed, yeast two-hybrid screening using the ANCO-1 ankyrin repeats as bait has discovered several other proteins,2 suggesting that ANCO-1 may play a role in integrating signals from multiple protein-protein interactions. The core domain of ankyrin repeat adopts a helix-loop-helix structure, and the repeats occur in a large number of functionally diverse proteins. Among the five ankyrin repeats, the first repeat is least conserved, and whether it would adopt the normal ankyrin structure is unclear since minimal ankyrin repeats can occur in four consecutive copies.

Immunofluorescence staining of ANCO-1 shows clear nuclear foci localization. Localization of ANCO-1 in the nucleus is consistent with the presence of multiple nuclear localization signals and its predicted function as a transcriptional coregulator. However, accumulation of ANCO-1 protein in nuclear foci was unexpected, but it provides an opportunity for analyzing in vivo protein-protein interactions through subcellular colocalization. We found that the ANCO-1 nuclear foci are distinct from other known nuclear structures, suggesting that ANCO-1 nuclear foci may represent novel compartments that are likely important for ANCO-1 function. Coexpression of ANCO-1 with RAC3 also shows colocalization (not shown), although RAC3 signal was less consistent due to its rapid degradation (39). Conceivably, it is possible that ANCO-1 nuclear foci may be associated with the DNA-bound NR/coactivator complex, although one cannot rule out the possibility that the ANCO-1 foci might act as protein depots for sequestrating coactivators or corepressors. Clear formation of ANCO-1 nuclear foci can be observed at low levels of ANCO-1 expression and also in stable clones, suggesting that the endogenous ANCO-1 may form nuclear foci.

The facts that ANCO-1 is a nuclear protein associated with p160 coactivators and the revelation that ANCO-1 also binds to HDAC corepressors implicate a potential corepressor function for ANCO-1. Indeed, the transient reporter assay establishes a strong inhibition of NR-mediated transcriptional activation by ANCO-1 (Fig. 5). Competition experiments further suggest an involvement of p160 coactivators in ANCO-1-mediated transcriptional inhibition. Thus, we believe that ANCO-1 functions as a negative transcriptional coregulator for NRs by interfering with p160 coactivators through the recruitment of HDACs. So far, we found no evidence of direct interactions between ANCO-1 and NRs, suggesting that ANCO-1 may be recruited to NR-targeted promoters indirectly by p160 coactivators. ACTR/RAC3 can be acetylated by CREB-binding protein/p300 and serves as a transcriptional termination signal for estrogen receptor (40). The GRIP1/TIF2 coactivator can also be phosphorylated by mitogen-activated protein kinase in response to growth factor signals (41). Conceivably, coactivator modifications may lead to recruitment or stabilization of the ANCO-1/HDAC complex. It will be interesting to analyze the molecular signals and sequence of events governing protein-protein interactions among p160 coactivators, HDAC corepressors, and ANCO-1.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY533563 [GenBank] (ANCO-1) and AY533564 [GenBank] (ANCO-2).

* This work was supported by grants from the National Institutes of Health (Grants DK52888 and DK52542) and the Leukemia and Lymphoma Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 595, New York, NY 10021. Back

§ Present address: Tuffs-New England Medical Center, Molecular Oncology Research Institute, 750 Washington St., NEMC Box 80, Boston MA 02111. Back

To whom correspondence should be addressed: Dept. of Pharmacology, UMDNJ-RWJMS, 661 Hoes Lane, Piscataway, NJ 08854-5635. E-mail: chenjd{at}umdnj.edu.

1 The abbreviations used are: NR, nuclear receptor; SRC, steroid receptor coactivator; RAC3, receptor-associated coactivator 3; ANCO-1/2, ankyrin repeats containing cofactors-1/2; GST, glutathione S-transferase; HDAC, histone deacetylase; DBD, DNA-binding domain; LBD, ligand-binding domain; bHLH, basic-helix-loop-helix; PAS, Per-Arnt-Sim; HA, hemagglutinin; PR, progesterone receptor; CREB, cAMP-response element-binding protein; Luc, luciferase; PML, promyelocytic leukemia protein; MMTV, mouse mammary tumor virus. Back

2 C.-W. Li, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank S. Schreiber, E. Seto, M. Lazar, and X.-J. Yang for plasmids, J. Zhang and A. Chen for technical help, and J. D. Fondell for critical comments on the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nawaz, Z., Baniahmad, C., Burris, T. P., Stillman, D. J., O'Malley, B. W., and Tsai, M. J. (1994) Mol. Gen. Genet. 245, 724-733[CrossRef][Medline] [Order article via Infotrieve]
  2. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schütz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., and Evans, R. M. (1995) Cell 83, 835-839[CrossRef][Medline] [Order article via Infotrieve]
  3. Chen, J. D., and Evans, R. M. (1995) Nature 377, 454-457[CrossRef][Medline] [Order article via Infotrieve]
  4. Horlein, A. J., Naar, A. M., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R., Ryan, A., Kamei, Y., Soderstrom, M., Glass, C. K., and Rosenfeld, M. G. (1995) Nature 377, 397-404[CrossRef][Medline] [Order article via Infotrieve]
  5. Park, E. J., Schroen, D. J., Yang, M., Li, H., Li, L., and Chen, J. D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3519-3524[Abstract/Free Full Text]
  6. Ordentlich, P., Downes, M., Xie, W., Genin, A., Spinner, N. B., and Evans, R. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2639-2644[Abstract/Free Full Text]
  7. Nagy, L., Kao, H. Y., Chakravarti, D., Lin, R. J., Hassig, C. A., Ayer, D. E., Schreiber, S. L., and Evans, R. M. (1997) Cell 89, 373-380[CrossRef][Medline] [Order article via Infotrieve]
  8. Guenther, M. G., Lane, W. S., Fischle, W., Verdin, E., Lazar, M. A., and Shiekhattar, R. (2000) Genes Dev. 14, 1048-1057[Abstract/Free Full Text]
  9. Li, J., Wang, J., Nawaz, Z., Liu, J. M., Qin, J., and Wong, J. (2000) EMBO J. 19, 4342-4350[CrossRef][Medline] [Order article via Infotrieve]
  10. Leo, C., and Chen, J. D. (2000) Gene (Amst.) 245, 1-11[CrossRef][Medline] [Order article via Infotrieve]
  11. Oñate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1995) Science 270, 1354-1357[Abstract/Free Full Text]
  12. Hong, H., Kohli, K., Trivedi, A., Johnson, D. L., and Stallcup, M. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4948-4952[Abstract/Free Full Text]
  13. Hong, H., Kohli, K., Garabedian, M. J., and Stallcup, M. R. (1997) Mol. Cell. Biol. 17, 2735-2744[Abstract]
  14. Voegel, J. J., Heine, M. J. S., Zechel, C., Chambon, P., and Gronemeyer, H. (1996) EMBO J. 15, 3667-3675[Medline] [Order article via Infotrieve]
  15. Li, H., Gomes, P. J., and Chen, J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8479-8484[Abstract/Free Full Text]
  16. Torchia, J., Rose, D. W., Inostroza, J., Kamei, Y., Westin, S., Glass, C. K., and Rosenfeld, M. G. (1997) Nature 387, 677-684[CrossRef][Medline] [Order article via Infotrieve]
  17. Anzick, S. L., Kononen, J., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X. Y., Sauter, G., Kallioniemi, O. P., Trent, J. M., and Meltzer, P. S. (1997) Science 277, 965-968[Abstract/Free Full Text]
  18. Chen, H., Lin, R. J., Schiltz, R. L., Chakravarti, D., Nash, A., Nagy, L., Privalsky, M. L., Nakatani, Y., and Evans, R. M. (1997) Cell 90, 569-580[CrossRef][Medline] [Order article via Infotrieve]
  19. Takeshita, A., Cardona, G. R., Koibuchi, N., Suen, C.-S., and Chin, W. W. (1997) J. Biol. Chem. 272, 27629-27634[Abstract/Free Full Text]
  20. Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997) Nature 387, 733-736[CrossRef][Medline] [Order article via Infotrieve]
  21. Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) Cell 95, 927-937[CrossRef][Medline] [Order article via Infotrieve]
  22. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman, R. A., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1996) Cell 85, 403-414[CrossRef][Medline] [Order article via Infotrieve]
  23. Blanco, J. C., Minucci, S., Lu, J., Yang, X. J., Walker, K. K., Chen, H., Evans, R. M., Nakatani, Y., and Ozato, K. (1998) Genes Dev. 12, 1638-1651[Abstract/Free Full Text]
  24. Xu, J., Qiu, Y., DeMayo, F. J., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1998) Science 279, 1922-1925[Abstract/Free Full Text]
  25. Xu, J., Liao, L., Ning, G., Yoshida-Komiya, H., Deng, C., and O'Malley, B. W. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6379-6384[Abstract/Free Full Text]
  26. Wang, Z., Rose, D. W., Hermanson, O., Liu, F., Herman, T., Wu, W., Szeto, D., Gleiberman, A., Krones, A., Pratt, K., Rosenfeld, R., Glass, C. K., and Rosenfeld, M. G. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13549-13554[Abstract/Free Full Text]
  27. Tikkanen, M. K., Carter, D. J., Harris, A. M., Le, H. M., Azorsa, D. O., Meltzer, P. S., and Murdoch, F. E. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12536-12540[Abstract/Free Full Text]
  28. Huang, W., Sun, G.-L., Li, X.-S., Cao, Q., Lu, Y., Jang, G.-S., Zahang, F.-Q., Chai, J.-R., Wang, Z.-Y., Waxman, S., Chen, Z., and Chen, S.-J. (1993) Blood 82, 1264-1269[Abstract/Free Full Text]
  29. Wu, X., Li, H., and Chen, J. D. (2001) J. Biol. Chem. 276, 23962-23968[Abstract/Free Full Text]
  30. Li, H., and Chen, J. D. (1998) J. Biol. Chem. 273, 5948-5954[Abstract/Free Full Text]
  31. Li, H., Leo, C., Zhu, J., Wu, X., O'Neil, J., Park, E. J., and Chen, J. D. (2000) Mol. Cell. Biol. 20, 1784-1796[Abstract/Free Full Text]
  32. Dyck, J. A., Maul, G. G., Miller, W. H., Jr., Chen, J. D., Kakizuka, A., and Evans, R. M. (1994) Cell 76, 333-343[CrossRef][Medline] [Order article via Infotrieve]
  33. Sedgwick, S. G., and Smerdon, S. J. (1999) Trends Biochem. Sci. 24, 311-316[CrossRef][Medline] [Order article via Infotrieve]
  34. Behrends, U., Schneider, I., Rossler, S., Frauenknecht, H., Golbeck, A., Lechner, B., Eigenstetter, G., Zobywalski, C., Muller-Weihrich, S., Graubner, U., Schmid, I., Sackerer, D., Spath, M., Goetz, C., Prantl, F., Asmuss, H. P., Bise, K., and Mautner, J. (2003) Int. J. Cancer 106, 244-251[CrossRef][Medline] [Order article via Infotrieve]
  35. Belandia, B., Orford, R. L., Hurst, H. C., and Parker, M. G. (2002) EMBO J. 21, 4094-4103[CrossRef][Medline] [Order article via Infotrieve]
  36. Kim, J. H., Li, H., and Stallcup, M. R. (2003) Mol. Cell 12, 1537-1549[CrossRef][Medline] [Order article via Infotrieve]
  37. Bernstein, E., Denli, A. M., and Hannon, G. J. (2001) RNA (N. Y.) 7, 1509-1521
  38. Powell, J. A., Gardner, A. E., Bais, A. J., Hinze, S. J., Baker, E., Whitmore, S., Crawford, J., Kochetkova, M., Spendlove, H. E., Doggett, N. A., Sutherland, G. R., Callen, D. F., and Kremmidiotis, G. (2002) Genomics 80, 303-310[CrossRef][Medline] [Order article via Infotrieve]
  39. Lonard, D. M., Nawaz, Z., Smith, C. L., and O'Malley, B. W. (2000) Mol. Cell 5, 939-948[CrossRef][Medline] [Order article via Infotrieve]
  40. Chen, H., Lin, R. J., Xie, W., Wilpitz, D., and Evans, R. M. (1999) Cell 98, 675-686[CrossRef][Medline] [Order article via Infotrieve]
  41. Lopez, G. N., Turck, C. W., Schaufele, F., Stallcup, M. R., and Kushner, P. J. (2001) J. Biol. Chem. 276, 22177-22182[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Physiol. GenomicsHome page
I. Barbaric, M. J. Perry, T. N. Dear, A. Rodrigues Da Costa, D. Salopek, A. Marusic, T. Hough, S. Wells, A. J. Hunter, M. Cheeseman, et al.
An ENU-induced mutation in the Ankrd11 gene results in an osteopenia-like phenotype in the mouse mutant Yoda
Physiol Genomics, February 19, 2008; 32(3): 311 - 321.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J. H. Kim, C. K. Yang, and M. R. Stallcup
Downstream signaling mechanism of the C-terminal activation domain of transcriptional coactivator CoCoA.
Nucleic Acids Res., January 1, 2006; 34(9): 2736 - 2750.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
Y.-H. Chen, J. H. Kim, and M. R. Stallcup
GAC63, a GRIP1-Dependent Nuclear Receptor Coactivator
Mol. Cell. Biol., July 15, 2005; 25(14): 5965 - 5972.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
X.-J. Yang and S. Gregoire
Class II Histone Deacetylases: from Sequence to Function, Regulation, and Clinical Implication
Mol. Cell. Biol., April 15, 2005; 25(8): 2873 - 2884.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/32/33799    most recent
M403997200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, A.
Right arrow Articles by Chen, J. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, A.
Right arrow Articles by Chen, J. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.