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Originally published In Press as doi:10.1074/jbc.M507356200 on September 26, 2005

J. Biol. Chem., Vol. 280, Issue 47, 39436-39447, November 25, 2005
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Microspherule Protein 1, Mi-2{beta}, and RET Finger Protein Associate in the Nucleolus and Up-regulate Ribosomal Gene Transcription*

Keiko Shimono{ddagger}§1, Yohei Shimono{ddagger}1, Kaoru Shimokata¶, Naoki Ishiguro§, and Masahide Takahashi{ddagger}||2

From the {ddagger}Department of Pathology, §the Department of Orthopedic Surgery, Department of Internal Medicine, and ||Department of Molecular Pathology, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

Received for publication, July 7, 2005 , and in revised form, September 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The nucleolus is the site of ribosomal DNA (rDNA) transcription and ribosome production. In exploring the role of nucleolar protein MCRS1 (microspherule protein1)/MSP58 (58-kDa microspherule protein), we found that Mi-2{beta}, a component of a nucleosome remodeling and deacetylase (NuRD) complex, RET finger protein (RFP), and upstream binding factor (UBF) were associated with MCRS1. Yeast two-hybrid assays revealed that MCRS1 bound to the ATPase/helicase region of Mi-2{beta} and the coiled-coil region of RFP. Interestingly, confocal microscopic analyses revealed the co-localization of MCRS1, Mi-2{beta}, RFP, and the rRNA transcription factor UBF in the nucleoli. We also found that MCRS1, Mi-2{beta}, and RFP were associated with rDNA using a chromatin immunoprecipitation assay. Finally, we showed that MCRS1, Mi-2{beta}, and RFP up-regulated transcriptional activity of the rDNA promoter and that ribosomal RNA transcription was repressed when MCRS1, Mi-2{beta}, and RFP expression was reduced using siRNA. These results indicated that Mi-2{beta} and RFP, known to be involved in transcriptional repression in the nucleus, co-localize with MCRS1 in the nucleolus and appear to activate the rRNA transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The nucleolus is a subnuclear compartment in eukaryotic cells responsible for rRNA synthesis and ribosome biogenesis. In actively growing animal and plant cells, rRNA synthesis accounts for about 50-80% of total cellular RNA production (1). Human diploid cells contain about 400 ribosomal genes organized as tandem repeats at nucleolus organizer regions encoded on chromosomes 13, 14, 15, 21, and 22 (1, 2). rRNA gene transcription varies according to the demand for ribosome production and protein synthesis, and in yeast and murine cells, only 30-50% of the rRNA genes are in an open structure that facilitates active transcription (3, 4). Transcription of rRNA is extensively regulated by a large number of proteins such as growth factors, CBP (cAMP-response element-binding protein (CREB)-binding protein), Rb, p53, and Myc (5-10). In addition, growing evidence indicates roles for different histone modification and chromatin remodeling complexes in the coordinated regulation of rRNA gene transcription. Nucleolar remodeling complex (NoRC) is a well recognized chromatin remodeling complex involved in rRNA gene silencing and is known to cooperate with DNA methyltransferases, histone deacetylases, histone methyltransferases, and TTF-I (transcription terminator factor) (11-14). In contrast, ribosomal DNA transactivation is associated with Tip60 histone acetyltransferase and histone acetylation (15, 16). However, the chromatin remodeling proteins that facilitate the establishment of the open state in rRNA genes remain to be characterized.

Mi-2 is a nuclear protein with chromatin remodeling activity and is the largest component of the nucleosome remodeling and deacetylase (NuRD)3 complex (17-20). The transcriptional repressive activity of Mi-2 is mediated by NuRD complex components, such as methyl CpG-binding protein MBD 2/3, histone deacetylases HDAC1/2, metastasis-associated protein MTA2/3, and RbAp46/48. NuRD complex and Mi-2 have been associated with the transcriptional repressive activities of KAP-1, RET finger protein (RFP), Trk69 (Tram-track69), and ROR{gamma} (21-24). In addition, MTA-3, another component of the NuRD complex, was reported to repress Snail expression, and the estrogen-dependent transcriptional control of MTA3, Snail, and E-cadherin was associated with breast cancer progression (25). However, Mi-2 also plays roles in transactivation outside the NuRD complex. A transactivating role of dMi-2 was suggested in studies of Drosophila salivary glands that showed the localization of dMi-2 to chromatin puffs (26). We recently reported that Mi-2{beta} possessed both transactivating and repressing subdomains (24). Furthermore, the study, based on a conditional Mi-2{beta} knock-out mouse line, clearly showed that Mi-2{beta} activated CD4 transcription in T cells by forming a complex with p300 histone acetyltransferase and the E box-binding protein HEB (27). Thus, Mi-2{beta} appears to be involved in both transcriptional repression and activation through the formation of distinct protein complexes in the nucleus.

The RFP was originally identified as a fusion protein with RET-tyrosine kinase (28). RFP mRNA is strongly expressed in a variety of human and rodent tumor cell lines. In addition, its protein expression is detected strongly in male germ cells and relatively weak in peripheral and central neurons, hepatocytes, and adrenal chromaffin cells (28, 29). RFP contains a tripartite motif consisting of a RING finger, a B-box zinc finger, a coiled-coil domain that is involved in protein-protein associations, and an RFP domain. RFP is a nuclear protein and was reported to partially co-localize with promyelocytic leukemia protein and int-6 in the nucleus (30, 31). We found that RFP exhibited transcriptional repressive activity and that it was associated and co-localized with Enhancer of Polycomb 1 and Mi-2{beta} in the nucleus (24, 32). We also showed that the subnuclear distribution of RFP was altered by small ubiquitin-like modifier modification by protein inhibitor of activated STAT (signal transducers and activators of transcription) (PIAS) proteins (33).

MCRS1 (microspherule protein 1)/MSP58 (58-kDa microspherule protein) and the related protein p78 were originally identified as proteins that interacted with nucleolar protein p120 and herpes simplex virus 1-infected cell protein 22 (ICP22) (34, 35). MCRS1 is a nucleolar protein that contains a bipartite nuclear localization motif, a nucleolar localization motif, a coiled-coil domain, and a forkhead associated domain. Several functions for MCRS1 and its splice variant MCRS2 have been reported, including a transforming activity, nucleolar sequestration activity, and telomerase inhibition (36-39). MCRS1 and TOJ3, a protein with high structural similarity to MCRS1, exhibit transforming activity, whereas phosphatase and tensin homologue (PTEN) suppresses the transforming activity of MCRS1 (36, 38). It has also been shown that MCRS1 can relieve the repressor activity of Daxx, an adaptor protein that links Fas signaling to the c-Jun NH2-terminal kinase pathway via a nucleolar sequestration mechanism (39). MCRS2 is involved in telomere shortening by associating with telomerase (37). In addition, binding and stabilization of MSP58 by transcription factor STRA13 has been observed (40). Although these findings suggest that MCRS1 may have several distinct functions, its transcriptional role in the nucleolus has not yet been fully characterized.

In this report we investigated the role of MCRS1 in ribosomal gene transcription and its association and co-localization with Mi-2{beta} and RFP in the nucleolus. Our results indicated that Mi-2{beta}, RFP, and nucleolar protein MCRS1 were involved in ribosomal gene transcription and suggested that Mi-2 may be a candidate for the unidentified chromatin remodeling protein that establishes the euchromatin structure of ribosomal genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—MCRS1/MSP58 cDNA (GenBankTM accession number NM_006337 [GenBank] ) was obtained by PCR using a human testis cDNA library as a template and sequenced. The same product was obtained using a human placenta cDNA library as a template. The full-length MCRS1 was cloned in-frame into pEGFP-C1 plasmid (Clontech).

Human ribosomal DNA promoter spanning -483 to +377 bp with respect to the transcription initiation site (GenBankTM accession number U13369 [GenBank] ) (41) was amplified from genomic DNA isolated from human embryo kidney (HEK) 293 cells and checked by sequencing. The internal ribosome entry site (IRES) from the encephalomyocarditis virus was amplified from pIRES2-EGFP vector (Clontech) and checked by sequencing. The Kozak sequence of the pGL3-basic vector was removed by digestion with NcoI and HindIII and replaced with the IRES sequence to generate the pGL3-IRES-Luc plasmid (42). The pGL3-GAL4-hrDNAP-IRES-Luc plasmid was generated by cloning the GAL4 DNA binding region (GAL4BD) fragment of pAS2-1 vector (Clontech) and the human ribosomal DNA promoter (-483 to +377) into the pGL3-IRES-Luc plasmid. The pcDNA3-GAL4BD plasmid was described previously (32), and the nucleolar localization signal (NoLS) of MCRS1 (32-57 amino acids) was inserted right after the GAL4BD sequence to generate the pcDNA3-GAL4BD-NoLS plasmid.

Antibodies—Anti-RFP polyclonal antibody was described previously (24, 32). Anti-Mi-2 polyclonal antibody was a kind gift from Dr. Wade at Emory University (43). Anti-MCRS1 polyclonal antibody was developed against the 19 carboxyl-terminal amino acids of MCRS1 (GenBankTM accession number NM_006337 [GenBank] ). Anti-FLAG (M2) monoclonal antibody was obtained from Sigma. Anti-upstream binding factor (UBF) monoclonal antibody was purchased from Santa Cruz Biotechnology. Anti-GFP polyclonal antibody was purchased from MBL (Japan). Alexa-conjugated secondary antibodies were purchased from Molecular Probes. Anti-MCRS1 polyclonal antibody was labeled with Alexa 594 using the Alexa Fluor 594 protein labeling kit (Molecular Probes).

Western Blotting and Co-immunoprecipitation Experiments—HEK 293 cells plated in 100-mm dishes were transfected with 4 µg of pFLAG-CMV2 expression vector or pEGFP-C1-MCRS1 expression vector using Lipofectamine PLUS (Invitrogen) according to the manufacturer's instructions. After 48 h of incubation, cells were washed twice with ice-cold washing buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) and resuspended in 1 ml of hypotonic buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride (PMSF)) containing Complete protease inhibitor mixture (Roche Diagnostics). The suspension was briefly sonicated and mixed with 160 µl of 2.5 M sucrose solution. After centrifugation at 1200 x g for 5 min at 4 °C, the pellet was lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM MgCl2, 0.5% Nonidet P-40, and 1 mM PMSF) containing Complete protease inhibitor mixture and centrifuged again at 12,000 x g for 25 min at 4 °C. The resultant supernatants were incubated with specific polyclonal antibodies or mouse anti-FLAG M2 antibody coupled with protein A-Sepharose or protein G-Sepharose (Sigma) for 5 h at 4°C. Co-immunoprecipitation, electrophoretic separation, and Western blotting were performed as previously described (24). The nuclear/cytosol fractionation kit (Bio Vision) was used to prepare nuclear extracts to check the specificity of anti-MCRS1 antibody.

Yeast Two-hybrid Assay—The SfiI/PmeI fragment of the pcDNAV5-HisC-HA Mi-2{beta} full was cloned in-flame into the SfiI/SmaI site of the pAS2-1 vector (Clontech), and fragments of Mi-2{beta} were cloned into the SfiI/SalI site of the pAS2-1 vector (24). pAS2-1-RFP constructs were as described previously (32). Fragments of MCRS1 were amplified by PCR and cloned into the pACT2 vector (Clontech). To characterize regions of Mi-2{beta} and RFP that mediated binding to MCRS1, pAS2-1-Mi-2{beta} or pAS2-1RFP constructs were co-transfected with the pACT2-MCRS1 vector into S. cerevisiae strain Y190 (Clontech). Positive interactions were determined by two criteria, expression of {beta}-galactosidase and growth on the triple drop-out medium without tryptophan, leucine, and histidine. Measurements were performed on at least three independent colonies.

Immunofluorescence and Confocal Microscopy—SW480 cells were grown on coverslips and washed twice with PBS (20 mM potassium phosphate, pH 7.4, 150 mM NaCl). To express FLAG-MCRS1, pCMV FLAG-MCRS1 vector was transfected into SW480 cells using Lipofectamine 2000 (Invitrogen). Cells were fixed with methanol (5 min, -20 °C) and acetone (30 s, -20 °C), washed 3 times with PBS, and incubated in nuclease digestion buffer (66 mM Tris-HCl, pH 7.4, 0.66 mM MgCl2, 1 mM 2-mercaptoethanol, 100 units/liter deoxyribonuclease I) for 30 min at 37 °C. After washing 3 times with PBS, cells were blocked with 10% goat serum in PBS and incubated with primary antibodies (1:100 dilution for anti-MCRS1 polyclonal antibody, 1:1000 for anti-RFP polyclonal antibody, 1:2500 for anti-Mi-2 antibody, 1:200 for anti-UBF antibody, and 20 µg/ml for anti-FLAG antibody), again washed 3 times in PBS, and then stained with Alexa Fluor 488-conjugated anti-rabbit IgG antibody and an Alexa Fluor 594-conjugated anti-mouse IgG antibody. The Alexa 594-conjugated anti-MCRS1 antibody was diluted to 1:100 and used. To enhance the signal of the anti-MCRS1 antibody, Can Get Signal solution (Toyobo) was used. Slides were mounted in PermaFluor (Shandon) and observed using a confocal microscope (Olympus).



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  		FIGURE 1.
MCRS1 interacts with Mi-2 and RFP. A, FLAG-MCRS1 expression in HEK 293 cells. HEK 293 cells were transfected with FLAG or FLAG-MCRS1 expression vector. Whole cell lysates were immunoprecipitated with an anti-FLAG antibody (Ab) and analyzed by Western blotting (IB). B, co-immunoprecipitation (IP) of Mi-2 and RFP with FLAG-MCRS1. Lysates from HEK 293 cells transfected with FLAG or FLAG-MCRS1 were immunoprecipitated with an anti-FLAG antibody and then immunoblotted with anti-Mi-2 or anti-RFP antibody. Input represents 2% of the lysate for immunoprecipitation. C, co-immunoprecipitation of Mi-2 with GFP-MCRS1. Lysate from HEK 293 cells transfected with GFP-MCRS1 was immunoprecipitated with an anti-Mi-2 antibody and then immunoblotted with anti-GFP or anti-MCRS1 antibody. D, HEK 293 cell nuclear extracts were analyzed by Western blotting using anti-MCRS1 rabbit polyclonal antibody. Nuclear extract from HEK 293 cells transfected with FLAG-MCRS1 was used to identify the position of MCRS1. E, association of Mi-2 and UBF with endogenous MCRS1. HEK 293 cell whole cell lysates were immunoprecipitated with anti-MCRS1 polyclonal antibody and then immunoblotted using anti-Mi-2 and anti-UBF antibodies.

 
Chromatin Immunoprecipitation (ChIP) Assay—After 48 h of incubation, HEK 293 cells plated in 100-mm dishes were fixed with 1% formaldehyde for 10 min at 37 °C and incubated with ice-cold glycine/PBS (125 mM glycine, 1 mM PMSF, and 1 µg/ml aprotinin in PBS) for 10 min at 4 °C. Cells were then washed twice with ice-cold PBS containing protease inhibitors (1 mM PMSF, 1 µg/ml aprotinin). After scraping and collecting cells by centrifugation at 500 x g for 3 min at 4 °C, cell pellets were resuspended in 300 µl of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1 mM PMSF, 1 µg/ml aprotinin) and incubated for 10 min on ice. Cell lysates were sonicated briefly 4 times on ice to shear the DNA and diluted with 2700 µl of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, 1 mM PMSF, 1 µg/ml aprotinin). Diluted cell suspensions were precleared with 80 µl of salmon sperm DNA/protein A-agarose (Upstate) for 1 h at 4 °C. After a brief centrifugation, supernatant fractions were immunoprecipitated overnight with specific antibodies or control normal rabbit immunoglobulin (Santa Cruz Biotechnology). After washing with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and LiCl buffer (0.25 M LiCl, 1% Nonidet P-40, 1% sodium deoxy-cholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and twice with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), pellets were eluted twice with 150 µl of elution buffer (1% SDS, 0.1 M NaHCO3). Cross-links were then reversed by adding 12 µl of 5 M NaCl and incubated overnight at 65 °C, after which 10% of the purified DNA was amplified by PCR (30 cycles of 30 s at 96 °C, 30 s at 55 °C for 28 S ribosomal DNA, 57 °C for rDNA non-coding region, 60 °C for rDNA promoter, or 65 °C for GAPDH, and 30 s at 72 °C).



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  		FIGURE 2.
Mapping of the MCRS1-interacting domains of Mi-2{beta} and RFP. A, schematic illustration of Mi-2{beta}, RFP, and MCRS1. Yeast two-hybrid assays were performed to reveal associations between the indicated regions of Mi-2{beta} and RFP cloned into pAS2-1 vectors and each region of MCRS1 cloned into pACT2 vector. a.a., amino acids; PHD domain, plant homeodomain; NoLS, nucleolar localization signal; NLS, nuclear localization signal; FHA domain, forkhead-associated domain. B, result of yeast two-hybrid assay. Associations were assessed by histidine expression and {beta}-galactosidase activity. The degree of association was graded as strong (2+), moderate (+), weak (+/-), or negative (-). Measurements were performed on at least three independent colonies.

 
PCR primer sets used were: rDNA promoter 1 (forward primer, 5'-GAGCGGTCCCTCTGCCGCGAT-3'; reverse primer, 5'-AGGCGCGACCTCTCGGGCC-3'), rDNA promoter 2 (forward primer, 5'-CCCTGCGTGTGGCACGGGC-3'; reverse primer, 5'-AGGAGCGCGGCCGGCTAGCC-3'), 28 S RNA (forward primer, 5'-GCGACCTCAGATCAGACGTG-3'; reverse primer, 5'-CTTAACGGTTTCACGCCCTC-3'), rDNA non-coding region 1 (forward primer, 5'-TGAACGTTTCTCTTGCCGTCTCCCG-3'; reverse primer, 5'-AGCAGCATCAACGCGGTTCCCCT-3'), rDNA non-coding region 2 (forward primer, 5'-GAGCCACACCTCATTCCGTGCCA-3'; reverse primer, 5'-CGTCTGTTCAGCTATTTTGCAGGAGTCG-3'), and GAPDH (forward primer, 5'-GAAGGTGAAGGTCGGAGTCAA-3'; reverse primer, 5'-GAGATGATGACCCTTTTGGCTC-3').

GAL4-fused Ribosomal DNA Promoter-targeted Transcription Assay—Mouse L cells were kindly provided by Dr. Kaibuchi at Nagoya University. HEK 293 cells and mouse L cells were cultured in 24-well tissue culture plates and co-transfected with 40 ng of GAL4-hrDNA promoter-IRES luciferase reporter plasmid, 40 ng of pRL-TK plasmid (Promega), and 320 ng of pcDNA3-GAL4BD Mi-2{beta}, RFP, or MCRS1 expression plasmids with or without NoLS using Lipofectamine PLUS (Invitrogen). Cells were harvested 48 h after transfection, and luciferase assays were performed as previously described (32). Co-transfection with the pRL-TK plasmid was used to normalize luciferase values. Values were expressed as the means ± S.D. of at least three independent experiments.

RNA-mediated Interference—MCRS1 and Mi-2{beta} siRNA and control siRNA (siControl non-targeting siRNA) were purchased from Dhamacon, and RFP siRNA was purchased from Qiagen. The siRNAs were transfected using X-tremeGENE siRNA transfection reagent (Roche Applied Science) according to the manufacturer's instructions.

Ribosomal RNA Expression Analysis—Total cellular RNA was isolated using the RNeasy Mini kit (Qiagen), and residual DNA was digested using RNase-free DNase (Qiagen). RNA was reverse-transcribed with MultiScribe reverse transcriptase (Applied Biosystems) for 30 min at 48 °C. The resultant cDNA was amplified by PCR for 12-40 cycles of 30 s at 96 °C, 30 s at 55 °C for 28 S ribosomal RNA and 47 S/45 S ribosomal RNA or 65 °C for GAPDH, and 30 s at 72 °C. PCR primer sets used for 47 S/45 S ribosomal RNA were forward primer, 5'-CGAAGAAGCGTCGCGGGTCT-3', and reverse primer, 5'-CCAAGTAGGAGAGGAGCGAGC-3', and primer sets for 28 S ribosomal RNA and GAPDH were the same as those used for the ChIP assay.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MCRS1 Interacts with Mi-2 and RFP—MCRS1 is a nucleolar protein that contains a bipartite nuclear localization motif, a nucleolar localization motif, a coiled-coil domain, and a forkhead-associated domain. MCRS1 is involved in the nucleolar sequestration of Daxx and was recently reported to have transforming activity (38, 39). In a survey of MCRS1-interacting proteins by immunoprecipitation of lysates from HEK 293 cells transfected with FLAG-MCRS1 (Fig. 1A), we repeatedly identified a 220-kDa protein that co-precipitated with FLAG-MCRS1. Because 220 kDa was consistent with the molecular mass of Mi-2 and mass spectrometric analysis of nucleolar proteins suggested the presence of Mi-2{beta} (CHD4) in the nucleolus (44), we investigated whether Mi-2 is associated with FLAG-MCRS1. As shown in Fig. 1B, we found that Mi-2 co-immunoprecipitated with FLAG-MCRS1. We then examined the possibility that RFP also interacts with MCRS1, as we had previously reported an association between Mi-2 and RFP (24). Immunoprecipitation experiments revealed an association between RFP and MCRS1 (Fig. 1B). To further confirm the association of MCRS1 and Mi-2, we next performed reciprocal immunoprecipitation by using anti-Mi-2 rabbit polyclonal antibody. GFP-MCRS1 was used to avoid the superimposition of the immunoglobulin band on that of MCRS1. Fig. 1C shows that GFP-MCRS1 co-immunoprecipitated with endogenous Mi-2.



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  		FIGURE 3.
Co-localization of Mi-2 and RFP with MCRS1 in the nucleolus. A, SW480 cells transfected with FLAG-MCRS1 expression plasmid were incubated with anti-RFP or anti-Mi-2 rabbit polyclonal antibodies and anti-FLAG mouse monoclonal antibody and then with Alexa 488-labeled anti-rabbit IgG and Alexa 594-labeled anti-mouse IgG secondary antibodies. The figure shows confocal microscopy images. The two images were merged digitally, and co-localization of the two proteins is indicated by the yellow color. B, co-localization of Mi-2 and RFP with endogenous MCRS1. SW480 cells incubated with anti-Mi-2 or anti-RFP rabbit polyclonal antibodies were incubated with Alexa 488-labeled anti-rabbit IgG secondary antibody and then stained with Alexa 594-labeled anti-MCRS1 polyclonal antibody. Confocal microscopy images are shown.

 
To characterize proteins that are associated with endogenous MCRS1, we raised an anti-MCRS1 polyclonal antibody and confirmed its specificity (Fig. 1D). As shown in Fig. 1E, Mi-2 was associated with endogenous MCRS1 in HEK 293 cells. Nucleolar transcription factor UBF also co-immunoprecipitated with endogenous MCRS1 (Fig. 1E). However, we could not investigate the association between RFP and endogenous MCRS1, as the RFP band coincided with the anti-MCRS1 rabbit polyclonal antibody band used for immunoprecipitation.

We next performed a yeast two-hybrid assay to map the binding domains. We performed PCR on human testis and placenta cDNA libraries to obtain full-length MCRS1/MSP58 cDNA (GenBankTM accession number NM_006337 [GenBank] ). We cloned the full-length MCRS1 cDNA into the pACT2 vector and constructed pAS2-1 vectors that carried different regions of Mi-2{beta} and RFP (Fig. 2A). Yeast strain Y190 was then transformed with the pAS2-1 and pACT2 constructs, and interactions between full-length MCRS1 and Mi-2{beta} or RFP fragments were assayed both by growth on selective medium lacking histidine, tryptophan, and leucine with 40 mM 3-amino-1,2,4-triazole and by {beta}-galactosidase activity.

As shown in Fig. 2B, full-length MCRS1 was specifically associated with the ATPase/helicase region of Mi-2{beta}. Deletion constructs of MCRS1 (Fig. 2A) mapped the central region (MCRS1-2) as the region that interacted both with the ATPase/helicase region and with the full length of Mi-2{beta}. The association between the full-length Mi-2{beta} and the full-length MCRS1 was relatively weak in this assay. We speculate that transcriptional repressive activity of the full-length Mi-2{beta} and steric constraint of the large GAL4 fusion protein may affect the expression of yeast histidine and {beta}-galactosidase genes.



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  		FIGURE 4.
Co-localization of endogenous MCRS1, Mi-2, and RFP with UBF. SW480 cells were incubated with anti-MCRS1, anti-Mi-2, or anti-RFP rabbit polyclonal antibodies followed by incubation with Alexa 488-labeled anti-rabbit IgG secondary antibodies. Nucleoli were then stained with anti-UBF mouse monoclonal antibody followed by Alexa 594-labeled anti-mouse IgG antibody. The two images were merged digitally.

 
Full-length MCRS1 and its central region (MCRS-2) also interacted with both full-length RFP and its coiled-coil region (Fig. 2B). Although the RFP coiled-coil region (RFP-2) interacted with both MCRS1-2 and the carboxyl-terminal region of MCRS1 (MCRS1-3), full-length RFP interacted with only MCRS1-2. Taken together, we concluded that MCRS1 interacted with Mi-2{beta} and RFP, with the central region of MCRS1 acting as the binding site for the ATPase/helicase region of Mi-2{beta} and the coiled-coil region of RFP.

MCRS1, Mi-2{beta}, and RFP Co-localize in the Nucleolus—Although MCRS1 localizes exclusively to the nucleolus (34), endogenous Mi-2 and RFP are distributed throughout the nucleoplasm in a fine granular pattern or as variously sized dot-like structures. To explore the possibility that some of these domain-like structures represented the nucleolar distribution of Mi-2 and RFP, we transfected human colorectal adenocarcinoma SW480 cells with the pFLAG-MCRS1 expression plasmid and checked for the co-localization of RFP and Mi-2 with MCRS1 in the nucleolus. In SW480 cells, Mi-2 and RFP appeared to co-localize with MCRS1 at the nucleolus, as revealed by the superimposition of confocal images (Fig. 3A). We also examined the localization of RFP, Mi-2, and MCRS1 in HeLa cells and obtained the same results (data not shown).

We then investigated the endogenous distribution patterns of MCRS1, Mi-2, and RFP and assessed their co-localization in the nucleolus. Immunofluorescence analysis of SW480 cells using anti-RFP or anti-Mi-2 polyclonal antibodies together with Alexa 594-conjugated-anti-MCRS1 antibody clearly showed the co-localization of these proteins in nucleoli (Fig. 3B). The distribution of RFP, Mi-2{beta}, and MCRS1 showed a relatively large fusing dot-like structure or a circular distribution of small fusing dot-like structure in the nucleoli of SW480 cells.

Because nucleolar protein UBF co-immunoprecipitated with MCRS1 using HEK 293 cell lysates (Fig. 1D), we further investigated whether MCRS1, Mi-2, and RFP also co-localized with UBF. Fig. 4 shows that MCRS1, RFP, and MCRS1 co-localized with UBF in the nucleoli of SW480 cells. It has been shown that UBF localizes to the dense fibrillar component and at the periphery of the fibrillar centers in the nucleoli, where active ribosomal DNA transcription occurs by associating with RNA polymerase I and by inducing the chromatin remodeling (1, 45, 46). Co-localization of MCRS1, Mi-2, and RFP with UBF in the nucleoli suggested that these proteins may be associated with the regulation of nucleolar activity and ribosomal gene transcription.

MCRS1, Mi-2, and RFP Are Associated with Human rDNA—To investigate the recruitment of MCRS1, Mi-2, and RFP to the ribosomal DNA regions, ChIP assays were performed. Ribosomal genes are aligned as tandem repeats at nucleolar organizer regions on chromosomes 13, 14, 15, 21, and 22 (2). Each ribosomal DNA repeating unit is about 43 kilobases in length, with about 13 kilobases of 47 S ribosomal RNA transcribed from each unit (Fig. 5A). We designed two sets of primers within the promoter region, one set of primers within the 28 S ribosomal RNA coding region and two sets of primers within the 3' non-coding region (Fig. 5A). As shown in Fig. 5B, Mi-2 and RFP were associated with both the promoter regions and the 28 S rRNA coding region. In addition, the proteins also bound to the 3' non-coding regions examined. These results suggested that Mi-2 and RFP bound to the entire rDNA region. The distribution profiles of Mi-2 and RFP matched the proposed distribution of UBF, an essential component of the RNA polymerase I initiation complex, which is distributed throughout the ribosomal gene repeats rather than restricted to the promoter region (47). However, MCRS1 was associated with both the promoter regions and the 28 S rRNA coding region but not with the 3' non-coding regions (Fig. 5C). This suggested that the co-operative role of MCRS1 with Mi-2 and RFP may be restricted to the modulation of rRNA transcription and that MCRS1 is not associated with Mi-2 and RFP in 3'-non-coding regions. However, because Mi-2 and RFP are associated with the entire ribosomal gene repeat, these proteins might also be involved in the modulation of chromatin structure, like UBF.

MCRS1, Mi-2{beta}, and RFP Activate rRNA Promoter Activity—The co-localization of MCRS1, Mi-2, and RFP with rRNA transcription factor UBF in the nucleolus together with the association of MCRS1, Mi-2, and RFP with ribosomal gene repeats strongly suggested that these proteins are involved in ribosomal gene transcription. To investigate this hypothesis, we cloned the human rRNA promoter region spanning from -483 to +377 bp and constructed the pGL3-GAL4-hrDNAP-IRES-Luc luciferase vector (Fig. 6A). To minimize a luciferase gene expression driven by RNA polymerase II, the Kozak sequence of the pGL3 vector was replaced by the IRES sequence as previously described (42, 48). It is known that the human rRNA promoter is not transcribed by the mouse pol I transcription machinery and vice versa (49). By taking advantage of the species specificity of pol I transcription, Ghoshal et al. (42) demonstrated the pol I-specific transcription of the luciferase reporter construct that contained the human rRNA promoter region (from -410 to +314) (42). We confirmed the pol I-specific transcription of our pGL3-GAL4-hrDNAP-IRES-Luc luciferase vector by comparing the luciferase activities in human HEK 293 cells and mouse L cells. Fig. 6B shows that the transcriptional activity of the pGL3-GAL4-hrDNAP-IRES-Luc luciferase vector is markedly lower in mouse L cells than in human HEK 293 cells, demonstrating the pol I-specific transcription of the pGL3-GAL4-hrDNAP-IRES-Luc vector.



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  		FIGURE 5.
Association of MCRS1, Mi-2, and RFP with human ribosomal DNA. A, schematic representation of the structure of a human rDNA-repeating unit region and the PCR primers used for the ChIP assay. B, association of Mi-2 and RFP with rDNA. HEK 293 cells were fixed with formaldehyde, and cross-linked chromatin was precleared and immunoprecipitated with anti-Mi-2 or anti-RFP rabbit polyclonal antibodies. Eluted DNA was decross-linked and amplified using specific primers. Input represents 1% of the sample for immunoprecipitation. C, association of MCRS1 with rDNA. HEK 293 cells were fixed and immunoprecipitated with anti-MCRS1 antibody. Eluted DNA was amplified as described above.

 
Transcriptional activity of the rRNA promoter was assessed by the transient transfection of GAL4-DBD-fused MCRS1, Mi-2{beta}, or RFP constructs with pGL3-GAL4-hrDNAP-IRES-Luc luciferase plasmid into HEK 293 cells. As shown in Fig. 6C, Mi-2{beta} strongly transactivated rRNA promoter activity by ~400%, whereas RFP and MCRS1 transactivated rRNA promoter activity by ~200%. We have previously shown that Mi-2{beta} and RFP repressed the RNA polymerase II-driven luciferase activity by about 70% in the nucleus (24, 32). Thus, our results indicated that Mi-2{beta} and RFP have distinct transcriptional activities in the nucleus and the nucleolus.

To further assess the transcriptional activity of each region of MCRS1, Mi-2{beta}, and RFP, we constructed a series of GAL4-DBD-NoLS-fused deletion constructs (Fig. 6D). The NoLS was used to ensure nucleolar localizations of the deletion constructs. As shown in Fig. 6E, both the central region of MCRS1 (i.e. the Mi-2{beta} and RFP binding site) and the carboxyl-terminal region that contains the forkhead-associated domain transactivated the rRNA promoter. In contrast, the amino-terminal region of MCRS1 showed no such transactivating activity. We recently reported that the amino-terminal region of Mi-2{beta} had strong transactivating activity, whereas the carboxyl-terminal region of Mi-2{beta} had repressing activity in the nucleus (24). Surprisingly, assessment of transcriptional activities of Mi-2{beta} deletion constructs revealed that the repressive activity of the carboxyl-terminal region was completely lost, such that all three regions examined transactivated the rRNA promoter (Fig. 6F). We speculate that the difference of associating proteins between nucleolar pol I transcription machinery and nuclear pol II transcription machinery may alter the transcriptional activity of Mi-2{beta} and its carboxyl-terminal region.

We next investigated the transcriptional activities of RFP deletion constructs. Fig. 6G demonstrates that the coiled-coil region of RFP (i.e. the Mi-2{beta} and MCRS1 binding site) transactivated the rRNA promoter, whereas the RFP domain region had weak transactivating activity. In contrast, the RING finger B-box region showed no transactivation function. These results suggested that the repressing functions of RFP and Mi-2{beta} subdomains were not observed in the nucleolus and that MCRS1, Mi-2{beta}, and RFP are involved in the transactivation of ribosomal gene transcription. siRNA-mediated Down-regulation of MCRS1, Mi-2{beta}, and RFP Inhibited rRNA Production—To determine the effect of MCRS1, Mi-2{beta}, and RFP on rRNA production in vivo, we treated HeLa cells with siRNA against the respective genes. Treatment of HeLa cells with MCRS1, Mi-2{beta}, and RFP siRNA resulted in significant reductions of the respective mRNAs and proteins compared with treatment with the control siRNA (Fig. 7A). We further confirmed that siRNAs used specifically repressed their target molecule and did not affect the expressions of other molecules such as MCRS1, Mi-2{beta}, RFP, and UBF (Fig. 7A).



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  		FIGURE 6.
Transactivating activities of MCRS1, Mi-2{beta}, and RFP on the rDNA promoter region. A, schematic representation of the pcDNA-V5-GAL4BD-MCRS1/Mi-2{beta}/RFP construct and pGL3-GAL4-hrDNAP-IRES-Luc reporter plasmid. A segment of the rDNA gene with promoter activity (-483 to +377 bp with respect to the transcriptional start site) was amplified and cloned into the modified pGL3 luciferase vector. IRES, internal ribosomal entry site of the encephalomyocarditis virus. B, pol I-dependent transcription of pGL3-GAL4-hrDNAP-IRES-Luc reporter. pGL3-GAL4-hrD-NAP-IRES-Luc reporter plasmid (RLU1: firefly) and the internal control plasmid pRL-TK (RLU2: Renilla) were co-transfected into HEK 293 cells or L cells. After 36 h, both firefly and Renilla luciferase activity were measured. The table represents the RLU1 and RLU2 luciferase activities in HEK 293 cells and L cells. A lower panel shows the graphical representation of the relative ratio of RLU1 to RLU2 activity in HEK 293 cells and L cells. Each value represents a result of three independent experiments. Error bars indicate S.D. of each value. RLU, relative luminescence units. C, MCRS1, Mi-2{beta}, and RFP transactivate the rDNA promoter. pGL3-GAL4-hrDNAP-IRES-Luc plasmid was co-transfected with pcDNA3-V5-GAL4-DBD effecter constructs that express GAL4-DBD fused with MCRS1, Mi-2{beta}, or RFP. Luciferase activity in cells transfected with control plasmid expressing GAL4-DBD alone was set at 100%, and luciferase activities in cells transfected with the indicated plasmids were expressed as average percentages of the control value. Each value represents a result of three independent experiments. Error bars indicate the S.D. of each value. D, schematic representations of MCRS1, Mi-2{beta}, and RFP regions analyzed by transcriptional assays. E-G, transcriptional activity of each region of MCRS1 (E), Mi-2{beta} (F), and RFP (G)on the rDNA promoter region. The indicated regions of MCRS1, Mi-2{beta}, and RFP were cloned into the pcDNA3-V5-GAL4-DBD effecter constructs. The luciferase activity of each construct was assayed as described above. NoLS, nucleolar localization signal; NLS, nuclear localization signal; BD, binding domain.

 




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  		FIGURE 7.
Effects of siRNA-mediated down-regulation of MCRS1, Mi-2{beta}, and RFP on rDNA transcription in vivo. A, down-regulation of MCRS1, Mi-2{beta}, and RFP by specific siRNA. HeLa cells were transfected with specific siRNA, and expression of MCRS1, Mi-2{beta}, RFP, and UBF was assayed by both reverse transcription (RT)-PCR and Western blotting. si, small interfering. B, schematic representation of pre ribosomal RNA processing pathway. 47 S rRNA is processed to generate 18 S, 5.8 S, and 28 S. The primer sets used for reverse transcription-PCR analysis are indicated. ETS, external transcribed spacer; ITS, internal transcribed spacer. C-D, HeLa cells were transfected with control siRNA or specific siRNA for MCRS1, Mi-2{beta}, and RFP. At 48 h after transfection the expressions of 28 S ribosomal RNA region that reflects subtotal rRNA (C) and 47 S/45 S ribosomal RNA (D) were analyzed by reverse transcription-PCR. GAPDH was amplified as a control. Samples were analyzed at indicated cycle numbers as shown on the right side of the panel. The numbers below the panel correspond to the intensity of each product as measured by WinROOF software. E, relative intensity of ribosomal RNA bands of cells treated with specific siRNAs. Amounts of 47 S/45 S ribosomal RNA at 37 cycles and 28 S ribosomal RNA region at 15 cycles were assayed as described above and normalized according to the intensity of the GAPDH amplified product. Results are expressed as average percentages of the value obtained from control siRNA-treated cells. Each value represents a result of three independent experiments. Error bars indicate S.D.

 
Finally we assessed the amounts of rRNA in HeLa cells treated with the specific siRNAs. Human rRNA is transcribed as a primary 47 S precursor and is subsequently processed to generate mature 28 S, 18 S, and 5.8 S through the activities of different RNA-processing enzymes (Fig. 7B). PCR primer sets specific for 5'ETS-18 S rRNA region and 28 S rRNA region were designed to measure the amounts of 47 S/45 S rRNA and subtotal rRNA, except for 18 S and 5.8 S, respectively (Fig. 7B). Reverse transcription-PCR experiments demonstrated that 28 S rRNA region production was inhibited by 24-43% when HeLa cells were treated with MCRS1, Mi-2{beta}, or RFP siRNAs (Fig. 7, C and E). The earlier processed 47 S/45 S rRNA production was more significantly reduced by 52-59% (Fig. 7, D and E). These results agreed with the transactivating activities of MCRS1, Mi-2{beta}, and RFP for the rRNA promoter as shown in Fig. 6 and indicated that MCRS1, Mi-2{beta}, and RFP are involved in the up-regulation of ribosomal gene transcription in vivo.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Transcriptional Function and Expression Profile of MCRS1 in the Nucleolus—MCRS1 is a nucleolar protein that is highly expressed in microspherules within the nucleolus. The numbers of microspherules and the size and density of the dense fibrillar centers in the nucleolus are associated with increased nucleolar activity and the production of pre-ribosomal RNA and pre-rRNP particles. Although the finding that UBF, RNA polymerase I, fibrillarin, and p130 along with MCRS1 localized to microspherules suggested an association between MCRS1 and ribosomal gene transcription or ribosomal subunit maturation (34, 50-53), the transcriptional roles of MCRS1 have not yet been elucidated.

In this report we examined the role of MCRS1/MSP58 in the transactivation of the ribosomal genes. MCRS1 was associated and co-localized with RFP and chromatin remodeling protein Mi-2{beta} in the nucleolus. MCRS1, Mi-2{beta}, and RFP up-regulated ribosomal gene promoter activity and co-localized with UBF, an essential component of the RNA polymerase I initiation complex and a transactivator of RNA polymerase I-mediated transcription. In addition, down-regulation of MCRS1, Mi-2{beta}, and RFP by siRNA treatment inhibited the rRNA production. These findings support the view that MCRS1, Mi-2{beta}, and RFP are involved in RNA polymerase I-mediated transcriptional activation.

Transactivating Role of Mi-2{beta} and RFP in the Nucleolus—Mi-2{beta} is a major component of the well characterized NuRD complex. Mi-2{beta} forms a complex with histone deacetylase HDAC1, methyl CpG-binding protein MBD2/3, metastasis-associated protein MTA 1/2/3, and histone-binding protein RbAp 48/46 and is involved in transcriptional repression in the nucleus. In a previous report we found that RFP acted as a transcriptional co-repressor that was associated with Polycomb-group protein, Enhancer of Polycomb 1, and Mi-2{beta} in the nucleus (24, 32).

We first speculated that MCRS1, Mi-2{beta}, and RFP formed a repressive complex in the nucleolus, as proposed in the nucleus. To our surprise, a transcriptional assay using a ribosomal gene promoter revealed that Mi-2{beta} and RFP exhibited transactivating activities in the nucleolus. In addition, the strong repressive activities of both the Mi-2{beta} carboxyl-terminal region and the RFP coiled-coil region were lost, with significant transactivation of the ribosomal gene promoter observed. Ribosomal RNA is transcribed via RNA polymerase I machinery, and messenger RNA is transcribed via RNA polymerase II machinery. Likewise, the processing of transcribed RNA is also different between the nucleus and the nucleolus. Our findings indicated that Mi-2{beta} and RFP had distinct transcriptional activities between the nucleus and the nucleolus and suggested that differences in associating proteins may explain these distinct transcriptional activities.

Role of Mi-2{beta} and RFP in Chromatin Structure Modulation within Ribosomal Gene Repeats—ChIP assays revealed that Mi-2{beta} and RFP were associated with the entire rDNA region, including the promoter region, the 47 S rRNA coding region, and the 3'-non-coding region. Human cells contain about 400 ribosomal genes in tandem repeats at nucleolus organizer regions encoded on five pairs of chromosomes 13, 14, 15, 21, and 22. Previous studies have indicated that only 30-50% of rRNA genes display an open accessible chromatin structure (3, 4).

Recent evidence has proposed that histone modification and chromatin remodeling proteins are highly associated with the formation of accessible or silent chromatin structures within the nucleolus organizer regions in the nucleolus in the same way as in the nucleus. Demethylation of histone H3 lysine 9 is associated with ribosomal gene silencing, whereas acetylation of H3 lysine 9 and additional histone modification are involved in transactivation (54). The chromatin remodeling complex NoRC, containing TIP5 and SNF2h, represses RNA polymerase I transcription by recruiting histone deacetylase complex containing HDAC1, Sin3A, TTF-I (transcription terminator factor), and RbAp46 (11, 13).

Although UBF was originally characterized as a transactivator of RNA polymerase I by associating with the TATA box-binding protein-containing complex SL1, recent studies have revealed that UBF is distributed throughout ribosomal DNA and activates ribosomal gene transcription through the induction of the chromatin decondensation (47, 55). The distribution of Mi-2{beta} and RFP throughout the entire ribosomal gene region and their co-localization with UBF within the nucleolus suggest that Mi-2{beta} and RFP may be involved both in the transactivation of ribosomal gene transcription and in the formation of active chromatin structures that induce chromatin decondensation in the nucleolus.

Activation of Ribosomal Gene Transcription—The transcription of ribosomal gene repeats is thought to be controlled by the coordination of activating and repressing chromatin remodeling proteins. Although the chromatin remodeling complex NoRC is a well recognized repressive complex (12-14), the chromatin remodeling protein that is involved in transactivation remains largely unknown. Although the SWI/SNF chromatin remodeling complex has been characterized as a transactivator in the nucleus, their components have not been associated with actively transcribed regions in the nucleolus (55).

Based on the results of our study, it is possible that Mi-2{beta} acts as a chromatin remodeling protein involved in ribosomal gene transactivation. Although Mi-2{beta} is a major component of the NuRD complex involved in transcriptional repression in the nucleus, we have previously shown that Mi-2{beta} contains both amino-terminal transactivating and carboxyl-terminal repressive regions (24). In addition, a recent report has revealed that Mi-2{beta} played a direct transactivating role in CD4 expression in T cells by associating with the E box-binding protein HEB and histone acetyltransferase p300 (27). These findings suggest that another complex other than NuRD might be involved in the formation of active transcriptional loci in ribosomal gene repeats. UBF and histone acetyltransferase Tip60 are reported to reside at sites of active ribosomal DNA transcription (16, 45), and our immunofluorescence experiments showed the co-localization of MCRS1, Mi-2{beta}, RFP, and UBF in the nucleolus. Thus, it is possible that Mi-2{beta} can facilitate the formation of an active chromatin structure in the nucleolus by remodeling chromatin and associating with histone acetyltransferases such as Tip60 or p300.

Our study investigated the transcriptional function of nucleolar protein MCRS1/MSP58 and its interaction with Mi-2{beta} and RFP. We identified the Mi-2{beta} as a candidate molecule for the chromatin remodeling protein that is involved in the formation of open chromatin structures that facilitate active rRNA gene transcription. Our results contribute to the understanding of active chromatin structure formation at ribosomal gene repeats in the nucleolus.


   FOOTNOTES
 
* This work was supported by grants-in-aid for the 21st Century Center of Excellence Research, Scientific Research on Priority Areas "Cancer" and Scientific Research (A) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Tel.: 81-52-744-2093; Fax: 81-52-744-2098; E-mail: mtakaha{at}med.nagoya-u.ac.jp.

3 The abbreviations used are: NuRD complex, nucleosome remodeling and deacetylase complex; MCRS1, microspherule protein1; MSP58, 58 kDa microspherule protein; RFP, RET finger protein; UBF, upstream binding factor; NoLS, nucleolar localization signal; IRES, internal ribosome entry site; GAL4BD, GAL4 DNA binding domain; ChIP, chromatin immunoprecipitation; HEK cells, human embryonic kidney cells; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; GFP, green fluorescent protein; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; rDNA, ribosomal DNA; pol I, polymerase I. Back


   ACKNOWLEDGMENTS
 
We thank K. Imaizumi, K. Uchiyama, and S. Kawai for excellent technical assistance.



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