Regulation of Osteocalcin Gene Expression by a
Novel Ku Antigen Transcription Factor Complex*
David M.
Willis
§,
Arleen P.
Loewy,
Nichole
Charlton-Kachigian,
Jian-Su
Shao,
David M.
Ornitz§, and
Dwight A.
Towler§¶
From the Departments of Medicine and
§ Molecular Biology and Pharmacology, Washington
University School of Medicine, St. Louis, Missouri 63110
Received for publication, June 30, 2002
 |
ABSTRACT |
We previously described an
osteocalcin (OC) fibroblast growth factor (FGF)
response element (FRE) DNA binding activity as a target of Msx2
transcriptional regulation. We now identify Ku70, Ku80, and Tbdn100, a
variant of Tubedown-1, as constituents of the purified OCFRE-binding
complex. Northern and Western blot analyses demonstrate expression of
Ku and Tbdn100 in MC3T3E1 osteoblasts. FGF2 treatment
regulates Ku, but not Tbdn100, protein accumulation. Gel supershift
studies confirm sequence-specific DNA binding of Ku in the OCFRE
complex; chromatin immunoprecipitation assays confirm association of Ku
and Tbdn100 with the endogenous OC promoter. In the
promoter region 
154 to 113, the OCFRE is juxtaposed to OSE2, an
osteoblast-specific element that binds Runx2 (Osf2, Cbfa1). Expression of the Ku·Tbdn100 complex up-regulates both the
basal and Runx2-dependent transcription driven by this
42-bp OC promoter element, reconstituted in CV-1 cells.
Synergistic transactivation occurs in the presence of activated FGF
receptor 2 signaling. Msx2 suppresses Ku- and
Runx2-dependent transcription; suppression is dependent
upon the Msx2 homeodomain NH2-terminal arm and extension. Pull-down assays confirm physical interactions between Ku and these
co-regulatory transcription factors, consistent with the functional
interactions identified. Finally, cultured Ku70 / calvarial cells
exhibit a profound, selective deficiency in OC expression
as compared with wild-type calvarial cells, confirming the biochemical
data showing a role for Ku in OC transcription. In
toto, these data indicate that a novel Ku antigen complex
assembles on the OC promoter, functioning in concert with
Msx2 and Runx2 to regulate OC gene expression.
| |
INTRODUCTION |
Expression of the osteoblast phenotype is regulated by
morphogenetic, metabolic, endocrine, inflammatory, and mechanical
signals that convey cognate physiological demands upon the skeleton
(1). The osteocalcin
(OC)1 promoter has
emerged as a powerful tool for identifying and characterizing nuclear
DNA-protein interactions mediating transcriptional responses to many of
these signals in the osteoblast (2, 3). To date, much of what we know
of the osteoblast transcriptional responses to the vitamin D receptor,
Runx2 (also known as Osf2, PEBP2A
, and Cbfa1), Msx2, Dlx5,
the glucocorticoid receptor, and AP1 family members derive from studies
of OC promoters cloned from human, rat, and mouse (OG2)
genomes (4-13). Except for the absence of the vitamin D
receptor-regulated enhancer in the mouse OG2 gene (14), key
features of OC promoter regulation are remarkably well
conserved across mammalian species (3).
The osteoblast homeoprotein Msx2 plays a crucial role in the regulation
of osteoblast proliferation and differentiation (15). Msx2
and Msx1 are absolutely required for craniofacial bone
formation (15). Msx2 controls the timing of osteoblast differentiation, including the temporospatial patterns of OC expression in the calvarium
and teeth (12, 16). Msx2 plays a global role in determination of
skeletal mass, elegantly demonstrated in murine genetic models;
moreover, as highlighted by Maas and colleagues (15), osteogenic
effects during development are dependent upon Msx
gene dosage, suggesting that stoichiometry is an important feature of
Msx2 action. Consistent with this, we (11, 12, 16-18) and others (2,
19) have shown that Msx2 and Msx1 participate in specific
protein-protein interactions that control gene transcription. In the
rat OC gene, transcriptional regulation by Msx2 converges on
a 42-bp region at nucleotides 154 to 113 relative to the transcription initiation site, encompassing an FGF responsive element
at nucleotides 142 to 136 (17). The OCFRE (OC FGF response element) is a GCAGTCA motif that confers both basal and FGF2-regulated expression of the OC gene in MC3T3E1
calvarial osteoblasts (17, 20, 21). A DNA binding activity up-regulated by FGF2 in MC3T3E1 cells recognizes this element and is constitutively expressed by MG63 osteosarcoma cells (17, 20). Msx2 exerts suppressive
actions on the OC promoter in part by inhibiting the OCFRE
DNA binding activity present in either MC3T3E1 cells or purified from
MG63 cells (17). By contrast, Msx2 does not alter vitamin D
receptor-dependent transcription or vitamin D receptor binding to its DNA cognate (17). Inhibition of OCFRE activity is
dependent upon key regulatory domains encoded in the Msx2 homeodomain NH2-terminal arm and extension (17). Interestingly, Stein
and co-workers (22, 23) identified that this same OC
promoter region mediates transcriptional repression in response to
transforming growth factor-
, and thus named this same element
the transforming growth factor- response element. They identified
that an AP1 complex mediates transforming growth factor- repression
in ROS17/2.8 cells. We have confirmed these
observations,2 however,
applying immunological and competition studies, we determined that the
Msx2-regulated DNA binding activity was not an AP1 family member (20)
(see below). Thus, the OCFRE mediates both positive and negative
regulation of OC gene transcription in osteoblasts.
To fully characterize the DNA binding activities that recognize and
regulate the OCFRE, we have purified the major homeoprotein-regulated OCFRE complex about 25,000-fold from MG63 human osteosarcoma cells, using DNA binding in gel shift as an assay. Ku70 and Ku80 (24-26) were
identified as the Msx2-regulated DNA binding constituents (17) in the
purified OCFRE binding complex by nanobore LC-MS/MS "sequencing" of
tryptic peptides (27). Additionally, we identify a novel 100-kDa
variant of Tubedown-1, hence, named Tbdn100, as a constituent of this
complex purified from this osteoblastic cell line. Tubedown is an
N-acetyltransferase initially identified in immature
endothelial cells (28); however, Northern blot analyses verify the
expression of Tbdn in MC3T3E1 calvarial osteoblasts, a useful and
relevant cell background for osteoblast gene expression studies, and in
many other tissues. Western blot analyses with antibodies raised to the
COOH-terminal domain of Tbdn100 confirm the accumulation of the 100-kDa
Tbdn100 variant in nuclear fractions prepared from MC3T3E1 osteoblasts.
Moreover, chromatin immunoprecipitation (ChIP) assays confirm the
association of Ku and Tbdn100 with endogenous OC promoter,
but not with the BSP promoter. Immunological supershift and
competition studies confirm sequence-specific association of Ku in the
nuclear complex assembled by the OCFRE. In transient transfection
assays, co-expression of the novel Ku complex constituents (Ku70, Ku80,
and Tbdn100) up-regulates basal OC promoter activity and
OCFRE-dependent transcription; by contrast, the
RSV promoter is not regulated. In the 42-bp promoter region
154 to 113, the OCFRE (20) is juxtaposed to OSE2 (29), an element
that binds the osteoblast transactivator Runx2 (also known as
Osf2 and Cbfa1) (3, 9). Runx2 and Ku:Tbdn100 synergistically
activate transcription driven by the OC promoter region
154 to 113 when placed upstream of the unresponsive RSV
promoter. Synergistic activation of OC-(154/113)-RSVLUC is markedly
augmented by FGFR2-ROS, a constitutively active FGF receptor (30, 31).
Importantly, Msx2 suppresses Ku- and Runx2-dependent transcription, consistent with our prior studies demonstrating that
OCFRE binding is inhibited by Msx2 (17). Pull-down assays confirm
physical interactions between Ku and Msx2, and Ku and Runx2. Finally,
we show that calvarial cells lacking Ku70 exhibit profound, selective
deficiencies in OC, but not OPN, gene expression. In toto, these data indicate that a novel Ku antigen complex
assembles on the OC promoter, functioning in concert with
Msx2 and Runx2 to regulate OC gene expression. Tbdn100 emerges as a
nuclear transcriptional regulator that associates with the
OC gene and promotes transactivation responses.
| |
EXPERIMENTAL PROCEDURES |
Reagents--
Tissue culture supplies and custom synthetic
oligodeoxynucleotides were obtained from Invitrogen. Reagents for
protein purification were obtained from Pierce, Sigma, and Fisher.
Radionuclides were obtained from Amersham Biosciences.
Purification of OCFRE DNA Binding Activity from MG63
Cells--
MG63 cells (ATCC number CRL-1427, Manassas, VA) were grown
at the Washington University Tissue Culture and Media Center in -modified Eagle's medium, supplemented with 5% fetal calf
serum, 5% newborn calf serum, 2 mM glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin. Pilot purification revealed
that more that half of the OCFRE-binding complex was present in the
insoluble nuclear matrix, released by timed digestion with DNase I or
fracture of nuclear matrix by vortexing with glass beads. Therefore,
cell extracts were made using a modification of a glass bead lysis protocol previously described (32), with all steps carried out at
4 °C. Briefly, the washed cell pellet (from 2 × 1010 MG63 cells grown in roller bottles) was resuspended in
5 volumes of extraction buffer (25 mM Hepes, pH 7.9, 0.5%
Triton X-100, 0.45 M NaCl, 25% (v/v) glycerin, 1 mM dithiothreitol, 0.5 mM EDTA, 0.1 mM vanadate, 0.1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 6 µg/ml soybean trypsin
inhibitor, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A). After
addition of prewashed glass beads (400-600 µm; Sigma), the
cell extract was prepared by homogenization using the BioSpec Bead
Beater (Bartlesville, OK). After centrifugation at 7500 rpm for 15 min
to clear the extract, ammonium sulfate fractionation and protein
purification chromatography were carried out using standard techniques
(32) as summarized in the legend to Fig. 1. Electrophoretic mobility
shift assay was used detect binding to the radiolabeled OCFRE as
previously detailed (20); the upper strand sequence of the
radiolabeled duplex oligo used to follow OCFRE binding activity is
5'-GGCAGCTGCAGTCACCGGCCC-3' (the OC promoter sequence
encompassing the OCFRE is underlined). Total protein recovery was
determined by the Pierce bicinchoninic acid assay after protein
precipitation and resolubilization as described (33). High performance
liquid chromatography (HPLC) was carried out using a Waters (Milford,
MA) AGC gradient controller with two model 515 pumps, fitted with a
preparative scale 10-ml Rheodyne injector (Rohnert Park, CA) and low
protein retention polyetheretherketone (PEEK) tubing. A Waters model
468 tunable ultraviolet detector set at 280 nm was used to follow
protein elution. A 5-ml Amersham Bioscience Mono Q FPLC column (20 mM Tris buffered, pH 8.0) was used for anion exchange, and
an Amersham Bioscience disposable 1-ml HiTrap heparin-Sepharose
affinity column (20 mM Hepes buffered, pH 7.9) were used
for final purification steps. For both HPLC applications, 1 ml/min flow
rates with a 14 mM/min NaCl gradient were used to elute
OCFRE binding activity; 0.1 mM EDTA and 1 mM
2-mercaptoethanol were included in all HPLC buffers. Proteins were
resolved by SDS-PAGE using an 11% 0.75-mm thick polyacrylamide gel
poured on methanol-cleaned virgin glass plates (Mini-Protean II;
Bio-Rad) and pre-run to clear oxyradicals. After electrophoresis,
fractionated proteins were visualized by staining in Coomassie
Brilliant Blue, briefly destained, and excised with a scalpel, reduced
and alkylated with dithiothreitol and iodoacetamide, respectively,
rinsed with 50% acetonitrile in deionized water with 0.1% acetic acid
to remove SDS, and speed evaporated dry. After pre-swelling gel
fragments in 50 mM NaHCO3, proteins were
digested with 1:20 (mass/mass) of acetylated trypsin (Promega), and
tryptic peptides were analyzed by nanobore LC-MS/MS at the Harvard
Microchemistry Facility (B. Lane, director) using a Finnigan ion trap
mass spectrometer (34). SEQUEST analysis (27) of the NCBI cDNA data
base identified Ku70 (24 unique peptides) and Ku80 (17 unique
peptides). Analysis of the translated EST data base in all 6 reading
frames was required to identify peptides from the unknown 100-kDa
subunit (8 unique peptides initially identified; 24 identified in final
analysis, Fig. 3).
Cloning of Tbdn100--
Because there were no matches for our
uLC-MS/MS tryptic peptide data in the nonredundant cDNA data base
for the 100-kDa protein, we searched the translated NCBI EST data base
with BLAST (35). We identified several ESTs which assembled into a
900-base pair contig containing a putative stop codon and providing a
contiguous open reading frame encoding 8 of the unique tryptic peptides
we identified from mass spectroscopy analysis of the purified 100-kDa subunit. EST analyses revealed expression in the heart (and
subsequently confirmed by Northern blot); therefore, the 900-bp
fragment was amplified from commercially available human heart cDNA
(Clontech Marathon Ready cDNA;
Clontech) and sequenced to verify correct assembly.
By applying successive rounds of 5'-rapid amplification of cDNA
ends from this Marathon Ready cDNA and iterative EST data base
searches, we assembled the complete sequences of both the mouse and
human cDNAs (Fig. 3). After obtaining the complete sequence, reanalysis of our raw LC-MS/MS data identified a total of 24 peptides, including one at our predicted translation start site without Met (Fig.
3). During our studies, a variant of this 100-kDa protein was cloned by
Gendron and colleagues (28) and named Tubedown-1. Because our protein
is 100 kDa in mass because of an additional 272 amino acids at the
amino terminus, we named our variant Tubedown-100 or Tbdn100 (Fig. 3;
GenBankTM number AY112670). Sequence analysis was performed
using ProfileScan software accessible from the ISREC Bioinformatics
home page (Swiss Institute for Experimental Cancer Research;
www.isrec.isb-sib.ch/index.html), Pfam Hidden Markov Models software
(36) accessible from the Washington University Genetics website
(pfam.wustl.edu/hmmsearch.shtml), and DNAMAN version 4.0 software for
Windows purchased from Lynnon BioSoft (Vandreuil, Quebec, Canada).
Electrophoretic Mobility Gel Shift Assays--
MC3T3-E1 mouse
calvarial osteoblasts were grown as described previously detailed (20)
using cells between passages 6 and 16 from our frozen stocks. Crude
nuclear extracts were prepared from control or FGF2- (3 nM)
and forskolin (1 µM)-treated MC3T3E1 cells as indicated
as previously detailed (Newberry et al. (21)); however, 1 nM okadaic acid and 10 nM sodium
orthovanadate were added to inhibit phosphoprotein phosphatases during
extraction. The radiolabeled OCFRE duplex oligo (see above) was used to
detect DNA-protein interactions. Gel supershifts and competition
experiments were carried out as previously detailed (20, 31).
Antibodies, Western Blot Analyses, and ChIP Assays--
The
Ku70:Ku80 heterodimer-specific monoclonal antibody clone 162 (37) was
obtained from Neo Markers (Fremont, CA). Anti-Tbdn100 antibodies were
commercially prepared (Zymed Laboratories Inc.) using
a conjugated synthetic peptide corresponding to the COOH-terminal 14 amino acids of Tbdn100 as the immunogen; monospecific polyclonal antibodies were prepared from immune serum using purified, recombinant Tbdn100 (expressed in pET23d; Novagen) conjugated to cyanogen bromide-activated Sepharose; these techniques have been previously detailed (11). Western blot analyses were carried out on crude or
fractionated cellular extracts essentially as previously detailed (11),
using commercially available polyclonal anti-Ku70 (Santa Cruz, San
Diego, CA, sc-1487/M19 and sc-1486/C19), anti-Ku80 (Santa Cruz number
sc-1485), anti-tubulin, or anti-Tbdn100 antibodies as indicated.
Proteins were resolved by SDS-PAGE (4% stacking gel, 11% resolving
gel), transferred to polyvinylidene difluoride membrane, and blocked
with 0.2% I-Block (Tropix, Bedford, MA) in Tris-buffered saline. The
membranes were then probed with polyclonal antibodies at 1:250 to
1:1000 dilutions. The bands were visualized using chemiluminescent
detection as previously described (18) using alkaline
phosphatase-conjugated rabbit anti-goat or goat anti-rabbit secondary
antibodies as appropriate. ChIP assays (see Ref. 38, for method review)
were carried out with differentiating MC3T3E1 calvarial osteoblasts
essentially as previously described by Hinds and colleagues (39) with
the following minor modification. Platinum Taq Hi Fi
polymerase (Invitrogen; 1 unit) with 30 ng of DNA template and 0.2 µM amplimers were used in a 50-µl reaction per the
manufacturer's instructions to carry out triphasic PCR for 35 cycles
of 94 °C × 30 s, 60 °C × 30 s,
68 °C × 30 s, preceded by heat activation (94 °C × 4 min) of the latent polymerase. PCR primers used for OC ChIP were
5'-TATGAGAGTTGGAGCCCAGTTTATC-3' and 5'-CCCTAGAGACAAGAACCCTATGCAT-3'
directed to the mouse OC (OG2) promoter region
384 to 298 (GenBankTM accession number U66848).
OC ChIP assays were quantified by fluorescence PCR with an
Applied Biosystems GeneAmp 5700 sequence detection system (see below).
Data were expressed as the mean ± S.D. enrichment by
immunoprecipitation from quadruplicate evaluations. Twenty nanograms of
processed DNA was used in each PCR reaction, and signal arising from 1 pg of genomic DNA was used as a reference. PCR primers used for
BSP ChIP were 5'-GGCATCAACTCATTTCCTAATTCAG-3' and
5'-CTTTCACACAGCACTGCATGGGTC-3' directed to the mouse BSP
promoter region 399 to 343 (GenBankTM accession number
AF071079).
Eukayrotic Expression Constructs, OC Promoter- Reporter
Constructs, and Transient Transfection Assays--
Plasmids containing
the human Ku70 and Ku80 cDNAs were the kind gift of Dr. Westley
Reeves (40); Ku70 and Ku80 cDNAs were subcloned into pcDNA3 for
expression and sequenced to verify fidelity. pCMV-Osf2 encoding
the Osf2 isoform of Runx2 was the generous gift of Dr. Gerard
Karsenty (Baylor College of Medicine); Runx2 was further subcloned into
pcDNA3. The synthesis and characterization of 0.2-kb OCLUC (OC
promoter 222 to +32, luciferase reporter) and RSVLUC (RSV minimal
promoter, luciferase reporter) in pGL2 Basic (Promega) has been
described in detail (20). OC-(154/113)-RSVLUC was synthesized by
inserting 7 copies of the duplex oligodeoxynucleotides encompassing the
OC promoter region 154 to 113
(5'-CCCGGCAGCTGCAGTCACCAACCACAGCATCCTTTGGGTTTG-3') upstream of RSVLUC; this FGF responsive, 42-bp OC promoter
element encompasses the juxtaposed OCFRE (GCAGTCA) and OSE2 (ACCACA)
cognates (20, 21) as underlined. All constructs were sequenced to
verify insert identity. As indicated, MC3T3E1 osteoblasts or CV1
fibroblastic cells were plated in either 6- or 12-well clusters and
transfected using LipofectAMINE Plus (Invitrogen). After 3 days,
cellular luciferase was measured as previously described (18) using a Berthold AutoLumat 953 luminometer (EG&G Instruments, Oak Ridge, TN).
Data are presented as the mean (± S.D.) activity observed in three
independent replicates. All data sets were repeated in a minimum of two
independent experiments to verify results.
Purification of Full-length Recombinant Ku70 and
Ku80--
Full-length recombinant Ku70 and Ku80 proteins were
expressed in BL21(DE3) as NH2-terminal T7-tagged and
COOH-terminal histidine-tagged proteins using Novagen pET23b vectors
applying techniques previously detailed (11). Full-length proteins were
isolated by sequential nickel-nitrilotriacetic acid chelate (Qiagen)
and anti-T7 Tag-agarose chromatography (Novagen T7 Tag affinity
purification kit) per the manufacturer's instructions with the
following modifications. After adjusting the Ku70 or Ku80 proteins
eluted from the nickel-nitrilotriacetic acid column with 0.2 M NDSB 195 as detailed by Ochem (41), samples were
equilibrated by dialysis in 1× Novagen T7 Tag binding buffer using
Slide-A-Lyzer 10K dialysis cassettes (Pierce), and purified on the T7
Tag affinity resin. T7-tagged Ku70 and Ku80 were then eluted in 0.1 M citric acid (pH 2.2), dialyzed in transcription buffer D
of Dignam et al. (42), and stored at 80 °C until used in pull-down assays.
Pull-down Assays of Protein-Protein Interactions--
Three
micrograms of purified T7-tagged, full-length recombinant Ku70 or Ku80
bound to 25 µl of T7 Tag-agarose (Novagen; anti-T7 antibody
conjugated to agarose; 50% slurry in pull-down buffer) in a total
volume of 160 µl was mixed with 40 µl of 10 mg/ml acetylated BSA
(Promega) and 200 µl of 2× pull-down buffer (2× pull-down buffer:
100 mM Tris, pH 8, 200 mM NaCl, 0.2 mM EDTA, 0.2% Nonidet P-40, 0.5 mg/ml BSA, 2× Sigma
protease inhibitor mixture number P2714). After binding of T7-Ku70 or
T7-Ku80 overnight with tumbling at 4 °C, the mixture was
centrifuged, and the pellet was washed 3 times with 500 µl of 1×
pull-down buffer. BSA was used as a negative control. Subsequently, 150 µl of 1× pull-down buffer was added to resuspend the respective
Ku70, Ku80, or null affinity resins, follow by the addition of 150 µl
of [35S]Met-radiolabeled protein (10 µl of a
TNT reaction diluted with 140 µl of 1× pull-down
buffer). Coupled transcription-translation reactions (50-µl total
volume per reaction; Promega reticulocyte lysate TNT kit)
were carried out as previously detailed (18) in the presence of
[35S]Met utilizing pcDNA3 expression vectors for
Msx2, Msx2-(2-208; del 132-148), Runx2, Tbdn100, Ku70, and Ku80 as
TNT templates (pcDNA3 supplies both T7 and
cytomegalovirus promoters) (18). After binding overnight, pellets were
washed 5× with 500 µl of 1× pull-down buffer, bound proteins were
eluted with 50 µl of 2× Laemmli buffer and heating (5 min at
95 °C), and proteins were resolved by SDS-PAGE. Radiolabeled
proteins were visualized by fluorography as previously described (43)
using Amplify (Amersham Biosciences) following the fixation,
infiltration, and drying of gels as per the manufacturer's instructions.
Preparation of Calvarial Osteoblasts from Neonatal Mice, and RNA
Analyses--
RNA extraction and Northern blot analysis was carried
out with random hexamer-primed radiolabeled human Tbdn100
cDNA probe using methods previously detailed (44). Primary mouse
calvarial osteoblasts were isolated from newborn mice as previously
detailed (45), and cultured in -minimal Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml
penicillin, 100 µg/ml streptomycin, 50 µg/ml ascorbic acid, and 3 mM -glycerol phosphate to induce differentiation. Cells
were passaged once, plated in 10-cm culture dishes, and maintained
under the above mineralizing culture conditions for 0, 5, 15, or 25 days, with refeeding every 2 days. RNA was extracted as previously
described (44). An Applied Biosystems GeneAmp 5700 sequence detection system using Sybr Green fluorescent dye binding to the PCR product was
used to quantify osteogenic mRNA accumulation via RT-PCR. The
technique of fluorescence RT-PCR has been recently reviewed (46).
Primer Express 1.0 software was used to design amplimers from murine
OC and osteopontin (OPN) cDNA sequences.
Amplimers for OC RT-PCR are: 5'-CAGCGGCCCTGAGTCGTA-3' and
5'-GCCGGAGTCTGTTCACTACCTTA-3'. Amplimers for OPN RT-PCR are:
5'-GTATTGCTTTTGCCTGTTTGG-3' and 5'-TGAGCTGCCAGAATCAGTCACT-3'.
Commercially available primers from Applied Biosystems were
used to quantify GAPDH expression for normalization in
RT-PCR (TaqMan Rodent GAPDH Control Reagent, PE Biosytems, Palo Alto,
CA). Standard curves were generated using appropriate cDNA
standards. Relative expression was normalized to GAPDH, and
reference to baseline levels observed for osteogenic mRNA
accumulation was measured at day 0 of wild-type calvarial cell
cultures. Data are presented as the mean ± S.D. obtained from two
independent experiments (4 replicates performed at each time point).
| |
RESULTS |
Purification of OCFRE-binding Complex from MG63 Osteosarcoma Cells
and Identification of Ku70, Ku80, and Tbdn100 as the Major
Constituents--
Previously we described the characterization of an
FGF2- and Msx2-regulated DNA-protein interaction at the OCFRE (17). We wished to identify the transcription factor complex binding to the
OCFRE. Gel filtration analyses indicated that the native binding activity was ~200 kDa (data not shown) and was likely to be a multiprotein complex. Therefore, we purified OCFRE binding activity 25,000-fold using classical biochemical techniques (32, 47), starting
with 2 × 1010 MG63 human osteosarcoma cells and
applying sequential ammonium sulfate precipitation, gel filtration,
diethylaminoethyl-Sephacryl chromatography, Mono Q FPLC, and
heparin-Sepharose FPLC techniques, following DNA binding in gel shift
as an assay (Fig. 1). This resulted in
about 4 µg of purified protein. As shown in Fig.
2A, Coomassie Brilliant Blue
staining of SDS-PAGE-fractionated protein reveals the presence of four
constituents of ~50, 70, 80, and 100 kDa in the fraction with peak
binding activity. Notably, the pattern of elution for the 70-, 80-, and
100-kDa proteins (arrowheads, Fig. 2A) coincide
with the elution of OCFRE DNA binding activity from the
heparin-Sepharose FPLC column (Fig. 2B). Therefore, the heparin-Sepharose column fraction containing the greatest OCFRE binding
activity and amounts of these three constituents was concentrated 50-fold and proteins were resolved by SDS-PAGE, the individual bands
were excised and digested in-gel with trypsin, and tryptic peptides
were analyzed by mass spectrometry. The 70- and 80-kDa proteins were
identified as Ku70 (GenBankTM accession number P12956) and
Ku80 (GenBankTM accession number P13010), the heterodimeric
constituents of nuclear Ku antigen. Initially, the 100-kDa protein
(Fig. 3; GenBankTM accession
number AY112670) was completely novel, i.e. did not match
any of the known proteins in the nonredundant GenBankTM
data base. However, this transcript recently was reported as a shorter
variant called Tubedown-1, a nuclear protein
N-acetyltransferase (28). Using 5'- and 3'-rapid
amplification of cDNA ends, we cloned the 100-kDa form of this
novel acetyltransferase from mouse and human heart cDNA. Sequencing
of both the human (866 codons) and mouse (865 codons) heart cDNAs
encoding Tbdn100 confirm the predicted protein sequence of
the longer variant (Fig. 3). Importantly, mass spectroscopy analysis
identified 23 polypeptides encoded by the human Tbdn100
cDNA, including polypeptides found in the extreme end of the novel
NH2 terminus. This confirms that the sequence predicted by
the cDNA sequence does encode a protein containing these sequences
and that the full-length, 100-kDa form of Tbdn is expressed both in the
MG63 osteoblasts (by LC-MS/MS protein sequencing) and heart (by
cDNA sequencing). We call the novel, longer variant we identify in
bone and heart "Tbdn100," reflecting its larger size
of Mr = 100,000 and relationship to the
murine Tbdn sequence. The novel subunit, Tbdn100, is
glutamate- and lysine-rich protein, has a putative bipartite nuclear
localization signal (48, 49) at residues 612-629, and multiple
NH2-terminal tetratricopeptide repeat motifs (Fig. 3).
Tbdn100 also shows distant homology to the GCN5 family of
N-acetyltransferases (50, 51), consistent with its role in
transcriptional regulation (see below). Northern blot analyses of
poly(A) mRNA (Fig. 4) confirms the
robust expression of Tbdn (see below) in mouse tissues. Four
transcripts of about 7, 5, 4, and 3 kb were visualized. The 5-kb
transcript was widely expressed, but robustly so in heart, liver,
kidney, testis, and MC3T3E1 murine calvarial osteoblasts (Fig. 4);
in situ hybridization studies also confirm the expression of
Tdbn in craniofacial osteoblasts (data not shown). The
expression of the 7-kb transcript was selective for heart and kidney,
whereas the smaller transcripts were most prominent in heart and testis (Fig. 4). Larger messages can be additionally identified in total RNA
from MC3T3E1 cells, possibly representing incompletely processed nascent transcripts (not shown). Thus, Ku70, Ku80, and Tbdn100 represent the proteinaceous constituents of the purified osteoblast OCFRE binding activity. Northern blot (Fig. 4) and Western blot (Fig.
5, see below) analyses confirm expression
of subunits in osteoblasts.
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Fig. 1.
Purification scheme for isolating the OCFRE
DNA-binding protein complex. Cellular protein was extracted from
2 × 1010 human MG63 osteosarcoma cells and OCFRE DNA
binding activity purified using standard protein chromatography
techniques, applying OCFRE binding in gel shift as an assay (17, 20).
Starting with 1 g of protein extract, ~4 µg of purified OCFRE
protein was obtained as eluted from HiTrap heparin-Sepharose HPLC. The
binding complex eluted with an apparent molecular mass of 200 kDa from
the Sephacryl S300 gel filtration chromatography.
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Fig. 2.
Elution profile of OCFRE binding activity and
proteins from HiTrap heparin FPLC chromatography column. Panel
A, proteins present in the 1-ml fractions eluted a with NaCl
gradient surrounding the peak of OCFRE binding activity, resolved by
SDS-PAGE, and visualized by Coomassie Brilliant Blue staining.
Panel B, elution profile of OCFRE binding activity. Note the
70-, 80-, and 100-kDa proteins (arrows, panel A)
that co-elute with OCFRE binding activity (panel B). See
text for details.
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Fig. 3.
The cDNA sequence and predicted protein
sequence of human Tbdn100. 5'- and 3'-rapid amplification
of cDNA end reactions were carried out with human heart cDNA to
obtain the sequence used to clone human Tbdn100 by PCR as
described in the text. The GenBankTM accession number for
human Tbdn100 is AY112670. The nuclear localization
consensus at residues 612-628 is in bold italics. The 23 unique peptides identified by micro LC-MS/MS from Tbdn100 purified from
human MG63 osteosarcoma cells are underlined. Affinity
purified antipeptide antibodies were generated against the
COOH-terminal 15 amino acids of Tbdn. Seven tetratricopeptide repeat
motifs are encoded by residues 46-79, 80-113, 114-147, 148-181,
224-257, 374-407, and 408-441.
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Fig. 4.
Northern blot analysis of Tbdn expression in
murine tissues. Commercially available polyadenylated RNA
(Clontech blot) isolated from the indicated murine
organs was analyzed for Tbdn100 (upper panel) or
-actin (lower panel) expression by Northern
blot. Polyadenylated RNA was also independently isolated from mouse
testis and MC3T3E1 osteoblasts. Note selective expression of the 7-kb
message in heart, skeletal muscle, and testis, with much lower levels
of expression in other tissues. A 5-kb message is widely expressed, but
at highest levels in heart, liver, kidney, testis, and MC3T3E1
osteoblasts. Smaller transcripts are also visualized primarily in
heart, testis, and MC3T3E1 cells.
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Fig. 5.
Immunoreactive Tbdn100 is detected in nuclear
fractions of MC3T3E1 cells. Panel A, nuclear (N)
and cytoplasmic (C) fractions were prepared from MC3T3E1
cellular extracts, and Western blot analyses was performed with
affinity purified Tbdn polyclonal antibody and tubulin antibody as
described under "Experimental Procedures." Note that 100- and
50-kDa proteins that localize primarily in the nuclear fraction are
immunovisualized with Tbdn100 antibody (lanes 1 and
2). By contrast, tubulin is localized in the cytoplasm
(lanes 3 and 4). Lanes 5 and
6, negative control lacking primary antibody. Panel
B, MC3T3E1 extracts from MC3T3E1 cells were analyzed for Ku70,
Ku80, and Tbdn100 expression by Western blot following treatment with
FGF2 for the indicated times. Treatment for 22-50 h up-regulates Ku80
and Ku70 (but not Tbdn100) protein accumulation about 2-3-fold. See
text for details.
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Expression of Tbdn100, Ku70, and Ku80 in MC3T3E1
Osteoblasts--
We wished to verify the expression of OCFRE
constituents in osteoblasts. Therefore, affinity purified anti-Tbdn
polyclonal antibodies were prepared, commercially available Ku70 and
Ku80 antibodies were obtained, and MC3T3E1 cellular extracts were
prepared for Western blot analyses as outlined under "Experimental
Procedures." The accumulation of 100- and 50-kDa proteins
selectively immunoreactive for affinity purified anti-Tbdn100 antibody
was detected in the nuclear fraction of MC3T3E1 calvarial osteoblasts
(Fig. 5A, lanes 1 and 2). Notably,
this confirms that the longer Tbdn100 variant protein is indeed
expressed in MC3T3E1 calvarial osteoblasts; the more rapidly migrating
form may represent either a proteolytic fragment encompassing the
COOH-terminal domain (antibody directed to the COOH terminus) or a
variant arising from alternative mRNA processing (Fig. 4).
Immunoreactivity for the cytoplasmic marker tubulin was present
predominantly in the cytoplasmic fraction (Fig. 5B,
lanes 3 and 4); no immunoreactive proteins were
observed in the absence of primary antibody (lanes 5 and
6). Western blot analyses also confirm the expression of
Ku70 and Ku80 in MC3T3E1 calvarial osteoblasts (Fig. 5B). As
also shown in Fig. 5B, FGF2 treatment of MC3T3E1 osteoblasts
for >20 h up-regulates Ku80 and Ku70 protein accumulation 2-3-fold,
as suggested from previous binding data (20). By contrast, total Tbdn
protein accumulation assayed by Western blot is not affected by FGF
treatment (data for Tbdn100 is shown in Fig. 5B). Thus,
Ku70, Ku80, and Tbdn100 represent the proteinaceous constituents of the
purified osteoblast OCFRE binding activity. The novel, lengthier
Tubedown variant Tbdn100 is expressed in MC3T3E1 calvarial osteoblasts,
and localizes to the nuclear fraction as predicted from the primary
structure of its cDNA.
Ku Antigen Complexes Assembled on OC Promoter Chromatin and the
OCFRE--
Our original analyses of OCFRE function and protein-DNA
interactions was carried out in MC3T3E1 osteoblasts. To show that Ku
binds endogenous OC chromatin in the osteoblast, we
performed ChIP assays (38) using anti-Ku70 and anti-Tbdn100 antibodies as outlined under "Experimental Procedures." As shown in Fig. 6A, anti-Ku70 and
anti-Tbdn100 antibodies recruited the OC gene to the pellets
in ChIP assays; by contrast, the BSP gene (encodes another
extracellular matrix protein) was not found in the pellet in ChIP using
either anti-Ku70 or anti-Tbdn100 antibodies. Two separate anti-Ku70
antibodies (see M19 and C19; see "Experimental Procedures") were
used that detected Ku antigen association with the OC gene
in ChIP assays (Fig. 6A, lower), whereas anti-GST and anti-Gal4 DNA-binding domain polyclonal antibodies (negative controls) precipitated relatively little OC chromatin. When
quantified by fluorescence PCR, a 15-30-fold enrichment of the
OC gene is observed with the two distinct anti-Ku70
antibodies (Fig. 6B). The background arising in ChIP
performed with nonspecific negative control antibodies (anti-FLAG,
anti-Gal4, and anti-GST) is negligible; moreover, no enrichment occurs
in ChIP assays with anti-Ku70 antibodies using Ku70 / primary
calvarial cell cultures as the source of chromatin (Fig.
6B). Using either MC3T3E1 (not shown) or COS-1 crude nuclear
extracts (Fig. 6C), specific DNA binding that contains Ku
antigen could be seen to be associated with the OCFRE. Competition studies confirmed binding sequence-specific binding; whereas cold homologous duplex OCFRE oligo (GCAGTCA cognate) can compete for binding
(Fig. 6B, lanes 2 and 3; compare with
lanes 1 and 6, no competitor), the unrelated
A/T-rich HOX sequence (CTAATTG cognate) (18) is without effect (Fig.
6C, lanes 4 and 5). Notably, the addition of the heterodimer-specific anti-human Ku antibody monoclonal antibody 162 (37) (Fig. 6C, lane 7) disrupts
formation of the complex. Disruption without supershift is observed
with endogenous binding activity in this monkey fibroblastic cell line
(Fig. 6C, lane 7). In COS-1 cells, markedly
increased OCFRE binding is observed following transient
co-transfection with pcDNA3-Ku70 with pcDNA3-Ku80 (human
cDNAs kind gift of Dr. Westley Reeves; Fig. 6C,
lane 8). Incubation of extracts from transfected cells with
monoclonal antibody 162 results in a visible supershifted complex (Fig.
6C, lane 9). Similar results are obtained in
MC3T3E1 osteoblasts (not shown). Notably, monoclonal antibody 162 does
not interact nonspecifically with the radiolabeled OCFRE probe (Fig.
6C, lane 10). Thus, Ku antigen-containing
complexes were detected bound to the endogenous OC gene in
MC3T3E1 cells, and in sequence-specific DNA-binding complexes assembled
by the OCFRE.
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Fig. 6.
Association of Ku complexes with the
OC promoter. Panel A, ChIP assays were
carried out as described under "Experimental Procedures." Data
are representative of results obtained in three independent
experiments. Note that although anti-Ku70 and anti-Tbdn antibodies
precipitate OC genomic DNA, BSP genomic DNA is
not precipitated (upper). Two distinct anti-Ku70 antibodies
(shown, lower panel) and an anti-Ku80 antibody (not shown)
recruit OC genomic DNA in ChIP; anti-FLAG, anti-glutathione
S-transferase (GST), and anti-Gal DNA-binding
domain antibodies do not (negative controls, lower). Panel
B, real time fluorescence PCR measurement of OC gene
enrichment in ChIP assays as described under "Experimental
Procedures." Note the 15-30-fold enrichment of the OC
gene by immunoprecipitation of MC3T3E1 chromatin by Ku70 antibodies.
Note that control antibodies (anti-FLAG, anti-Gal4, and anti-GST) do
not immunoprecipitate OC chromatin, and give a background
signal similar to that arising from ChIP performed with Ku70 / cells. Panel
C, gel shift assays were carried out using the radiolabeled duplex
oligonucleotide encompassing the OCFRE as described under
"Experimental Procedures." Extracts were prepared either from
CV-1 cells (lanes 1-7) or COS-1 cells transfected with
human Ku70 and Ku80 expression constructs. No extract was added in
lane 10. Lanes 1-5, competition assays with cold
oligos; lanes 6-10, immunological supershift assays. Note
that although cold homologous OCFRE duplex oligo can compete for
complex formation (lanes 2 and 3), the A/T-rich
HOX cognate does not (lanes 4 and 5). The anti-Ku
antibody disrupts formation of this endogenous complex (lane
7 versus lane 6). Extracts from COS-1 cells expressing
human Ku subunits yield a partial supershift in this assay (lane
9). Note that the anti-Ku antibody alone does not interact with
the probe (lane 10). See text for details.
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Ku Up-regulates Basal and Runx2-activated Transcription Directed by
the OC Proximal Promoter Region 154 to 113--
We wished to
confirm a functional role for the Ku·Tbdn complex in
OCFRE-dependent transcription. Therefore, we transfected expression constructs for Ku70, Ku80, and Tbdn100 with the 0.2-kb OC promoter-reporter (0.2-kb OCLUC; 222 to +32) in
MC3T3E1 calvarial osteoblasts using techniques previously described. As
shown in Fig. 7A,
co-expression of Ku subunits with or without Tbdn100 up-regulates basal
OC promoter activity. Moreover, transcriptional activation
can be recapitulated in heterologous cellular and promoter contexts.
The CV-1 fibroblastic cell background was chosen because these cells
lack significant endogenous Runx2/Osf2 expression (see below),
and are routinely used for detailed studies of transcriptional co-regulatory interactions. A reporter was constructed by placing 7 copies of the OC promoter fragment 154 to 113
(encompassing the OCFRE) upstream of RSVLUC (unresponsive RSV minimal
promoter with the luciferase gene (20)). As seen in Fig. 7B,
the Ku:Tbdn subunits augment OC-(154/113)-RSVLUC activity about
3-fold (p < 0.03). By contrast, Ku:Tbdn does not
activate RSVLUC, demonstrating that responsiveness is conveyed by the
42-bp OC promoter fragment 154 to 113, and is
promoter-specific (Fig. 7, A versus
B). In the OC promoter, the OCFRE is immediately
juxtaposed to OSE2, an important regulatory element recognized by the
osteoblast transactivator Runx2. We wished to evaluate potential
interactions between complexes bound by these elements. Therefore, we
co-transfected CV-1 cells with pcDNA3 (18) eukaryotic expression
constructs encoding subunits of the FGF responsive element-binding
complex (Ku70, Ku80, and Tbdn100) with or without Runx2 (Osf2;
kind gift of Dr. Gerard Karsenty). Runx2/Osf2 significantly
augments OC-(154/113)-RSVLUC about 7-fold, but not RSVLUC activity
(Fig. 8A).
Notably, Ku·Tbdn augments Runx2 transcription (p < 0.02), with the combination providing a 14-fold induction from basal
activity (Fig. 8A). This synergistic interaction was even
more pronounced in the presence of activated FGF receptor 2 signaling
(Fig. 8B). FGFR2-ROS (30) is a constitutively active FGF
receptor oncoprotein isolated from ROS17/2.8 osteosarcoma cells that
stimulates OCFRE-dependent transcription (31).
Co-expression of FGFR2-ROS augments OC-(154/113)-RSVLUC about
20-fold in the absence of added co-regulators (Fig. 8, B versus A). With coexpression of FGFR2-ROS, marked
synergistic (greater than additive) activation of the
OC-(154/113)-RSVLUC is observed between the Ku·Tbdn complex and
Runx2 in transient co-transfections (Fig. 8B); a 10.7-fold
induction is elicited. The heterologous RSV minimal promoter
is not synergistically induced. The individual contributions of Runx2,
Ku, and Tbdn100 were examined further in the presence of FGFR2-ROS
signaling; both Ku and Tbdn100 augment Runx2-dependent
transactivation (Fig. 8C). Whereas Ku uniformly enhances
Runx2 activation of OC-(154/113)-RSVLUC either in the presence or
absence of FGFR2-ROS, Tbdn100 augmentation is pronounced only in the
presence of FGFR2-ROS (Fig. 8C and data not shown). Notably,
this functional interaction is promoter-specific, because
RSV (Figs. 7 and 8) and matrix metalloproteinase 1 promoter (data not shown) basal promoters are not activated. Thus, these data
demonstrate the functional interactions between Ku, Tbdn100, and Runx2
in transcription driven by the 42-bp OC promoter region 154 to 113, and confirm the role of the novel Ku complex in OC promoter regulation. Ku and Tbdn100 serve to augment
Runx2 transcription, enhanced in the presence of activated FGF receptor signaling.
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Fig. 7.
Regulation of OC proximal promoter activity
by Ku antigen and Tbdn100. Cells were transiently transfected with
the indicated promoter-reporter constructs and combinations of Ku70,
Ku80, and Tbdn100 pcDNA3 expression constructs as detailed under
"Experimental Procedures"; pcDNA3 was used to maintain
uniform DNA content in each transfection. Luciferase activity was
measured after 3 days as previously detailed (18, 20). Data are
presented as the mean (± S.D.) activity observed in three independent
replicates. Panel A, MC3T3E1 cells were transfected with the
0.2-kb OC promoter-luciferase reporter (0.2-kb OCLUC) and expression
plasmids for Ku70, Ku80, and Tbdn100. Note that the combination of Ku
with Tbdn100 up-regulates OC promoter activity 7-fold. Panel
B, CV-1 cells were transfected with pcDNA3, Ku (Ku70 + Ku80), or Tbdn100 (Tbdn) in the indicated combinations
either with RSVLUC or OC-(154/113)-RSVLUC (42-bp OC promoter region
154 to 113 placed upstream of RSVLUC) as described under
"Experimental Procedures." Note that although the RSV promoter is
unresponsive, OC-(154/113)-RSVLUC is up-regulated by expression of
Ku + Tbdn100. See also Fig. 8, and text for details.
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Fig. 8.
Runx2, Ku, and Tbdn100 synergistically
activate transcription driven by the OC promoter region 154 to
113. Transfections were carried out as described in the legend
to Fig. 7 and under "Experimental Procedures." Panel
A, CV-1 cells transfected with Runx2 alone or in combination with
Ku + Tbdn expression plasmids (as in Fig. 7), using either RSVLUC or
OC-(154/113)-RSVLUC as the reporters. Note that Runx2 activation is
further enhanced another 2-fold by co-expression of Ku:Tbdn components.
The RSV basal promoter is unresponsive. Panel B, same
transfection protocol as outlined in panel A but in the
presence of FGFR2-ROS, a constitutively active FGF receptor. Note that
the synergy observed is enhanced in the presence of FGF receptor
activation (11-fold overall induction). The RSV promoter is not
synergistically induced. Panel C, regulation of
OC-(154/113)-RSV LUC transcription in CV-1 cells by Tbdn100 in the presence of
FGFR2-ROS. Tbdn100 augments OC-(154/113)-RSVLUC transcription
activated by Runx2 or the Runx2·Ku complex. See Fig. 11 for summary,
and text for details.
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Functional and Physical Interactions between Ku, Runx2, and
Msx2--
The OC promoter region 154 to 113 is a
target for Msx2-dependent suppression and
Runx2-dependent activation. A characteristic feature of
OCFRE-dependent transcription is suppression of
transcription driven by the OCFRE by Msx2. To confirm this functionally
important hallmark, we examined the effects of Msx2 on the activation
of OC-(154/113)-RSVLUC by Ku:Tbdn and Runx2. As shown in Fig.
9A, Msx2 inhibits
transcription via this element. As previously described (17),
suppression was not dependent upon the Msx2 COOH-terminal domain, but
requires the intact Msx2 homeodomain NH2-terminal arm and
extension missing in Msx2-(2-208; 
132-148) (Fig. 8A). Consistent with a role for Msx2 protein-protein interactions in this
regulation, the histone deacetylase inhibitor trichostatin A does not
alter Msx2- dependent suppression; moreover, the Msx2 variant
Msx2(T147A) that cannot bind DNA (18) still suppresses activation (data
not shown). Pull-down assays confirm protein-protein interactions
between these OC regulatory factors. As shown in Fig. 9B,
[35S]Met-radiolabeled Msx2 associates with T7-tagged Ku70
(lane 1) and Ku80 (lane 2) bound to the T7
affinity resin, but not to the BSA-coated anti-T7 antibody-coated
Sepharose resin itself (lane 3). About 5% of input
(lane 4) was recovered. By contrast, a Msx2 variant lacking
the NH2-terminal arm and extension (17) does not interact
with either Ku70 (lane 5) or Ku80 (lane 6).
Radiolabeled Runx2 avidly binds to both Ku70 (Fig. 9B,
lane 9) and Ku80 (lane 10), whereas interacting
minimally with the anti-T7 antibody resin (lane 11),
consistent with the functional interactions we observe in transient
co-transfection assays (see above). Under these pull-down assay
conditions, Tbdn100 interacts albeit less robustly with the individual
Ku70 (Fig. 9B, lane 13) and Ku80 (lane
14) subunits but not at all with anti-T7 antibody-coated resin
(lane 15). However, the interaction observed is similar to
the heterodimeric interactions noted with the radiolabeled Ku subunits,
with immobilized Ku70 (lanes 17-19) or Ku80 (lanes
20-22). USF1 also does not interact with Ku antigen in this
assay, and Dlx5 selectively interacts with Ku70 but not Ku80 (data not
shown). Thus, both functional (Figs. 8 and 9A) and physical
(Fig. 9B) interactions exist between components of the
Ku·Tbdn complex and the osteoblast transcription factors, Msx2 and
Runx2.
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Fig. 9.
Functional and physical interaction between
Ku antigen, Msx2, and Runx2. Panel A,
OC-(154/113)-RSVLUC was up-regulated by transient transfection of
the Runx2 and Ku complex transactivators as described in the legend to
Fig. 8 in the presence of the indicated amounts of pcDNA3-Msx2,
pcDNA3-Msx2-(2-208), or pcDNA3-Msx2-(2-208; 132-148);
pcDNA3 was used to maintain uniform DNA input. Note that as
observed in the native OC promoter context (17), OC promoter activation
via the region 154 to 113 is inhibited by Msx2 or Msx2-(2-208).
Further note that Msx2-(2-208; 132-148), lacking key residues
necessary to inhibit OCFRE binding activity (17), fails to suppress
OC-(154/113)-RSVLUC activation. Panel B, protein-protein
interactions were analyzed by pull-down assays as detailed in the text.
Anti-T7 Tag affinity resins containing immobilized Ku70 (lanes
1, 5, 9, 13, and 17),
Ku80 (lanes 2, 6, 10, 14,
and 20), or BSA (lanes 3, 7,
11, 15, 18, and 21) were used to
bind the indicated radiolabeled proteins. The amount of protein bound
is referenced to 5% of the input of radiolabeled proteins (lanes
4, 8, 12, 16, 19, and
22), and the relative signal obtained with the known
interaction between Ku subunits (lanes 17-22). Note that
Msx2 and Runx2 robustly interact with Ku subunits. By contrast,
Msx2-(2-208; 132-148) does not bind either Ku70 or Ku80. Tbdn100
weakly interacts with Ku. USF1 also does not interact with Ku antigen
in this assay, and Dlx5 selectively interacts with Ku70 but not Ku80
(data not shown). Bkgnd, background binding to the T7
antibody-coated affinity resin.
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Calvarial Cells Isolated from Ku70 / Mice Exhibit
Profound Deficiencies in OC but Not OPN Gene Expression--
Alt and
co-workers (52) recently generated Ku70-deficient mice for studies of
lymphocyte biology. To characterize the effects of Ku complexes on
OC expression, we isolated calvarial osteoblasts from
wild-type and Ku70 / neonatal mice, and evaluated the expression of
the OC gene when calvarial cell cultures were grown under
mineralizing conditions. We applied quantitative fluorescence RT-PCR to
the analyses of OC (osteoblast-specific terminal
differentiation marker (16)) and OPN (extracellular matrix
protein expressed in osteoblasts, monocytes, and migrating vascular
smooth muscle cells (53)) gene expression in these cultures.
OC and OPN levels were normalized to the
expression of the constitutive housekeeping gene GAPDH. As
shown in Fig. 10A, with time
in mineralizing conditions, expression of OC is up-regulated
600-fold in wild-type cultures, heralding osteoblast terminal
differentiation. However, in Ku70 / calvarial cell cultures
OC gene expression is markedly perturbed, as demonstrated by
the greater than 90% decrement in OC gene expression. By
contrast, expression of the gene for OPN was not reduced; in
fact, at later stages of culture, OPN expression in Ku70
/ cultures slightly exceeds that of wild-type cultures (Fig.
10B), indicating selective perturbation of OC
expression. Thus, biochemical analyses, transient transfection studies,
and quantitative evaluation of gene expression of Ku70-deficient
calvarial cells converge to indicate a role for Ku antigen complexes in
the expression of the OC gene in calvarial osteoblasts.
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Fig. 10.
Selective perturbation of OC
gene expression in differentiating calvarial cells lacking
Ku. Primary calvarial cell cultures were prepared from wild-type
and Ku70 / mice, and maintained under mineralizing conditions.
Total RNA was extracted from these cultures at the times indicated, and
analyzed for expression of OC (panel A) and
OPN (panel B) using real-time fluorescence RT-PCR
as described under "Experimental Procedures." Expression levels
are normalized to GAPDH, and referenced to baseline in
cultures of wild-type calvarial cells to facilitate comparisons. Note
that during early culture periods, the expression of OC is
low. With differentiation, OC mRNA accumulation is
up-regulated ~600-fold (panel A), as previously identified
by Northern blot analyses (10). Further note the profound deficiency in
OC mRNA accumulation in Ku70 / cells (up-regulated
only 10-fold with differentiation) compared with wild-type cell
cultures. By contrast, expression of OPN is not reduced at
these later stages in Ku70 / calvarial cells (panel
B).
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DISCUSSION |
The OCFRE confers both positive (20, 21) and negative (17, 22)
regulation on the OC promoter, and functions as a target of
Msx2 action (12, 17) via the OC promoter region 154 to 113. We have identified Ku70, Ku80, and Tbdn100 as the constituents of the major homeoprotein-regulated OCFRE complex in MG63 osteosarcoma cells (12, 17) by protein purification and nanobore LC-MS/MS analysis.
Applying immunological reagents in ChIP and gel shift assays, we
confirm the association of Ku and Tbdn100 with the OC
promoter. In transient transfection assays, we show that Ku:Tbdn expression functions to activate the OCFRE in concert with Runx2, a
master regulator of osteoblast-specific gene expression in mesenchymal cells (3). Importantly, mutation of the OCFRE precludes both basal
activity and factor-stimulated transcriptional activation driven by the
FGF-responsive OC promoter fragment 154 to 113 (21) (and
data not shown). Profound reduction in OC gene expression is
observed in cultures of calvarial osteoblasts from Ku70 / mice as
compared with wild-type cells; the deficiency in OC
expression that arises in the absence of this OCFRE-binding protein
subunit provides additional evidence for its function in OC
transcription. Ku participates in distinctive patterns of
protein-protein interactions with Msx2, Runx2, and Tbdn100. Based upon
our cumulative data, our working model of OC promoter
regulation by Msx2 (Fig. 11) reflects the inhibition of Runx2 and Ku DNA binding activity via these protein-protein interactions. ChIP assays confirm that Msx2 inhibits Runx2 association with OC chromatin.2 Future
experiments will identify the precise structural motifs present in Msx2
necessary for interaction with each Ku subunit, and the effects of Msx2
on formation of the Ku·Runx2 and Ku·Tbdn complexes. Preliminary
structure-function analysis suggests that, in addition to the
homeodomain NH2-terminal arm, the COOH terminus of Msx2
stabilizes physical interactions with Ku.
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Fig. 11.
Working model for transactivation of the
OC promoter region 154 to 113 and inhibition by
Msx2. OC promoter activity is supported by protein-DNA
interactions at adjacent OCFRE and OSE2 cognates bound by Ku and Runx2,
respectively. Note that unlike most Runt domain proteins, Runx2 does
not dimerize with CBF- (82). FGF receptor signaling enhances
transcriptional interactions between these factors via mechanisms yet
to be detailed, but may function to stabilize assembly of the complex
via Tbdn. Msx2 inhibits transcription driven by this complex via
inhibitory protein-protein interactions that regulate DNA binding of
transactivators (17) and the basal transcriptional machinery (18). See
text for details.
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A more general theme of functional interactions between Ku antigen and
homeodomain transcriptional regulators is emerging. We previously
described the OCFRE as a DNA binding activity inhibited by Msx2 (12),
and show physical interactions between Msx2 and Ku subunits in this
report. Recently, Hache and co-workers (54) identified by the yeast
two-hybrid cloning strategy that Dlx1 and Dlx2, homeoproteins highly
related to Dlx5 and Msx2, interact with the COOH-terminal domain of
Ku70 via protein-protein interactions with a trihelical DNA binding
motif uniquely found in Ku70 but not Ku80. We also observe Dlx5
interactions with Ku70 but not Ku80 (data not shown). Because Msx2
interacts with both Ku subunits, this suggests a fundamental difference
in the molecular biology of Msx and Dlx interactions with Ku antigen.
It is tempting to speculate that cross-talk occurs between Ku and Msx2
signaling during osteoblast development, coordinating proliferation and the timing of terminal differentiation. The regulation of Ku activities by Msx2 during DNA repair (55), Werner protein RecQ helicase stimulation (56, 57), and telomerase targeting (58) have yet to be
examined. If regulated by the osteoblast homeoproteins, these latter
activities may contribute to the net actions of Msx2 on skeletal
growth, osteoblast senescence, and sarcomatous transformation (59,
60).
The physical and functional interactions identified between Ku and
Runx2 are intriguing. Like Runx2 (61), Ku70 is required for robust
OC gene expression. The precise mechanisms whereby Ku and
Runx2 synergistically activate OC transcription are now being examined.
Possibilities include promotion of inward DNA transactions,
stabilization of DNA binding to certain cognates, and nuclear matrix
targeting. Dynan and co-workers (62) recently showed that Ku plays a
distinct role in the reinitiation phase of gene transcription. Based
upon their new data, one attractive mechanism for the observed synergy
would be that Runx2 promotes initiation whereas Ku augments
Runx2-directed re-initiation of OC gene transcription.
Potential differences arising from the Runx2 variants that are
differentially expressed in osteoblasts (63, 64) will be evaluated. The
effects of Msx2 and Ku antigen on the assembly and stabilization of
promoter-specific Runx2-chromatin complexes is being examined in ChIP
assay; Msx2 selectively inhibits assembly of these complexes on the
endogenous OC chromatin.2 Because Ku antigen and Runx2
appear important for both normal T-cell function (52, 65, 66) and
osteoblast gene expression (63, 67), functional interactions between Ku
and Runx2 may occur in lymphocytes as well.
It is interesting to note that the extended OCFRE cognate (CTGCAGTCACC,
upper strand; GGTGACTGCAG, lower strand) closely resembles that of
several other Ku responsive elements previously identified, including
the NRE1 from the mouse mammary tumor virus promoter (26), and the
strict late UL38 promoter element of HSV1 (68). The steps of
transcriptional activation (69-71), reinitiation (62), and suppression
(26, 72-74) have been shown to be regulated Ku dependent upon
promoter context. Other targets of Ku action in the osteoblast are not
known; however, a Ku cognate mediating transcriptional suppression of
the parathyroid hormone (75) and parathyroid hormone-related
polypeptide (76) promoters have been described that mediate responses
to calcium and the calcitropic hormone, vitamin D. Whether the
cell-autonomous perturbations in osteoblast functions that we have
identified in calvarial cell cultures from Ku70-deficient mice are
additionally influenced in vivo by dysregulated calcium
homeostasis remains to be tested.
Recently, the crystal structure of the Ku70:Ku80 heterodimer bound to
DNA was published (77). The Ku heterodimer forms a proteinaceous ring
around the co-crystalized stem-loop DNA structure, with contacts made
primarily in the major and minor grooves of the double helix (77). As
pointed out by the authors, this raises a number of topological
conundrums that must be answered. For example, if the ring conformation
identified in the crystal structure is adopted by Ku during DNA
ligation reactions, how is Ku released from DNA following the ligation
event? A similar question arises in the promoter-specific regulation of
gene transcription and chromatin remodeling by Ku in vivo
and in transient transfection assays that utilize circular, supercoiled
DNA promoter-reporter plasmids. A second COOH-terminal
helix-strand-helix DNA-binding domain recently characterized by nuclear
magnetic resonance (78) is present in Ku70 residues 536-609 that is
disordered in the above crystal structure, and does not require
heterodimerization with Ku80 for interaction with DNA (40). Notably,
this COOH-terminal domain interacts with homeodomain proteins, and
mutations as Lys residues 595 and 596 in human Ku70 abrogate
heterodimerization with the homeodomain (54). It is intriguing to
speculate that this COOH-terminal DNA binding module may confer a
second, distinct set of DNA-protein interactions in concert with other
interacting DNA-binding proteins. Future studies will detail the
potential roles for OC promoter DNA configuration in the
recognition by Ku, and outline the structure-function relationships for
Ku-dependent transcription modulated by associated
co-regulatory molecules such as Runx2 and Msx2.
In a recent report by Hasty and co-workers (79), the Ku80 (also known
as Ku86) knockout mice have been shown to have osteoporosis as well as
other phenotypes consistent with precocious aging, characterized by
excessive tissue apoptosis. These authors point to the differences
between Ku80 / mice and Ku70 / mice;
specifically, they note the lower incidence of neoplasms in
Ku80 / versus Ku70 / animals.
Ku70 typically heterodimerizes with Ku80; however, as noted above, Ku70
also exhibits DNA binding activity as a monomer (40) in part via the
unique COOH-terminal DNA-binding domain that mediates protein-protein
interactions with homeoproteins of the Dlx family (54). Systematic
comparisons of the skeletal phenotypes and osteogenic gene regulatory
programs elaborated in Ku70 /, Ku80 /,
and Ku70 /:Ku80 / animals will be
required to determine whether the Ku subunits serve distinct functions in skeletal physiology.
Our data demonstrate for the first time that Tbdn100 is expressed in
the MC3T3E1 osteoblast and functions as a transcriptional co-regulator,
cooperating with Ku antigen and Runx2 to up-regulate transcription from
the OC promoter. The poly(A)+ mRNA for the
lengthier variant we identify is widely expressed. Gendron and
co-workers (28) recently identified Tubedown-1 as an FGF2- and leukemia
inhibitory factor-regulated transcript in endothelial cells; moreover,
they showed that the shorter variant of murine Tbdn can undergo
autoacetylation. Thus, Tbdn100 may function as a co-adapter that
mediates transactivation in part via regulation of chromatin
acetylation, as described for P/CAF, the nuclear receptor p160
co-regulators, and cAMP-response element-binding protein/p300 (51).
Tbdn100 possesses an additional 4 NH2-terminal tetratricopeptide repeat motifs that are lacking in the shorter variant
of Tbdn previously reported. Our Western blot analyses clearly
demonstrate the nuclear localization of Tubedown, and the accumulation
of full-length Tbdn100 predicted from our cDNA sequence (100 kDa)
as well as a smaller 50-kDa protein. The ability of Tbdn100 to promote
activation of OC-(154/113)-RSVLUC is pronounced in the presence of
FGFR2-ROS. This suggests that post-translational modifications
initiated by FGFR2 activation may promote stable association of Ku and
Runx2 complexes via Tbdn. Notably, phosphorylation (80) and
N-acetylation (81) have been shown to enhance functionally important transcription factor protein-protein interactions. A series
of systematic structure-function studies are underway to: (a) identify the Tbdn100 domain structures necessary for
transcriptional regulation, nuclear protein acetylation,
protein-protein interactions, and responses to FGFR2-ROS; and
(b) characterize the role and regulation of different
Tubedown isoforms in osteoblast gene expression.
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ACKNOWLEDGEMENTS |
We thank Bill Lane and Eric Spooner (Harvard
Microchemistry) for assistance in the characterization of purified
DNA-binding proteins by mass spectroscopy, Dr. Fred Alt for generously
providing the Ku70 / mice, Heather Walker for technical assistance
with mouse husbandry, and Drs. Gerard Karsenty
(Osf2/Cbfa1/Runx2) and Westley Reeves (Ku70, Ku80) for the
generous gifts of the indicated cDNAs.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants R01 DK52446 and R01 AR43731 (to D. A. T.) and a
pre-doctoral research fellowship from the National
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ABSTRACT
INTRODUCTION
