![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Previous Article | Next Article 
Molecular and Cellular Biology, May 2007, p. 3337-3352, Vol. 27, No. 9
0270-7306/07/$08.00+0 doi:10.1128/MCB.01544-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Suk Hee Lee,1
Matthew Mandeville,1
Brian Weiner,1
Martin Montecino,2
Andre J. van Wijnen,1
Janet L. Stein,1
Gary S. Stein,1 and
Jane B. Lian1*
Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655,1 Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias Biologicas, Universidad de Concepcion, Concepcion, Chile2
Received 18 August 2006/ Returned for modification 4 October 2006/ Accepted 9 February 2007
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Mammalian Hox genes (homologues of the Drosophila homeotic genes) encode transcription factors crucial in regional development along the anterior-posterior axis during embryogenesis (44, 64). The mouse and human genomes contain 39 Hox genes, which are grouped into four clusters, Hoxa, Hoxb, Hoxc, and Hoxd, positioned on four separate chromosomes in 13 paralogs (38, 44). Hoxa10 is a member of the Abdominal B (Abd B) class of homeobox protein-encoding genes, representing the most 5' genes in the cluster consisting of paralog genes from Hoxa 9 to Hoxa 13 (4, 44, 50). The corresponding class of homeobox proteins binds preferentially to a consensus core of TTAT or TTAC, which is distinct from the TAAT homeodomain consensus core binding site recognized by MSX and DLX proteins (4). HOXA10 DNA binding is influenced by flanking sequences and the formation of complexes with HOXA10-interacting proteins of the MEIS and PBX classes of transcription factors, as well as other coregulatory proteins, such as histone deacetylase 2 (13, 51, 60, 72, 74, 81).
Several of the Hox genes are essential for normal skeletal development (31, 64). The nonparalogous Hoxa10 and Hoxd11 genes cooperate in the development of the forelimbs and axial skeleton and are required to globally pattern the mammalian skeleton (6, 7, 26, 68, 80). Inactivation of the paralogous Hoxa10 and Hoxd10 genes results in alterations in the formation of the forelimbs and hind limbs. Hoxa10–/– mice revealed an active role for the gene in modeling the femur, tibia, and fibula (12, 79). Transgenic expression of HOXA10 in presomitic mesoderm of the mouse resulted in vertebrae without ribs (11). HOXA10 is expressed in the presomitic mesoderm, which develops into the axial skeleton and cooperates with other Hox genes (e.g., Hoxd11) for normal skeletal development (11, 26, 31, 64). Despite the considerable genetic evidence that HOXA10 has critical skeletal functions, target genes of HOXA10 in bone have not been identified.
In this study we have characterized HOXA10 regulation of the key osteogenic factor Runx2, as well as RUNX2 target genes, identifying Hoxa10-specific regulatory elements in promoters of the Runx2, osteocalcin (OC), alkaline phosphatase, and bone sialoprotein osteoblast-related genes. This discovery of a Hox regulatory factor in activating Runx2 provides novel insights for a mechanism that regulates a transcription factor essential for bone formation (43, 58). Although Runx2 is rapidly induced in response to BMP2 and present in developing limbs, somites, and mesenchymal condensations prior to chondrogenic and osteoblast differentiation (23, 46, 76), a Smad-responsive element has not been defined. Thus, BMP2-induced HOXA10 represents a key regulator of Runx2 transcription during embryogenesis. Our studies also show that HOXA10 can regulate osteoblast genes independent of RUNX2. We have thus identified an additional role for HOXA10 in postnatal bone formation and maintenance of the osteoblast phenotype. We propose that HOXA10 functions in two capacities: as a component of a BMP2 signaling cascade prior to RUNX2 to mediate the developmental induction of osteogenesis and during osteoblast differentiation to regulate the temporal expression of bone phenotypic genes to drive osteoblast maturation through mechanisms involving chromatin remodeling of gene promoters.
(Brian Weiner's contribution to this paper is in fulfillment of a Worcester Polytechnic Institute undergraduate thesis project.)
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-MEM supplemented with 10% FBS. Primary rat osteoblast cells were isolated from calvaria according to the procedures described previously (34). The rat osteosarcoma cell line ROS 17/2.8, representing a mature osteoblast phenotype, was maintained in F-12 medium supplemented with 5% FBS (70). Cells were cultured under osteogenic conditions with MEM (GIBCO-BRL) supplemented with 10% FBS, 50 µg of ascorbic acid/ml, and 10 mM ß-glycerol phosphate. Bone marrow stromal cells were isolated from 8-week-old C57BL6 mice and cultured in MEM containing 10% FBS (29). Runx2 null cells were isolated from calvarial tissues of mouse embryos (17.5 days postcoitum) of the Runx2 null mouse and immortalized using mouse telomerase (TERT). Characterization of this cell line has been described previously (1). The BMP2 used in these studies was a kind gift from John Wozney (Wyeth Research, Women's Health and Musculoskeletal Biology).
Transfection and reporter assays. The Hoxa10 expression clone containing the mouse cDNA (1.2 kb) of the Hoxa10-1 variant (55 kDa) containing the transactivation domain was kindly provided by Richard L. Maas (Harvard Medical School, Boston, MA) (4). Transient transfections were performed in six-well plates at 50 to 70% confluence with 5 µl of FuGENE6 transfection reagent with wild-type (WT) and deleted promoter reporter DNA according to the manufacturer's instructions (Roche, Indianapolis, IN). A tagged Hoxa10 expression vector (pcDNA3.1-Xpress-Hoxa10) was constructed was used along with a Runx2 expression vector (pcDNA3.1-HA-Runx2) (85) in this study. For control of expression, vector pcDNA3.1 was transfected according to the experimental condition. The following Runx2 WT and deletion promoter reporter plasmids (21) were used: the WT Runx2 0.6-kb fragment and the deletion series (–490, –458, –351, –288, –128) fragments. These fragments were cloned in pGL3 basic luciferase vector (Promega, Madison WI). Cells transfected with either Runx2 promoter luciferase or –208 OC promoter chloramphenicol acetyltransferase (CAT) reporter constructs along with Hoxa10, Runx2, or control vector were harvested 24 to 36 h after transfection, and all lysates were assayed for luciferase or CAT activity according to the manufacturer's instructions (Promega, Madison, WI). All results were normalized to the luciferase activity resulting from transfection of the promoterless pGL3 luciferase construct (Promega, Madison, WI). The OC promoter activity was assayed by using the –208-bp promoter DNA fragment from rat OC genes cloned in the pCAT basic vector (Promega, Madison, WI) (32). The percent CAT conversion was the average of values for six similar transfection samples.
cDNA synthesis and QPCR. RNA was isolated from cultures of MC3T3, NIH 3T3, and C3H10T1/2 cells by use of TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. cDNAs were synthesized with oligo(dT) primers by use of a SuperScript first-strand cDNA synthesis kit (Invitrogen) according to the manufacturer's protocol. Gene expression was assessed by real-time quantitative PCR (RT-QPCR) using Power SYBR green PCR master mix (Applied Biosystems, California). Primers used for PCRs are listed in Table 1.
|
-32P]ATP by use of T4 polynucleotide kinase (New England Biolabs, Massachusetts). The detailed procedures have been described previously (34). Anti-HOXA10 (N20) or a nonspecific antibody (either -actin or normal immunoglobulin G [IgG] as indicated) (Santa Cruz) were used for the immunoshift studies. Complexes were visualized by autoradiography of a 6.5% acrylamide gel. Immunoblotting. Each well of a six-well plate was lysed in 50 µl lysis buffer (2% sodium dodecyl sulfate [SDS], 10 mM dithiothreitol, 10% glycerol, 12% urea, 10 mM Tris-HCl [pH 7.5], 1 mM phenylmethylsulfonyl fluoride, 1x protease inhibitor cocktail [Roche], 25 µM MG132 [proteosome inhibitor from Calbiochem]) and boiled for 5 min. Equal amounts of total protein were analyzed by SDS-polyacrylamide gel electrophoresis and probed with suitable antibodies. Immunocomplexes were detected using Western Lightning chemiluminescence reagent (Perkin Elmer, Boston, MA).
Antibodies.
The following antibodies were purchased from Santa Cruz Biotechnology. HOXA10 N20 (SC-17158) was for chromatin immunoprecipitation (ChIP) and EMSA, and A20 (5C-17159) was for Western blotting. RUNX2 antibodies were PEBP2A (M-70 [SC-10758]), PEBP2A (C-19 [SC-8566]), and actin (I-19 [SC-1616]). Mouse monoclonal RUNX2 antibody was a generous gift from Yoshi Ito and Kosei Ito (National University, Singapore, Republic of Singapore). Anti-hyperacetylated histone H4 (Penta) and anti-trimethyl histone H3 (Lys4), clone MC315, were purchased from Upstate Cell Signaling Solutions (Charlottesville, VA). The mouse monoclonal anti-Xpress antibody was obtained from Invitrogen (Carlsbad, CA). Mouse monoclonal antibody against RNA polymerase II (Pol II) (clone 8WG16) was obtained from Covance (Princeton, NJ) and used in ChIP studies.
ChIP assays. The procedure for ChIP in primary rat osteoblasts has already been described (34). Control primer pairs from 3' untranslated regions (UTR) of the genes were used to verify specific and nonspecific binding of DNA fragments (Table 1). IgG antibody was used as a control for nonspecific pull-down of immunocomplexes. Sequential ChIP studies were performed using the primary pull-down from one antibody, which was divided into equal aliquots for the second pull-down with antibodies specific for coregulatory molecules. Instead of being eluted in 1% SDS and 100 mM Na2HCO3 after cross-linking and washing, immunocomplexes were eluted in 10 mM dithiothreitol. The eluate was further diluted 1:40 in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM Tris-HCl [pH 8.1], 167 mM NaCl) and used for the second immunoprecipitations. Aliquots (2 to 3 µl) of DNA samples from different pull-downs were assayed by either radioactive labeling or RT-QPCR using Power SYBR green PCR master mix (Applied Biosystems, California) for the detection of specific DNA fragments with primers in the proximal promoters of bone-related genes that encompass the Hoxa10 binding sites (Table 1).
Immunohistochemistry and immunofluorescence. Long bones from normal newborn mice were fixed with 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, for 48 h, dehydrated, and embedded in paraffin by standard procedures. Paraffin-embedded tissues (5-µm sections) were immunoperoxidase labeled and blocked by 0.3% H2O2 in absolute methanol for 30 min at room temperature. To reveal antigens, sections were put in a 1 mM Tris solution, pH 9.0, supplemented with 0.5 mM EGTA. The sections for RUNX2 immunohistochemistry were heated in a microwave oven for 10 min after EGTA treatment, while a normal steaming antigen retrieval method was used for HOXA10. Nonspecific immunoglobulin binding was prevented by incubating the sections in 50 mM NH4Cl for 30 min followed by blocking with phosphate-buffered saline supplemented with 1% bovine serum albumin, 0.05% saponin, and 0.2% gelatin. Serial sections were incubated overnight at 4°C with anti-rabbit RUNX2 (M-70 and C-19) or anti-goat HOXA10 (N20) antibody diluted (1:100) in phosphate-buffered saline supplemented with 0.1% bovine serum albumin and 0.3% Triton X-100. Equal amounts of the respective blocking peptides (2 µg/ml) for HOXA10 and RUNX2 were also used. Normal IgG was used as a nonspecific control. Immunolabeling controls were performed by using antibodies preabsorbed with immunizing peptides. Labeling was visualized with the horseradish peroxidase-conjugated secondary antibody (P448, 1:200; Dako).
MC3T3 cells were plated at a density of 0.6 x 105 cells/well on gelatin-coated coverslips in six-well plates. Cells were processed for in situ immunofluorescence analyses, which were carried out as described previously (39). HOXA10 was detected by a goat polyclonal antibody at a dilution of 1:100 (Santa Cruz Biotechnology). The secondary antibody used was Alexa 488-anti-goat antibody (Molecular Probes) at a dilution of 1:800. For overexpressed HOXA10 protein, HeLa cells at 0.5 x 105cells/well on gelatin-coated coverslips were transfected with 0.5 µg of Hoxa10 expression construct, and 24 h after transfection the coverslip was processed for immunofluorescence study (39). Xpress-HOXA10 was detected by a mouse monoclonal antibody against the Xpress tag at a dilution of 1:3,000 (Invitrogen). The secondary antibody used was Alexa 568-anti-mouse antibody (Molecular Probes, Eugene, OR) at a dilution of 1:800. Slides were examined on a Zeiss Axioplan 2 microscope fitted with epifluorescence (Carl Zeiss, Jena, Germany) attached to a charge-coupled-device camera. Images were saved and processed using Metamorph imaging software, version 6.1 (Universal Imaging, Downingtown, PA).
RNA interference (RNAi) of Hoxa10. The mouse MC3T3-E1 osteoblastic cells at 30 to 50% confluence were transfected using Oligofectamine (Invitrogen Life Technologies) with small interfering RNA (siRNA) duplexes specific for murine Hoxa10 r(CCA AAU UAU CCC ACA ACA A)dTdT and r(UUG UUG UGG GAU AAU UUG G)dCdG obtained from QIAGEN Inc. (Stanford, CA). Six different sets of siRNA duplexes at different concentrations were used to evaluate the target specificity and knockdown efficiency. The cells were also transfected with control siRNA duplexes specific for green fluorescent protein by use of the same concentrations to check the transfection efficiency. The siRNA experiment was carried out for 72 h. Total RNA and proteins from the specific siRNA oligonucleotide-treated, untreated, Oligofectamine-treated and nonspecific-oligonucleotide-treated cells were analyzed by Western blotting and RT-QPCR. To study chromatin modifications after Hoxa10 siRNA treatment, cells were harvested 72 h posttransfection and used for immunoprecipitation with trimethyl histone H3K4 and hyperacetylated H4 antibody for ChIP analyses.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
To appreciate the role of HOXA10 in osteogenesis in vivo, we examined its expression in bone cell populations in situ in relation to endochondral bone formation and skeletal development. Sections of long bones and vertebrae from newborn mice were treated with specific antibodies to detect RUNX2 and HOXA10 (Fig. 2). We found strong expression of HOXA10 in the periosteum, the hypertrophic chondrocyte zone of the growth plate, and osteoblasts on all bone surfaces. RUNX2 was also expressed in these cell populations. Weak expressions of HOXA10 and RUNX2 were found in the flattened cells that represent the prehypertrophic chondrocyte phenotype. It is noteworthy that both HOXA10 and RUNX2 were absent from the epiphyseal chondrocytes that represent permanent hyaline cartilage. Similar observations were made for vertebrae tissue. We conclude from these studies that RUNX2 and HOXA10 are expressed in osteogenic lineage cells and maturing chondrocytes at the growth plate in both the axial and appendicular skeletons.
|
The coordinate BMP2 induction of Hoxa10 and Runx2 in response to BMP2-induced osteogenesis in several cell models, taken together with the profiles of expression of these genes during the maturation of MC3T3 cells as well as their coexpression in osteogenic lineage cells, suggests a functional relationship between RUNX2 and HOXA10.
The P1 bone-related promoter of Runx2 and the endogenous Runx2 gene are regulated by HOXA10. As activating signals on the Runx2 promoter during early stages of embryonic development are limited, we pursued the characterization of a Hoxa10 regulatory element in the bone-related Runx2 P1 promoter. Examination of the Runx2 0.6-kb promoter, which has been studied for its regulation in osseous and nonosseous cells, revealed the presence of three putative Hox consensus sequences. Two TTAT-containing core sequences (4, 24) and one sequence with a TTAC core (72), which binds HOXA9 well but HOXA10 with a low frequency (4, 73), are present in the proximal promoter (Fig. 3A). These sequences differ from the TAAT binding site for homeodomain proteins of the MSX and DLX classes (34, 45). To identify the HOXA10-responsive regulatory element(s) in the Runx2 gene, deletion mutation analyses were performed with both NIH 3T3 (nonosseous) and MC3T3 (osseous) cells cotransfected with Runx2 promoter deletion fragments and HOXA10. The endogenous protein levels of HOXA10 are slightly higher in the MC3T3 preosteoblasts than in the NIH 3T3 fibroblasts (Fig. 3B, control lanes) which may be contributing to the basal expression of the promoter constructs in MC3T3 cells, where the –351 fragment is reduced 20% (Fig. 3C). In the presence of exogenous HOXA10, a 2- to 2.5-fold increase in promoter activity of the –600, –490, and –458 fragments is observed, suggesting that the proximal TTAT site 1 is sufficient for HOXA10 regulation of Runx2 (Fig. 3C). Furthermore, complete loss of HOXA10 responsiveness occurs with the –351 Runx2-Luc fragment, which lacks the putative site 1 for HOXA10 binding. Furthermore, mutation of site 1 blocked HOXA10-mediated transcriptional activation (Fig. 3D). The site 2 mutation had no effect on HOXA10 induction of Runx2, i.e., the construct retained the same 2.5-fold activation level as the WT. The TTAC core site, located at –420/–417, cannot be functional, as the site 1 mutation showed no HOXA10-induced change in promoter activity. Taken together, these results are consistent with the location of a functional Hoxa10 site in the proximal promoter fragment at –353/–351, a regulatory element mediating HOXA10 activation of the Runx2 P1 promoter.
|
|
|
Functional recruitment of HOXA10 to promoters of Runx2 and other osteoblast-related genes. To gain further insight into the in vivo regulation of Runx2 transcription by HOXA10 during the development of the osteoblast phenotype, we performed ChIP assays with primary rat calvarial osteoblasts, which undergo a well-documented sequence of maturation stages (59). Western blot analysis for RUNX2 and HOXA10 protein levels during the differentiation time course shows that a low basal level of HOXA10 protein is present in proliferative osteoblasts (day 4), while RUNX2 protein is not observed until day 6 (Fig. 6A). When bone-like nodules form on day 10, HOXA10 is upregulated twofold, increases again on day 12, and remains at a constitutive level into the mineralization stage (day 20). RUNX2 protein levels increase continuously during osteoblast maturation but are reduced on day 20 (heavily mineralized cultures). These findings emphasize that HOXA10 is present in osteoprogenitors prior to RUNX2 protein appearance and that HOXA10 may function throughout differentiation.
|
ChIP studies allow for assays of transcription factors associated with different promoters in the same sample. We therefore examined the promoters of several osteoblast genes for potential Hoxa10 binding sites and designed primers to amplify the DNA of the appropriate promoter domain. Table 2 shows the putative sequences. We examined the direct regulation of the osteoblast-specific OC gene, which is highly regulated by RUNX2 (40). A Hox binding site was located in the proximal –208 promoter segment, which exhibits tissue-restricted expression (37) (Fig. 7A). Using a –208 OC-CAT reporter construct, we found a two- to threefold increase in promoter activity in response to HOXA10 in multiple experiments (Fig. 7B). By ChIP analysis (Fig. 7C), we found that HOXA10 is associated with OC chromatin 24 h prior to RUNX2 association. During osteoblast differentiation, both HOXA10 and RUNX2 binding were enhanced on days 8 and 9, when OC mRNA first began to accumulate (Fig. 7D), consistent with increased Pol II binding on day 9 (Fig. 7C). After this initial induction of OC, HOXA10 association with the OC promoter diminished in the matrix maturation and mineralization stages, while RUNX2 remained present on OC chromatin. The coordinate increase in HOXA10 and RUNX2 binding to OC prompted us to examine the combined effects of HOXA10 and RUNX2 on OC promoter activity. We found an additive effect on promoter activity as a result of the coexpression of the factors (Fig. 7B), suggesting that the Hoxa10 and Runx2 regulatory elements function independently on the OC promoter, not synergistically. Taken together, these results demonstrate HOXA10 supports the regulation of OC expression earlier than RUNX2 and significantly contributes to OC-induced mRNA.
|
|
|
HOXA10 regulates osteoblast genes both dependent on (in MC3T3 cells) and independent of (in Runx2 null cells) RUNX2. The ChIP studies (Fig. 6, 7, and 8) suggest that HOXA10 may regulate osteoblast genes that are significant functional components of bone, either synergistically, by increasing Runx2 expression, or directly, through Hox regulatory motifs. We therefore examined the consequences of the depletion of Hoxa10 on the endogenous expression of these skeletal markers of osteoblast differentiation by RT-QPCR (Fig. 9A). MC3T3 cells were treated with Hoxa10 siRNA for 48 h, which reduced Runx2 gene expression threefold. Dose-dependent decreases of the early osteoblast markers ALP and BSP were also observed, as was a twofold reduction in OC mRNA. Exogenous expression of HOXA10 in MC3T3 cells (Fig. 9B) shows that Runx2 and ALP expression is increased twofold, while the mature osteoblast-related genes OC and BSP are induced between five- and eightfold.
|
HOXA10 activation of osteogenic genes OC, ALP, and BSP in Runx2 null cells, taken together with the association of HOXA10 with binding elements in these promoters, supports a RUNX2-independent direct role for HOXA10 in promoting osteoblast differentiation. However, these promoters are also Runx2 responsive, and HOXA10 directly induces Runx2, thereby contributing to HOXA10 RUNX2-dependent transcription of osteoblast genes. The BMP2-initiated induction of Hoxa10, Runx2, and other transcriptional factors (e.g., DLX3 and DLX5) indicates that these factors all contribute specific regulatory functions to support bone formation. This combinatorial control is schematically illustrated in Fig. 9D.
HOXA10 contributes to chromatin modification of osteoblast target genes for induced transcription. The early recruitment of HOXA10, prior to RUNX2, on the OC, ALP, and BSP gene promoters suggests that HOXA10 may contribute to remodeling the chromatin of these phenotypic genes. The remodeling of chromatin structure is mediated in part by enzymes that topologically alter DNA interactions with histones or that covalently modify the core histone proteins H3 and H4 (41). Acetylation of histone H4 and H3K4 methylation are modifications that strongly correlate with transcriptionally active chromatin (10, 75, 77). To test this hypothesis, we examined the effects of Hoxa10 depletion on histone acetylation and H3K4 methylation of gene promoters, which reflect transcriptionally active chromatin. Cells were treated with Hoxa10-specific siRNA for 72 h (Fig. 10A), and these chromatin modifications were assayed by ChIP analysis for the four bone phenotypic genes (Fig. 10B). We confirmed a 50% knockdown of Hoxa10 by siRNA and a 50% reduction in HOXA10 binding to OC, ALP, and BSP promoters but a modest decrease in the recruitment of HOXA10 to the RUNX2 promoter (Fig. 10B, left panels). We observed a significant 50% reduction in the acetylation of OC, BSP, and Runx2 chromatin but a modest decrease in ALP chromatin acetylation in cells treated with Hoxa10 siRNA relative to control levels (nonsilencing siRNA) (Fig. 10B). On the other hand, we found in multiple studies (n = 3) a significant decrease in the H3K4 methylation of ALP and BSP, while the methylation of OC and Runx2 chromatin was less decreased.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Our findings suggest HOXA10 functions in a linear pathway with RUNX2 to promote bone formation as well as independently of RUNX2. Our model (Fig. 9D) proposes that HOXA10 functions as an immediate early response gene to the BMP2 signal for the initiation of osteogenic gene regulation and provides an amplification step for RUNX2-induced expression to establish the osteoblast phenotype. As other BMP2-induced transcription factors regulate Runx2 and osteoblastic genes (e.g., homeodomain proteins [MSX2, DLX3, and DLX5], TCF/LEF1, AP-1, OSTERIX, CCAAT/enhancer-binding proteins [encoded by C/EBP], and ATF4) (22, 28, 45, 82, 83, 86), a compelling question is that of the hierarchy of HOXA10, RUNX2, and these other factors in regulating gene expression for bone formation. Null mouse models have identified OSTERIX function later than RUNX2 (56). From a developmental perspective, BMP/TGFß, Wnt, and HOX signaling interactions are documented (5, 62), and all of these proteins contribute to bone development. From a gene regulation perspective, a hierarchy of transcriptional control can be better deduced by understanding the ordered recruitment of factors to gene promoters and the temporal levels of the regulatory factors during osteoblast differentiation. Abd B class Hoxa10, Msx2, and the distal-less Dlx3 genes are induced within a few hours, while other factors (e.g., Dlx5 and C/EBPß) are induced later (after 8 h) in the C2C12 model. Recent studies provide evidence from ChIP studies for a homeodomain protein regulatory network for osteoblast commitment and differentiation (35). Here we show HOXA10 to be associated with bone promoters prior to RUNX2, suggesting that it contributes to the initiation of the osteoblast phenotype. However, increased HOXA10 binding appears to be coordinated with increased recruitment of Runx2, homeodomain proteins, and other factors postproliferatively for accumulation of mRNA, thereby establishing the bone phenotype (35). The selective temporal binding of HOXA10 to promoters reflects binding and dissociation during differentiation. This is in contrast to the continual increase in RUNX2 binding to promoters, suggesting that HOXA10 has a very dynamic regulatory function for regulating osteogenic gene expression during osteoblast differentiation.
We found modifications of histones at the level of chromatin in response to altered HOXA10 expression, indicating a remodeling of the transcriptional machinery that is necessary for in vivo gene regulation. HOXA10 recruitment to bone promoters prior to a significant accumulation of mRNA but after Pol II association emphasizes a functional role for HOXA10 increasing transcription through chromatin remodeling. This is supported by knockdown of HOXA10 reducing histone acetylation and H3K4 methylation, which are associated with active transcription and by HOXA10 and p300/CBP coassociation on the Runx2 and OC promoters (ChIP-reChIP studies). Earlier studies have reported that HOX factors can modify transcriptional activation and repression by interacting with p300/CBP (14, 71). Our studies have focused on HOXA10 in supporting chromatin remodeling for the activation of osteoblast-related genes, as was shown for p21 (9). Equally important is the fact that HOXA10 can promote histone deacetylation by recruiting histone deacetylase 2 and SIRT2 histone deacetylases to repress gene transcription (67). Histone deacetylase-HOXA10 interactions would reverse HAT activity and could be operative in osteoblasts for HOXA10 to mediate either activation or attenuation of gene expression. Such mechanisms are important for HOXA10 suppressor function in undifferentiated myeloid cells and HOXA9 regulation of neovascularization (2, 51, 65, 78). In addition to coregulatory factors that modify chromatin, the other well-characterized partner proteins for the Abd B HOX proteins, PBX1 and MEIS, both having homeodomain protein modules, form complexes with HOX proteins and also bind to DNA to regulate repressor and enhancer activities of Hox genes. HOXA9, HOXA10, HOXA11, HOXD12, and HOXB13 each have different properties of interaction with these coregulators (52, 61, 72-74). For example, high-affinity DNA binding can be achieved when HOXB9 and HOXA10 proteins are dimerized with PBX1. While our forced-expression and siRNA knockdown studies show that HOXA10 has anabolic activity on osteoblast gene expression, how these endogenous partner proteins influence the HOXA10-mediated expression of osteoblast genes is a provocative question.
The specificity of HOXA10 for regulating osteoblast differentiation should be considered in relation to Hox nonparalogous and paralogous genes, which are known to overlap in expression in some tissues but can have preferential activities (15, 17, 27). For example, HOXA10 and HOXD10 regulate the skeleton but may exhibit specific gene regulation properties (36, 49, 79). Also, HOXA10 and HOX11 homeobox proteins are equivalent for axial but not appendicular skeletal development (87). The TTAT regulatory element is recognized by several HOX proteins; however, protein-DNA interactions as well as protein-protein interactions are dependent on the contextual sequence of the core TTAT motif (8, 13, 57). Our analysis of the functional specificity of the Runx2 Hoxa10 site 1 and no other core motifs in the 0.6-kb Runx2 promoter is consistent with other studies evaluating the specificities of Hoxa10 sites (4, 72, 74). Although several HOX proteins are likely present in osteoblast nuclear extracts (e.g., HOXA9), our findings revealed a supershifted/block shifted complex by the HOXA10 antibody. However, it is possible that other HOX protein interactions may occur at this and potentially other HOX motifs within the Runx2 gene. Our data do support the concept that the Runx2 site 1 (TTAT) has a high degree of specificity for HOXA10 binding. Interestingly, the OC gene is characterized by a TTAC core motif, implicating this motif in the formation of complexes that support a high degree of OC expression in mature osteoblasts. It is also noteworthy that the flanking nucleotides are distinct among the four target genes studied here (Table 2). This finding implies that their sequences may hold information for the dynamic association of HOXA10 with an individual gene throughout differentiation.
HOXA10 is expressed in many different tissues in which its functions have been addressed and may be related to its activities in bone tissue. Progesterone and 17ß-estradiol, hormones that regulate turnover of adult bone (53, 63), increase Hoxa10 expression (16, 48). HOXA10 regulates hematopoietic differentiation in part via activation of p21 (WAF1/CIP1), the cyclin-dependent kinase inhibitor (9, 25). In the spinal cord, expression of Hoxa10 is confined to the postmitotic cell population (15). We find that HOXA10 is expressed in proliferating cells and upregulated in postproliferative mature osteoblasts in vitro. HOXA10 is also a target of induction by vitamin D3 (66), a hormone that increases the expression of osteoblast-related genes. Thus, HOXA10 appears to have a broad role in gene regulation for contributing to cell differentiation. Our studies have added to the growing recognition that Hox genes have important functions in the adult, including now the skeleton (55).
In conclusion, our characterization of HOXA10 positive regulation of genes that represent major functional components for bone formation, including those encoding RUNX2 (a bone essential transcription factor), alkaline phosphatase (required for matrix mineralization), bone sialoprotein (an important cell matrix-binding phosphoprotein with hydroxyapatite nucleation capabilities), and OC (the calcium binding bone-specific protein) (47), solidifies the concept that HOXA10 is an important regulator of gene expression throughout the progression of bone formation. These novel functions for HOXA10 in regulating target genes for osteoblast differentiation and bone formation in the postnatal skeleton have broad implications for HOXA10 functions in normal bone metabolism and bone-related disorders.
|
ACKNOWLEDGMENTS |
|---|
The contents of this work are solely our responsibility and do not necessarily represent the official views of the National Institutes of Health.
We thank Richard L. Maas (Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA) for reagents, Anthony Imbalzano (University of Massachusetts Medical School) for helpful discussions, Dana Fredericks for technical assistance, and Judy Rask for manuscript preparation.
|
FOOTNOTES |
|---|
Published ahead of print on 26 February 2007.
Present address: University Orthopedics, Bone and Joint Research Group, General Hospital, Southampton, United Kingdom.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and 1,25(OH)2D3. J. Cell. Biochem. 100:434-449.[CrossRef][Medline]2. Bae, N. S., M. J. Swanson, A. Vassilev, and B. H. Howard. 2004. Human histone deacetylase SIRT2 interacts with the homeobox transcription factor HOXA10. J. Biochem. (Tokyo) 135:695-700.
3. Balint, E., D. Lapointe, H. Drissi, C. van der Meijden, D. W. Young, A. J. van Wijnen, J. L. Stein, G. S. Stein, and J. B. Lian. 2003. Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP-2-mediated osteoblast differentiation. J. Cell. Biochem. 89:401-426.[CrossRef][Medline]
4. Benson, G. V., T. H. Nguyen, and R. L. Maas. 1995. The expression pattern of the murine Hoxa-10 gene and the sequence recognition of its homeodomain reveal specific properties of Abdominal B-like genes. Mol. Cell. Biol. 15:1591-1601.[Abstract]
5. Bondos, S. 2006. Variations on a theme: Hox and Wnt combinatorial regulation during animal development. Sci. STKE 2006:e38.
6. Boulet, A. M., and M. R. Capecchi. 2002. Duplication of the Hoxd11 gene causes alterations in the axial and appendicular skeleton of the mouse. Dev. Biol. 249:96-107.[CrossRef][Medline]
7. Boulet, A. M., and M. R. Capecchi. 2004. Multiple roles of Hoxa11 and Hoxd11 in the formation of the mammalian forelimb zeugopod. Development 131:299-309.
8. Brake, R. L., U. R. Kees, and P. M. Watt. 2002. A complex containing PBX2 contributes to activation of the proto-oncogene HOX11. Biochem. Biophys. Res. Commun. 294:23-34.[CrossRef][Medline]
9. Bromleigh, V. C., and L. P. Freedman. 2000. p21 is a transcriptional target of HOXA10 in differentiating myelomonocytic cells. Genes Dev. 14:2581-2586.
10. Bulger, M. 2005. Hyperacetylated chromatin domains: lessons from heterochromatin. J. Biol. Chem. 280:21689-21692.
11. Carapuco, M., A. Novoa, N. Bobola, and M. Mallo. 2005. Hox genes specify vertebral types in the presomitic mesoderm. Genes Dev. 19:2116-2121.
12. Carpenter, E. M., J. M. Goddard, A. P. Davis, T. P. Nguyen, and M. R. Capecchi. 1997. Targeted disruption of Hoxd-10 affects mouse hindlimb development. Development 124:4505-4514.[Abstract]
13. Chang, C. P., L. Brocchieri, W. F. Shen, C. Largman, and M. L. Cleary. 1996. Pbx modulation of Hox homeodomain amino-terminal arms establishes different DNA-binding specificities across the Hox locus. Mol. Cell. Biol. 16:1734-1745.[Abstract]
14. Chariot, A., C. van Lint, M. Chapelier, J. Gielen, M. P. Merville, and V. Bours. 1999. CBP and histone deacetylase inhibition enhance the transactivation potential of the HOXB7 homeodomain-containing protein. Oncogene 18:4007-4014.[CrossRef][Medline]
15. Choe, A., H. Q. Phun, D. D. Tieu, Y. H. Hu, and E. M. Carpenter. 2006. Expression patterns of Hox10 paralogous genes during lumbar spinal cord development. Gene Expr. Patterns 6:730-737.[CrossRef][Medline]
16. Daftary, G. S., and H. S. Taylor. 2006. Endocrine regulation of HOX genes. Endocr. Rev. 27:331-355.
17. Davis, A. P., D. P. Witte, H. M. Hsieh-Li, S. S. Potter, and M. R. Capecchi. 1995. Absence of radius and ulna in mice lacking hoxa-11 and hoxd-11. Nature 375:791-795.[CrossRef][Medline]
18. de la Fuente, L., and J. A. Helms. 2005. Head, shoulders, knees, and toes. Dev. Biol. 282:294-306.[CrossRef][Medline]
19. Depew, M. J., C. A. Simpson, M. Morasso, and J. L. Rubenstein. 2005. Reassessing the Dlx code: the genetic regulation of branchial arch skeletal pattern and development. J. Anat. 207:501-561.[Medline]
20. Dobreva, G., M. Chahrour, M. Dautzenberg, L. Chirivella, B. Kanzler, I. Farinas, G. Karsenty, and R. Grosschedl. 2006. SATB2 is a multifunctional determinant of craniofacial patterning and osteoblast differentiation. Cell 125:971-986.[CrossRef][Medline]
21. Drissi, H., Q. Luc, R. Shakoori, S. Chuva de Sousa Lopes, J.-Y. Choi, A. Terry, M. Hu, S. Jones, J. C. Neil, J. B. Lian, J. L. Stein, A. J. van Wijnen, and G. S. Stein. 2000. Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene. J. Cell. Physiol. 184:341-350.[CrossRef][Medline]
22. Drissi, H., A. Pouliot, J. L. Stein, A. J. van Wijnen, G. S. Stein, and J. B. Lian. 2002. Identification of novel protein/DNA interactions within the promoter of the bone-related transcription factor Runx2/Cbfa1. J. Cell. Biochem. 86:403-412.[CrossRef][Medline]
23. Ducy, P., R. Zhang, V. Geoffroy, A. L. Ridall, and G. Karsenty. 1997. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747-754.[CrossRef][Medline]
24. Ekker, S. C., D. G. Jackson, D. P. von Kessler, B. I. Sun, K. E. Young, and P. A. Beachy. 1994. The degree of variation in DNA sequence recognition among four Drosophila homeotic proteins. EMBO J. 13:3551-3560.[Medline]
25. Eklund, E. A. 2006. The role of HOX genes in myeloid leukemogenesis. Curr. Opin. Hematol. 13:67-73.[Medline]
26. Favier, B., F. M. Rijli, C. Fromental-Ramain, V. Fraulob, P. Chambon, and P. Dolle. 1996. Functional cooperation between the non-paralogous genes Hoxa-10 and Hoxd-11 in the developing forelimb and axial skeleton. Development 122:449-460.[Abstract]
27. Fromental-Ramain, C., X. Warot, S. Lakkaraju, B. Favier, H. Haack, C. Birling, A. Dierich, P. Dollé, and P. Chambon. 1996. Specific and redundant functions of the paralogous Hoxa-9 and Hoxd-9 genes in forelimb and axial
