Molecular Switches Involving Homeodomain Proteins, HOXA10and RUNX2 Regulate Osteoblastogenesis

Mohammad Q. Hassan1, Sharanjot Saini1, Jonathan A. R. Gordon1, Andre J. van Wijnen1,Martin Montecino2, Janet L. Stein1, Gary S. Stein1, and Jane B. Lian1,*

1 Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester,MA 01655

2 Universidad de Concepcion, Facultad de Ciencias Biologicas, Departamento de Bioquimica y BiologiaMolecular, Concepcion, Chile

AbstractThe osteoinductive BMP2 signal facilitates commitment to the osteoblast phenotype by inducingseveral classes of early response genes. Among these are bone related HOX factors, Homeodomain(HD), RUNX and OSTERIX proteins. Here we demonstrate molecular events among BMP2 inducedtranscription factors that constitute a network of molecular switches on promoters of bone relatedgenes to coordinate their temporal expression during cellular differentiation. Our studies provideevidence for (i) selective association of HOXA10, MSX2, DLX3 and DLX5 homeodomaintranscription factors on Runx2 and Osteocalcin genes at stages of osteoblast maturation; and (ii)participation of these factors with RUNX2 in chromatin remodeling of bone specific genes forrepression, activation, and attenuation of transcription. These findings reveal the requirement formultiple levels of control for the appropriate timing of osteoblast related gene expression.

KeywordsOsteoblasts; Gene Regulation; Homeodomain; Chromatin Remodeling; Runx2; MSX2; DLX3;DLX5

INTRODUCTIONAmong the principal regulatory cascades for skeletal development are Hox genes thatdetermine the timing, position and shape of a tissue element during embryogenesis. Hox andother homeodomain (HD) transcription factors also function in the adult skeleton to supportosteoblast differentiation (Zakany and Duboule, 2007; Depew et al., 2005). The BMP2osteogenic signal activates several Hox and HD proteins as early response genes. HOXA10and DLX-3 and -5 HD proteins were characterized as enhancers of osteoblast gene transcriptionon gene promoters (Hassan et al., 2007; Hassan et al., 2006). RUNX2, the transcription factoressential for bone formation, interacts with genes on mitotic chromosomes as an epigeneticmechanism for development of the osteoblast phenotype (Young et al., 2007b), attenuatesglobal protein synthesis (Young et al., 2007a), and integrates osteogenic signals throughphosphorylation of Runx2 and formation of complexes with the coregulatory proteins thattransduce the response to gene targets (Lian et al., 2006). Here we have used two signatureosteoblast specific gene promoters (Runx2 and OC) as models to probe interactions of

*Address Correspondence to: Jane B. Lian, PhD, Department of Cell Biology, University of Massachusetts Medical School, 55 LakeAvenue North, Worcester, MA 01655. Tel: 508–856–5625; Fax: 508–856–6800; Email: Jane.Lian@umassmed.edu.

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Published in final edited form as:Cells Tissues Organs. 2009 ; 189(1-4): 122–125. doi:10.1159/000151453.

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HOXA10, MSX2, DLX3, DLX5 and RUNX2 with chromatin of target genes for their inducedand physiological expression during stages of osteoblastogenesis.

MATERIALS and METHODSWestern Blotting

Electrophoresis conditions are previously described (Hassan et al., 2006). ChIP assays. Theprocedures for chromatin immunoprecipitation (ChIP) in primary rat osteoblasts are detailedelsewhere (Hassan et al., 2004; Hassan et al., 2006). The crosslinked rat calvarial osteoblastcells were lysed and sonicated using appropriate conditions. Precleared soluble chromatinswere immunoprecipitated with specific antibodies. DNA fragments were purified from theimmunocomplexes and specific promoters analyzed by PCR. siRNA: HOXA10 siRNA wasobtained from Qiagen and protein knockdown tested against a non-specific siRNA duplex oligousing antibodies to HOXA10 from Santa Cruz (Hassan et al., 2007).

RESULTSTranscriptional mechanisms that program osteoblast growth and differentiation include DNAbinding of regulatory complexes and chromatin modifications for activation and repression ofa gene during stages of differentiation (Fig 1A). Western analyses reveal changing proteinlevels of the transcription factors during osteoblast maturation (Fig 1B). Although MSX2decreases, RUNX2, DLX3, and DLX5 increase to maximal levels with formation of bonenodules (day 12 in primary rat calvarial cells). The ChIP studies define the in vivo occupancyof the factors on the respective gene promoters (Fig 1C,D). We find a temporal recruitment ofHD proteins in relation to Runx2 (Fig 1C) and OC (Fig 1D) transcription. MSX2 is associatedwith both promoters only in the proliferation period (day 4) and is no longer bound to both ofthem by day 12 despite significant MSX2 cellular protein levels (Fig 1B). On day 12 bothDLX3 and DLX5, together with RUNX2, are recruited to the Runx2 and OC promoters. Aconcomitant increase in RNA POL II occurs at this time reflecting induced transcription.However, DLX3 binding to Runx2 is transient in the matrix maturation stage of differentiationwith preferential binding of DLX5 at later stages. This suggests an important role for DLX5in the HD network to regulate terminal differentiation. In the mineralization stage, an increasedassociation of RUNX2 to OC is accompanied by maximal expression of the gene. However,the increased binding of RUNX2 to its own promoter results in a slight decrease in cellularprotein; yet, transcription is maintained as indicated by RNA POL II binding and likelysustained by DLX5 binding. Thus Runx2 and OC are examples of a coordinated temporalregulation by an HD network, and with RUNX2, these factors support the timing of expressionand physiological levels of target genes during osteoblast differentiation.

The early association of HOXA10, prior to RUNX2, on bone related gene promoters whenexpression is very low (Runx2) or not detected (BSP, OC), indicates a specialized function (Fig1C,D). We had reported the effects of HOXA10 depletion (by siRNA) on histone acetylationand H3K4 methylation of gene promoters, which reflect transcriptionally active chromatin(Hassan et al., 2007). Our findings showed a significant decrease in acetylation of Runx2,BSP and OC chromatin in the absence of HOXA10 in osteoblasts. From these studies, a patternemerges that defines functional activities of HOXA10, HD proteins and RUNX2 and amechanism for activating gene transcription and regulating their temporal expression profileduring stages of osteoblast maturation.

DISCUSSIONThere is a requirement for chromatin remodeling of genes silenced by condensed chromatinfor BMP2 induction of osteogenesis (Young et al., 2005). The signals for induction of tissue

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specific genes occur at multiple levels of control as indicated by the presented studies (Fig 2).RUNX2 functions in bone specific activation by supporting chromatin modification. This wasestablished by mutation of the Runx2 sites in the OC gene (Javed et al., 1999). We proposethat the recruitment of HOXA10 to gene promoters prior to RUNX2 binding may fulfill severalroles. HOXA10 complexes with the co-repressors PBX and MEIS and co-activators such asp300 (Capellini et al., 2006; Saleh et al., 2000). Its binding to bone promoters prior to Runx2suggests an epigenetic modification without transcription that later contributes to activation.Another level of gene regulation is the need for physiological control of transcription drivenby cellular representation of the factors and formation of heterodimeric complexes with co-regulatory factors (Fig 2) that may alter DNA binding affinity. We postulate that the MSX andDLX HD proteins function to regulate the timing of stage specific gene expression coordinatedwith requirement for RUNX2 and HOXA10. In summary, combinatorial mechanisms areoperative for regulated transcription of osteoblast genes through diversification of bothsequence-specific activators/repressors that contribute to patterns of gene expression and themultistep process of programming involved in bone formation.

AcknowledgementsThis study was supported by National Institutes of Health grants DE12528 and AR39588. The contents of thismanuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NationalInstitutes of Health.

AbbreviationsOC

Osteocalcin

BSP Bone Sialoprotein

HDAC Histone deacetylases

BMP bone morphogenetic protein

HD homeodomain proteins

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Fig 1. Regulation of Stage Specific Expression of HD proteins (MSX2, DLX3, DLX5), HOXA10and RUNX2 Transcription Factors during Osteoblast DifferentiationA. Schematic of osteogenic lineage cells and expression of regulatory and signaling factors atdifferent stages of osteogenesis. B. HD proteins exhibit different temporal expression profilesduring development of the osteoblast phenotype. C and D. Molecular switches and temporalrecruitment of HD (MSX2, DLX3 and DLX5), HOXA10 and RUNX2 and coordinate changesin binding of RNA Polymerase II (Pol II) to the Runx2 (C) and OC (D) promoters. Stagesshown are Growth (day5), Matrix formation (day12) and Mineralization (day20) of primaryfetal rat calvarial cell cultures.

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Fig 2. Mechanisms for Coordinated Control of Transcription Factors and Chromatin Modifiersfor Regulating the Program of Osteoblast DifferentiationA general model is proposed for the sequential expression of osteoblastic genes. Inosteoprogenitor cells, the initial transcription complexes (e.g., MSX2; HOXA10) arecomposed of transcription factors partnering with repressor coregulatory proteins. RNA Pol IIbinding is presented as a result of epigenetic modifications to poise the genes for activatedtranscription in osteoblasts. For induced transcription, HOXA10 and RUNX2 recruit differentcoregulatory factors to become activation complexes and DLX3 and DLX5 become bound tothe genes. This unique mechanism of switching occupancy of transcription factor complexesregulates gene specific expression during osteoblast differentiation.

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