YY2 Promotes Osteoblast Differentiation by Upregulating Osterix Transcriptional Activity

Yin Yang 2 (YY2) is a paralog of YY1, a well-known multifunctional transcription factor containing a C-terminal zinc finger domain. Although the role of YY1 in various biological processes, such as the cell cycle, cell differentiation and tissue development, is well established, the function of YY2 has not been fully determined. In this study, we investigated the functional role of YY2 during osteoblast differentiation. YY2 overexpression and knockdown increased and decreased osteoblast differentiation, respectively, in BMP4-induced C2C12 cells. Mechanistically, YY2 overexpression increased the mRNA and protein levels of Osterix (Osx), whereas YY2 knockdown had the opposite effect. To investigate whether YY2 regulates Osx transcription, the effect of YY2 overexpression and knockdown on Osx promoter activity was evaluated. YY2 overexpression significantly increased Osx promoter activity in a dose-dependent manner, whereas YY2 knockdown had the opposite effect. Furthermore, vectors containing deletion and point mutations were constructed to specify the regulation site. Both the Y1 and Y2 sites were responsible for YY2-mediated Osx promoter activation. These results indicate that YY2 is a positive regulator of osteoblast differentiation that functions by upregulating the promoter activity of Osx, a representative osteogenic transcription factor in C2C12 cells.


Introduction
Bone development is maintained by the formation and resorption of osteoblasts. Disruptions in the dynamic balance of formation and resorption, such as excessive resorption, can cause loss of bone mass, density, and strength, resulting in systemic diseases such as osteoporosis [1,2]. Therefore, promoting the formation of osteoblasts and inhibiting excessive resorption by osteoclasts are important, effective strategies for preventing and treating osteoporosis. However, the regulatory mechanism of bone formation and resorption remains under investigation.
Osteoblasts are the main cell type responsible for bone formation. They are derived from mesenchymal stem cells (MSCs), which differentiate into myoblasts, chondrocytes, adipocytes, or osteoblasts, depending on the conditions [3]. MSC differentiation into osteoblasts is controlled by several signaling pathways, including the bone morphogenetic protein (BMP), Wnt, and Notch signaling pathways, and by transcriptional regulators, mainly runt-related transcription factor (Runx2; Cbfa1/AML3), distal-less homeobox 5 (Dlx5), and Osterix (Osx) [4][5][6]. Particularly, Osx plays an important role in the proliferation and differentiation of osteoblasts. Osx, also known as Sp7, has three Cys2His2 (C2H2; Kruppel-type) zinc finger structures at the C-terminus and is highly homologous to the zinc finger structures of other speckled protein (SP) family members (such as Sp1 and Sp4) [7][8][9]. Osx-deficient mice exhibit failed bone formation and osteoblast differentiation and lack the expression of osteoblast-specific markers, such as bone sialoprotein (BSP) and osteonectin. In contrast, cells stably transfected with Osx clearly exhibit an osteoblastic phenotype, suggesting that Osx plays an important role in osteoblast differentiation. The mechanism of Osx function in osteoblast differentiation occurs via the binding of a GC-rich domain in the osteoblast marker gene promoter region [10]. Osx transcription is absent in Runx2-null mice. In contrast, the expression levels of Runx2 in Osx-null mice are similar to those in wild-type mice, suggesting that Runx2 is an important upstream regulator of Osx in the differentiation pathway [11].
Yin Yang 2 (YY2) is a member of the Yin Yang (YY) family and has a highly conserved DNA-binding zinc finger structural domain at the C-terminus. YY2 is highly homologous to another member of the YY family, Yin Yang 1 (YY1). YY2 may function as a transcriptional regulator, similar to YY1 [12,13]. However, unlike YY1, YY2 is not ubiquitously expressed; therefore, the biological function of YY2 has not been identified. Recently, YY2 has been shown to be involved in tumorigenesis as a novel tumor suppressor, with a role in cell cycle regulation and cell proliferation [14]. YY2 is also involved in the response to immune diseases, regulating the promoter activity of interleukin 4 (IL-4) by exerting an antagonistic effect that opposes the effect of YY1 [15]. During osteoblast differentiation, YY1 regulates Runx2 transcriptional activity and inhibits osteoblast differentiation [16]. YY2 is reportedly involved in the pathogenesis of various diseases, but its role in osteoblast differentiation remains unclear.
Here, we present evidence of the role of YY2 in osteoblast differentiation. YY2 promoted BMP4-induced osteoblast differentiation by regulating the promoter activity of Osx and increasing Osx expression.

YY2 Promoted BMP4-Induced Osteoblast Differentiation
To elucidate the role of YY2 in osteoblast differentiation, we first examined the effect of YY2 on BMP4-induced alkaline phosphatase (ALP) expression in C2C12 cells. ALP is an enzyme that is involved in bone mineralization so it is commonly used as an early marker for osteoblast differentiaion [17]. Under the influence of BMP4 in vitro, the myoblast cell line (C2C12) undergoes osteoblast differentiation [18]. Overexpression of YY2 significantly enhanced BMP4-induced ALP activity in a dose-dependent manner ( Figure 1A,B). We also examined the effect of YY2 on the expression of osteogenic markers. Overexpression of YY2 increased the mRNA levels of ALP, osteocalcin (OC) after 2 days of treatment with BMP4 ( Figure 1C,D). These results suggest that YY2 overexpression promotes osteoblast differentiation.

Knockdown of YY2 Attenuated Osteoblast Differentiation
To confirm our findings, we examined the effect of YY2 knockdown on osteob differentiation. The efficiency of the inhibition of endogenous YY2 was described p ously [19]. First, YY2 knockdown dramatically reduced BMP4-induced ALP act (Figure 2A,B). Second, YY2 knockdown significantly decreased the mRNA expressio osteoblast differentiation markers, such as ALP and OC after 2 days of treatment BMP4 ( Figure 2C,D). These results suggest that YY2 knockdown inhibits osteoblast ferentiation.

Knockdown of YY2 Attenuated Osteoblast Differentiation
To confirm our findings, we examined the effect of YY2 knockdown on osteoblast differentiation. The efficiency of the inhibition of endogenous YY2 was described previously [19]. First, YY2 knockdown dramatically reduced BMP4-induced ALP activity (Figure 2A,B). Second, YY2 knockdown significantly decreased the mRNA expression of osteoblast differentiation markers, such as ALP and OC after 2 days of treatment with BMP4 ( Figure 2C,D). These results suggest that YY2 knockdown inhibits osteoblast differentiation.

YY2 Postively Regulates the Promoter Activity of Osteoblast-Specific Marker Genes
To investigate whether YY2 can increase the mRNA levels of osteogenic genes Data are expressed as mean ± SEM of at least three experiments. * p < 0.05, and ** p < 0.01.

YY2 Postively Regulates the Promoter Activity of Osteoblast-Specific Marker Genes
To investigate whether YY2 can increase the mRNA levels of osteogenic genes in a transcription level, we examined whether YY2 could regulate the promoter activity of osteoblast-specific genes such as ALP, BSP, and OC using luciferase reporter, which contains each promoter region. Interestingly, YY2 enhanced the promoter activity of ALP, BSP, and OC in a dose-dependent manner ( Figure 3A). In contrast, the knockdown of YY2 inhibited the promoter activity of ALP, BSP, and OC ( Figure 3B). These results suggest that YY2 positively regulates osteogenic genes in BMP4-induced osteoblast differentiation.

YY2 Upregulats the Expression of Osx
To examine the underlying mechanism of how YY2 does positively regulates the teogenic gene transcription, we investigated the relationship between YY2 and os blast differentiation-related transcription factors such as Runx2 and Osx. First, the ef of YY2 on mRNA levels of Runx2 and Osx. Of note, overexpression of YY2 increased mRNA levels of Osx ( Figure 4A). In contrast, the knockdown of YY2 decreased mRNA levels of Osx ( Figure 4B). Interestingly, neither overexpression nor knockdow YY2 significantly affected the mRNA expression of Runx2. Overexpression of YY2 creased exogenous Osx but did not affect exogenous Runx2 ( Figure 4C). Similarly, O expression of YY2 enhanced endogenous Osx protein expression while it did not af endogenous Runx2 protein expression during osteoblast differentiation (Figure 4 These results indicate that YY2 affects osteoblast differentiation by upregulating the pression of the specific transcription factor Osx.

YY2 Upregulats the Expression of Osx
To examine the underlying mechanism of how YY2 does positively regulates the osteogenic gene transcription, we investigated the relationship between YY2 and osteoblast differentiation-related transcription factors such as Runx2 and Osx. First, the effect of YY2 on mRNA levels of Runx2 and Osx. Of note, overexpression of YY2 increased the mRNA levels of Osx ( Figure 4A). In contrast, the knockdown of YY2 decreased the mRNA levels of Osx ( Figure 4B). Interestingly, neither overexpression nor knockdown of YY2 significantly affected the mRNA expression of Runx2. Overexpression of YY2 increased exogenous Osx but did not affect exogenous Runx2 ( Figure 4C). Similarly, Overexpression of YY2 enhanced endogenous Osx protein expression while it did not affect endogenous Runx2 protein expression during osteoblast differentiation ( Figure 4D). These results indicate that YY2 affects osteoblast differentiation by upregulating the expression of the specific transcription factor Osx.

YY2 Activates the Osx Promoter Activity
Osx exhibits specific expression during in vivo embryonic development and in vitro cell culture, indicating that Osx transcription is tightly controlled via promoter activity [20]. To investigate YY2-mediated regulation of Osx expression, we determined whether YY2 stimulates Osx transcription by using a 3.0 kb (from −3064 to −64) upstream regulatory sequence of the human Osx gene to analyze Osx promoter activity ( Figure 5A). Overexpression of YY2 significantly enhanced Osx promoter activity in a dosedependent manner ( Figure 5B). It was reported that YY2 and YY1 bind to the same DNA region, which contains the YY1 consensus motif (CGCCATnT) [21]. We found that the promoter region of Osx contained two YY1 consensus sequences, aacctCCATattccttgtc (Y1 site) and ctgctCCATttgctgagct (Y2 site) using genematix software. To determine whether the effect of YY2 on Osx occurs at the YY1 consensus motif, we used a reporter assay to analyze the effects of deletions in the 3.0 kb Osx promoter ( Figure 5C). The promoter activity of the 1.4 kb (from −1441 to −64) and 0.7 kb (from −766 to −64), which contained a single consensus site, was significantly decreased compared to that of the 3.0 kb, which contained two consensus sites ( Figure 5D). This suggests that the effect of YY2 on Osx occurs at the YY1 consensus site.

YY2 Activates the Osx Promoter Activity
Osx exhibits specific expression during in vivo embryonic development and in vitro cell culture, indicating that Osx transcription is tightly controlled via promoter activity [20]. To investigate YY2-mediated regulation of Osx expression, we determined whether YY2 stimulates Osx transcription by using a 3.0 kb (from −3064 to −64) upstream regulatory sequence of the human Osx gene to analyze Osx promoter activity ( Figure 5A). Overexpression of YY2 significantly enhanced Osx promoter activity in a dose-dependent manner ( Figure 5B). It was reported that YY2 and YY1 bind to the same DNA region, which contains the YY1 consensus motif (CGCCATnT) [21]. We found that the promoter region of Osx contained two YY1 consensus sequences, aacctCCATattccttgtc (Y1 site) and ctgctC-CATttgctgagct (Y2 site) using genematix software. To determine whether the effect of YY2 on Osx occurs at the YY1 consensus motif, we used a reporter assay to analyze the effects of deletions in the 3.0 kb Osx promoter ( Figure 5C). The promoter activity of the 1.4 kb (from −1441 to −64) and 0.7 kb (from −766 to −64), which contained a single consensus site, was significantly decreased compared to that of the 3.0 kb, which contained two consensus sites ( Figure 5D). This suggests that the effect of YY2 on Osx occurs at the YY1 consensus site.

YY2 Regulates Osx Promoter by Binding YY1 Consensus Motif
To further demonstrate the importance of the YY1 consensus site for the YY2 function, we constructed each single-mutation plasmid (Y1-mt and Y2-mt) ( Figure 6A). Overexpression of YY2 exerted a significantly weaker effect on the mutant Osx promoters than on the wild-type promoters ( Figure 6B). These results indicate that YY2mediated upregulation of Osx expression stimulates Osx promoter activity at the YY1 consensus motif. The above results indicated that YY2 stimulates the Osx promoter at the YY1 consensus motif. To investigate whether YY2 could bind to the proposed binding site (Y2), we performed a ChIP assay using Myc antibody and a primer set flanking the binding site ( Figure 6C). We found that YY2 binds Y2 sites on the Osx promoter ( Figure 6D). These results suggest that YY2 acts as a novel regulator of Osx by binding to the YY1 consensus motif on the Osx promoter.

YY2 Regulates Osx Promoter by Binding YY1 Consensus Motif
To further demonstrate the importance of the YY1 consensus site for the YY2 function, we constructed each single-mutation plasmid (Y1-mt and Y2-mt) ( Figure 6A). Overexpression of YY2 exerted a significantly weaker effect on the mutant Osx promoters than on the wild-type promoters ( Figure 6B). These results indicate that YY2-mediated upregulation of Osx expression stimulates Osx promoter activity at the YY1 consensus motif. The above results indicated that YY2 stimulates the Osx promoter at the YY1 consensus motif. To investigate whether YY2 could bind to the proposed binding site (Y2), we performed a ChIP assay using Myc antibody and a primer set flanking the binding site ( Figure 6C). We found that YY2 binds Y2 sites on the Osx promoter ( Figure 6D). These results suggest that YY2 acts as a novel regulator of Osx by binding to the YY1 consensus motif on the Osx promoter.

Discussion
Bone formation is divided into endochondral ossification and intramembranous sification. These two different patterns of bone development involve a variety of tr scription factors and secreted molecules, indicating the complexity of the bone forma process and the diversity of the regulatory processes [22]. Bone contains two specific types, osteoblasts, and osteoclasts. Osteoblasts are responsible for bone formation. V ous cytokines, growth factors, hormones, signaling pathways, and specific transcrip factors, such as Runx2 and Osx, are involved in the bone formation process by regu ing the proliferation and differentiation of osteoblasts. Mice deficient in either Runx Osx show a complete lack of intramembranous and endochondral ossification, dem strating the importance of both transcription factors in osteoblast differentiation [11].
Sp7/Osx is an osteoblast-specific transcription factor that regulates bone forma and osteoblast differentiation in vivo and in vitro. Osx was concluded to be a do stream factor of Runx2 due to the absence of Osx expression in Runx2-null mice. Rec ly, BMP was found to induce Osx expression in a Runx2-dependent or non-Run dependent manner [23,24]. Osx expression is also regulated by IGF [25], the inflamm ry cytokine TNF-α [26], and parathyroid hormone [27]. This evidence suggests that

Discussion
Bone formation is divided into endochondral ossification and intramembranous ossification. These two different patterns of bone development involve a variety of transcription factors and secreted molecules, indicating the complexity of the bone formation process and the diversity of the regulatory processes [22]. Bone contains two specific cell types, osteoblasts, and osteoclasts. Osteoblasts are responsible for bone formation. Various cytokines, growth factors, hormones, signaling pathways, and specific transcription factors, such as Runx2 and Osx, are involved in the bone formation process by regulating the proliferation and differentiation of osteoblasts. Mice deficient in either Runx2 or Osx show a complete lack of intramembranous and endochondral ossification, demonstrating the importance of both transcription factors in osteoblast differentiation [11].
Sp7/Osx is an osteoblast-specific transcription factor that regulates bone formation and osteoblast differentiation in vivo and in vitro. Osx was concluded to be a downstream factor of Runx2 due to the absence of Osx expression in Runx2-null mice. Recently, BMP was found to induce Osx expression in a Runx2-dependent or non-Runx2-dependent manner [23,24]. Osx expression is also regulated by IGF [25], the inflammatory cytokine TNF-α [26], and parathyroid hormone [27]. This evidence suggests that the regulation of Osx expression by various signal transduction proteins and transcription factors is important for the regulation of osteoblast differentiation [28].
YY2 is a paralog of YY1 with a highly similar structure. Therefore, YY2 exhibits either transcriptional activation or transcriptional repression. Although the C-terminal region of YY2 is similar to that of YY1, the N-terminal region is very different, suggesting that the YY2 protein functions differently from the YY1 protein [12,29,30]. YY2 recognizes the same DNA sequence as YY1 in vitro but exhibits different DNA binding affinities [21]. For example, interleukin (IL)-4, share the DNA binding sequence of YY1 and YY2 but have opposite biological functions [15]. To date, while the regulatory role of YY1 is well understood, the role of YY2 in cellular biological activity, particularly osteoblast differentiation, requires elucidation.
In the present study, our results emphasized the essential role of YY2 in osteoblast differentiation. YY2 overexpression dramatically increased BMP4-induced osteoblast differentiation and stimulated mRNA expression of the osteoblast differentiation markers ALP, OC, and the transcription factors Runx2 and Osx. YY2 also significantly upregulated the promoter activity of ALP, BSP, and OC. Therefore, YY2 and YY1 exhibited opposite phenotypes during osteoblast differentiation. To clarify the molecular mechanisms of YY2-mediated promotion of osteoblast differentiation, we further investigated the relationship between YY2 and osteoblast transcription factors. YY2 specifically stimulated the expression of the transcription factor Osx during osteoblast differentiation. While YY2 did not affect both mRNA and protein levels of Runx2.
Based on our hypothesis that YY2-mediated gene regulation is promoter-dependent, we constructed a luciferase reporter plasmid containing the human Osx promoter. In agreement with our hypothesis, YY2 upregulated Osx promoter activity in a dose-dependent manner. Using Genomatix software, we identified two YY1 consensus sequences located in the Osx promoter region. Two YY1 consensus sequences were identified as essential for YY2-mediated regulation of the Osx promoter. More importantly, YY2 regulated Osx promoter activity in a YY1-independent manner. Subsequent deletions in the Osx promoter revealed that the reduction in promoter activity at the Y1 site in response to YY2 overexpression was significantly smaller than that in the wild-type promoter. Nevertheless, the presence of the Y2 site allowed the activity of the deleted promoter to be maintained. This suggests that the two YY1 consensus sequences are essential for YY2-mediated regulation of the Osx promoter.
To further demonstrate the importance of the consensus sequences, mutations were introduced into the Y1 and Y2 sites, separately and simultaneously. Consistent with our hypothesis, the promoter activity associated with mutations in the Y1 or Y2 sites was significantly reduced. Furthermore, we found that YY2 binds to the Y2 site in the promoter region of Osx, but not the Y1 site. The mutated Y1 site loses the induction of YY2 and we retain the possibility that there are other factors on the Y1 site involved in the regulation of the Osx promoter by YY2 that require further elucidation. These findings highlighted the importance of the YY1 consensus motif for YY2-mediated regulation of the Osx promoter, and we determined that YY2 regulated Osx promoter activity and expression by binding to the YY1 consensus motif, especially the Y2 site. Moreover, YY2 knockdown decreased ALP activity and osteogenic gene expression, resulting in the suppression of osteoblast differentiation.
In summary, we provide novel evidence of the essential role of YY2 in osteoblast differentiation. Our findings also indicate that Osx is a major target of YY2. YY2 increased osteoblast differentiation by binding with the Osx promoter to regulate promoter activity and gene expression.

Alkaline Phosphatase (ALP) Staining
C2C12 cells were cultured for 72 h in 2% FBS medium containing BMP4 to induce differentiation. The cells were washed twice with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde at room temperature (RT) for 15 min, washed twice again with PBS, and then incubated in BCIP/NBT substrate (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at RT. Quantification of ALP staining was performed at an absorbance of 480 nm.

Luciferase Assay
C2C12 cells were seeded in 24-well plates at a density of 2 × 10 4 cells/well. The cells were transfected with a CMV promoter-driven β-galactosidase reporter gene (pCMV-βgal) (0.05 µg) and luciferase reporter gene (0.3 µg), and the cell lysates were analyzed for luciferase activity 36 h after differentiation. A luciferase reporter gene assay kit (Promega, Madison, WI, USA) was used to measure luciferase activity on the TriStar 2 Multimode Reader platform. All experiments were performed in triplicate.

RNA Isolation and RT-qPCR
Total mRNA was extracted from cultured C2C12 cells using RNAiso Plus (Total RNA Extraction Reagent; TaKaRa, Tokyo, Japan). cDNA was synthesized from 1 µg of total RNA using Oligo dT primers and reverse transcriptase (Promega, Madison, WI, USA). cDNA was synthesized using SYBR Premix Ex Taq kit (TaKaRa) on a CFX96 real-time PCR system(Bio-Rad Laboratories, Hercules, CA, USA). The following PCR conditions were used: initial denaturation at 94 • C for 3 min, 40 cycles of denaturation at 94 • C for 30 s, and annealing at the optimal temperature for each primer pair for 30 s. The primer sequences used for PCR were as follows: mALP forward 5 -ATC TTT GGT CTG GCT CCC ATG-3 and reverse 5 -TTT CCC GTT CAC CGT CCA C-3 ; mOC forward 5 -GCA ATA AGG TAG TGA ACA GAC TCC-3 and reverse 5 -GTT TGT AGG CGG TCT TCA AGC-3 ; mRunx2 forward 5 -CCT GAA CTC TGC ACC AAG TCC T-3 and reverse 5 -TCA TCT GGC TCA GAT AGG AGG G-3 ; mOsx forward 5 -TCG CAT CTG AAA GCC CAC TT-3 and reverse 5 -CTC AAG TGG TCG CTT CTG GT-3 ; GAPDH forward 5 -AGG TCG GTG TGA ACG GAT TTG-3 and reverse 5 -GGG GTC GTT GAT GGC AAC A-3 .