Human study protocols and patient consents were reviewed and approved by the Institutional Review Board at the University of Alabama at Birmingham. CCD-011 dental pulp cells and age- and sex-matched control dental pulp cells from non-affected healthy individual were grown in αMEM supplemented with 10% FBS, ascorbic acid (50 μg/ml), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C with 5% CO2. For cell differentiation, cells were cultured with growth media above supplemented with 10 mM β- glycerophosphate for indicated time.

Total RNA from CCD-011 and control pulp cells were isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocols. Next generation RNA Sequencing (RNA-Seq) was performed at the Heflin Center for Genomic Sciences Genomic Core. Briefly, mRNA sequencing was performed on Illumina HiSeq2000 platform. Total RNA was assessed using the Agilent 2100 Bioanalyzer followed by 2 rounds of poly A+ selection and conversion to cDNA. The TruSeq RNA Library Prep Kit were followed according to the manufacturer’s instructions (Illumina, San Diego, CA, United States). Library construction consisted of random fragmentation of the polyA mRNA, followed by cDNA production using random primers. The ends of the cDNA were repaired and A-tailed, and adaptors were ligated for indexing (up to 12 different barcodes per lane) during the sequencing runs. The cDNA libraries were quantitated using quantitative PCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA, United States) prior to cluster generation. Clusters yielded approximately 725K–825K clusters/mm². Cluster density and quality was determined during the run after the first base addition parameters were analyzed. Paired end 2 × 50 bp sequencing runs were run to align the cDNA sequences to the reference genome. Because CCD sample with allelic RUNX2 deletion is very rare, we have collected one sample (CCD-011) so far which was used in this experiment. The data has been deposited to the sequence read archive¹.

Image files from the sequencer were converted to raw sequence fastq files using the Illumina compute server running CASAVA version 1.8.2. Quality control of fastq files was checked with FastQC version 0.10.1 and found that no trimming or removal of poor quality sequences was needed. Alignments of the raw sequence reads to the UCSC human hg19 reference genome was performed using TopHat version 2.0.9 with the following parameters: –library-type fr-unstranded; -r 150. Transcript abundances was calculated using Cufflinks version 2.1.1 with the following parameters: -g; -b; -u. Cuffmerge was then used to merge the two transcript abundance files together, followed by pairwise differential expression with Cuffdiff (parameters used: -u; -b).

The gene lists from Cuffdiff were uploaded to Ingenuity Pathway Analysis and the Core Analysis was used to identify significant interactions, downstream effects, and pathways.

Total RNA were isolated from indicated cells as described as above. Single-strand cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., United States). Primer sets for IGFBP2, GREM1 (Gremlin 1, DAN Family BMP Antagonist), BARX1 (BarH-like homeobox 1), and ALPL (alkaline phosphatase, liver/bone/kidney) were purchased from the Integrated DNA Technologies (IDT, Coralville, Iowa., United States). Other primer sets were synthesized by IDT as follows: TFAP2A(transcription factor AP-2 alpha)-For: 5′-GAGCCATGGCACGCACGAGACGGTATCTA-3′, TFAP2A-Rev: 5′-GAGCTCGAGCTCGCAGTCCTCGTACTTGA-3′; LYPD6B (LY6/PLAUR Domain Containing 6B)-For: 5′-GTTTCCTGACCCGTGAAATG-3′, LYPD6B-Rev: 5′-GTCCCGTCCAGATGTTGG-3′; RUNX2-For: 5′-TTACTTACACCCCCCAGTC-3′, RUNX2- Rev: 5′-CACTCTGGCTTTGGG AAGAG-3′;and endogenous control GAPDH-For: 5′-AGGTCGGAGTCA ACGGATTTG-3′, GAPDH-Rev: 5′-GGGGTAACTGTGC-CTATTCG-3′. Quantitative PCR using SYBR Green SuperMix (Qiagen) was performed as we previously described (Lu et al., 2014). The level of mRNA expression was measured using threshold cycle (CT) according to the ΔΔCT method (Livak and Schmittgen, 2001).

Recombinant pLenti-RUNX2 was customarily generated by VectorBuilder (Santa Clara, CA) by inserting normal full length of human RUNX2 into lentiviral expression empty vector. Lentivirus were prepared by transfection of expression vector, either pLenti-RUNX2 or Lentiviral empty vector together with packaging vectors (pMD2.G and psPAX.2; Addgene, Cambridge, MA, United States) into HEK-293T cells. The viruses were concentrated and titered. CCD-011 dental pulp cells were infected with empty lentivirus (lenti-Con) or recombinant lentivirus (lenti-RUNX2), respectively at the MOI of 0.5. Cells were further selected using G418 (20 μg/ml) for 1 week.

A 1.5 kb fragment of the proximal IGFBP2 promoter was amplified with the following primer set: IGFBP2-For: 5′-CAGGGTACCCTGTGCCCTTGCTAACCGCCCATTTC-3′, IG FBP2-Rev: 5′-CAGGCTAGCCGGGTCCTAAGGGCCGGCTTCTCC-3′ (restriction enzyme site KpnI and NheI were underlined). The DNA fragment was inserted into the luciferase reporter vector pGL4.18 [luc2P/Neo] (Promega Corporation, Madison, Wisconsin, United States) by KpnI and 3′ NheI (IGFBP2-pGL4.18). For luciferase reporter assay, 5 × 10⁴ HEK293T cells were cultured in 12-well plates overnight. Next day, cells were transfected with 1 μg of with lentiviral empty vector or lenti-RUNX2 together with 20 ng of IGFBP2-pGL4.18 and Renilla luciferase pGL4.74 [hRluc/TK] plasmid (Promega, Madison, WI, United States) using PolyJet^TM In Vitro DNA Transfection Reagent (SignaGen Laboratories, Rockville, MD, United States). At 48 h post transfection, luciferase activity in each well was measured by Dual-Luciferase^® Reporter Assay System (Promega) and normalized to Renilla.

Histological sections (5 μm) of postnatal day 5 mice tissue samples were prepared for immunohistochemistry as previously described (MacDougall et al., 1998). Immunostaining was performed using primary antibodies against IGFBP2 (Cell Signaling Technologies, Danvers, MA, United States). After deparaffinization, heat induced citrate antigen retrieval and blocking with 3% goat serum, 1% bovine serum albumin, and 0.5% tween in phosphate-buffered saline for 20 min at RT, mouse tooth sections were incubated with primary antibody for 1 hour at room temperature, followed by horseradish peroxidate (HRP) poly conjugate for 10 min and 3,3′-diaminobenzidine for optimal time. Images were visualized and captured by Nikon Eclipse TE2000-E microscope (Nikon Instruments, Melville, NY, United States).

In order to identify novel RUNX2 target genes involved in tooth formation and signaling pathways that potentially contribute to the dental phenotypes seen in CCD, Next-Generation RNA Sequencing was performed on CCD-011 dental pulp cells carrying a total RUNX2 deletion in one allele and compared to one age- and sex-matched control pulp cells. Of the 25,643 genes analyzed, 11,039 genes had no detectable signal in both CCD and control samples tested, leaving 14,604 genes that were evaluated for differential gene expression. In the detectable genes, 60 transcripts (4.1%) were found to be statistically significantly dysregulated with 63% upregulated and 27% downregulated (fold change ≥2; q-value < 0.05). The top 10 genes with differential change are listed in Table 1. Analysis of isoform differential expression revealed 35% downregulated and 65% upregulated transcripts. The isoforms with twofold statistically significant change are listed in Supplementary Table 1. Ingenuity Pathway Analysis revealed top up- and down-regulated genes delineating multiple putative RUNX2 targets genes both upstream and downstream (Supplementary Figures 1, 2).

Among 60 genes identified by RNA-Seq, six genes (Figure 1A), TFAP2A, GREM1, BARX1, ALPL, LYPD6B, and IGFBP2 have been previously reported to be associated with osteogenesis and/or craniofacial development (Eckstein et al., 2002; Stoetzel et al., 2009; Tekin et al., 2009; Chung et al., 2012; Leijten et al., 2013; Nichols et al., 2013; Gasque et al., 2015). Their dysregulation in CCD-001 pulp cells was further confirmed by qPCR (Figure 1B). Four genes: TFAP2A, GREM1, BARX1, ALPL have been reported to be involved in craniofacial and/ or tooth development (Milunsky et al., 2008; Mitsiadis and Drouin, 2008; Nagatomo et al., 2008; Li et al., 2013; Liu et al., 2014). However, the remaining two genes, LYPD6B and IGFBP2, have not been described in tooth development.

To investigate the role of RUNX2 in the regulation of target genes involved in CCD, CCD-011 pulp cells were transduced with lenti-RUNX2 to introduce the full length of human RUNX2 (Figure 2A). Cells transduced with empty lentivirus was used as control. As seen in Figure 2B, there was almost fivefold higher level of RUNX2 in CCD-011 pulp cells transduced with Lenti-RUNX2 in comparison to that in cells transduced with empty lentivirus. To investigate if RUNX2 introduction could rescue the dysregulation of the six genes identified by RNA-Seq in CCD-011 pulp cells, their expression was quantitatively analyzed. As seen in Figure 2C, the expression of TFAP2A, LYPD6B, and ALPL was increased in CCD-011 pulp cells after introducing normal human RUNX2, indicating the positive regulation of their expression by RUNX2. This finding also suggests that their up-regulation in CCD-011 dental cells seen in Figure 1 may be due to individual variation. However, the expression of IFGBP2, GREM1, and BARX1 was down-regulated in CCD-011 pulp cells with normal RUNX2 introduction. Under differentiation condition, IGFBP2 expression was further down-regulated by RUNX2 overexpression distinct from the up-regulation of ALPL (Figure 2D). Taken together, these findings indicate that introduction of normal human RUNX2 gene into CCD-011pulp cells can partially rescue the dysregulation of gene expression in these cells.

IGFBP2 is a highly conserved family of six IGFBPs that circulate in serum and local biological fluid at relatively high concentrations. IGFBP2 serves as carrier proteins through high binding to IGFs and regulates their bioactivity (Ehrenborg et al., 1991; DeMambro et al., 2008). To determine if RUNX2 directly regulates IGFBP2, the human IGFBP2 promoter was first analyzed for putative RUNX2 binding sites based on previous reports (Ducy et al., 1997; Chen et al., 2002). As shown in Figure 3A, in the 1.45 kb segment of the proximal promoter, there are total of four potential RUNX2 binding sites. This promoter fragment was then amplified and inserted into luciferase reporter vector (IGFBP2-pGL4.18). HEK293T cells were transfected with lentiviral empty vector or lenti-RUNX2 together with luciferase IGFBP2-pGL4.18 and Renilla vector for luciferase assay. As shown in Figure 3B, there was significant lower level of luciferase activity in HEK293T cells with lenti-RUNX2 compared with that in cells with lentiviral empty vector, demonstrating that IGFBP2 is a direct target of RUNX2.

To determine the protein expression of IGFBP2 in the dental tissue during tooth development, postnatal day-1 and -5 mouse tooth sections were analyzed by immunohistochemistry. IGFBP2 was detected in skeletal muscle, alveolar bone, the odontoblasts, throughout the dental pulp, and within the oral epithelium (Figure 4). Staining was also seen within the stellate reticulum region associated with blood vessels. No staining was detectable within the epithelial components of the tooth including the ameloblasts, stellate reticulum, and outer enamel epithelium. These findings strongly suggest IGFBP2 may play a functionally important role during odontogenesis associated with dentinogenesis.