MAST4 regulates stem cell maintenance with DLX3 for epithelial development and amelogenesis

The asymmetric division of stem cells permits the maintenance of the cell population and differentiation for harmonious progress. Developing mouse incisors allows inspection of the role of the stem cell niche to provide specific insights into essential developmental phases. Microtubule-associated serine/threonine kinase family member 4 (Mast4) knockout (KO) mice showed abnormal incisor development with low hardness, as the size of the apical bud was decreased and preameloblasts were shifted to the apical side, resulting in amelogenesis imperfecta. In addition, Mast4 KO incisors showed abnormal enamel maturation, and stem cell maintenance was inhibited as amelogenesis was accelerated with Wnt signal downregulation. Distal-Less Homeobox 3 (DLX3), a critical factor in tooth amelogenesis, is considered to be responsible for the development of amelogenesis imperfecta in humans. MAST4 directly binds to DLX3 and induces phosphorylation at three residues within the nuclear localization site (NLS) that promotes the nuclear translocation of DLX3. MAST4-mediated phosphorylation of DLX3 ultimately controls the transcription of DLX3 target genes, which are carbonic anhydrase and ion transporter genes involved in the pH regulation process during ameloblast maturation. Taken together, our data reveal a novel role for MAST4 as a critical regulator of the entire amelogenesis process through its control of Wnt signaling and DLX3 transcriptional activity.

Horseradish peroxidase-conjugated antibodies (Millipore) were used as secondary antibodies.The peroxidase reaction products were visualized with WESTZOL (Intron).All signals were detected by Amersham Imager 600 (GE Healthcare Life Sciences).

Real-Time quantitative PCR (RT-qPCR)
For the RT-qPCR, the total RNA of the cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA).The extracts were reverse transcribed using Maxime RT PreMix (#25081, iNtRON, Korea).RT-qPCR was performed using a StepOnePlus Real-Time PCR System (Applied BioSystems, USA).The amplification program consisted of 40 cycles of denaturation at 95°C for 15 s and annealing at 60 ℃ for 30 s.The expression levels of each gene are expressed as normalized ratios against the B2m housekeeping gene.The oligonucleotide primers for RT-qPCR are described in Supplementary Table 1.

Chromatin IP (ChIP) assay
Cells were cross-linked with 4% paraformaldehyde for 10 minutes at room temperature.Glycine was added to a final concentration of 125 mM for 5 minutes to quench the formaldehyde crosslinks.Cells were washed with ice-cold phosphate buffered saline, harvested by scraping, pelleted, and resuspended in SDS lysis buffer (50 mM Tris-HCl [pH 8.1], 1% SDS, 10 mM EDTA) with complete protease inhibitor cocktail (Roche).Cell extracts were sonicated with a Bioruptor TOS-UCW-310-EX (output, 250W; 25 cycles of sonication with 30seccond intervals; Cosmo Bio).Samples were centrifuged at 14,000 rpm at 4℃ for 10 minutes, and the supernatants were diluted 10-fold in dilution buffer (20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 1% Triton X-100, 150 mM NaCl, and complete protease inhibitor cocktail).Chromatin samples were precleared with protein Aagarose beads (Santa Cruz) for 2h before immunoprecipitation against Flag (Sigma-Aldrich) antibodies overnight at 4 ℃.Immune complexes were collected with protein A-agarose beads.Samples were washed five times (first wash with low salt immune complex wash buffer [20 mM Tris-HCl, pH.8.0, 2 mM EDTA, 1% Triton X-100, 0.1 % SDS, and 150 mM NaCl], second wash with high salt immune complex wash buffer [20 mM Tris-HCl, pH.8.0, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, and 500 mM NaCl], third wash with LiCl immune complex wash buffer [10 mM Tris-HCl, pH.8.0, 1 mM EDTA, 250 mM LiCl, 1% NP-40, and 1% Na-deoxycholate], and the last two washes with TE buffer).Immunoprecipitated samples were eluted with buffer containing 1% SDS and 100 mM NaHCO3 at room temperature.Eluates were heated overnight at 65℃ to reverse crosslinks after adding NaCl to a final concentration of 100 mM.Genomic DNA was extracted with a PCR purification kit (GeneAll).Precipitated chromatin by real-time PCR and the readouts were normalized using 5% input chromatin for each sample.The experiments were repeated two or more times.
Primers for ChIP-PCR.

Site-directed mutagenesis
Using the Flag-tagged wild-type Dlx3 plasmid (Flag-DLX3 or DLX3 WT ) as a template, PCR-based mutagenesis was performed using a primer containing the desired mutation site (T134, S136, and S137).We generated mutations that mimicked either inactive phosphorylation status (serine/threonine to alanine) or activated phosphorylation status (serine/threonine to glutamic acid).The completed PCR product was cut with DpnI for 2 h and transformed into E. coli DH5alpha competent cells by heat shock.Colonies from each plate were grown and DNA was extracted.Mutations were verified by sequencing.

Luciferase assay
The HEK293T cells were transiently transfected with 3X DRE-Luc and HA-MAST4-Full, 3Flag-DLX3 and series of mutation constructs using polyethylenimine (Polysciences, Inc).After 24 h, cells were lysed and the luciferase activities were analyzed using the Luciferase Assay System kit (Promega) according to the manufacturer's protocol.All assays were done in triplicate, and all values were normalized for transfection efficiency against β-galactosidase activities.

CA6
AGC AAA GGG TGG TTT AGC C AGG AAC AGG GAC AGC AGA AG CA12 GCT ACA ACT CAC CTA GGA TCT GG CTT CGC TCT CCT GGC TTC SLC24A1 GAG GGA ACA ACG CAC ATT CT CCC AGT CTC TGC TTT CAA GG SLC26A1 TCC TAC AAG CCC CTG ATT ACA GTC TGT GCC CTG GAC TCT G AMELX TTT CTT TCT CTG CAT TTC TTT T GAC AAG CAC TTT GTT GCA TT ENAM TAG GTT TCA GCT CCC AGG TT TGA GTT TTT CCC CCT TTA CTT