Biomedical Research (Tokyo) 41 (5) 217–225, 2020 Intracellular zinc-dependent TAS2R8 gene expression through CTCF activa- tion 1 1, 2 2 1, 3 2, 4 Tsuyoshi KOJIMA , Toyonobu MAEDA , Atsuko SUZUKI , Tetsuo YAMAMORI , and Yasumasa KATO Departments of 1 Oral Rehabilitation and 4 Oral Physiology and Biochemistry, Ohu University Graduate School of Dentistry, Koriyama 963-8611, Japan; Departments of 2 Oral Function and Molecular Biology and 3 Prosthetic Dentistry, Ohu University School of Dentistry, Koriyama 963-8611, Japan (Received 21 April 2020; and accepted 7 June 2020) ABSTRACT Taste-2 receptors (TAS2Rs), which belong to the G-protein coupled receptor (GPCR) family, are receptors for bitter taste perception. The aim of this study was to investigate whether zinc defi- ciency affects the expression of TAS2R genes. The promoter activity of the TAS2R7, TAS2R8, and TAS2R42 genes were determined in Ca9-22 oral squamous cell carcinoma cells cultured in the presence or absence of zinc. Luciferase reporter assays showed that zinc deprivation inhibited TAS2R8 promoter activity, but not the promoter activity of the other two genes. Treatment of the cells with N,N,N’,N’-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), an intracellular chela- tor of Zn2+, in the presence of 10% fetal bovine serum reduced TAS2R8 promoter activity. Trunca- tion/deletion mutants of TAS2R8 promoter-luciferase constructs showed that the region from nucleotide −1152 to nucleotide −925 was critical for intracellular zinc dependency and contained a CCCTC-binding factor (CTCF) binding motif. A chromatin immunoprecipitation (ChiP) assay showed that CTCF bound specifically to this region, a binding abrogated by zinc deficiency, sug- gesting that CTCF plays a critical role in zinc-dependent bitter taste perception through TAS2R8. TAS2R, which belong to the G-protein coupled re- INTRODUCTION ceptor (GPCR) family. TAS1R and TAS2R each has Basic taste signals are categorized into five groups, isotypes, with various monomers and heterodimers salt, sour, sweet, bitter, and umami, with these sen- functioning as different types of taste receptors. For sory systems evolving to allow nutrition and avoid example, the heterodimers TAS1R1/TAS1R3 and potentially noxious and/or poisonous chemicals TAS1R2/TAS1R3 act as taste receptors for umami (Chandrashekar et al. 2006). In humans, taste is an and sweet, respectively (Li et al. 2002; Nelson et al. important sensory system for enjoyment of food and 2002), and all TAS2R monomers, comprised of ≈30 drink. Taste perceptions are associated with different subtypes, are utilized for bitter taste perception receptor molecules, including sodium channels for (Martin and Dupré 2016). A comparison of TAS2R8 salt, polycystic kidney disease 2-like 1 (PKD2L1) and TAS2R39 showed that they differ in recogniz- for sour, and taste receptors (TASRs) for sweet, bit- ing specific bitter tastes, with each TAS2R subtype ter and umami (Martin and Dupré 2016). TASRs having high ligand specificity (Ueno et al. 2011). can be categorized into two groups, TAS1R and Because zinc is an important co-factor for matrix metalloproteinases (MMPs) and zinc finger proteins, Address correspondence to: Yasumasa Kato, Department its deprivation for long periods of time reduces the of Oral Function and Molecular Biology, Ohu Universi- ty School of Dentistry, Koriyama 963-8611, Japan The work is part of the Ph.D. thesis of Tsuyoshi Tel: +81-24-932-8978 Kojima at Ohu University Graduate School of Dentist- E-mail: [email protected] ry, Koriyama, Japan. 218 T. Kojima et al. activity of these proteins. Zinc finger is a protein dual-luciferase® reporter assay system was from motif that stabilizes protein folding by coordination Promega (Madison, WI, USA). The intracellular with one or more Zn2+ ions. The nuclear protein membrane-permeable ion chelator N,N,N’,N’- CCCTC-binding factor (CTCF), an 11-zinc finger tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) protein, functions as an insulator (Ong and Corces and the insoluble formazan form of 3-(4,5-dimeth- 2014). Insulators mediate intra- and inter-chromo- ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide somal interactions and play a role in allowing or (MTT) were from Dojindo (Kumamoto, Japan). preventing three-dimensional folding between en- Xfect transfection reagent, PrimeSTAR GXL and hancers and promoters in distal regions, thereby in- In-Fusion® HD Cloning Kit were from Takara (To- ducing or repressing gene transcription. CTCF null kyo, Japan). mice are embryonically lethal (Moore et al. 2012), suggesting that CTCF-target molecules have played Antibodies. Anti-CTCF rabbit monoclonal antibody important roles in morphogenesis during evolution. (mAb) was purchased from abcam (Cambridge, UK) CTCF promotes alternative splicing by RNA poly- and mouse IgG1 (isotype control) was from MBL merase II through a process of exon inclusion (Nagoya, Japan). (Shukla et al. 2011) and also contributes to genomic imprinting (Fedoriw et al. 2004). In breast cancer, a Cells and cell culture. HEK293 cells, which were CTCF binding motif was found in the 3’ flanking derived from human embryonic kidney, were pur- region of the MMP7 gene, and a single nucleotide chased from the Japanese Collection of Research polymorphism (SNP) in this motif was shown to be Bioresources (JCRB) Cell Bank (Osaka, Japan), and associated with reduced breast cancer susceptibility human gingival squamous carcinoma (Ca9-22) cells and MMP7 promoter activity (Beeghly-Fadiel et al. were the kind gift of Dr. Kimiharu Hirose (Ohu 2008). CTCF bound to several molecules, including University School of Dentistry, Koriyama, Japan). transcription factor II-I (TFII-I) (Peña-Hermández et Both cell lines were grown in DMEM (high glu- al. 2015), the TATA-binding protein associated fac- cose) supplemented with 10% fetal bovine serum tor 3 (TAF3) (Liu et al. 2011), coheshin (Phillips- (FBS) and subcultured by treatment with 0.25% Cremins et al. 2013), and Smad2 (Van Bortle et al. trypsin/0.02% EDTA. After reaching confluency, the 2015), was shown to function as a transcriptional cells were washed twice with Mg2+ and Ca2+-free activator. Thus, CTCF functions as an architectural phosphate buffered saline (PBS(–)) and maintained protein in response to intracellular signal transduc- in serum-free DMEM (zinc-deprived medium) or in tion. serum-free DMEM supplemented with 15.3 μM Although zinc deprivation disrupts taste percep- ZnSO4 to yield a final concentration of 100 μg/dL, tion, the association of zinc deprivation with TAS2R the serum concentration of zinc in healthy males expression has not been determined. Although tran- (zinc-adequate medium) (Buxaderas and Farre-Rovira scription factors involving TAS2R gene regulation 1985), whose dose was confirmed to be within pre- by in silico analysis using cardiac gene expression vious reports (Deters et al. 2003; Sharif et al. 2012; data, were investigated, regulation of promoter ac- Takeda et al. 2018). To chelate intracellular zinc, tivity of the bitter taste TAS2R7, TAS2R8, TAS2R42 TPEN, a cell-permeable Zn2+ chelator (Treves et al. genes is not analyzed in-depth due to its low ex- 1994; Kolenko et al. 2001), was added to a final pression level in the heart (Foster et al. 2015). Be- concentration of 20 μM; as a vehicle control, etha- cause they are major molecules expressed in the nol was added to a final concentration <0.1%. taste buds (Hevezi et al. 2009), we focused to ex- amine their involvement in bitter taste disorder asso- Cloning, vector construction, transfection. Genome ciated with zinc deficiency. This study reports that DNA was extracted from CA9-22 cells using a Wiz- TAS2R8 expression is upregulated by CTCF finger eard genomic DNA purification kit (Promega). The protein. DNA sequences of TAS promoter regions were am- plified by polymerase chain reaction (PCR) using PrimeSTAR GXL with the primer sets shown in Ta- MATERIALS AND METHODS ble 1 and cloned into pGL4.20 vector (Promega) us- Reagents. Dulbecco’s Eagle Medium (DMEM) was ing an In-Fusion® HD Cloning Kit with the primer purchased from Sigma-Aldrich (Merck KGaA, sets shown in Table 2. Mutant expression vector was Darmstadt, Germany); fetal bovine serum (FBS) was constructed similarly using the primer sets in Ta- from Hyclone (South Logan, UT, USA); and the ble 3. TAS2R8 expression via CTCF 219 Table 1 PCR primer sets for amplifying gene promoter region Genes Sequences F: 5’- TAG CAA ACT ACT GAA TAC ATC TTT TCT ATC -3’ TAS2R7 R: 5’- TTC TTA GAT TTT GAT GTA GTT TTC TTT ACC -3’ F: 5’- CAT TTT CTC TTA TAT GCT ATT GGA AGT CAT -3’ TAS2R8 R: 5’- GTT TGT AGA GAG AAC AAT CTG ATT TCA AAT -3’ F: 5’- CTC CAG AGA CAA AAA AAT CCA AGT TTT TAA -3’ TAS2R42 R: 5’- CTC CAG AGA CAA AAA AAT CCA AGT TTT TAA -3’ F, forward; R, reverse. Table 2 In-Fusion primer sets for cloning the amplified promoter region of TAS2R8 into pGL4.20 vector Genes Sequences (Region, size) TAS2R7 F: 5’- ATC AAA ATC TAA GAA ATG GAA GAT GCC AAA -3’ (−3285 – +1, 3.3 kb) R: 5’- TTC AGT AGT TTG CTA AGG CCA GAG AAA TGT-3’ TAS2R8 F: 5’- GTT CTC TCT ACA AAC ATG GAA GAT GCC AAA -3’ (−2080 – +1, 2.1 kb) R: 5’- ATA TAA GAG AAA ATG AGG TAC CGG CCA GTT -3’ TAS2R8 F: 5’- CCT TGA CAG AGA GAG GAG GCA CAG TTA TGT -3’ (−1572 – +1, 1.6 kb) R: 5’- CTC TCT CTG TCA AGG TAC CGG CCA GTT A -3’ TAS2R42 F: 5’- TTT TTG TCT CTG GAG ATG GAA GAT GCC AAA -3’ (−4873 – +1, 4.9 kb) R: 5’- TTC CAA TAA GAC ATT TCC TCG AGG CTA GCG -3’ F, forward; R, reverse. Table 3 In-Fusion primer sets for cloning the deletion mutant of TAS2R8 pro- moter