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Article BTG4 is A Novel Target That Inhibits Cell Growth and Induces Apoptosis

Na Zhang 1,2, Tinghui Jiang 1,2, Yitao Wang 1,2, Lanyue Hu 1,2 and Youquan Bu 1,2,*

1 Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016, China; [email protected] (N.Z.); [email protected] (T.J.); [email protected] (Y.W.); [email protected] (L.H.) 2 Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, China * Correspondence: [email protected]; Tel.: +86-23-68485991

 Received: 30 January 2020; Accepted: 17 February 2020; Published: 19 February 2020 

Abstract: BTG4 is the last cloned and poorly studied member of BTG/Tob family. Studies have suggested that BTG4 is critical for the degradation of maternal mRNAs in mice during the process of maternal-to-zygotic transition, and downregulated in cancers, such as gastric cancer. However, the regulatory mechanism of BTG4 and its function in cancers remain elusive. In this study, we have for the first time identified the promoter region of the human BTG4 gene. Serial luciferase reporter assay demonstrated that the core promoter of BTG4 is mainly located within the 388 bp region near its transcription initiation site. binding site analysis revealed that the BTG4 promoter contains binding sites for canonical transcription factors, such as Sp1, whereas its first intron contains two overlapped consensus p53 binding sites. However, overexpression of Sp1 has negligible effects on BTG4 promoter activity, and site-directed mutagenesis assay further suggested that Sp1 is not a critical transcription factor for the transcriptional regulation of BTG4. Of note, luciferase assay revealed that one of the intronic p53 binding sites is highly responsive to p53. Both exogenous p53 overexpression and adriamycin-mediated endogenous p53 activation result in the transcriptional upregulation of BTG4. In addition, BTG4 is downregulated in lung and colorectal cancers, and overexpression of BTG4 inhibits cell growth and induces apoptosis in cancer cells. Taken together, our results strongly suggest that BTG4 is a novel p53-regulated gene and probably functions as a tumor suppressor in lung and colorectal cancers.

Keywords: BTG4; p53; promoter; proliferation; apoptosis

1. Introduction The mammalian BTG (B-cell translocation gene)/Tob (Transducer of ErbB2) family comprises six members, including BTG1, BTG2/PC3/Tis21, BTG3/ANA, BTG4/PC3B, Tob1/Tob, and Tob2 [1,2]. Among the six members, BTG2, previously named as PC3 or Tis21, is the first to be identified independently by two groups [3,4]. So far, accumulating studies have demonstrated that the BTG/Tob family plays important roles in regulating various biological processes, including progression, stress response, , and tumorigenesis [1,2]. The BTG/Tob are structurally characterized by the highly conserved N-terminal BTG domain, which contains two conserved motifs, BOX-A and BOX-B, whereas their C-terminal regions are less conserved. Biochemical data revealed that the conserved BTG domain prominently function as a –protein interaction module. It has been reported that the BTG domain can interact with several DNA-binding transcription factors to mediate its role in transcriptional regulation. For example, BTG1 and BTG2 can interact with Hoxb9 [5], BTG3 interacts with E2F1 [6], and BTG1 can interact with the nuclear receptor TRa and the myogenic factor MyoD [7]. The BTG domain is also capable of

Genes 2020, 11, 217; doi:10.3390/genes11020217 www.mdpi.com/journal/genes Genes 2020, 11, 217 2 of 14 interacting with CNOT7 and CNOT8, two deadenylase subunits of the Ccr4-Not complex involved in regulating mRNA deadenylation and turnover [8–10]. Studies suggest that BTG/Tob proteins play important roles in tumorigenesis and cancer. Suzuki et al. reported that Tob1 inhibits cell growth by decreasing expression, and phosphorylation-mediated inactivation of Tob1 by Erk1 and Erk2 is required for Ras-mediated cell proliferation and transformation [11]. Mice lacking Tob1 are prone to spontaneous carcinogenesis with high frequencies of tumors [12]. BTG1 and BTG2 have been found to be frequently downregulated or mutated in several types of cancers, and the decreased expression of BTG1 and BTG2 is positively associated with malignant cell behavior and poor outcome in cancer patients [2,13]. BTG3 has also been shown to be downregulated in several cancers, and its expression might predict survival and prognosis in cancer patients [14–16]. Mice lacking BTG3 also display an increased incidence of lung tumors [1,14]. BTG4 is the last cloned BTG/Tob family member, and thus remains poorly studied [17]. Two recent studies reported that BTG4 is highly expressed and critical to degrade maternal mRNAs in mice during the highly conserved process of maternal-to-zygotic transition (MZT) [18–20]. BTG4 fosters maternal RNA degradation by interacting with CNOT7 and CNOT8, two components of the RNA de-adenylation complex CCR4-NOT. Three studies also suggested that BTG4 is downregulated in gastric cancer, chronic lymphocytic leukemia, and colorectal cancer [21–23]. Those studies highlighted the physiological and pathological significances of BTG4 expression. However, the regulatory mechanism of BTG4 still remains elusive, which needs further exploration. In the present study, we have for the first time cloned and identified the promoter region of the human BTG4 gene. We also found that BTG4 is a p53-regulated gene and probably functions as a tumor suppressor, at least in lung and colorectal cancers.

2. Materials and Methods

2.1. Cell Culture Human lung cancer cells H1299 and A549 and colon cancer cells HCT116 were obtained from Cell Resource Center, Shanghai Institutes for Biological Science, Chinese Academy of Sciences. H1299 were cultured in RPMI 1640 (Hyclone), and A549 were cultured in DME-F12 (Hyclone), and HCT116 were cultured in DMEM (Hyclone) supplemented with 50 units/mL penicillin, 50 mg/mL streptomycin, and 10% (v/v) FBS (Invitrogen). These cells were cultured in a 37 ◦C incubator in a humidified atmosphere containing 5% CO2.

2.2. Cloning of BTG4 Gene Promoter Region The luciferase reporters BTG4-P1837 ( 1611/+226), BTG4-P647 ( 421/+226), BTG4-P412 ( 421/ 9), − − − − and BTG4-p388 ( 397/ 9) were established by cloning the BTG4 promoter region into pGL3-basic − − vector using a seamless cloning kit (Novorec PCR NR001, Novoprotein, Shanghai, China). The primer sequences as well as restriction enzymes are provided in Table1. The cloned fragments were verified by direct DNA sequencing. Genes 2020, 11, 217 3 of 14

Table 1. The primer sequences for BTG4 promoter reporter construction.

Constructs Primer Sequences and Templates

F:50-GCTAGCCCGGGCTCGAGATCTACTGTGGCAGGAACTGAGATGGA-30 Bgl II BTG4-P1837( 1611/+226) R:5 -CAGTACCGGAATGCCAAGCTTGCTACCCAAGCCCACCTCCATTT-3 Hind III − 0 0 PCR Template: human genomic DNA F:50-GCACAACACGCGTAGCTGGGCCA-30 BTG4-P647( 421/+226) R:5 -GGTACCTATCGATAGAGAAATGTTCTGGC-3 Kpn I − 0 0 PCR Template: BTG4-P1837 F:50-AAGCTTGGCATTCCGGTACTGTTGGTAA-30 Hind III BTG4-P412 ( 421/ 9) R:5 -CAGCGCTTTCTCTCAGCCTCCTCC-3 − − 0 0 PCR Template: BTG4-P647 F:50-GGGCGGCGCTGCCGGGGAGGA-30 BTG4-P388( 397/ 9) R:5 -GGTACCTATCGATAGAGAAATGTTCTGGC-3 Kpn I − − 0 0 PCR Template: BTG4-P412 F:50-AGGCATGCCTGGAGACCAGGGAGCACAACACGCGTAGCTGGGCCA-30 BTG4-P388-p53BS2 R:50-GGTACCTATCGATAGAGAAATGTTCTGGC-30 Kpn I PCR Template: BTG4-P388 F:50-GTTCATGTCTAGGCATGCCTGGGCGGCGCTGCCGGGGAGGA-30 BTG4-P388-p53BS1 R:50-GGTACCTATCGATAGAGAAATGTTCTGGC-30 Kpn I PCR Template: BTG4-P388 F:50-TCTACTCCAACCGTCCTTGGAACCC-30 BTG4-P412-Sp1-M1 R:50-CCGCCACAGTCACTCGGCCGCTCAG-30 PCR Template: BTG4-P412 F:50-TTGAGGGGCGGGCGCGAGACGGGGC-30 BTG4-P412-Sp1-M2 R:50-CTCGGCCCGCGGGGTTCCAAGGACG-30 PCR Template: BTG4-P412 F:50-CTATGGGCGCGAGACGGGGCGGGTG-30 BTG4-P412-Sp1-M3 R:50-CGCCCCTCGGCCCGCGGGGTTCCAA-30 PCR Template: BTG4-P412 F:50-TTCGGGGATGGGGGGGGGGAGGAGG-30 BTG4-P412-Sp1-M4 R:50-CCCACCCGCCCCGTCTCGCGCCCGC-30 PCR Template: BTG4-P412 The underlined sequences represent the restriction enzyme sites.

2.3. Site-Directed Mutagenesis The luciferase reporters P412-M1, P412-M2, P412-M3, and P412-M4 were constructed by a site-directed mutagenesis kit (TOYOBO, Osaka, Japan) based on the parental construct BTG4-P412 (-421/-9) according to the manufacturer’s instruction. For M1 mutant, the putative Sp1 binding site of GGGCGA at 115 bp was mutated to TCTACT. For M2 mutant, the putative Sp1 binding site − of GGGGCG at 80 bp was mutated to GTTGAG. For M3 mutant, the putative Sp1 binding site − of GGGGCG at 75 bp was mutated to GCTATG. For M4 mutant, the putative Sp1 binding site of − GGGAGG at 48 bp was mutated to GTTCGG. All the mutations were confirmed by DNA sequencing. − The primer sequences are provided in Table1.

2.4. Transfection and Luciferase Reporter Assay For the luciferase reporter assays, cells were seeded in 12-well plates in triplicate and then co-transfected with the indicated plasmids using Neofect DNA® transfection reagent (Neofect, Beijing, China). The pRL-TK vector (Promega, Madison, America) encoding Renilla luciferase was used as an internal control to monitor the transfection efficiency. Forty-eight hours after transfection, cells were lysed, and luciferase activity was determined using the Dual-Luciferase assay system (Promega, Madison, America) as described previously [24,25].

2.5. DNA Sequence Alignment and Database Mining The BTG4 mRNA and promoter sequences were acquired from the NCBI and UCSC database (https://genome.ucsc.edu/). Transcription factor binding sites were analyzed by online software MatInspector. Alignment for the BTG4 gene promoters from the human, mouse, and rat were carried out by online software Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Genes 2020, 11, 217 4 of 14

2.6. RNA Extraction and Quantitative Real-Time RT-PCR Total RNA was extracted from cells using TRIzol (Sigma, Tokyo, Japan) according to the manufacturer’s instruction. cDNA was synthesized from 500 ng of total RNA using a PrimeScript™ RT Master Mix kit (TaKaRa, Daliang, China). Real-time PCR was conducted by using the SYBR Premix Ex Taq (Perfect Real Time, TAKARA) as described previously [2,26]. The sequences of the primers were Forward: 5’-AGAAGTCACTGGCACTCTGA-3’ and Reverse: 5’-CCATTCCTCCCATCTGCCTT-3’ for BTG4, and Forward: 5’-ACCTGACCTGCCGTCTAGAA-3’ and Reverse: 5’-TCCACCACCCTGTTGCTGTA-3’ for GAPDH as an internal control for quantitative RT-PCR.

2.7. Cell Proliferation Assay HCT116 cells were transfected with empty vector (pcDNA3.0-Flag) and BTG4 expression plasmid (pcDNA3.0-Flag-BTG4). Forty-eight hours later, cells were seeded into 96-well plates at a density of 3000 cells per well. The CCK8 reagent was added at 0, 24, 48, 72, and 96 h after seeding and incubated for 1 h at 37 ◦C. The resultant optical densities (ODs) were measured at 450 nm.

2.8. Wound-Healing Assay H1299 cells were transfected with empty vector (pcDNA3.0-Flag) and BTG4 expression plasmid (pcDNA3.0-Flag-BTG4). Forty-eight hours later, cells were scratched with a 10-µL pipette tip, and washed twice with PBS, and finally incubated in serum-free RPMI-1640. Images were taken at 0 and 24 h with a Leica light microscope (DM4B, Leica Corporation, Wetzlar, Germany). The migration rate was quantified by calculating the wound area changes at the indicated time points.

2.9. Apoptosis Assay An annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Pharmingen, Franklin lake, America) was used to determine apoptosis according to the manufacturer’s instructions. In brief, cells were collected, washed twice in ice-cold 1 PBS, and resuspended in binding buffer at a × concentration of 1 106 cells/mL. Annexin V-FITC and propidium iodide (PI) were then added and × incubated for 15 min at room temperature in the dark. Cells were finally analyzed for apoptosis by flow cytometry.

3. Results

3.1. Gene Organization and Chromatin State of the BTG4 Gene To better understand the genomic organization and chromatin state of the human BTG4 gene, we retrieved the UCSC genome browser (http://genome.ucsc.edu/). As shown in Figure1, the BTG4 gene is mapped at chromosome11q23.1 and comprises six exons and five introns. A classical CpG island exists immediately upstream of the BTG4 transcription start site. The CpG island overlaps with regions marked by DNase I hypersensitivity and enriched with H3K4me3 marks (a hallmark of transcriptional initiation), suggesting that the CpG island harbors the promoter of the BTG4 gene. Interestingly, two human miRNA genes of miR-34b and miR-34c are located upstream of BTG4 and oriented in a head-to-head manner, implying that the BTG4 promoter might function as a bidirectional promoter. Genes 2020, 11, 217 4 of 13

2.7. Cell Proliferation Assay HCT116 cells were transfected with empty vector (pcDNA3.0‐Flag) and BTG4 expression plasmid (pcDNA3.0‐Flag‐BTG4). Forty‐eight hours later, cells were seeded into 96‐well plates at a density of 3000 cells per well. The CCK8 reagent was added at 0, 24, 48, 72, and 96 h after seeding and incubated for 1 h at 37 °C. The resultant optical densities (ODs) were measured at 450 nm.

2.8. Wound‐Healing Assay H1299 cells were transfected with empty vector (pcDNA3.0‐Flag) and BTG4 expression plasmid (pcDNA3.0‐Flag‐BTG4). Forty‐eight hours later, cells were scratched with a 10‐μL pipette tip, and washed twice with PBS, and finally incubated in serum‐free RPMI‐1640. Images were taken at 0 and 24 h with a Leica light microscope (DM4B, Leica Corporation, Wetzlar, Germany). The migration rate was quantified by calculating the wound area changes at the indicated time points.

2.9. Apoptosis Assay An annexin V‐fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Pharmingen, Franklin lake, America) was used to determine apoptosis according to the manufacturer’s instructions. In brief, cells were collected, washed twice in ice‐cold 1× PBS, and resuspended in binding buffer at a concentration of 1 × 106 cells/mL. Annexin V‐FITC and propidium iodide (PI) were then added and incubated for 15 min at room temperature in the dark. Cells were finally analyzed for apoptosis by flow cytometry.

3. Results

3.1. Gene Organization and Chromatin State of the BTG4 Gene Locus To better understand the genomic organization and chromatin state of the human BTG4 gene, we retrieved the UCSC genome browser (http://genome.ucsc.edu/). As shown in Figure 1, the BTG4 gene is mapped at chromosome11q23.1 and comprises six exons and five introns. A classical CpG island exists immediately upstream of the BTG4 transcription start site. The CpG island overlaps with regions marked by DNase I hypersensitivity and enriched with H3K4me3 marks (a hallmark of transcriptional initiation), suggesting that the CpG island harbors the promoter of the BTG4 gene. Interestingly, two human miRNA genes of miR‐34b and miR‐34c are located upstream of BTG4 and orientedGenes 2020 , in11, 217a head‐to‐head manner, implying that the BTG4 promoter might function as5 of 14a bidirectional promoter.

FigureFigure 1. 1. BTG4BTG4 gene gene structure structure and and chromosomal chromosomal state. state. The The human human genomic genomic region region harboring harboring the the BTG4BTG4 gene gene (chr11:111,3327,050 (chr11:111,3327,050-111,394,285‐111,394,285 67,236 67,236 bp, bp, human human species species genomic genomic assembly assembly version, version, GRCh37/hg19)GRCh37/hg19) is is schematically schematically represented represented with with the the indicated indicated tracks tracks (http// (http// genome.ucsc.edu/).genome.ucsc.edu/). As As forfor the the chromatin chromatin state state segmentation, segmentation, the the active active promoter promoter is is shown shown in in bright bright red, red, inactivated/poised inactivated/poised promoter in purple, strong enhancer in orange, insulator in blue, transcriptional elongation in deep green, and weakly transcribed region in light green.

3.2. Identification of the BTG4 Promoter Region To identify the potential proximal promoter region of the human BTG4 gene, a series of luciferase reporters, including BTG4-P1837( 1611/+226), BTG4-P647( 421/+226), BTG4-P412( 421/ 9), − − − − and BTG4-P388( 397/ 9), were established by cloning the different sizes of 5 -flanking genomic − − 0 fragments of the BTG4 gene into a pGL-3-basic promoter-less vector (Figure2a). As displayed in Figure2b, the luciferase reporter assay showed that the luciferase activities of all four aforementioned constructs are significantly enhanced when compared to pGL3-basic in both HCT116 and H1299 cells. Among the four constructs, the activity of BTG4-P388( 397/ 9) is the highest while that of − − BTG4-P1837( 1611/+226), BTG4-P647( 421/+226), and BTG4-P412( 421/ 9) are relatively lower in both − − − − HCT116 and H1299 cells, suggesting that the core promoter region of the BTG4 gene is mainly located in the 5 -flanking 412-bp region corresponding to the BTG4-P412( 421/ 9) construct. Intriguingly, 0 − − the shortest construct BTG4-P388( 397/ 9) showed much higher promoter activity in both cell lines − − than that of BTG4-P412( 421/ 9), which contains the promoter fragment only 24 bp longer than that − − of BTG4-P388( 397/ 9). This result strongly suggests that the 24-bp fragment from 421 to 397 bp − − − − might contain a strong silencer element. As the gene organization analysis revealed that BTG4 and its adjacent miR-34b/c are oriented head to head, we established an overlapped promoter reporter for miR-34b/c to determine whether the intergenic region has bidirectional promoter activity. As shown in Figure2b, miR-34b /c-P400 shows remarkable promoter activity comparable to that of the shortest construct BTG4-P388 ( 397/ 9). This − − intriguing result suggests that BTG4 and miR-34b/c might share an intergenic bidirectional promoter. Genes 2020, 11, 217 5 of 13

promoter in purple, strong enhancer in orange, insulator in blue, transcriptional elongation in deep green, and weakly transcribed region in light green.

3.2. Identification of the BTG4 Promoter Region To identify the potential proximal promoter region of the human BTG4 gene, a series of luciferase reporters, including BTG4‐P1837(−1611/+226), BTG4‐P647(−421/+226), BTG4‐P412(−421/−9), and BTG4‐P388(−397/−9), were established by cloning the different sizes of 5ʹ‐flanking genomic fragments of the BTG4 gene into a pGL‐3‐basic promoter‐less vector (Figure 2a). As displayed in Figure 2b, the luciferase reporter assay showed that the luciferase activities of all four aforementioned constructs are significantly enhanced when compared to pGL3‐basic in both HCT116 and H1299 cells. Among the four constructs, the activity of BTG4‐P388(−397/−9) is the highest while that of BTG4‐P1837(−1611/+226), BTG4‐P647(−421/+226), and BTG4‐P412(−421/−9) are relatively lower in both HCT116 and H1299 cells, suggesting that the core promoter region of the BTG4 gene is mainly located in the 5ʹ‐flanking 412‐bp region corresponding to the BTG4‐P412(−421/−9) construct. Intriguingly, the shortest construct BTG4‐P388(−397/−9) showed much higher promoter activity in both cell lines than that of BTG4‐P412(−421/−9), which contains the promoter fragment only 24 bp longer than that of BTG4‐P388(−397/−9). This result strongly suggests that the 24‐bp fragment from −421 to −397 bp might contain a strong silencer element. As the gene organization analysis revealed that BTG4 and its adjacent miR‐34b/c are oriented head to head, we established an overlapped promoter reporter for miR‐34b/c to determine whether the intergenic region has bidirectional promoter activity. As shown in Figure 2b, miR‐34b/c‐P400 shows remarkable promoter activity comparable to that of the shortest construct BTG4‐P388

Genes(−397/2020−9)., 11 This, 217 intriguing result suggests that BTG4 and miR‐34b/c might share an intergenic6 of 14 bidirectional promoter.

Figure 2.2. IdentificationIdentification of the BTG4 promoter region.region. ( (aa)) Schematic diagram of the BTG4 genegene promoter reporterreporter constructs.constructs. TheThe positionspositions relativerelative toto thethe majormajor BTG4BTG4 transcriptiontranscription startstart sitesite ((+1)+1) are indicated.indicated. The firstfirst and second exons of BTG4 are displayed by twotwo openopen boxes.boxes. The two overlapped p3 bindingbinding sitessites (p53BS1(p53BS1/2)/2) areare shownshown byby graygray boxes.boxes. MiR34bMiR34b andand miR34cmiR34c are transcribedtranscribed in thethe opposite orientation from BTG4.BTG4. The CpG island is indicated by thickthick blackblack lines.lines. (b) LuciferaseLuciferase assay. HCT116HCT116 andand H1299H1299 cellscells were transientlytransiently co-transfectedco‐transfected withwith thethe indicatedindicated luciferase reporter constructs togethertogether withwith pRL-TK. pRL‐TK. Forty-eight Forty‐eight hours hours later, later, luciferase luciferase activities activities were were determined determined by theby Dualthe Dual Luciferase Luciferase Assay Assay System System (Promega). (Promega). Data Data are are shown shown as theas the fold fold induction induction compared compared to thatto that of theof the empty empty pGL3-basic pGL3‐basic vector. vector. The The results results are are presented presented as theas the mean mean and and standard standard deviation deviation (S.D.) (S.D.) of triplicatesof triplicates from from a representative a representative experiment. experiment. 3.3. Sequence Analysis of the BTG4 Promoter Region

To understand the transcriptional regulatory mechanism of the human BTG4 gene, we used the online software MatInspector to predict potential transcriptional factor binding sites in the promoter region of the BTG4 gene. As displayed in Figure3, the BTG4 gene lacks the classical TATA box but contains potential transcription factor binding sites, such as Sp1, E2F4, etc. (Figure3a). Multiple sequence alignment analysis revealed that the four Sp1 binding sites are evolutionarily conserved across several species, including the human, mouse, and rat (Figure3b), suggesting that Sp1 might play an important role in regulating the transcription of the human BTG4 gene. Genes 2020, 11, 217 6 of 13

3.3. Sequence Analysis of the BTG4 Promoter Region To understand the transcriptional regulatory mechanism of the human BTG4 gene, we used the online software MatInspector to predict potential transcriptional factor binding sites in the promoter region of the BTG4 gene. As displayed in Figure 3, the BTG4 gene lacks the classical TATA box but contains potential transcription factor binding sites, such as Sp1, E2F4, etc. (Figure 3a). Multiple sequence alignment analysis revealed that the four Sp1 binding sites are evolutionarily conserved across several species, including the human, mouse, and rat (Figure 3b), suggesting that Sp1 might Genes 2020, 11, 217 7 of 14 play an important role in regulating the transcription of the human BTG4 gene.

Figure 3. Nucleotide sequences of the human BTG4 gene promoter. (a) The putative transcription factor bindingFigure sites3. Nucleotide in the promoter sequences region of the of thehuman BTG4 BTG4 gene gene were promoter. predicted and(a) The underlined. putative Nucleotidestranscription arefactor numbered binding based sites onin thethe majorpromoter transcription region of start the siteBTG4 of BTG4gene were (+1). predicted (b) Conservative and underlined. analysis ofNucleotides the BTG4 promoter are numbered sequence. based Sequenceon the major alignment transcription of the start nucleotide site of BTG4 sequences (+1). of(b) partial Conservative BTG4 geneanalysis promoters of the fromBTG4 the promoter indicated sequence. species wasSequence conducted alignment by the of online the nucleotide software Clustal sequences Omega. of partial The putativeBTG4 gene Sp1-binding promoters sites from are boxed. the indicated Identical species bases among was conducted different species by the are online marked software with stars Clustal (*). Omega. The putative Sp1‐binding sites are boxed. Identical bases among different species are 3.4. Functional Analysis of Sp1 Binding Sites in the BTG4 Gene Promoter marked with stars (*). As Sp1 binding sites are prominently enriched in the proximal promoter of the BTG4 gene, we decided3.4. Functional to further Analysis verify of their Sp1 roles Binding in BTG4 Sites in transcription the BTG4 Gene regulation. Promoter To this end, wildtype BTG4-P412 was usedAs Sp1 as abinding template sites to are establish prominently four mutant enriched reporter in the proximal constructs, promoter P412-Sp1-M1, of the BTG4 P412-Sp1-M2, gene, we P412-Sp1-M3,decided to further and P412-Sp1-M4, verify their in roles which in the BTG4 four evolutionallytranscription conserved regulation. putative To this Sp1 end, binding wildtype sites inBTG4 the BTG4‐P412 proximalwas used promoter as a template region wereto establish disrupted four by mutant site-directed reporter point constructs, mutation, P412 respectively‐Sp1‐M1, (FiguresP412‐Sp13b‐M2, and 4P412a).‐ AsSp1 shown‐M3, and in FigureP412‐Sp14b,‐M4, disruption in which of eachthe four of the evolutionally four Sp1 binding conserved sites putative did notSp1 attenuate binding thesites constitutive in the BTG4 promoter proximal activity promoter of the region BTG4 genewere indisrupted both HCT116 by site and‐directed H1299 cells.point Intriguingly,mutation, respectively disruption of(Figures Sp1BS1 3b even and caused 4a). As a shown remarkable in Figure increase 4b, indisruption the promoter of each activity of the of four the BTG4-P412Sp1 binding reporter sites did in bothnot attenuate HCT116 andthe H1299constitutive cells, promoter suggesting activity that Sp1BS1 of the might BTG4 function gene in asboth a potentialHCT116 silencer. and H1299 In addition, cells. Intriguingly, exogenous disruption overexpression of Sp1BS1 of Sp1 even had negligible caused a eremarkableffects on the increase promoter in activitythe promoter of wildtype activity BTG4-P412 of the BTG4 as well‐P412 as mostreporter of the in fourboth mutantHCT116 reporter and H1299 constructs, cells, suggesting P412-Sp1-M1, that P412-Sp1-M2, P412-Sp1-M3, and P412-Sp1-M4. These results together strongly suggest that Sp1 is not a critical transcription factor for the transcriptional regulation of BTG4. Genes 2020, 11, 217 7 of 13

Sp1BS1 might function as a potential silencer. In addition, exogenous overexpression of Sp1 had negligible effects on the promoter activity of wildtype BTG4‐P412 as well as most of the four mutant reporter constructs, P412‐Sp1‐M1, P412‐Sp1‐M2, P412‐Sp1‐M3, and P412‐Sp1‐M4. These

Genesresults2020 together, 11, 217 strongly suggest that Sp1 is not a critical transcription factor for the transcriptional8 of 14 regulation of BTG4.

FigureFigure 4. 4. FunctionalFunctional analysisanalysis ofof Sp1Sp1 bindingbinding sitessites in in the the BTG4 BTG4 promoter. promoter. ( a(a)) Schematic Schematic diagram diagram of of site-directedsite‐directed mutagenesis mutagenesis of of Sp1 Sp1 binding binding sites sites in the in BTG4-P412the BTG4‐P412 luciferase luciferase reporter reporter harboring harboring the BTG4 the promoter.BTG4 promoter. The four The potential four potential Sp1 binding Sp1 binding sites are sites indicated are indicated as open boxesas open (Sp1BS1, boxes (Sp1BS1, Sp1BS2, Sp1BS3,Sp1BS2, andSp1BS3, Sp1BS4). and TheSp1BS4). indicated The point indicated mutations point are mutations denoted by are a crossdenoted at the by potential a cross Sp1at the binding potential sites. Sp1 (b) Luciferasebinding sites. reporter (b) assays.Luciferase The indicatedreporter assays. luciferase The reporter indicated constructs luciferase were reporter transfected constructs into HCT116 were andtransfected H1299 cells into togetherHCT116 withand H1299 empty cells vector together or with with Sp1 empty expression vector plasmid. or with Sp1 Forty-eight expression hours plasmid. later, theirForty luciferase‐eight hours activities later, their were luciferase determined, activities as in Figure were 3determined,d. as in Figure 3d. 3.5. Identification of a p53-Responsive Element with the First Intron of the BTG4 Gene 3.5. Identification of a p53‐Responsive Element with the First Intron of the BTG4 Gene To further explore the transcription factors that regulate BTG4 gene expression, we retrieved the To further explore the transcription factors that regulate BTG4 gene expression, we retrieved transcription factor binding sites in the whole genomic sequences of the BTG4 gene. The analysis the transcription factor binding sites in the whole genomic sequences of the BTG4 gene. The revealed that the first intron of the BTG4 gene contains two overlapped p53 binding sites, namely analysis revealed that the first intron of the BTG4 gene contains two overlapped p53 binding sites, p53BS1 and p53BS2 (Figures2a and5a). Of note, the two p53 binding sites are highly conserved namely p53BS1 and p53BS2 (Figures 2a and 5a). Of note, the two p53 binding sites are highly among the human, mouse, and rat (Figure5a). To determine whether the two p53 binding sites are conserved among the human, mouse, and rat (Figure 5a). To determine whether the two p53 responsive to p53 expression, the two sites were cloned into the BTG4-P388 reporter to establish the binding sites are responsive to p53 expression, the two sites were cloned into the BTG4‐P388 BTG4-P388-p53BS1 and BTG4-P388-p53BS2 constructs (Figure5b). The luciferase assay revealed that reporter to establish the BTG4‐P388‐p53BS1 and BTG4‐P388‐p53BS2 constructs (Figure 5b). The exogenous overexpression of p53 significantly enhanced the promoter activity of BTG4-P388-p53BS1 luciferase assay revealed that exogenous overexpression of p53 significantly enhanced the promoter but not BTG4-P388-p53BS2, suggesting that only the first p53 binding site is responsive to p53. activity of BTG4‐P388‐p53BS1 but not BTG4‐P388‐p53BS2, suggesting that only the first p53 binding 3.6.site P53is responsive Induces BTG4 to p53. Transcription

3.6. P53Next, Induces we decided BTG4 toTranscription determine whether BTG4 transcription can be regulated by p53. As shown in Figure5c, exogenous overexpression of p53 caused a significant increase of BTG4 expression at the mRNANext, level we in decided H1299 cells. to determine In support whether of this, BTG4 BTG4 transcription transcription can was be also regulated induced by byp53. adriamycin As shown treatmentin Figure in5c, A549 exogenous and HCT116 overexpression cells. Adriamycin of p53 caused is a chemotherapeutic a significant increase agent, of BTG4 which expression is known to at bethe able mRNA to cause level endogenous in H1299 activationcells. In support of p53. Therefore, of this, BTG4 the results transcription suggest thatwas p53 also induces induced BTG4 by transcription. In addition, our preliminary experiment using HCT116 wildtype and p53-/- cells also suggested that the transcription of BTG4 was induced in a manner dependent on the presence of p53 (Supplementary Figure S1). Genes 2020, 11, 217 8 of 13

adriamycin treatment in A549 and HCT116 cells. Adriamycin is a chemotherapeutic agent, which is known to be able to cause endogenous activation of p53. Therefore, the results suggest that p53 induces BTG4 transcription. In addition, our preliminary experiment using HCT116 wildtype and Genesp532020‐/‐ ,cells11, 217 also suggested that the transcription of BTG4 was induced in a manner dependent9 of 14 on the presence of p53 (Supplementary Figure S1).

FigureFigure 5. BTG45. BTG4 gene gene contains contains a p53 a p53 response response element element and and is inducedis induced by by p53. p53. (a) ( Partiala) Partial sequences sequences of of thethe first first intron intron of theof the BTG4 BTG4 gene gene were were aligned aligned across across the the human, human, mouse, mouse, and and rat rat by by online online software software ClustalClustal Omega. Omega. The The position position is relativeis relative to to the the transcription transcription start start site site of BTG4.of BTG4. The The two two overlapped overlapped p53p53 binding binding sites sites are indicatedare indicated as p53BS1 as p53BS1 and p53BS2. and p53BS2. (b) The (b two) The overlapped two overlapped p53 binding p53 binding sites were sites clonedwere into cloned the wildtype into the reporter wildtype BTG4-P388, reporter respectively.BTG4‐P388, Therespectively. indicated The reporters indicated were cotransfectedreporters were intocotransfected H1299 cells withinto emptyH1299 orcells Flag-p53 with empty expression or Flag plasmids‐p53 expression and pRL-TK. plasmids The luciferase and pRL activities‐TK. The wereluciferase determined activities as described were determined in Figure2 as. The described two potential in Figure p53 2. binding The two sites potential of p53BS1 p53 binding and p53BS2 sites of comparedp53BS1 toand the p53BS2 consensus compared p53 binding to the site consensus (p53 CBS). p53 R, binding purine; site Y, pyrimidine; (p53 CBS). R, W, purine; adenine Y, or pyrimidine; thymine. (c) H1299 cells were transiently transfected with empty vector (pcDNA3.0-Flag) or pcDNA3.0-Flag-p53 W, adenine or thymine. (c) H1299 cells were transiently transfected with empty vector expression plasmid. Forty-eight hours later, cells were collected and subjected to RT-PCR analysis. (pcDNA3.0‐Flag) or pcDNA3.0‐Flag‐p53 expression plasmid. Forty‐eight hours later, cells were A549 and HCT116 cells were treated with adriamycin at the final concentration of 1µM. Cells were then collected and subjected to RT‐PCR analysis. A549 and HCT116 cells were treated with adriamycin at collected and subjected to RT-PCR analysis at the indicated time points. the final concentration of 1μM. Cells were then collected and subjected to RT‐PCR analysis at the 3.7. BTG4indicated Inhibits time Cell points. Proliferation and Migration, and Induces Apoptosis

We then determined whether BTG4 could affect cancer cell proliferation, migration, and apoptosis. To this end, HCT116 as well as H1299 cells were transiently transfected with BTG4 expression plasmid. As shown in Figure6a, exogenous overexpression of BTG4 significantly inhibited cell Genes 2020, 11, 217 9 of 13

3.7. BTG4 Inhibits Cell Proliferation and Migration, and Induces Apoptosis We then determined whether BTG4 could affect cancer cell proliferation, migration, and Genes 2020, 11, 217 10 of 14 apoptosis. To this end, HCT116 as well as H1299 cells were transiently transfected with BTG4 expression plasmid. As shown in Figure 6a, exogenous overexpression of BTG4 significantly inhibitedproliferation cell in proliferation HCT116 cells. in Annexin HCT116 V-FITC cells. Annexin and PI double V‐FITC staining and PI with double flow cytometrystaining with was flow then cytometryconducted was to determine then conducted apoptotic to determine cells. As apoptotic shown in cells. Figure As6 b,shown approximately in Figure 6b, 9.39% approximately and 4.74% + ＋ 9.39%of BTG4-overexpressed and 4.74% of BTG4 cells‐overexpressed displayed an earlycells apoptoticdisplayed (FITC an early/PI −apoptotic) and late (FITC apoptotic/PI−/)secondary and late apoptotic/secondarynecrotic phenotype 48 necrotic hours after phenotype transfection, 48 hours whereas after onlytransfection, 5.86% and whereas 3.05% ofonly control 5.86% cells and showed 3.05% ofthis control phenotype. cells showed As shown this in phenotype. Figure6c, exogenousAs shown in overexpression Figure 6c, exogenous of BTG4 overexpression remarkably suppressed of BTG4 remarkablymigration in suppressed H1299 cells migration as detected in H1299 by the cells wound as detected healing assay.by the Ofwound note, healing as the BTG assay./Tob Of family note, ashas the been BTG/Tob shown family to play has important been shown roles to in play regulating important cell roles cycle in progression regulating [cell1,2], cycle the proliferation progression [1,2],inhibition the andproliferation apoptosis inhibition induction causedand apoptosis by BTG4 overexpressioninduction caused is probably by BTG4 preceded overexpression by potential is probablycell cycle preceded arrest. by potential cell cycle arrest.

Figure 6. BTG4BTG4 inhibits inhibits cell cell proliferation and migration, and induces apoptosis. ( (aa)) HCT116 cells were transientlytransiently transfected transfected with with empty empty vector (pcDNA3.0-Flag)vector (pcDNA3.0 or‐Flag) pcDNA3.0-Flag-BTG4 or pcDNA3.0‐Flag expression‐BTG4 expressionplasmid, and plasmid, then subjected and then tosubjected cell proliferation to cell proliferation assay. (b) assay. HCT116 (b) cellsHCT116 were cells transfected were transfected as in (a). asForty-eight in (a). Forty hours‐eight later, cellshours were later, harvested cells were and subjectedharvested to and apoptosis subjected assay. to The apoptosis lower-left assay. quadrant The + lowerrepresents‐left quadrant viable cells represents (FITC−/PI −viable); lower cells right (FITC represents−/PI−); lower early apoptoticright represents cells (FITC early/PI apoptotic−); upper rightcells + + (FITCrepresents＋/PI−); late upper apoptotic right represents and secondary late apoptotic necrotic and cells secondary (FITC /PI necrotic). (c) H1299 cells (FITC cells＋ were/PI＋). transiently (c) H1299 cellstransfected were withtransiently empty vectortransfected (pcDNA3.0-Flag) with empty or vector pcDNA3.0-Flag-BTG4 (pcDNA3.0‐Flag) expression or pcDNA3.0 plasmid,‐Flag and‐BTG4 then expressionsubjected to plasmid, the wound and healing then assay.subjected Cell to migration the wound status healing was monitored assay. Cell under migration a microscope status at was the monitoredindicated time under points, a microscope and subsequently at the indicated quantified. time points, and subsequently quantified.

Genes 2020, 11, 217 11 of 14 Genes 2020, 11, 217 10 of 13

3.8.3.8. The The Expression Expression of of BTG4 BTG4 in in Normal Normal and and Cancer Cancer Tissue Tissue Finally,Finally, wewe examinedexamined whether whether the the expression expression of of BTG4 BTG4 is is aberrantly aberrantly changed changed in cancerin cancer tissues. tissues. As Asshown shown in Figure in Figure7, BTG4 7, BTG4 mRNA mRNA was significantly was significantly decreased decreased in both in lung both and lung colon and cancer colon samples cancer samplescompared compared with their with normal their counterpart normal counterpart tissues. Of note, tissues. in lung Of cancernote, in tissues, lung thecancer expression tissues, levels the expressionof BTG4 mRNA levels were of BTG4 significantly mRNA were associated significantly with the associated clinical stage. with the clinical stage.

Figure 7. The expression of BTG4 mRNA in lung and colon cancers. BTG4 expression levels in normal Figure 7. The expression of BTG4 mRNA in lung and colon cancers. BTG4 expression levels in and cancerous tissues were analyzed with quantitative RT-PCR using OriGene TissueScan cancer panels normal and cancerous tissues were analyzed with quantitative RT‐PCR using OriGene TissueScan (CSRT101). 0 denotes normal lung or colorectal tissues. I, II, III, IV represent different clinical stages of cancer panels (CSRT101). 0 denotes normal lung or colorectal tissues. I, II, III, IV represent different lung and colorectal cancers. clinical stages of lung and colorectal cancers. 4. Discussion 4. Discussion In the present study, we firstly identified the promoter region of the human BTG4 gene, which mightIn notthe bepresent mainly study, regulated we firstly by Sp1 identified although the it promoter contains region several of GC the boxes. human Our BTG4 results gene, strongly which mightsuggest not that be BTG4mainly is aregulated novel target by Sp1 gene although of p53 as it evidencedcontains several by the GC fact boxes. that its Our first results intron strongly contains suggesta canonical that p53BTG4 response is a novel element target and gene its of transcription p53 as evidenced could by be the induced fact that by its p53. first As intron a well-known contains atumor canonical suppressor, p53 response p53 mainly element functions and its as transcription transcription could factor be to induced regulate by the p53. expression As a well of‐ variousknown tumordownstream suppressor, target p53 genes, mainly consequently functions participating as transcription in diverse factor to biological regulate processes,the expression including of various DNA downstreamdamage response, target cellgenes, cycle consequently progression, participating apoptosis, autophagy, in diverse metabolism,biological processes, etc. [27– 29including]. Thus, DNAidentification damage of response, novel p53-target cell cycle genes progression, would provide apoptosis, greater autophagy, insight intometabolism, the molecular etc. [27–29]. mechanisms Thus, identificationthat govern the of tumornovel suppressor p53‐target functionsgenes would of p53. provide Previously greater reported insight well-known into the p53molecular targets mechanismsinclude Bax and that PUMA govern involved the tumor in apoptosis, suppressor CDKN1A functions in of cell p53. cycle Previously arrest, TIGAR reported in the well metabolism‐known p53of glycolysis, targets include and DRAM Bax and in autophagy PUMA involved [27]. It hasin apoptosis, been reported CDKN1A that both in BTG2cell cycle and arrest, BTG3 are TIGAR directly in theregulated metabolism by p53 of and glycolysis, implicated and in DRAM the DNA in autophagy damage response [27]. It pathwayhas been as reported well as apoptosisthat both BTG2 [6,30]. andAs our BTG3 results are directly demonstrated regulated that by BTG4 p53 isand induced implicated upon in the the treatment DNA damage of adriamycin, response whichpathway could as wellcause as DNA apoptosis double [6,30]. breaks, As it our is of results interest demonstrated to further investigate that BTG4 the is role induced of BTG4 upon in the the DNA treatment damage of adriamycin,response pathway. which could cause DNA double breaks, it is of interest to further investigate the role of BTG4Our in the data DNA also damage found thatresponse the expression pathway. of BTG4 is downregulated in lung and colon cancers, and overexpressionOur data also found of BTG4 that can the inhibit expression proliferation of BTG4 and is migration,downregulated and induce in lung apoptosis, and colon suggesting cancers, andthat similaroverexpression to BTG2 andof BTG4 BTG3, can BTG4 inhibit might proliferation also act as a and potential migration, tumor suppressor.and induce Ourapoptosis, results suggestingare consistent that with similar the previousto BTG2 reportand BTG3, by Toyota BTG4 et might al., who also found act as the a 5potential0-flanking tumor region suppressor. of BTG4 is Ourhypermethylated results are consistent and overexpression with the ofprevious BTG4 inhibits report theby colonyToyota formation et al., who ability found in colorectal the 5ʹ‐flanking cancer regioncells [23 of]. AlthoughBTG4 is hypermethylated Ou et al. reported and that overexpression BTG3 binds to of and BTG4 inhibits inhibits E2F1 the through colony its conservedformation abilityN-terminal in colorectal BTG domain, cancer our cells preliminary [23]. Although results Ou revealed et al. that reported BTG4 that does BTG3 not bind binds to E2F1 to and in ainhibits similar E2F1manner. through As the its recent conserved studies N have‐terminal demonstrated BTG domain, that BTG4 our preliminary could interact results with CNOT7revealed and that CNOT8 BTG4 doesto foster not maternalbind to E2F1 mRNA in degradationa similar manner. during As maternal-to-zygotic the recent studies transition have demonstrated [18–20], further that study BTG4 is couldneeded interact to investigate with whetherCNOT7 BTG4 and regulatesCNOT8 mRNAto foster deadenylation maternal throughmRNA a similardegradation mechanism during in maternaltumorigenesis.‐to‐zygotic Of note, transition Liu et al. [18–20], and Yu etfurther al. also study revealed is thatneeded BTG4 to transcripts investigate are whether highly enriched BTG4 regulatesin the ovary mRNA and testis deadenylation rather than through somatic a tissues. similar We mechanism also found in that tumorigenesis. BTG4 is expressed Of note, at a Liu very et low al. and Yu et al. also revealed that BTG4 transcripts are highly enriched in the ovary and testis rather than somatic tissues. We also found that BTG4 is expressed at a very low level in somatic cells and

Genes 2020, 11, 217 12 of 14 level in somatic cells and its expression is difficult to detected by routine immunoblotting. Thus, highly sensitive methods for detecting BTG4 protein expression need to be developed in the future. However, the current data do not rule out the possibility that the BTG4 gene might execute its functions only through its RNA transcripts rather than proteins. Of note, two microRNAs miR34b and miR34c exist upstream of the BTG4 gene (Figure2). Our results suggested that BTG4 and miR34b/c share a bidirectional promoter and constitute a head-to-hear gene pair [24] (Figure3). Interestingly, previous reports showed that miR34b /c is directly regulated by p53 and implicated in cancer development and apoptosis [31]. Thus, the BTG4 and miR34b/c gene pair could be coordinately regulated by p53 as well as other transcription factors. Our results suggested that the 24-bp region from 421 to 397 bp might contain a strong silencer element − − (Figure2a,b). Transcription factor binding analysis revealed that this 24-bp region contains a high-scored cis-acting element for PLU1 or JARID1B, now officially named lysine demethylase 5B (KDM5B). KDM5B is a lysine-specific histone demethylase that belongs to the jumonji/ARID domain-containing family of histone demethylases. It is capable of demethylating tri-, di-, and monomethylated lysine 4 of histone H3. Studies showed that KDM5B is upregulated in certain cancers and plays roles in the transcriptional repression of tumor suppressor genes, such as BRCA1 [32,33]. Thus, we speculated that KDM5B could be a transcriptional repressor for the BTG4 gene. Mutation of the first Sp1 binding site at 113 to − 118 bp (BTG4-P412-Sp1-M1) also caused remarkably increased promoter activity compared with the − wildtype reporter BTG4-P412, strongly suggesting a robust silencer(s) exists within or overlaps with this Sp1 binding site. Transcription factor binding analysis indicated that the first Sp1 binding site also serves as a high-scored cis-acting element for transcriptional repressors, such as ZNF219, ZNF300, and INSM1 [34–37]. Of note, INSM1 (insulinoma-associated 1) has been shown to be implicated in some cancers, and function as a transcriptional repressor [34–37]. Collectively, future works are needed to investigate whether these transcriptional repressors, especially KDM5B and INSM1, regulate the transcription of BTG4 as well as miR34b/c.

5. Conclusions Human BTG4 gene contains several potential Sp1 and E2F binding sites in its promoter region, and one functional p53 consensus binding site in its first intron. Sp1 might not directly regulate BTG4 transcription. Overexpression of BTG4 inhibits cell growth and induces apoptosis in cancer cells. BTG4 is downregulated in lung and colorectal cancers. BTG4 is a novel p53-regulated gene and probably functions as a tumor suppressor in lung and colorectal cancers.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/2/217/s1, Figure S1: The constitutive and induction of BTG4 transcription is dependent on p53. Author Contributions: Conceptualization, Y.B.; investigation, N.Z., T.J., Y.W., L.H. and Y.B.; methodology, N.Z.; validation, N.Z., T.J., Y.W. and L.H.; writing—original draft preparation, N.Z.; writing—review and editing, Y.B.; supervision, Y.B.; funding acquisition, Y.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China, grant number 81672301, and the Basic Sciences and Advanced Technology Key Project of CQ CSTC, grant number cstc2017jcyjBX0069. Acknowledgments: We thank all our lab members for their helpful and valuable discussions. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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