LIU Xiumei ,LI Zan ,YAN Weijie ,ZHAO Haitao ,LIU Yuxiang ,HAN Miao ,WANG Xubo,HE Yan,YU Haiyang,and ZHANG Quanqi,

1) College of Life Sciences, Yantai University,Yantai 264005,China

2) Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China,Qingdao 266003,China

3) Dongying Municipal Bureau of Marine Development and Fisheries, Dongying 257000, China

4) Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237,China

Abstract GATA4,as a member of the GATA transcription factor family,plays a significant role in lineage specification and transdifferentiation of cells by regulating target gene expression.In this study,the potential transcription factor binding sites in the promoter of Paralichthys olivaceus sox2 gene were analyzed,and a GATA4-binding motif was identified.A cleaved amplified polymorphic sequence-based binding assay experiment was performed,and the results showed a significant binding process between the Homo sapiens GATA4 recombinant protein and the DNA fragment of P.olivaceus sox2 promoter.Luciferase reporter assay revealed that GATA4 could improve the activity of the P.olivaceus sox2 promoter.Overexpression of P.olivaceus GATA4 could also increase the sox2 mRNA expression level in flounder brain cells.Overall,our results indicate that sox2 is a target gene of GATA4 in P.olivaceus.Sox2 is implicated in the maintenance of stem cell pluripotency and can reprogram differentiated cells to iPS cells.In addition,sox2 is involved in the development of the central nervous system (CNS).Therefore,this study may provide a reference for exploring the function of P.olivaceus GATA4 in the regulation of pluripotent stem cells and the development of the CNS.

Key words Paralichthys olivaceus;GATA4;sox2;pluripotency;CNS

1 Introduction

Transcription factors (TFs) are at the center of the broad regulatory network orchestrating gene expression programs that elicit different biological responses.The Sox family,as a class of TFs,is widely distributed across the animal kingdom.According to sequence similarity and gene structure analysis,the Sox family is subdivided into Groups A through I (Bowleset al.,2000).As a member of the Sox B1 subgroup,sox2plays an important role in cell fate regulation.During embryonic development,sox2functions as a temporary marker for germ layer selection and is critical for neural ectodermal formation (Sarkar and Hochedlinger,2013).It is also important for maintaining the pluripotent state of embryonic and neural stem cells (Avilionet al.,2003;Adachiet al.,2010;Bertoliniet al.,2019).

Pluripotent stem cells hold great promise for regenerative medicine.Embryonic stem (ES) cells,which are obtained from the inner cell mass of blastocysts,can grow indefinitely while maintaining pluripotency and can differentiate into cells of all three germ layers.In 1981,researchers successfully isolated and cultured ES cells for the first time and found that multiple TFs,includingoct4andsox2,were expressed in such ES cells (Martin,1981).In 2006,Yamanakaet al.(2006) demonstrated the induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by ectopic expression ofoct4,sox2,Klf4,andc-Myc(OSKM) under ES cell culture conditions.Later research found that single-locus targeting ofsox2was sufficient to activatesox2,which triggered pluripotency induction (Liuet al.,2018a).These induced pluripotent stem cells(iPSCs) have characteristics similar to those of ES cells.

For years,pluripotency-associated factors and lineage specifiers have been generally considered to determine the identities of pluripotent and differentiated cells,respectively.However,several studies showed that lineage specifiers(GATA3,GATA4,and GATA6) could take the place ofoct4to complete the induction of pluripotency (Shuet al.,2013).In addition,all mouse GATA family members could inhibit the over-represented ectodermal-lineage genes and substitute foroct4to induce pluripotency (Shuet al.,2015).Then,researchers came up with a ‘seesaw’ model,which points out that members of the GATA4 family can participate in pluripotency reprogramming by balancing the forces of lineage-specifying or directly activating pluripotencyassociated genes (Shuet al.,2015).

GATA-binding proteins (GATA1–GATA6) are a family of common TFs that are widely distributed among eukaryotes.Members of this family,which share conserved zinc fingers in their DNA-binding domains,are characterized by their ability to recognize and bind the specific DNA sequence (A/T)GATA(A/G) (Merika and Orkin,1993;Lowry and Atchley,2000).The GATA family plays an important role in embryo development and organogenesis.GATA1/2/3 are involved in the differentiation of mesoderm-and ectoderm-derived tissues,such as those of the hematopoietic system.During mouse embryonic development,the loss of GATA1/2/3 could cause embryonic death due to defects in the hematopoietic system (Tsaiet al.,1994;Pandolfiet al.,1995;Simon,1995;Fujiwaraet al.,1996).GATA4/5/6 are required for the development and differentiation of endoderm-and mesoderm-derived tissues,including cardiovascular embryogenesis and induction of differentiation of ES cells (Jianget al.,1998;Nemer and Nemer,2003;Laforest and Nemer,2011).Decades ago,researchers implemented the transformation of cell lineages by expressing specific TF (Daviset al.,1987).More studies have been carried out to investigate the roles of GATA-binding proteins in cell lineage specification and transdifferentiation.The overexpression of GATA1 could suppress myelomonocytic markers and induce the reprogramming of myeloblasts into cells resembling either transformed eosinophils or thromboblasts(Kulessaet al.,1995).GATA4,together with the TFs MEF2C and TBX5,could directly reprogram mouse fibroblasts into differentiated cardiomyocytes that exhibit the physiological characteristics of normal cardiomyocytes (Iedaet al.,2010).

The functions of mammalian GATA4 in embryonic development and the induction of pluripotency have been well studied,especially in humans and mice.However,functional studies of GATA4 in nonmammals are rarely reported,especially in teleosts.Paralichthys olivaceusis an important marine commercial fish that has been widely cultured in northern Asia.With the development and processing of selective breeding,researchers have made efforts to preserve genetically improved strains,and pluripotent stem cells are considered the best candidates.Thus,the analysis of closely related regulators of pluripotency is essential.Our laboratory previously identified and characterized thesox2andgata4genes inP.olivaceus(Gaoet al.,2014;Liuet al.,2018b).In this study,a GATA4-binding site in theP.olivaceussox2promoter was identified.In addition,the interaction between GATA4 andsox2was analyzed,and the regulatory action of GATA4 onP.olivaceus sox2was investigated.The results showed that GATA4 could increase the expression ofP.olivaceussox2.Our findings provide a reference for investigating the functions of marine teleost GATA4 in regulating cell fate.

2 Materials and Methods

2.1 Cell Culture

All researches were conducted according to the Institutional Animal Care and Use Committee of the Ocean University of China and the China Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing,Research,and Training (State Science and Technology Commission of the People’s Republic of China for No.2,October 31,1988).

Adherently growing HEK293T (human embryonic kidney [HEK] cell line HEK293T/17) were cultured in DMEM containing 10% (v/v) fetal bovine serum (FBS),100 μg mL-1of streptomycin,and 100 U mL-1of penicillin in the incubater with 5% carbon dioxide and 95% humidity at 37℃.The flounder brain cell (FBC) line established by our laboratory was maintained in DMEM with 5% (v/v) FBS without carbon dioxide at 24℃.The FBCs were fibroblastic in morphology and subcultured for more than 200 passages.All reagents and media were purchased from Gibco (Thermo Fisher Scientific Inc.).

2.2 Total RNA,Genomic DNA Isolation,and cDNA Synthesis

Total RNA was isolated from cells using an EasyPure RNA kit (TransGen Biotech,Beijing,China) in accordance with the manufacturer’s instructions.First-stand cDNA synthesis was performed with 1 μg of total RNA and the Prime-ScriptTMRT reagent kit with gDNA Eraser (Takara,Dalian,China) following the manufacturer’s instructions.Genomic DNA was extracted from muscle tissueviaphenol-chloroform extraction (Sambrooket al.,1982).The qualities of total RNA and genomic DNA were evaluated by 1.5% agarose gel electrophoresis,and the quantity was measured using a Nanophotometer Pearl (Thermo Scientific,Carlsbad,CA,USA).

2.3 Analysis of the P.olivaceus sox2 5’ Regulatory Region

Thesox2gene 5’ regulatory region was obtained from theP.olivaceusgenome,which was sequenced previously by our laboratory (Zhanget al.,2016;GenBank project accession PRJNA352095).Prediction and analysis of its transcription factor binding sites (TFBS) were performed using the online program MatInspector (http://www.genomatix.de/matinspector.html) (Carthariuset al.,2005).

2.4 Semiquantitative Analysis of GATA4-sox2 Interactions with Cleaved Amplified Polymorphic Sequence (CAPS)-Based Binding Assay (CBA)

A 497 bp fragment (corresponding to the positions–1195 to–699 bp from the start codon [ATG]) of thesox2promoter containing a GATA4-binding site was amplified fromP.olivaceusgenomic DNA using primerssox2-pro-Fw1/Rv1(Table 1).Then,seamless cloning PCR with primerssox2-pro-mut-Fw/Rv (Table 1) was performed to generate the mutant containing an artificialEcoR I restriction enzyme site(RES).The resultant 497 bp mutant DNA fragment was then used for CBA (Xieet al.,2016).CBA was performed following the methods described by Liuet al.(2018b).First,200 ng of target DNA,3 μg ofH.sapiensrecombinant GATA4 protein (Cloud-Clone Corp,USA),1.5 μL of CutSmart buffer (NEB),and,distilled water were combined to prepare a mixture with a final volume of 15 μL.The same amount of bull serum albumin (BSA) was used as a control.Then after the mixture was preincubated at 37℃ for 30 min,1 unit ofEcoR I -HF? (NEB) was added to the mixture and incubated for 5 min at 37℃ for digestion.Finally the denatured digested DNA mixture was loaded onto a 1.5%agarose gel for electrophoresis analysis.The experiment was repeated thrice.

Table 1 Primer sequences

2.5 Plasmids

The recombinant expression plasmids pEGFP-C1-GATA4,sox2-pEGFP-1,and sox2-pGL3-basic used in this study were constructed previously by our laboratory (Liuet al.,2016;Liuet al.,2018b).pEGFP-C1,pEGFP-1,and pGL3-basic were used as the corresponding controls for transfection.

2.6 Transfection,GFP,and Luciferase Reporter Assay

Dissociated cells were allocated to 24-well cell culture plates (NEST Biotechnology,Wuxi,China) (approximately 2 × 105per well) and cultured for 8 h under suitable conditions.Once the cells were 90% confluent,they were transfected with the plasmids with the TransIntro EL Transfection Reagent (TRANS),according to the manufacturer’s instructions.

In the case of the GFP reporter assay,the cells were transfected with the promoterless vector (pEGFP-1) as the negative control.The expression of GFP in transfected cells was observed using an inverted fluorescence microscope at 48 h after transfection.

In the case of the dual luciferase reporter assay (Promega),Renilla luciferase driven by the HSV-TK promoter(pRL-TK,Promega) (10 ng per well) was co-transfected with the sox2-pGL3-basic to normalize the variability of transfection efficiency.At 48 h after transfection,luciferase activity was measured using a GloMax 20/20 Luminometer (Promega) according to the manufacturer’s instructions.Luciferase activity was detected by calculating the ratio of firefly to Renilla luciferase.All data were measured from at least six independent experiments.

2.7 Treatment with H.sapiens Recombinant GATA4 Protein in Cells

HEK293T cells were treated with 1 μg mL-1ofH.sapiensrecombinant GATA4 protein 6 h after transfection with sox2-pGL3-basic plasmid.Luciferase activity was measured at 48 h after transfection.

2.8 qRT-PCR

qRT-PCR was performed in a 20 μL solution containing 10 ng of template cDNA and 2×SYBR Green qPCR Master Mix Plus (US Everbright?Inc,Suzhou,China) using LightCycler 480 (Roche,Switzerland) at 95℃ for 5 min pre-incubation,followed by 45 cycles of 95℃ for 15 s and 60℃ for 45 s.Finally,the melting curve was analyzed to detect single amplification.Fluorescent signal accumulation was recorded at the 60℃ 45 s phase during each cycle under the control of LightCycler 480 Software 1.5 (Fanet al.,2015).β-actinwas used as the reference gene.

2.9 Statistical Analysis

All data were tested using SPSS 20.0 software (IBM,New York,USA).Significant differences between samples were analyzedviaone-way ANOVA followed by the LSD test.Data were considered significantly different whenP<0.05.Data from qRT-PCR were expressed as mean ± standard error of the mean (SEM).Data from the luciferase reporter assay were expressed as means ± standard deviation (SD).

3 Results

3.1 Analysis of the P.olivaceus sox2 5’ Regulatory Region

The cDNA sequence ofP.olivaceus sox2was successfully identified by previous research (Gaoet al.,2014),and the GenBank accession number is KF709692.1.No insertion of introns in theP.olivaceus sox25’ regulatory region was found by blasting the cDNA sequence with theP.olivaceusgenome (Fig.1A).Thus,the sequence with a length of 3000 bp upstream of the initiation codon was considered the promoter region ofP.olivaceus sox2.It was analyzed using the online program MatInspector and many TFBSs were found.GATA4 TFBS (–854 to–866) was found with a core binding motif (GATA) located in–858 to–861 bp of theP.olivaceus sox2promoter (Fig.1B).

Fig.1 The gene structure analysis and promoter sequence of P.olivaceus sox2.(A) Gene structure analysis of sox2.Black boxes,gray boxes,and lines represent CDS,UTRs,and introns,respectively;(B) The 5’ regulatory sequence of sox2.The sequence shaded in gray is the 5’ UTR of sox2.The red letters indicate the initiation codon.Letters in blue are the predicted recognition and binding sequences of the GATA4 protein.Sequences marked with a red line correspond to the primer pair Sox2-pro-Fw1/Rv1.Sequences marked with a blue line correspond to the primer pair Sox2-pro-Fw2/Rv2.CDS=coding sequence;UTR=untranslated region.

3.2 Binding of H.sapiens Recombinant GATA4 Protein to the P.olivaceus sox2 Promoter Sequence

CBA was performed to verify whether GATA4 binds to thesox2promoter.The artificial RESEcoR I in the DNA fragment was selected using DNAstar software.The nucleotides 18 bp upstream of the GATA4 core binding motif to the artificialEcoR I recognition site (Fig.2A) were mutated,and the mutant DNA sequence for the CBA experiment was obtained.TheH.sapiensGATA4 recombinant protein was selected for the CBA experiment because GATA4 is highly conserved between vertebrates,especially the N-terminal and C-terminal zinc finger domains of its major functional regions.Fig.2B shows that the MT DNA fragments treated withH.sapiensGATA4 recombinant protein in Lane 1 were protected from cleavage byEcoR I,but the MT DNA fragments treated with the control protein BSA in Lane 2 were cleaved byEcoR I.Thus,theH.sapiensGATA4 recombinant protein did bind to the DNA fragment.

Fig.2 Validation of the interaction between GATA4 protein and P.olivaceus sox2 DNA by CBA.(A) Diagram of the wildtype (WT) sox2 DNA,the mutated DNA (MT) and related RESs for CBA.The gray oval indicates the binding protein GATA4 and the occupied region of sox2.EcoR I is an artificial RES created by base changes (lowercase letters) in the flanking sequence of the mutated DNA.(B)The results of CBA showed that the artificial EcoR I site in MT is protected by binding of the GATA4 protein.However,the artificial EcoR I site is cut in the presence of BSA protein.CBA,cleaved amplified polymorphic sequencebase binding assay;RES,restriction enzyme site;BSA,bovine serum albumin.

3.3 Analysis of P.olivaceus sox2 Promoter Activities

The results of the CBA experiment indicated thatP.olivaceussox2is the target gene of GATA4.The recombinant expression plasmids sox2-pEGFP-1 and sox2-pGL3-basic were constructed using the promoter sequence shown in Fig.1B to explore the regulatory relationship between GATA4 and theP.olivaceus sox2gene.EGFP and luciferase reporter assay were performed to detect the activity of theP.olivaceus sox2promoter.TheP.olivaceus sox2promoter could drive the expression of EGFP in FBCs and HEK293T cells (Fig.3A).In addition,theP.olivaceus sox2promoter could drive the expression of firefly luciferase in HEK293T cells (Fig.3B).

Fig.3 Determination of P.olivaceus sox2 promoter activity.(A) EGFP reporter assay of P.olivaceus sox2 promoter in cells.The P.olivaceus sox2 promoter could drive the expression of EGFP in both flounder brain cells and HEK293T cells.The length of the scale bars is 200 μm.(B) Luciferase reporter assay of P.olivaceus sox2 promoter in HEK293T cells.HEK293T cells were co-transfected with the constructed plasmids sox2-pGL3-basic and pRL-TK.After 48 h,we detected the relative luciferase activities (firefly/Renilla).Data are shown as mean ± SD (n=6).Asterisks indicate significant differences (P <0.01).

3.4 GATA4 Enhanced the Expression of P.olivaceus sox2

Luciferase reporter assay was performed to explore the effect of GATA4 onsox2promoter activity.Fig.4A shows that,compared with the control group,the relative luciferase activity significantly increased in HEK293T cells incubated withH.sapiensrecombinant GATA4 protein.Moreover,the overexpression ofP.olivaceusGATA4 evidently improved the relative luciferase activity in HEK293T cells(Fig.4B).Overall,the results implied that GATA4 could increase the activity of thesox2promoter.Then,qRT-PCR was performed to detect thesox2mRNA level in FBCs that overexpressedP.olivaceusGATA4.The transcripts ofsox2significantly increased 48 h afterP.olivaceusGATA4 overexpression,compared with the control group (Fig.4C).

Fig.4 GATA4 enhanced the expression of P.olivaceus sox2.(A) H.sapiens recombinant GATA4 protein increased the activity of the sox2 promoter.HEK293T cells co-transfected with the constructed plasmids sox2-pGL3-basic and pRL-TK were incubated with H.sapiens recombinant GATA4 protein.After 42 h,we detected the relative luciferase activities (firefly/Renilla).Data are shown as means ± SD (n=6).Asterisks indicate significant differences (P <0.05).(B) P.olivaceus GATA4 increased the activity of the sox2 promoter.HEK293T cells were co-transfected with the constructed plasmids pEGFPC1-GATA4,sox2-PGL3-basic,and pRL-TK.After 48 h,the relative luciferase activities (firefly/Renilla) were detected.Data are shown as means ± SD (n=6).Asterisks indicate significant differences (P <0.01).(C) Flounder brain cells were transfected with the plasmids pEGFP-C1 and pEGFP-C1-GATA4,respectively.After 24 h and 48 h,the relative expression levels of sox2 mRNA were detected,respectively.Data are shown as mean ± SEM (n=3).Asterisk indicates a significant difference (P <0.05).

4 Discussion

Lineage specifiers and pluripotency-associated factors have been considered the key elements in deciding cell fate.With the function of them,the stem cells either differentiate into specific cell types,or maintain pluripotency.

GATA4,as a lineage specifier,plays an important role in regulating cell fate (Huanget al.,2014).Previous studies showed that vertebrate GATA4 is expressed in many tissues of mesodermal and endodermal origin,such as the heart,intestine,liver,and gonads (Kuoet al.,1997;Holtzinger,2005;Lentjeset al.,2016).GATA4 is a cardiac muscle-restricted transcription factor,and its properties suggest an important regulatory role in heart development.Pluripotent P19 embryonal carcinoma cells can differentiate into beating cardiac muscle cells with the effect of GATA4 overexpression (Iedaet al.,2010).Researchers successfully induced mouse tail-tip fibroblasts into functional hepatocyte-like (iHep) cells by transduction of GATA4,Hnf1a,and Foxa3.The iHep cells rescued almost half of fumarylacetoacetate-hydrolase-deficient (Fah2/2) mice from death by restoring liver function (Huanget al.,2011).Researchers recently showed that mouse fibroblasts could be reprogrammed into Leydig-like cells (LCs) with functions similar to those of adult LCs by expressing three transcriptional factors,namely,Dmrt1,GATA4,and Nr5a1 (Yanget al.,2016).

Sox2has become widely known for its role in the maintenance of pluripotent stem cells of the early embryo and for its ability to reprogram differentiated cells to iPS cells.Completesox2knockout in the mouse led to loss of pluripotent cells of the epiblast (Avilionet al.,2003).Sox2together withoct4,Klf4,andc-Mychave been identified primarily as reprogramming factors for inducing pluripotency(Takahashi,2006).Subsequently,many research groups reported that some of them could be replaced by other pluripotency-associated factors.Traditionally,the overexpression of lineage specifiers counteracts pluripotency networks(Loh and Lim,2011).In recent years,several lineage specifiers were reported to be able to replace particular Yamanaka factors during the induction of iPS cells (Montserratet al.,2013;Shuet al.,2013,2015).This finding suggests a ‘seesaw’ model wherein pluripotency-associated proteins can function as lineage specifiers and differentially direct cell fate.Pluripotency is maintained as a consequence of the balance of different lineage-specifying forces.Among these lineage specifiers,all GATA family members can substituteoct4to induce pluripotency.Furthermore,the pluripotency-related genesall4is considered the target gene of GATA family members during reprogramming,and it serves as a bridge linking the lineage-specifying GATA family to the pluripotency circuit (Shuet al.,2015).

While the essential role of mammalian sox2 in maintaining the pluripotency of stem cells and in neurogenesis has been well studied,information from nonmammalian vertebrate species,especially marine teleosts,is relatively limited.In the past,when our team explored the functions of marine fish,P.olivaceussox2 in early embryonic development and neurogenesis,theP.olivaceus sox2gene was found to have high homology with the mammaliansox2and a relatively high expression level in the blastocyst and brain (Gaoet al.,2014).Moreover,the promoter ofP.olivaceussox2 could drive the specific expression of GFP in theDanio reriobrain (Liuet al.,2016).In addition,ES cells are derived from the inner cell mass of blastocysts,and the brain is an important part of the CNS.All these results suggest thatP.olivaceus sox2may have a conserved function in regulating cell fate.Its TFBS was analyzed by using the online program MatInspector,which identifies TFBS in nucleotide sequences using a large library of weight matrices to explore the upstream regulatory TFs ofP.olivaceus sox2in the above functions.Unexpectedly,binding sites of the lineage specifier GATA4 were found in theP.olivaceus sox2promoter.Thus,whether sox2 is the target gene of GATA4 during regulation of cell fate inP.olivaceusneeds to be determined.Then,a CBA experiment was performed to investigate whether GATA4 can bind to theP.olivaceus sox2promoter.As shown in Fig.2,theH.sapiensGATA4 recombinant protein did bind to the DNA fragment of theP.olivaceus sox2promoter,confirming thatP.olivaceus sox2is indeed the target gene of GATA4.

Luciferase reporter assay was performed to explore the regulation of GATA4 onP.olivaceus sox2.The activity of theP.olivaceus sox2promoter was analyzed using GFP and luciferase reporter assay,respectively.Fig.3 shows that the promoter that could drive the expression of GFP and firefly luciferase in the HEK293T cells ofP.olivaceus sox2was selected as well as in the FBCs (Liuet al.,2016).Subsequently,a luciferase reporter assay was performed to investigate the regulatory effect of GATA4 onP.olivaceus sox2promoter activity.The findings showed thatH.sapiensrecombinant GATA4 andP.olivaceusGATA4 improved its activity (Figs.4A and 4B).Likewise,theP.olivaceus sox2transcripts were increased afterP.olivaceusGATA4 overexpression in the FBCs (Fig.4C).From the above results,we infer that GATA4 could enhance the expression ofP.olivaceus sox2.Previously,we reported thatP.olivaceusgata4transcripts were detected in oogonia with reprogramming capability (Liuet al.,2018b).Furthermore,P.olivaceus sox2was found to be expressed in oogonia (the result was unpublished).Thus,P.olivaceusGATA4 may participate in deciding the fate of pluripotent stem cellsviathe regulation ofsox2.

Sox2 is not only the master regulator of pluripotent stem cells,but also plays an important role during embryonic development.During the development of the embryo,the expression ofsox2marks the developing CNS from the earliest stages of embryogenesis (Cavallaroet al.,2008).Generation of hypomorphic and conditional knockout mutations in mouse and loss-and gain-of-function experiments performed in mouse and other animal models uncovered unique functions ofsox2in CNS development (Avilionet al.,2003).Likewise,mammalian GATA4 is a regulator of astrocyte cell proliferation and apoptosis in the embryonic and adult CNS (Agnihotriet al.,2009).In addition,one line of evidence indicates that human GATA4 regulates apoptosis-related genes in cultured glioblastoma multiforme cell lines (Agnihotriet al.,2011).During the embryogenesis ofP.olivaceus,gata4andsox2have a high level of expression in the neurula stage (Gaoet al.,2014;Liuet al.,2018b).Thus,P.olivaceusGATA4 may be involved in the development of CNSviaregulation ofsox2.

5 Conclusions

In this study,the TFBS in the promoter ofP.olivaceus sox2was analyzed,and theP.olivaceus sox2promoter harbored a GATA4-binding motif.Subsequently,a CBA experiment showed thatH.sapiensGATA4 recombinant protein could bind to the DNA fragment of theP.olivaceus sox2promoter,indicating thatP.olivaceus sox2is the target gene of GATA4.Luciferase reporter assay and overexpression experiments confirmed that GATA4 could increase the expression ofsox2inP.olivaceus.Our findings provide a reference for exploring the functions of GATA4 in the regulation of pluripotent stem cells and the development of the CNS inP.olivaceus,as well as the regulation ofsox2expression in other vertebrates.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (No.31672646),the National Key R&D Program of China (No.2018YFD0900101),the Natural Science Foundation of Shandong Province (No.ZR2018BC052),and the Science and Technology Project of Yantai University (No.2219008).The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.