The 7-day-old (n = 3) and 1-year-old (n = 6) Jianzhou Daer male goats were used as experimental models, and the goats were purchased from Sichuan Jianyang Dageda Animal Husbandry Co., Ltd (Sichuan, China). Animal studies were approved by the Institutional Animal Care and Use Committee, Southwest Minzu University (Chengdu, China). One-year-old goats were slaughtered by using carotid bleeding. The heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi, biceps femoris, and triceps brachii were harvested and stored at liquid nitrogen. Seven-day-old goats were slaughtered on an empty stomach and washed twice with 10 mg/L of benzalkonium bromide and 75% ethyl alcohol. The longissimus dorsi was isolated under sterile conditions, washed thrice in phosphate-buffered saline (PBS) (HyClone, Logan, UT, United States) supplemented with 3% penicillin/streptomycin and minced. Triploid collagenase type II (Sigma-Aldrich Corp., St. Louis, MO, United States) was added to the minced tissues at 37°C for 1 h with gentle agitation to sufficiently digest. The same volume of DMEM/F12 (HyClone) containing 10% fetal bovine serum (FBS) was used to terminate enzymatic digestion. The cell suspension was filtrated through 75-μm mesh and centrifuged at 2,000 r/min for 5 min, and then the suspension was disposed with red blood cell (RBC) lysed solution. After centrifugation at 2,000 r/min for 5 min, the intramuscular preadipocytes were re-suspended in DMEM/F12 supplemented with 10% FBS and seed in cell culture flask. These cells were cultured in an incubator with 5% CO₂ at 37°C.

Based on the predicted sequence of goat KLF4 gene at GenBank with accession number: XM_005684390.1, clonal primers were designed with Primer Premier 5.0 software, which are KLF4-KS: 5′-TACCCCTTCTGCTTCGGA-3′ and KLF4-KA: 5′-TGTGGGTCACATCCACTGTT-3′. A 2 × GC-rich PCR MasterMix (TIANGEN, Beijing, China) and AG 22331 Hambury PCR (Thermo Fisher Scientific, Waltham, MA, United States) were used to perform polymerase chain reaction (PCR). The right separated DNA was purified by 1% agarose gel and connected with pMD-19T vector. These recombinants were converted into Escherichia coli DH5α (Tiangen, China). Finally, certified recombinants were submitted to TSINGKE (Chengdu, Sichuan, China) for sequencing in two directions. The tools for sequence analysis, including ORF Finder, DNAMAN, Version 5.2.10) and the Conserved Domains (CD) Search Service as described (Qing et al., 2018b), were used to analyze the open reading frame (ORF), the homology of derived amino acid sequences, and protein domain, respectively. The protein interaction network and neighbor-joining (NJ) phylogenetic tree were constructed by using STRING and MEGA7.0, respectively.

The coding sequence (CDS) of KLF4 was cloned into the pHBAD-EF1-MCS-3flag-CMV-GFP vector, named as pHBAD-KLF4. The SnapGene program was used for designing the primers, KLF4-Eco/Eco-Fg: gtgaccggcgcctacgccaccATGAGGC AGCCACCTGGC, KLF4-Eco/Eco-Rg: ggatcccgcccggggAAAGT GCCTTTTCATATGT (lowercase letters represent the sequence of vector). The pHBAD-KLF4 was transformed into DH5α and screened by PCR amplification. pHBAD-KLF4 and pHBAD-BHG were co-transfected in HEK293A cells by Lipofectamine^TM 3000 (Invitrogen, Carlsbad, CA, United States) to package adenovirus. Following multiplication in 293A cells, high titer of adenovirus was harvested for KLF4 expression. The adenovirus titer was measured by TCID₅₀ method. An adenovirus expression green fluorescent protein was used as the control (vector) and was stored in our laboratory.

Three gene-specific siRNAs for KLF4 were designed and synthesized by Invitrogen according to the sequence of goat KLF4 (KU041754.1), named as KLF4 siRNA-1 (5′-GACCUGGACUUUAUCCUCUCCAACUdTdT-3′), KLF4 si RNA-2 (5′-CCUACACGAAGAGUUCUCAUCUCAAdTdT-3′), and KLF4 siRNA-3 (5′-ACCACCUCGCCUUACAUAUGAAdT dT-3′). The sequence of negative control was 5′-UUCUCC GAACGUGUCACGUdTdT-3′.

The goat intramuscular preadipocytes whose confluence reached 80% were adipogenic inducted by DMEM/F12 supplemented with 10% FBS and 100 μM of oleic acid (Sigma) as described (Shang et al., 2014). For KLF4 knockdown, intramuscular preadipocytes at 80% confluence were transfected by siRNAs via Lipofectamine^® RNAIMAX Reagent (Invitrogen). Ad-GFP [negative control, (NC)] or Ad-KLF4 was used to infect cells to perform the experiment to overexpress KLF4. The cells were collected and monitored at day 1 with an analysis by qPCR, Oil Red O staining, or Bodipy staining after adipogenic differentiation.

The cells were washed twice with PBS and fixed with 500 μl of 10% formaldehyde for 30 min. Then, the cells were washed and stained using the Oil Red O or Bodipy working solutions for 20 min. Cells were observed and photographed with an Olympus TH4-200 microscope after staining and washing. Finally, Oil Red O dye was extracted with 1 ml of isopropanol and the Oil Red signal was quantified by measuring the absorbance at 490 nm (OD 490) to determine the extent of differentiation.

Total RNA was extracted from 1-year-old goats using TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer. The integrality and concentration of total RNA from cultured cell samples or tissues were detected by 1% agarose gel electrophoresis and ultraviolet spectrophotometer. Total RNA of 1 μg for each sample were reverse-transcribed by RevertAid First Strand cDNA Synthesis Kit (Thermo) according to the instructions. Peptidylprolyl isomerase A (PPIA) was selected to normalize the expression levels. qPCR was by using TB Green^TM Premix EX Taq^TM (Tli RNase H Plus) (Takara) and CFX96 (Bio-Rad, Hercules, CA, United States). Relative mRNA expressions were normalized by UXT. The primers’ information for qPCR is listed in Table 1. The 2^–ΔΔCt method was used to analyze the relative expression level of each gene (Livak and Schmittgen, 2001).

The cells were washed twice with PBS. Total protein was extracted from cells using 150 μl of radioimmunoprecipitation assay (RIPA) (Biosharp, Hefei City, Anhui, China) solution supplemented with 1% phenylmethylsulfonyl fluoride (PMSF) for each well. The protein concentrations were measured by bicinchoninic acid (BCA) protein quantitation assay (KeyGen Biotech, Nanjing, China) according to the instructions. Total proteins of 40 μg were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) (Roche Diagnostics, Basel, Switzerland) membrane. The membrane was blocked with 5% fat-free milk and then incubated for 2 h with antibodies to C/EBPβ (WanleiBio, Shenyang, China; WL01710)/β-actin (Abcam, Cambridge, United Kingdom; ab176323), which were diluted to 1:500. The membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (diluted to 1:1,000) for 1 h. Subsequently, an immunodetection was performed using Clarity^TM Western ECL Substrate (Bio-Rad) and analyzed by FluorChem R System (ProteinSimple, San Jose, CA, United States).

The promoter of goat C/EBPβ containing KLF4 target sites was cloned from goat genome DNA using primers tagged with XhoI and HindIII restriction sites (sense primer: 5′-CCGCTCGAGCACAATCGGCCATCCCAGG-3′ and antisense primer: 5′-CCCAAGCTTTAACTGAAGGCGGGAATGGG-3′). The wild-type promoter fragment was cloned into pGL3-basic vector, named as pGL3-C/EBPβ. The constructed plasmids of (pGL3-C/EBPβ)/pGL3-basic and pHBAD-KLF4/vector were co-transfected into goat intramuscular preadipocytes using Lipofectamine^® 3000 (Invitrogen). Then the cells were induced to adipogenic differentiation for 48 h; after that, the cells were harvested, and the luciferase activity was detected by an automated microplate reader.

Phylogenetic tree constructed based on the deduced KLF4 amino acid sequences with NJ method in MEGA v7.0.14 (Kumar et al., 2016). The GeneBank accession number: Ovis aries (ALI16866.1), Bos taurus (NP_001098855.1), Sus scrofa (NP_001026952.2), Equus caballus (XP_005605741.1), Homo sapiens (ABG25917.1), Macaca mulatta (NP_001136265.1), Rattus norvegicus (NP_446165.1) Mus musculus (NP_034767.2) Canis lupus familiaris (XP_005627053.1), Felis catus (NP_001166915.1), Oryctolagus cuniculus (XP_017202748.1), Gallus gallus (XP_004949426.1), and Danio rerio (NP_001106955.1).

All data were presented as “means ± SD.” The variance of data was analyzed by SPSS; to evaluate the significance of the differences between two groups, the means were compared using Student’s t test. Multiple-group comparisons were performed by Duncan’s multiple comparisons test. P < 0.05 was considered to be significant difference. All experiments in our study were carried out three times at least.

Previous studies reported that the function discrepancies of KLF4 were observed in different animal species and cell models (Birsoy et al., 2008; Park et al., 2017). To further explore its role in goat intramuscular adipogenesis, the subcutaneous fat tissue from 1-year-old Jianzhou Daer male goats was harvested. By means of PCR amplification, the complete CDS of KLF4 (GenBank No. KU041754.1) was obtained, which contains 1,461 bp and codes 311 amino acids (Supplementary Figure 1A). The deduced protein contains three zinc finger structures, which were the representative structure of KLFs (Supplementary Figure 1B). The amino acid sequence of goat KLF4 shows a similarity of 97.94, 97.94, 92.61, and 92.39% with O. aries, B. taurus, H. sapiens, and M. musculus, respectively (Supplementary Figure 2). According to the corresponding amino acid sequences, an NJ phylogenetic tree was constructed, which suggested that the KLF4 protein in goat has higher genetic relationship with O. aries and B. taurus (Supplementary Figure 2).

To explore the characteristics of KLF4 expression, qPCR was performed to detect its expression in various goat tissues. The result showed that KLF4 expression was the highest in the lung, whereas it was of middle expression level in the spleen and subcutaneous white adipose tissue with ∼13.7- and 18.6-fold change to that of the heart, respectively (Figure 1A). Alongside that, KLF4 presents a similar mRNA level in the heart, longissimus dorsi, biceps femoris, and triceps brachii, where the expression level was the lowest (Figure 1A). In view of subcutaneous white adipose tissue will be fractionated into mature adipocytes and stromal vascular fraction and the skeletal muscle tissues containing minimal intramuscular adipocytes, we isolated intramuscular preadipocytes from longissimus dorsi and induced them to adipogenic differentiation. The expression of KLF4 during differentiation showed that KLF4 mRNA abundance ratio was downregulated for the previous 36 h during differentiation and exhibited an increasing trend from 36 to 124 h (Figure 1B). All the above suggest that KLF4 might regulate the adipogenic differentiation of goat intramuscular preadipocytes.

To reveal the function of KLF4 on intramuscular preadipocyte differentiation, three independent siRNAs were transfected into preadipocytes to knockdown of KLF4 expression. The interference efficiency assay suggested that the siRNAs dramatically decreased the expression of KLF4 (Supplementary Figure 3). Interference efficiency reached 71.62, 85.11, and 61.50% compared with negative control (siNC) for siRNA1, siRNA2, and siRNA3, respectively (Supplementary Figure 3). Because of the prominent effect of siRNA2, the subsequent KLF4 knockdown experiment was performed by siRNA2; we named it siKLF4 (Figure 2A). At morphological observation, which was performed by Oil Red O staining and Bodipy staining, we found that not only lipid accumulation was augmented but also lipid droplets were increased, both of which were caused by knockdown of KLF4 expression (Figures 2B–D). Consistently, the Oil Red O signal was significantly increased in siKLF4 group compared with that of siNC as shown in Figure 2C. These data suggest that knockdown of KLF4 expression can increase intramuscular preadipocyte lipid accumulation.

Preadipocyte differentiation required the expression of numerous lipogenic genes, and then the cells present lipid accumulation and cellular morphology change (Ali et al., 2013; Cohen and Spiegelman, 2016). To explore the enhancive lipid content in KLF4 knockdown intramuscular preadipocytes whether caused by lipolysis inhibiting or promoting differentiation into adipocytes, we further detected the expression changes of several adipogenic and lipolysis genes, including C/EBPα, PPARγ, SREBP1, aP2, and Pref1. We found that the interference of KLF4 upregulated the mRNA level of PPARγ, which increased by ∼2.6-fold in siRNA2-treated cells (Figure 3A). Moreover, the mRNA level of C/EBPα, cooperatively active with PPARγ, was also upregulated in KLF4 knockdown group as shown in Figure 3B. However, the mRNA levels of aP2, SREBP1, and Pref1 were comparable with those of control cells (Figures 3C–E). These evidences suggest that loss function of KLF4 promotes adipogenic genes expression of PPARγ and C/EBPα and may promote goat intramuscular preadipocyte differentiation.

To further illustrate the role of KLF4 in the differentiation of goat intramuscular preadipocytes, stable KLF4 overexpression (KLF4) and the negative control (vector) cells were established by adenovirus-mediated technique. Overexpression gave rise to ∼71.3-fold increase of KLF4 mRNA level when compared with vector (Figure 4A). In addition, overexpression of KLF4 substantially inhibited lipid contents based on the evaluation of Oil Red O staining and Bodipy staining (Figures 4B–D). Consistently, Oil Red O signal measurement showed that the OD value at 490 nm was decreased in overexpression group (Figure 4C). These data demonstrated that gain of function KLF4 can inhibit intramuscular preadipocyte differentiation.

The efficient evidences between gain and loss function of KLF4 in differentiated intramuscular preadipocytes confirmed our initial speculation. And the speculation was further confirmed by detecting the expression of adipogenic and lipolysis genes as described above. Overexpression of KLF4 significantly downregulated the mRNA level of PPARγ, as shown in Figure 5A. Surprisingly, aP2 and Pref1, two key genes in TG synthesis and differentiation, were also inhibited by KLF4 overexpression (Figures 5C,E). However, no obvious change was observed about mRNA level of C/EBPα and SREBP1 when compared with the control group (Figures 5B,D). All the above suggest that KLF4 inhibits intramuscular preadipocyte differentiation and gene expression of PPARγ, aP2, and Pref1.

The internal regulation of KLF family members has been reported to be a well-orchestrated multistep process (Eaton et al., 2008; Guo et al., 2018). To determine whether the expression of the other members of KLF family will be affected by KLF4 in goat intramuscular preadipocytes, the mRNA levels of KLF family members were measured by qPCR technique. We first noticed the expression of KLF1, KLF2, and KLF17 belonging to the same subfamily with KLF4. Knockdown of KLF4 significantly enhanced the mRNA level of KLF2 as shown in Figure 6A. Similarly, overexpression of KLF4 suppressed the mRNA level of KLF2 by ∼0.31-fold compared with control group (Figure 6B). However, knockdown or overexpression of KLF4 did not affect the expression of KLF1 and KLF17 (data was not shown). KLF5, KLF6, and KLF7, belonging to the same subfamily of KLF, exhibited a dramatically higher mRNA level in KLF4 knockdown preadipocytes and a markedly lower mRNA level in KLF4 overexpression cells (Figures 6A,B). No significant change was recorded of KLF3, KLF4, KLF8, KLF9, KLF10, KLF11, KLF12, KLF13, KLF14, KLF15, and KLF16 (data not shown). On the whole, these data imply that KLF4 inhibits the expression of KLF2, KLF5, KLF6, and KLF7 in goat intramuscular preadipocytes.

Based on the fact that KLF4 inhibits goat preadipocyte differentiation and regulates the expression of several adipogenic genes, the potential downstream targets of KLF4 should be identified. Interestingly, we found that C/EBPβ expression was opposite with KLF4 during goat intramuscular preadipocyte differentiation (Xiong et al., 2018). Combining that C/EBPβ is a very important gene in regulating preadipocyte differentiation and bioinformatic analysis showed that C/EBPβ may be a target of KLF4 stimulated us to further investigate the regulation between C/EBPβ and KLF4 expression. We found that both mRNA and protein level of C/EBPβ were significantly upregulated by KLF4 interference when compared with the control groups (Figures 7A,B). Consistently, KLF4 overexpression visibly downregulated both the mRNA and the protein level of C/EBPβ (Figures 7A,B). Thus, we speculated that C/EBPβ might be a potential target gene of KLF4. To confirm this hypothesis, we first analyzed the transcriptional binding DNA motif of KLF4 using JASPAR software¹. The result showed that KLF4 binding sequence is TGGGTGGGGC as shown in Figure 7C. Then we analyzed the promoter region of C/EBPβ and found 77 potential binding sites of KLF4, and the top 12 is shown in Figure 7D. Further, through dual-luciferase reporter assay, we showed that the transcriptional activity of C/EBPβ in cells was inhibited by KLF4 by about 73.18% when compared with the control group (Figure 7E). These data jointly suggest that KLF4 inhibits goat intramuscular preadipocyte differentiation through targeting C/EBPβ directly.