Genome-wide phylogenetic analysis, expression pattern, and transcriptional regulatory network of the pig C/EBP gene family

doi:10.21203/rs.3.rs-96827/v1

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Genome-wide phylogenetic analysis, expression pattern, and transcriptional regulatory network of the pig C/EBP gene family

https://doi.org/10.21203/rs.3.rs-96827/v1

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Background: The vertebrate C/EBP transcription factors regulate many important biological processes, such as cell proliferation, differentiation, signal transduction, inflammation, and energy metabolism. The first C/EBP protein was identified in rat liver nuclei. Development of sequencing technology resulted in identification of the C/EBP genes in various species. In this study, a bioinformatics approach was used to determine the distribution of the members of the C/EBP family in vertebrates. A phylogenetic tree was constructed to analyze the C/EBP genes in vertebrates. Based on RNA-seq data, the expression patterns of pig C/EBP members in various tissues were analyzed. In addition, a gene transcription regulatory network was constructed with pig C/EBP members as the core.

Results: We identified a total of 92 C/EBP genes in 17 vertebrate genomes. Phylogenetic analysis showed that all C/EBP TFs were classified into two groups; group I contained C/EBPβ TFs, and group II contained the remaining C/EBP TFs. The C/EBPα, C/EBPβ, C/EBPδ, C/EBPγ, and C/EBPζ genes were expressed ubiquitously with inconsistent expression patterns in various tissues. Moreover, a pig C/EBP regulatory network was constructed, including C/EBP genes, TFs, and miRNAs. A total of 39 FFL motifs were detected in the pig C/EBP regulatory network. Based on the RNA-seq data, gene expression patterns related to this FFL sub-network were analyzed in 27 adult Duroc tissues. Certain FFL motifs may be tissue specific. Functional enrichment analysis indicated that C/EBP and its target genes are involved in many important biological pathways.

Conclusions: These results provide valuable information that clarifies the evolutionary relationships of the C/EBP family and contributes to the understanding of the biological function of C/EBP genes.

Animal Science

Biotechnology and Bioengineering

EBP family

Pig

Phylogenetic analysis

Transcriptional regulatory network

Expression pattern

Feed-forward loop

The CCAAT/enhancer binding protein (C/EBP) family includes important regulators that activate or inhibit gene transcription in many tissues and is a subfamily of the basic leucine zipper (bZIP) transcription factor (TF) superfamily [1]. The C/EBP family consists of 6 genes: C/EBP alpha (C/EBPα), C/EBP beta (C/EBPβ), C/EBP delta (C/EBPδ), C/EBP epsilon (C/EBPε), C/EBP gamma (C/EBPγ), and C/EBP zeta (C/EBPζ) [2].

The first C/EBP protein was identified in rat liver nuclei [3]. Five additional C/EBP TFs had been identified in humans and rats [2]. Advances in the sequencing techniques resulted in the identification of the C/EBP genes in various species and investigation of the evolutionary relationship among the C/EBP genes. Qiu et al (2018) identified the C/EBPα, C/EBPβ, and C/EBPδ genes genome-wide in 20 vertebrate genomes, including Atlantic salmon (Salmo salar), Japanese frog (Nanorana pleskei), southern painted turtle (Chrysemys picta bellii), duck (Anas platyrhynchos), and human (Homo sapiens), and analyzed the evolutionary relationships among the C/EBPα, C/EBPβ, and C/EBPδ genes [4]. Thus, understanding of the evolution and function of the C/EBP genes requires genome-wide comparative studies.

Transcription factors play an important regulatory role at the transcriptional level by activating or repressing target genes to regulate gene expression [5]. The C/EBP TFs in the vertebrates regulate many important biological processes, such as cell proliferation, differentiation, signal transduction, inflammation, and energy metabolism [1, 4]. C/EBPα, C/EBPβ, and C/EBPδ are involved in the regulation of adipocyte differentiation [6] and mammary development [7]. In the pulmonary epithelium, C/EBPα regulates proliferation and differentiation dependent gene expression, whereas C/EBPβ and C/EBPδ regulate the expression of differentiation markers and are involved in the responses to injury and hormones [8].

The complex gene transcription regulatory network includes transcriptional regulation and post-transcriptional regulation [9]. MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression at the post-transcriptional level by selectively targeting complementary mRNAs for degradation. MiR31 directly binds to the 3'-untranslated region (3'-UTR) of C/EBPα and inhibits its expression to mediate the adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells [10]. In mouse dendritic cells, miR369 suppresses C/EBPβ to regulate the inflammatory cascade in the chronic inflammatory response [11]. Transcription factors and miRNAs often synergize in the regulation the same target gene, defined as feed-forward loop (FFL). For example, miR223-LMO2-C/EBPβ FFL plays an important role in human hematopoiesis [12].

In this study, we identified C/EBP genes in 17 vertebrates and analyzed physicochemical properties of the proteins, conservative motifs, gene structure, and phylogenetic relationships. Subsequently, the expression patterns of the C/EBP genes were analyzed. A transcriptional regulatory network containing the C/EBP genes was constructed to investigate the functions of the C/EBP genes. Our study provides a comprehensive analysis of the pig C/EBP gene family and important information for subsequent investigation of the functions in pig.

Identification of C/EBP TFs

The General Feature Format Version 3 (GFF3) profiles and the genome and protein sequence files of 17 vertebrates were downloaded from the ENSEMBL database (http://asia.ensembl.org/index.html) [13]. The vertebrates included zebrafish (Danio rerio), tropical xenopus (Xenopus tropicalis), Chinses soft-shell turtle (Pelodiscus sinensis), chicken (Gallus gallus), platypus (Ornithorhynchus anatinus), cat (Felis catus), dog (Canis familiaris), horse (Equus caballus), pig (Sus scrofa), cow (Bos taurus), goat (Capra hircus), sheep (Ovis aries), mouse (Mus musculus), rat (Rattus norvegicus), macaque (Macaca mulatta), chimpanzee (Pan troglodytes), and human (Homo sapiens). The hmmbuild tool of HMMER 3.0 [14] was used to build the Hidden Markov Model (HMM) of the C/EBP family. The hmmsearch tool search for all putative TFs of the C/EBP family was performed (cutoff value <1E-20). The Conserved Domain Database of NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/ wrpsb.cgi) [15] and the SMART tool (http://smart.embl-heidelberg.de/) [16] were used to determine the conserved domains of each C/EBP protein. The longest protein of each C/EBP TF was retained.

Physicochemical parameters, exon-intron structure, and motif analysis

ProtParam (http://web.expasy.org/protparam/) [17] was used to analyze the physicochemical parameters of the C/EBP proteins, including molecular weight (MW), aliphatic index, isoelectric point (pI), and grand average of hydropathicity (GRAVY). The conserved motifs of the C/EBP proteins were identified by the online platform MEME (http://meme-suite.org/) [18]. The exon and intron structures of the C/EBP genes were obtained from the ENSEMBL gene annotation information. The diagrams of the gene structures were generated using the Gene Structure Display Server (GSDS) 2.0 (http://gsds.gao-lab.org/) [19].

Phylogenetic analysis

Maximum likelihood (ML) phylogenetic tree of C/EBP TFs was constructed using the Jones-Taylor-Thornton with Gamma Distributed (JTT+G) substitution model and 1,000 replicate bootstrap tests, and a cutoff bootstrap value of 65 was set to define the clades using MEGA (v10.0) [20]. Representations of the phylogenetic tree were constructed using the iTOL tool (https://itol.embl.de/) [21].

Construction of the transcriptional regulatory networks of the pig C/EBP genes

The Position Weight Matrix (PWM) of transcription factors for Sus scrofa was downloaded from the cisbp database (http://cisbp.ccbr.utoronto.ca/) [22]. The genomic sequences 2 kb upstream of the translation start site (TSS) and 3'-UTR were extracted by the R package biomaRt [23] for all coding genes. The TFBSTools package of R language [24] was used to predict the target genes of C/EBPs and the transcription factors regulating C/EBP genes with a threshold relScore value of 0.85. The sequences of miRNAs were downloaded from the miRBase database (http://www. mirbase.org/) [25]. The miRanda software [26] was used to predict the miRNA targets with a threshold TotScore value of 120. Cytoscape 3.7.2 [27] was used to visualize the gene regulatory network, and the NetworkAnalyzer tool was used to calculate the network topology properties, including the clustering coefficient, network centralization, and network heterogeneity. The R software package basicTrendline [28] was used to analyze the network node degree distribution.

Expression analysis of the pig C/EBP genes and target genes

The raw RNA sequencing (RNA-seq) data obtained in 27 adult Duroc pig tissue samples were downloaded from the NCBI Sequence Read Archive with the BioProject number PRJNA392949 [29]. The tissues included fat, thyroid, lymph, ovary, uterus, breast, spleen, lung, liver, placenta, etc. Purification of the total RNA from the mixture of equally unrelated pig pool tissues was performed.

After QC step with FastQc v0.11.8 (http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/), clean data were mapped and genome indexed with Hisat 0.1.6-beta 64-bit [30] to the pig genome (Sus scrofa 11.0). To obtain the expression levels of the genes across 27 tissues, the fragments per kilobase of exon model per million mapped reads (FPKM) values were calculated using Stringtie 1.0.4 (Linux x86 64) [31]. The TBtools software [32] was used to visualize the heatmaps of the gene expression profiles in 27 tissues.

The tissue specificity index (τ) of the C/EBP genes was also investigated. The tissue specificity index is defined as where x_iis the expression profile component normalized by the maximal component value and N is the number of tissues [33].

Functional enrichment analysis

The Database for Annotation, Visualization, and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/) [34] was used to annotate the functions of C/EBPs and their target genes, which includes gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.

Identification of C/EBP genes in the vertebrates

A total of 92 C/EBP genes were identified in 17 vertebrate genomes. The number of C/EBP genes in each species was slightly different as shown in Table 1. In total, the C/EBP family included C/EBPα, C/EBPβ, C/EBPδ, C/EBPε, C/EBPγ, and C/EBPζ. In particular, the C/EBPα and C/EBPγ genes of all species are located on the same chromosome within a distance of less than 100 kb, except for Xenopus tropicalis.

Various physicochemical properties of each C/EBP TF were calculated and are shown in Table 2 and Table S1. The C/EBPζ protein contains the highest number of amino acids and has the highest molecular weight, and the C/EBPγ protein consists of the least number of amino acids with the lowest molecular weight. The aliphatic index, a measure of thermostability, ranged from 60.36 to 76.17. There are significant differences (p<2e-16) in the aliphatic index between C/EBP TFs. The GRAVY values of C/EBP proteins are negative indicating hydrophilic properties. The GRAVY values vary from -1.4060 to -0.4360. The pI values of C/EBPβ, C/EBPε, and C/EBPγ proteins in each species are higher than 7, and the pI values of C/EBPζ are less than 7. The C/EBPα protein of zebrafish is acidic, and C/EBPα proteins of other species are alkaline. The C/EBPδ protein is acidic in 1/3 species and alkaline in the remaining 2/3 species.

Phylogenetic relationship analysis

To analyze the phylogenetic relationships between 92 C/EBP genes in 17 species, an unrooted maximum likelihood phylogenetic tree was constructed as shown in Fig 1. All C/EBP TFs were classified into two groups. Group I contains 16 vertebrate animal C/EBPβ TFs and was named C/EBPβ. Group II contains the remaining C/EBP TFs, which can be divided into five clades. Following the nomenclature, we named the clades of group II as C/EBPα, C/EBPδ, C/EBPε, C/EBPγ, and C/EBPζ. C/EBP genes were detected in most vertebrates indicating that the C/EBP family members originated in the early stage of vertebrate evolution. In each clade, C/EBP genes of the same order tend to cluster together indicating higher similarity to each other than to other orders of C/EBP genes. C/EBP orthologs of the order primates includes human, macaque, and chimpanzee; the order artiodactyls includes pig, cattle, and goat; the order carnivore includes cat and dog; and the order rodents (rat and mouse) genes are clustered together.

To investigate the structural features of the C/EBP members, the gene structure and conserved motifs were evaluated by the phylogenetic analysis, as shown in Fig 2. The number of exons in C/EBPζ genes, which contained 15 or 16 exons, was higher than that in the other C/EBP genes. Most of the C/EBPα, C/EBPβ,C/EBPδ, C/EBPε, and C/EBPγ genes contained one or two exons. A total of 10 conserved motifs were identified in C/EBP proteins. All C/EBP TFs contain motif 2, motif 3, and motif 6. Motifs 8 and 10 are unique motifs in C/EBPζ proteins and may be associated with the clade-specific functions of the C/EBP proteins.

Expression analysis of the C/EBP genes

The expression of C/EBP genes was compared in 27 adult Duroc pig tissues. The 6 C/EBP genes from pig were classified into two groups based on the cluster analysis of the C/EBP gene expression levels in various tissues, as shown in Fig 3. Group I contains only the C/EBPε gene, and other genes are included in group II. The expression levels of the C/EBPε gene were low in all tissues (FPKM<5), and the gene is expressed only in the intestine, salivary gland, thyroid, uterus, and lymph. Although C/EBPα, C/EBPβ, C/EBPδ, C/EBPγ, and C/EBPζ are expressed ubiquitously, the expression patterns in various tissues are inconsistent; the tissue specific index values (τ) are 0.862, 0.700, 0.654, 0.499 and 0.433, respectively. C/EBPα was expressed at the high levels in the thyroid, liver, lung, and adipose tissues. C/EBPβ was expressed at the high levels in the thyroid, adrenal gland, lung, adipose, liver, and ovary tissues. C/EBPδ was expressed at the high levels in the thyroid, gall bladder, ovary, and uterus. C/EBPγ and C/EBPζ are widely expressed in other tissues at similar levels.

Additionally, the results indicate that the expression patterns in certain tissues, such as brain and spinal cord in the nervous system and ovary and uterus tissues in the female reproductive system, are similar.

Construction of a transcriptional regulatory network of the C/EBP gene family

The C/EBP family is an important family of transcription factors that regulate the expression of the target genes by binding to the promoter regions to maintain the normal physiological processes in vivo. According to the PWMs of the C/EBPα, C/EBPβ, C/EBPδ, C/EBPε, and C/EBPγ genes from the cisbp database, we predicted 4,662, 3,164, 8,383, 7,278, and 1,604 target genes regulated by these genes, respectively (see Table 3). A total of 10,270 target genes are regulated by the C/EBP genes for a total of 25,091 regulatory relationships. Binding sites for other transcription factors and miRNAs are present in the regulatory regions of the C/EBP genes.

In this study, the C/EBP genes are predicted to be regulated by 423 TFs forming 1,582 regulatory relationships; mir503 and mir7140 are predicted to regulate C/EBPβ and C/EBPγ, respectively. Additionally, C/EBPβ and C/EBPγ are regulated by C/EBPα, C/EBPδ, and C/EBPε and C/EBPβ and C/EBPε, respectively. Interestingly, C/EBPβ self-regulation is also predicted. Thus, we constructed a pig C/EBP regulatory network (summarized in Fig 4) that includes C/EBP genes, TFs, miRNAs, and target genes. These genes were defined as a node, and the distribution of node degree approximately follows the power-law distribution indicating that the gene regulatory network is a scale-free network. Certain network concepts, including the clustering coefficient, network centralization, and network heterogeneity, were calculated to be 0.1890, 0.8160, and 25.1730, respectively.

FFLs related to the C/EBP genes

A total of 27 miRNA-FFL motifs were identified in the pig C/EBP regulatory network. According to the sequences, we predicted that mir503 and mir7140 regulate C/EBPβ and C/EBPγ, respectively. The miR503 and C/EBPβ genes coregulate 14 target genes forming 14 miR503-C/EBPβ-target gene FFL motifs, and miR7140 and C/EBPγ coregulate 11 target genes, including 11 miR7140-C/EBPγ-target gene FFL motifs. Additionally, miR503-ELF3-C/EBPβ and miR7140-ARID5B-C/EBPγ motifs were identified. The miR503-ELF3-C/EBPβ motif is involved in the miR503 and ELF3 coregulation of the C/EBPβ gene, and the miR503 gene targets the ELF3 and C/EBPβ genes. The miR7140-ARID5B-C/EBPγ motif is included in 3 regulatory relationships: miR7140→ARID5B, ARID5B→C/EBPγ, and miR7140→C/EBPγ. The C/EBPβ-binding sites in 5'-untranslated region (5'-UTR) of C/EBPγ, and C/EBPβ and C/EBPγ co-regulate 10 target genes forming 10 TF-FFL motifs. Thus, the combinations of all FFL motifs were used to construct the FFL sub-network (see Fig 5).

Based on the data of RNA-seq, gene expression patterns related to this FFL sub-network were analyzed in 27 adult Duroc tissues. The results indicate that target genes regulated by C/EBPβ and C/EBPγ have variable expression patterns in various tissues. The ATP synthase F1 subunit alpha (ATP5F1A) gene is expressed ubiquitously, and the glutamate decarboxylase-like protein 1 (GADL1) and Slit-Robo GTPase activating protein 3 (SRGAP3) genes are expressed at the high levels in the muscle and brain, respectively. We suggest that the C/EBPβ-C/EBPγ-GADL1 FFL motif may play an important role in the brain. Some FFL motifs may be tissue-specific. Based on the target genes expression pattern, we estimated that the number of FFL motifs in each tissue may be significantly different (see Table 4); however, miRNA expression patterns were not evaluated in the present study.

The dN and dS analysis of the C/EBP genes and target genes

The data on the nonsynonymous (dN) and synonymous (dS) substitution rates between the human and pig sequences were downloaded from the Ensembl database. The dN/dS values of the C/EBP genes ranged from 0.02 to 0.21 indicating that pig C/EBP genes underwent purifying selection. The dN+dS value of the C/EBPδ gene was 1.52, which was higher than that of five other C/EBP genes (0.31 ~ 0.67) indicating that the C/EBPδ gene evolved rapidly and had an increased mutation rate.

The dN+dS mean values of the target genes of C/EBPα, C/EBPβ, C/EBPδ, C/EBPε, and C/EBPγ are 0.58, 0.48, 0.56, 0.53, and 0.51, respectively. The dN/dS mean values of the target genes are 0.166, 0.175, 0.17, 0.177, and 0.166, respectively. The dN/dS and dN+dS mean values of the target genes of each C/EBP gene were compared using Kolmogorov-Smirnov (KS) test. The results indicate that the dN/dS distributions of the target genes of C/EBPα are similar to that of C/EBPβ, C/EBPε and C/EBPγ (p<0.05), respectively. The very low dN/dS values suggest strong negative selection on all C/EBP genes, which may remain due to genetic drift or persistence. The dN+dS value distribution of the C/EBPα target genes is similar to that of the C/EBPδ target genes and is significantly higher than that of other C/EBP target genes (P<0.05). The results indicate that the target genes of C/EBPα appear to be evolving rapidly.

Functional enrichment analysis of the C/EBP genes and target genes

We used the DAVID software to analyze the functions of the pig C/EBP genes. The results indicate that the functions are associated with many biological processes, including macrophage differentiation (GO: 0030225), inner ear development (GO: 0048839), positive regulation of osteoblast differentiation (GO: 0045669), transcriptional misregulation in cancer pathways (ssc05202), and tuberculosis pathways (ssc05152) (Table 5).

The functional enrichment analysis of the target genes regulated by the C/EBP genes showed that the target genes of C/EBPα, C/EBPβ, C/EBPε, and C/EBPγ are associated with nucleoplasm (GO: 0005654) and extracellular exosome (GO: 0070062). The target genes of C/EBPδ and C/EBPγ are involved in the transforming growth factor beta (TGFβ) receptor signaling pathway. The target genes of C/EBPδ are involved in the platelet-derived growth factor receptor signaling (GO: 0048008) (Table 6).

Improved genome sequencing and annotation enabled identification of all C/EBP TFs in the vertebrate genomes. This study identified all C/EBP TFs in 17 vertebrates and found that the C/EBP genes are ubiquitous in the majority of the vertebrates. The molecular phylogenetic tree showed that the C/EBP family members originated in the early stage of vertebrate evolution. Similarly, Qiu et al. analyzed the evolution of the C/EBPα, C/EBPβ, and C/EBPδ genes in 20 vertebrate genomes and demonstrated that the C/EBP TFs originated early in vertebrate evolution [4].

According to the phylogenetic tree, the C/EBPβ orthologs are clustered into a single group, and other C/EBP genes are clustered into another group. However, Qiu et al. analyzed the phylogenetic trees of C/EBPα, C/EBPβ, and C/EBPδ homologs in 2 fish, 2 amphibian, 2 reptile, 11 avian, and 3 mammalian species. The results indicated that the C/EBPα and C/EBPβ genes are clustered into one group. This difference may be due to different species and members of the C/EBP family.

Based on the data of RNA-seq, the C/EBPα, C/EBPβ, C/EBPδ, C/EBPγ, and C/EBPζ genes are expressed in all 27 adult Duroc tissues, and C/EBPε is expressed only in 5 tissues. Summers et al. (2020) created a pig gene expression atlas based on the meta-analysis and demonstrated that C/EBPε is expressed only in a few tissues, and other C/EBP genes are expressed in the majority of tissues [35]. Uhlén et al. (2015) studied the expression patterns of the C/EBP genes in 32 human tissues and demonstrated that the C/EBPβ, C/EBPδ, C/EBPγ, and C/EBPζ genes are expressed ubiquitously [36].

The differences in the exon-intron, motif structures and gene expression patterns among various clades provide some indications that the C/EBP genes may have a variety of physiological functions. Our data indicate that C/EBPα and C/EBPβ are expressed at the high levels in the adipose, ovarian, and hepatic tissues. Chen et al (2017) demonstrated that C/EBPα is the lipogenic marker of the porcine adipocytes that promotes adipocyte differentiation [37]. Wang et al. (2015) reported that C/EBPβ is expressed in porcine adipocytes to regulate adipocyte differentiation [38]. Gillio-Meina et al. (2005) reported that C/EBPα and C/EBPβ in the ovary regulate the differentiation and proliferation of granulosa cells [39]. Tang et al. (2015) demonstrated that C/EBPβ regulates insulin-like growth factor 1 (IGF1) expression in the crossbred porcine (Yorkshire * Erhualian pigs) liver during prenatal and postnatal development [40].

The C/EBP genes perform many important biological functions. Functional enrichment analysis demonstrated that pig C/EBP genes are involved in the inner ear development and positive regulation of osteoblast differentiation biological processes and the tuberculosis pathway. C/EBP genes are also involved in various biological functions. For example, C/EBPζ is involved in cochlear cell apoptosis in rats [41] and may coregulate the inner ear development with GATA binding protein 2 (GATA2) [42]; Hata et al (2005) reported that C/EBPβ promotes the differentiation of mouse mesenchymal cells into the osteoblast lineage in cooperation with Runt-related transcription factor 2 (Runx2) transcription factor [43]. Allele-specific induction of Interleukin 1 beta (IL-1b) expression by C/EBPβ contributes to increased tuberculosis susceptibility in humans [44].

Complex regulatory mechanisms of gene expression in eukaryotes control the development, physiology, and pathology [45]. In this study, we constructed a C/EBP gene regulatory network to provide some information to explain the regulatory mechanisms of the pig C/EBP genes. The results indicate that the C/EBP gene regulatory network is a typical scale-free network. A gene regulatory network influencing triple-negative breast tumors [46] and a human TF and miRNA regulatory network [47] were reported to correspond to approximately scale-free topology.

In the regulatory gene network, transcription factors play an important role at the gene transcription level due to the regulation of certain target genes. Each TF contains different conservation motifs to target various genes. In this study, the target genes of the porcine C/EBP TFs were different. In the Database of Human Transcription Factor Targets [48], the target genes of the C/EBP TFs are also different. Comparison with the target genes of the human C/EBP TFs indicated that porcine C/EBPδ regulates more target genes, but the number of the target genes of other porcine C/EBP TFs is less than that in humans. Selection of different target genes during evolution to meet the needs of normal homeostatic development and growth or to adapt to various environmental stresses may differ in different species.

The biological network is composed of motifs, and the FFL is an important motif. In this study, 39 FFLs were detected in the C/EBP genes of the transcriptional regulatory sub-network. The FFL-containing C/EBP genes play important roles in multiple tissues. Sun et al. (2010) reported that miR223->C/EBPβ-> LMO2 FFL in human myeloid cells can regulate cell proliferation and maintain normal differentiation and development [12]. Ponomarev et al. (2011) have reported that MiR124-C/EBPα-PU.1 FFL is related to the transition between an activated phenotype and a quiescent state of macrophages [49]. Shi et al. (2018) reported that 28 FFLs of C/EBPα, C/EBPβ, and C/EBPδ may be associated with occurrence of human hypertrophic cardiomyopathy [50].

In the present study, we identified all C/EBP TFs of 17 vertebrate species and demonstrated that the C/EBP genes are ubiquitous in the majority of the vertebrates. All C/EBP genes were classified into six subgroups with similar number of gene exons and motif compositions within each subgroup. The porcine C/EBP genes are expressed generally, except C/EBPε gene, and participate in many important biological functions. The C/EBP gene regulatory network was constructed to provide information on the regulatory mechanisms of the pig C/EBP genes. A total of 39 FFL motifs were identified with variable expression patterns in various tissues. Some FFL motifs may be tissues-specific. Thus, our study provides potential functional insight into the roles of the C/EBP genes in pig.

τ: tissue specificity index; 3'-UTR: 3'-untranslated region; 5'UTR: 5'-untranslated region; ARID5B: AT-rich interactive domain-containing protein 5B; ATPSF1A: ATP synthase F1 subunit alpha; BP: biological processs; bZIP: basic-leucine zipper; C/EBP: CCAAT/enhancer binding proteins; CC: cell component; DAVID: Database for Annotation, Visualization, and Integrated Discovery; dN: nonsynonymous; dS: synonymous; ELF3: E-74 like factor 3; FFL: feed-forward loop; FPKM: fragments per kilobase of exon model per million mapped reads; GADL1: glutamate decarboxylase-like protein 1; GATA2: GATA binding protein 2; GFF3: General Feature Format Version 3; GO: gene ontology; GRAVY: grand average of hydropathicity; GSDS: Gene Structure Display Server; HMM: Hidden Markov model; IL-1b: Interleukin 1 beta; IGF1: insulin-like growth factor 1; JTT + G: Jones-Taylor-Thornton with Gamma; KEGG: Kyoto Encyclopedia of Genes and Genomes; KS: Kolmogorov-Smirnov; LMO2: LIM domain only 2; MF: molecular function; miRNA: MicroRNA; ML: Maximum likelihood; MW: molecular weight; PI: isoelectric point; PU.1: Spi transcription factor; PWM: Position Weight Matrix; RNA-seq: RNA sequencing; Runx2: Runt-related transcription factor 2; SRGAP3: SLIT-ROBO Rho GTPase activating protein 3; TF: transcription factor; TGFβ: transforming growth factor beta; TSS: translation start site.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was financially supported by Natural Science Foundation of China (No. 32070571), the Academic Backbone Project of Northeast Agricultural University (No.15XG14), NEAU Research Founding for Excellent Young Teachers (2010RCB29).

Authors’ contributions

ZPW and CXZ conceived the study, and ZPW, CXZ, TW and SWL participated in its design. BZ, XL, JT and PW were involved in the acquisition of data, CXZ and TW performed all data analysis. ZPW and CXZ drafted the manuscript, and SWL, YYG, BZ, XL, JT and PW contributed to the writing and editing. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Authors' details

¹College of Animal Science and Technology, Northeast Agricultural University, Harbin 150000, China. ²Bioinformatics Center, Northeast Agricultural University, Harbin 150000, China. ³HeiLongJiang provincial Husbandry Dapartment, Harbin 150000, China.

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Table.1 Genomic distribution of C/EBP genes on vertebrates genome.

Taxonomy			Genes						Total
Class	Order	Species	C/EBPα	C/EBPβ	C/EBPδ	C/EBPε	C/EBPγ	C/EBPζ	Total
Pisces	Cypriniformes	Zebrafish	1	1	1	1	1	1	6
Amphibians	Anuras	Tropical xenopus	1	1	1	1	1	1	6
Reptiles	Chelonias	Chinses soft-shell turtle	1	1	1	1	1	1	4
Fishs	Galliformes	Chicken	1	1	1	1	1	1	4
Mammalias	Monotrematas	Platypus	1	1	1	1	1	1	5
	Carnivoras	Cat	1	1	1	1	1	1	6
	Carnivoras	Dog	1	1	1	1	1	1	5
	Perissodactylas	Horse	1	1	1	1	1	1	6
	Artiodactylas	Pig	1	1	1	1	1	1	6
		Cattle	1	1	1	1	1	1	4
		Goat	1	1	1	1	1	1	6
		Sheep	1	1	1	1	1	1	6
	Rodents	Mouse	1	1	1	1	1	1	6
	Rodents	Rat	1	1	1	1	1	1	6
	Primates	Macaque	1	1	1	1	1	1	5
		Chimpanzee	1	1	1	1	1	1	5
		Human	1	1	1	1	1	1	6

Table 2 The physicochemical properties of C/EBP proteins.

Proteins	Number of amino acids	MW (mean)	pI	Aliphatic index (mean)	GRAVY (mean)
C/EBPα	135~395	32.81	6.81~10.78	61.69	-0.77
C/EBPβ	126~348	32.16	8.3~10.37	60.74	-0.63
C/EBPδ	99~302	26.99	5.43~10.73	60.36	-0.73
C/EBPε	146~460	30.65	9.02~10.29	67.94	-0.74
C/EBPγ	137~465	19.02	9.43~11.26	70.16	-0.95
C/EBPζ	998~1074	119.81	5.31~6.03	76.17	-0.71

Table 3 Regulatory relationships involved in C/EBP family members.

C/EBP	Target genes				Factors regulating C/EBP
C/EBP	Total	Non-TF genes	TF (gene family)	C/EBP	TF (gene family)	miMRA
C/EBPα	4662	4466	196 (31)	C/EBPβ	311 (29)	-
C/EBPβ	3164	3059	105 (34)	C/EBPβ; C/EBPγ	293 (29)	miR503
C/EBPδ	8383	8063	320 (36)	C/EBPβ	336 (32)	-
C/EBPε	7278	7027	221 (35)	C/EBPβ; C/EBPγ	310 (30)	-
C/EBPγ	1604	1541	63 (19)	-	325 (32)	miR7140

Table 4 Distribution of FFLs in various tissues. In the each subnetworks, red represents higher expression levels; blue represents lower expression levels.

Due to technical limitations, table 4 is only available as a download in the Supplemental Files section.

Table 5 The results of functional enrichment analysis of porcine C/EBP family members.

Category	ID	Term	P value	C/EBP Genes
GO_MF	GO:0001077	Transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding	3.60E-07	C/EBPα, C/EBPε, C/EBPδ, C/EBPγ, C/EBPζ
GO_MF	GO:0000978	RNA polymerase II core promoter proximal region sequence-specific DNA binding	1.40E-06	C/EBPα, C/EBPε, C/EBPδ, C/EBPγ, C/EBPζ
GO_BP	GO:0030225	Macrophage differentiation	3.10E-03	C/EBPα, C/EBPε
	GO:0048839	Inner ear development	9.60E-03	C/EBPα, C/EBPδ
	GO:0045669	Positive regulation of osteoblast differentiation	1.40E-02	C/EBPα, C/EBPδ
	GO:0051091	Positive regulation of sequence-specific DNA binding transcription factor activity	2.20E-02	C/EBPβ
KEGG_PATHWAY	ssc05202	Transcriptional misregulation in cancer	4.00E-05	C/EBPα, C/EBPβ, C/EBPε
KEGG_PATHWAY	ssc05152	Tuberculosis	3.60E-03	C/EBPβ, C/EBPγ

Table 6 The results of functional enrichment analysis of porcine C/EBP target genes.

	Category	ID	Term	P value
C/EBPα target genes	GO_CC	GO:0005654	Nucleoplasm	1.37E-09
		GO:0070062	Extracellular exosome	8.76E-09
		GO:0005634	Nucleus	1.81E-05
		GO:0005737	Cytoplasm	5.53E-05
	GO_MF	GO:0044822	Poly(A) RNA binding	1.49E-05
	GO_MF	GO:0003824	Catalytic activity	5.88E-05
	KEGG_PATHWAY	ssc01100	Metabolic pathways	4.17E-06
C/EBPβ target genes	GO_CC	GO:0070062	Extracellular exosome	3.63E-09
	GO_CC	GO:0005654	Nucleoplasm	7.83E-06
	KEGG_PATHWAY	ssc01100	Metabolic pathways	4.21E-06
	KEGG_PATHWAY	ssc00310	Lysine degradation	1.40E-04
C/EBPδ target genes	GO_BP	GO:0001649	Osteoblast differentiation	5.31E-05
		GO:0000122	Negative regulation of transcription from RNA polymerase II promoter	2.02E-04
		GO:0045944	Positive regulation of transcription from RNA polymerase II promoter	2.91E-04
		GO:0007179	Transforming growth factor beta receptor signaling pathway	4.21E-04
		GO:0098779	Mitophagy in response to mitochondrial depolarization	6.24E-04
		GO:0001525	Angiogenesis	8.71E-04
		GO:0007420	Brain development	1.06E-03
		GO:1901998	Toxin transport	1.04E-03
		GO:0007507	Heart development	1.49E-03
		GO:0048008	Platelet-derived growth factor receptor signaling pathway	2.18E-03
		GO:0007059	Chromosome segregation	2.01E-03
		GO:0007519	Skeletal muscle tissue development	3.54E-03
		GO:0060325	Face morphogenesis	2.18E-03
		GO:0070372	Regulation of ERK1 and ERK2 cascade	1.89E-03
		GO:0045732	Positive regulation of protein catabolic process	3.25E-03
		GO:0006096	Glycolytic process	4.34E-03
		GO:0022008	Neurogenesis	5.97E-03
		GO:0032212	Positive regulation of telomere maintenance via telomerase	3.82E-03
		GO:0043967	Histone H4 acetylation	4.59E-03
		GO:0010628	Positive regulation of gene expression	8.20E-03
C/EBPε target genes	GO_CC	GO:0070062	Extracellular exosome	1.88E-12
		GO:0005654	Nucleoplasm	1.81E-12
		GO:0005737	Cytoplasm	2.14E-07
		GO:0005829	Cytosol	1.75E-07
		GO:0016020	Membrane	7.11E-06
	GO_MF	GO:0044822	Poly(A) RNA binding	2.81E-07
	GO_MF	GO:0005524	ATP binding	8.89E-06
	KEGG_PATHWAY	ssc01100	Metabolic pathways	9.13E-09
C/EBPγ target genes	GO_CC	GO:0005654	Nucleoplasm	3.28E-05
	GO_CC	GO:0070062	Extracellular exosome	9.08E-05
	GO_BP	GO: 0030512	Negative regulation of transforming growth factor beta receptor signaling pathway	1.29E-05

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Genome-wide phylogenetic analysis, expression pattern, and transcriptional regulatory network of the pig C/EBP gene family

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