1. Introduction

Stem cells are defined as undifferentiated cells which have the potential for self-renewal and develop into various cell types. In particular, lung stem cells are capable of carrying out different functions because of their ability of self-renewal and pluripotency. The lung has a much slower turnover of putative lung progenitor cells, unlike other epithelial tissues that undergo rapid regeneration [1,2].

Transcription factors plays an essential roles in regulation of gene expression [3]. TF genes of EYA1, SIX1, SOX9 have recently attracted the attention due to their multiple roles as transcriptional factors (TF) in controlling the transcription of DNA to mRNA by promoting or blocking the recruitment of RNA polymerase to specific genes [4]. EYA1-4 and sine oculis family genes (SO; which is a member of the SIX family of homeobox transcription factors, are unable to initiate eye development in non-retinal tissues) [5] exhibited interactions to regulate the development of many organs [6-9]. The eyes absent proteins act as transcriptional co-activators [10].

EYA1 switches SIX1 function from suppression to activation condition in the nucleus, resulting in transcriptional activation through recruitment of co-activators regulating precursor cell proliferation and survival during organogenesis [11]. EYA1 and SIX1 are important transcription factors for the lung epithelial stem/progenitor cells maintenance. Mice, deficient in EYA1 or SIX1, do not have the epithelial progenitor cell markers; they highly express differentiation markers in their lungs. In addition, their lungs are markedly hypo plastic with decreased epithelial branching capacity and intensive mesenchymal cellularity [12,13].

SIX1 are responsible for developing skeletal muscles and supports gene expressions related to functioning of developing progenitor cells. SIX1 is a critical TF in skeletal muscle development, and also in eye and kidney development. Numerous studies have demonstrated that SIX gene family play an important role in organogenesis [11,14-16]. Moreover, SIX1 is able to drive the transformation of slow-twitch fibers to the fast-twitch phenotype in synergy with its cofactor EYA1 in muscles of adult mice [5].

SOX9 is a member of high mobility group-box class DNA binding protein family of transcription factors, it is required for branching morphogenesis in the lungs and for controlling alveolar epithelial progenitor cell proliferation [17,18]. Upregulation of SOX9 expression was reported to increase cell proliferation [19-21] associated with a poor lung ADC (Antibody-Drug Conjugate) survival [1]. However, the functional role of SOX9 in lung cancer has not been fully elucidated. Furthermore, the upstream oncogenic molecular pathways regulating SOX9 overexpression in lung ADC have not been completely delineated.

SOX9 is highly expressed in the distal epithelial progenitors in embryonic lung, nonetheless conditional deletion of SOX9 does not affect progenitor cell behavior in lung [17,22,23].

In this study, genes and proteins sequences of identified EYA1, SIX1, SOX9 transcriptional factors in twelve different organisms were analyzed. Constructed Phylogenetic trees of TFs protein sequences showed their cladistics relation based on their structure and function. Moreover, TFs protein domains, alternative splicing [24], expression patterns, and interaction network were analyzed to elucidate these TFs genes in human tissues. This investigations shwed the importance of these genes in stem cell differentiation and their relations in many syndromes and functional disorders. Those genes were chosen to provide more knowledge about their role not only in human but also in other living organisms to investigate their functional roles. The results of this investigation may open a new venue to repair lung defects due to cancer or other disorders.

2. Material and Methods

Data resources

The EYA1, SIX1, and SOX9 TF protein sequences used for analysis and phylogenetic tree construction were obtained from the Uniprot database (). Transcript data used for analyzing the alternative splicing and the functional protein domains were obtained from NCBI database (http://www.ncbi.nlm.nih.gov). Expression analysis datasets were obtained from BioGPS database (http://Biogps.org/#goto).

Protein interaction networks were obtained from Gene Card human gene database (http://www.genecards.org/).

Identification of protein sequences and phylogenetic tree construction

EYA1, SIX1 and SOX9 TF protein sequences were obtained from the Uniprot database (http://www.uniprot.org//) and used as templates from twelve different spices To understand how different evolutionary changes of the structure did not affect conservative function of Homo sapiens (Human), Pan troglodytes (common chimpanzee), Macaca mulatta (Rhesus monkey), Gorilla gorilla (western gorilla), Mus musculus (mouse), Rattus norvegicus (Norway rats), Cavia porcellus (domestic guinea pig), Canis lupus (gray wolf); bird Gallus gallus (red junglefowl); frog Xenopus laevis (African clawed frog); fish Danio rerio (zebrafish); and roundworm Caenorhabditis elegans (nematode). Data were downloaded, analyzed, and phylogenetic trees were constructed using MEGA6 by the neighbor-joining method.

Protein Domains analysis and identification of alternative splicing

The functional TF protein domains of EYA1, SIX1 and SOX9 were obtained from NCBI database and analyzed by using the Pfam sequence tool (protein family database which contains information about protein domains and families) (https://www.ncbi.nlm.nih.gov/Structure/cdd). Alternative splicing of each gene was obtained from NCBI database.

Expression analysis in human body tissues

The gene expression analysis for TF EYA1, SIX1and SOX9 was obtained from BioGPS database (http://Biogps.org/#goto) representing samples of Human tissues at normal phase. More than 76 samples of tissues were measured.

Protein interaction network construction

Protein interaction network were obtained from Gene Card (Human gene database) (http://www.genecards.org/), and were illustrated the interacting proteins searched for. This search was based on published papers experimentally proven and accepted.

3. Results

Identification of TF proteins in species and phylogenetic tree construction

Different methods were used with all sequences to generate a multiple sequence alignment using [25,26]. In order to analyze TF gene sequences, we used MEGA6 program [27] to further analyze the function and conservation levels of these transcription factors, and to explain the evaluation between species. By using Neighbor-joining (NJ) method we constructed the phylogenetic tree of these genes. We assumed that the three TF exist in all species, however, in contrast to the expectation, SOX9 was not found in Caenorhabditis elegans and Cavia porcellus.The same results were obtained using Phylip [28]. This observation indicated that the function of the SOX9 gene in this species may be regulated by other genes, or may be lost during differentiation due to their live cycle. The role of gene as sex determinate not as transcriptional factor may indicate developmental differences between vertebrates and invertebrates (Table 1).

Table 1. Accession numbers of the three transcription factor genes investigated obtained from Uniprot database

In EYA1, the constructed protein phylogenetic tree showed that Rattus norvigicus was the most distant vertebrate from Homo sapiens, which reveals that the function of EYA1 may be depleted in rat in contrast to other mammals (Fig. 1a). However, from all studied species Caenorhabditis elegans EYA1 protein was showed the most distal phylogenetic differences from Homo sapiens, Gorilla gorilla showed the closest similarity to Homo sapiens followed by Pan troglodytes.

In SIX1, the phylogenetic tree showed no difference from EYA1 phylogenetic tree except that Danio rerio was the most different from Homo sapiens. Caenorhabditis elegans also showed large difference from Homo sapiens, which may indicate that the mechanism of SIX1 as regulator can participate in skeletal muscle development, which may depleted in fishes and invertebrates, or regulated by other genes, which construction is confirmed by conserved domains (Fig. 1b).

The SOX9 phylogenetic tree showed different results. Pan troglodytes, Macaca mulatta were the closest to Homo sapiens; Danio rerio, Xenopus laevis and Gallus gallus, showed the largest difference from Homo sapiens (Fig. 1c), which may be caused due to the evolutionary differences among these species, for the evolution of mammals made them to have sex determining genes. In contrast, Cavia porcellus and Caenorhabditis elegans do not have SOX9 gene but we found that the function of SOX9 is regulated by another gene from SOX family. e.g SOX10 which is a gene from the SRY (sex determining region Y-box) group. gene function were found to be changed during evolution of this two spices, which reveals that SOX9 function may be regulated by another gene. This observation can be applied as developmental differences between vertebrates and invertebrates; however this needs more studies to confirm this hypothesis.

Figure 1. Protein phylogenetic trees of transcription factors of (A) EYA1, (B) SIX1 and (C) SOX9 of the taxa studied. The evolutionary history was inferred using the Neighbor-Joining method (37). The optimal tree with branch lengths (the sum = 0.46071988) is shown (next to the branches). The evolutionary distances were computed using the Poisson correction method (38) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 395 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 (27). As SOX9 is missing from Caenorhabditis elegans and Cavia porvellus the tree C shows ten of the twelve taxa

Protein domains analysis and identification of alternative splicing

Protein domains are conserved parts of a given protein sequence that often form a functional unit [29]. Many proteins consist of several structural domains, and a given domain may appear in a variety of different proteins. Protein domains vary in length from about 25 amino acids (aa) up to 500 aa. In a multiple domain protein, each domain may fulfill its own function independently [30]. Molecular evolution research uses domains as building blocks and may recombine in different arrangements to form proteins with different functions.

Role of EYA1, SIX1 and SOX9 transcription factors in DNA repair and regeneration of lung epithelial progenitor cells

In EYA1, the conserved domain which shared in all species studied was found to be EYA-cons_domains super family [d11769]). Its length was 274 aa, and was common in all EYA homologs. Metazoan Eye’s also contain a variable N-terminal domains, consisting largely of low-similarity sequences to Homo sapiens, which had two conserved domains (EYA-cons_domains super family [d11769]) and (duf1421) (Fig. 2a).

In SIX1, conserved domains were found to common in the majority of species studied (Homeodomain[cd00086]), with 59 aa in length. In Danio rerio, it had one conserved domain (ham1[cd00515]) super family. In Caenorhabditis elegance there were other two conserved domains (pcl[Pfam01399] and Rpn6[cog5159]). These differences may cause the different phylogenetic tree constructions. In Homo sapiens a conserved domain of (Homeodomain [cd00086]) was identified (Fig. 2a).

SOX9 conserved domains (SOX_N [Pfam12444] and SOX-TCF_HMG-box [cd01388]) were found to be shared in all studied species. The (SOX-TCF_HMG-box [cd01388]), with length = 72 aa, included SRY and its homologs in insects and vertebrates as a class I member of the HMG-box superfamily of DNA-binding proteins. These proteins contain a single HMG box, and bind to the minor groove of DNA in a highly sequence-specific manner. The ([SOX_N [Pfam1244]) with length = 77 aa was found in eukaryotes. This family was found in association with Pfam00505. There were two conserved sequence motifs of YDW and PVR (not presented). This family contains SOX8, SOX9 and SOX10 proteins which have structural similarity to Homo sapiens conserved domains (Fig. 2a).

Analysis of Alternative splicing

Our alternative splicing analysis of EYA1 showed that each studied species expressed a wide range of isoforms and that EYA1 genes code for multiple proteins. Of note, most of the EYA1 isoforms in Homo sapiens have the same conserved domains. The close homology of EYA1 and SIX1 could explain the wide distribution of EYA1 in Homo sapiens (Fig. 2b). Alternative isoforms of EYA1 in Homo sapiens were analyzed to investigate the conserved domains of this gene. SIX1 showed less conserved domains in the isoforms and In SOX9 the alternative splicing was completely different with less isomorphs, which have been detected in species but more conserved domains were detected in these isoforms. That revealed that SIX1and SOX9 have less isoforms and less distributed proteins (table 2 a, b and c).

Figure 2. (A) Conserved domains of transcription factors (A) EYA1, (B) SIX1, and (C) SOX9 of Homo sapiens. Functional protein domains were obtained from NCBI database, and analyzed by using the Pfam sequence tool. (B), (C) and (D) Alternatively spliced isoforms of Human TFs of (B) EYA1, (C) SIX1 and (D) SOX9 (data were obtained from NCBI database). Each line represents an individual transcript. Exons that have at least one alternative splicing are indicated by red boxes; black boxes represent unspliced exons that had no change during transcription; introns are indicated by lines. Gene Bank Accession numbers of alternatively spliced exons are indicated

Table 2. The accession number of conserved domain of EYA1 isoforms, SIX1 isoforms and SOX9 isoforms obtained from Uniprot database

Microarray analysis of the three TFs expressed in Human tissues

We conducted systematical investigations on the expression of the three TFs in human body tissues using specific patterns of mRNA expression, which can indicate the important clues about gene functions. For microarray analysis of mRNA samples, more than 79 Human tissues from different organs were analyzed. The data was obtained from BioGPS database to explain the expression patterns.

The expression of EYA1 gene in Human tissues and organs with Median level (M) 4.6, showed only one exception in Pineal gland, it is above 10xM (10 times above the M) in night; and above 3xM in day (Fig. 3a).

The SX1 expression showed balanced levels in all tissues with Median level 5.28, and with some slight exceptions in Pituitary gland (below 3xM), Skeletal Muscles (about 2xM), and Subthalamic nucleus of the brain (below 2xM) (Fig. 3b). The SOX9 expression showed the most complex patterns with Median level 10.1, and with several extremely high expression levels in lung’s Trachea (above 10xM), Colorectalaadenocarcinoma (above 20xM), Prostate (above 20xM), Retina (10xM), and Prefrontal Cortex of the Brain (above 20xM) (Fig. 3c). Of note, we found high levels of SOX9 in male reproductive organs and tissues (above 10xM in testis) but with lower levels in other tissues. These data are in line with the notion that SOX9 gene contributes to sex determination in Human and that it functions with other genes to produce AMH (Anti-Mullerian Hormone) in Sertoli cells to inhibit the development of female reproductive tissues in particular ovary (Fig. 3c).

Figure 3. Microarray data of gene exprission levels of the three TFs studied in human body tissues and organs of (A) EYA1, (B) SIX1, and (C) SOX9. The expression analysis was obtained from BioGPS database representing samples of human tissues at normal phase. More than 76 samples of Human tissues were analyzed (39)

Protein Interaction network constructions

The function and activity of a protein is often modulated by other proteins, that are interacting and cooperating together to perform their functions. EYA1 and SIX1 were found to have the strongest interactions.

We found that the switch of SIX1, SIX2-4, and SIX6 transcription factors from the suppressive forms form into the activated forms depends on EYA1 gene family, including EYA2, EYA3 and EYA4. For instance, EYA1 activates SIX1 function in the nucleus and turns it from the repression to activation through recruitment of co-activators. It provides a mechanism for activation of specific gene targets, including those regulating precursor cell proliferations during organogenesis [11].

SOX9 interaction network was found widely distributed through different families of genes of PDIA2 (Protein Disulfide Isomerase-Associated 2), HERC1 (HECT and RLD domain containing E3 ubiquitin protein ligase family member 1), TRAF2 (TNF receptor (TNFR) associated factor 2), SMAD2 (Similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' (Mad) and the C. elegans gene Sma), SRA1 (Steroid Receptor RNA Activator 1), and POU3F3 (POU Class 3 Homeobox 3). The HERC1 were found to have different roles ranging from transcriptional activator and co-activator to intra-cellular signal transducer and transcriptional modulator (Fig. 4c). The search was based on earlier works experimentally proven and accepted [33,34].

Figure 4. The TF proteins interaction networks of (A) EYA1, (B) SIX1, and (C) SOX9. Data were obtained from Gene Card (human gene database) and illustrated the interacting proteins

4. Discussion

The phylogenetic trees mainly followed the evolutionary relationships among the species. We found that SOX9 doesn’t exist in Cavia porcellus, Caenorhabditis elegans, which may indicate that the function of SOX9 is regulated by other TFs in these organisms. Conserved domains of EYA1, SIX1, and SOX9 were found. EYA1 showed more than 36 alternative isoforms, which indicated that alternative splicing is a very important source of protein diversity, and accounts for the majority of different protein functions [31,32]. Expression analysis of genes in human body tissue showed normal levels detected in EYA1, and SOX9. However, high levels of expressions were detected in sex organs. High levels of SIX1 expression were detected in all body tissues, which indicate that SIX1 is mainly responsible for all organ development in order to its somatic function. These results support the relation with function of developing progenitor cells associated with tumor progression and metastasis, which provides a mechanism for activation of specific gene targets, including those regulating precursor cell proliferation during organogenesis [11,14,35,36]. Interaction network of the three TF genes showed strong interactions, and confirmed that EYA1 and SIX1 interact in a complex way, and SIX1 interacts with EYA1 in the development of several organs in the human body.

5. Conclusions

The three transcription factors were found to have crucial roles in the development and function of organs in animal bodies. Focusing to lung, our results may provide new knowledge about these TF genes and open new prospects to further research which can help to understand repairs of lung defects in order to cancer or other diseases.

ACKNOWLEDGEMENTS

This work has been supported by a grant (ID# 5245) funded from the Science and Technology Development Fund (STDF), Ministry of Scientific Research, Egypt to prof. Mohamed L. Salem, the Principal investigator of this project.