MAGE genes encoding for embryonic development in cattle is mainly regulated by zinc finger transcription factor family and slightly by CpG Islands

Melanoma Antigen Genes (MAGEs) are a family of genes that have piqued the interest of scientists for their unique expression pattern. The MAGE genes can be classified into type I MAGEs that expressed in testis and other reproductive tissues while type II MAGEs that have broad expression in many tissues. Several MAGE gene families are expressed in embryonic tissues in almost all eukaryotes, which is essential for embryo development mainly during germ cell differentiation. The aim of this study was to analyze the promoter regions and regulatory elements (transcription factors and CpG islands) of MAGE genes encoding for embryonic development in cattle. The in silico analysis revealed the highest promoter prediction scores (1.0) for TSS were obtained for two gene sequences (MAGE B4-like and MAGE-L2) while the lowest promoter prediction scores (0.8) was obtained for MAGE B17-like. It also revealed that the best common motif, motif IV, bear a resemblance with three TF families including Zinc-finger family, SMAD family and E2A related factors. From thirteen identified TFs candidates, majority of them (11/13) were clustered to Zinc-finger family serving as transcriptionally activator role whereas three (SP1, SP3 and Znf423) of them as activator or repressor in response to physiological and pathological stimuli. On the other hand we revealed slightly rich CpG islands in the gene body and promoter regions of MAGE genes encoding for embryonic development in cattle. This in silico analysis of gene promoter regions and regulatory elements in MAGE genes could be useful for understanding regulatory networks and gene expression patterns during embryo development in bovine.

predicting the promoter region or the transcription start site in investigating the functional roles of gene.
CpG islands are known to regulate gene expression through transcriptional silencing of the corresponding gene. DNA methylation at CpG islands is crucial for gene expression and tissue-specific processes [7]. About half of all CGIs self-evidently contain TSSs, as they coincide with promoters of annotated genes [8]. According to Deaton and Bird [9], most CGIs are sites of transcription initiation including distantly located from annotated promoters.
The melanoma associated antigen (MAGE) genes are conserved in all eukaryotes and lower eukaryotes to 40 genes in humans and mice [10]. They share common MAGE homology domain with high sequence similarity [11]. Some of MAGE genes are ubiquitously expressed in tissues; others are expressed in only germ cells [11]. Flork et al. [10] and Tacer et al. [12] reported that MAGE proteins regulate diverse cellular and developmental pathways and protect the germ-line from environmental stress.
Majority of the MAGE genes are located on the X chromosome and expressed in early spermatogenesis [13]. The MAGE gene can be classified into type I and type II based on their tissue expression pattern [11]. The type I MAGEs have expression restricted to testis and other reproductive tissues [12]. On the other hand, type II MAGEs that have broad expression in many tissues [11,13]. Several studies reported that MAGE genes play important roles during embryogenesis and germ cell genesis [11][12][13][14]. Although studies are conducted on the evolution and biological functions of MAGE genes, there is a limited data on the regulatory mechanisms of this gene during embryo formation in large mammals. Therefore, the aim of this study was to predict promoter and regulatory elements of MAGE genes encoding for embryonic development in cattle (Angus*Brahman F1) thereby provide basic information for improving reproductive efficiency and fertility in cattle.

Identification of TSS and promoter regions of MAGE genes
Promoter region analysis of MAGE genes encoding for embryonic development showed a small variation in the number of TSS where we revealed that 68.42% of the sequences had single TSS ( Table 1). The current study also revealed that eight (42.1%) TSSs are located at a distance below -500 bp when checked from the start codon even though TSSs of MAGE genes encoding for embryonic development were mostly located in the upstream region of − 137 to − 1782 bp.

Common candidate motifs and associated transcription factors in the promoter regions of MAGE genes
The present analysis discovered five binding motifs from which three motifs (I, III and V) were equally shared (50%) by all MAGE genes encoding for embryonic development in cattle ( Table 2). The candidate motif IV was revealed as the best common promoter motif for 66.67% of cattle MAGE genes encoding for embryonic development that serves as binding sites for TFs involved in the expression regulation of these genes. The present analysis revealed that majority (61.36%) of the candidate motifs were located and distributed between -700 bp to -200 bp with the reference to the transcription start site region (Fig. 1). The higher distributions of motifs were found in positive than in negative strands.
To address the information content, MEME created sequence logo for the best common motif, motif IV, which resulted in different characters of motif alignment columns, where the height of the letter represents how frequently that nucleotide is expected to be observed in that particular position (Fig. 2). Motif IV motif was compared with other registered motifs in publically available databases motif in order to explore matched motifs using TOMTOM web application. As a result, motif IV matched with thirteen (13) known motifs found in databases ( Table 3).
The present analysis revealed that the best common motif, motif IV, bear resemblance with three transcription factor families: Zinc-finger family, SMAD family and E2A related factors; where majority (84.6%, 11/13)  of them belong to Zinc-finger transcription family. The current study revealed SP1 and SP3 transcription factors activate or repress transcription and have major role in embryonic eye, placenta and skeletal system development as we revealed from Uniprot database. The findings from UniProt database also revealed that KLF1, KLF5, TCF4 and EGR3 transcription factors were transcriptionally activator and has role in utero embryonic development, intestinal epithelial cell development and nervous system development, muscle spindle development, respectively. Likewise, the transcription factor candidate EGR1 had function in the oocyte maturation.

Investigation for CpG islands in cattle MAGE genes
To further explore the regulatory elements that are involved in nineteen (19) MAGE genes encoding for embryonic development in cattle, CpG islands were investigated in both promoter and gene body regions using two algorithms. Using Takai and Jones' algorithm, we found six (6) CpG islands in promoter and five (5) CpG islands in gene body regions (Table 4). In this study, investigation of the CGIs indicated that MAGE genes encoding for embryonic development in cattle have slightly rich CGIs in their promoter and gene body regions.
Analysis for CpG islands on both promoter region and gene body region using restriction enzyme MspI was also conducted ( Table 5). The in silico digestion results revealed more CpG islands in gene body region compared to promoter region; and one gene (LOC113887988) contain two fragment sizes: 113 and 103 bps in gene body region and promoter region, respectively. In the present analysis, about six CGIs and three CGIs were found in gene body region and promoter region, respectively. The results indicated that cattle MAGE genes encoding for embryonic development in cattle are slightly few in CpG islands which is in agreement with the first method, Takai and Jones' algorithm.

Discussion
The retrieved sequence data from NCBI database were used to identify and characterize the promoter regions and regulatory elements of MAGE genes. The findings revealed that promoter region analysis of MAGE genes   [16,17]. The current study also revealed that TSSs of MAGE genes encoding for embryonic development was mostly located in the upstream region of -137 to -1782 bp. This result is in agreement with Mu et al. [18] who reported transcriptional initiation site location of -515 bp for ovine DKK1 gene and Pokhriyal et al. [19] who reported TSS location at 235 bp, 156 bp and 92 bp for BICP0, BICP4 and BICP22 in bovine genes, respectively.
The current analysis discovered multiple binding motifs for MAGE genes, which is significant to find all possible binding motifs for the same TF and co-factor binding motifs [20]. Likewise, the analysis revealed multiple  binding sites in the promoter region of candidate motifs, which could be used to strengthen binding interactions and different regulatory effect [21]. The majority of candidate motifs in the promoter regions of MAGE genes are located and distributed between -700 bp to -200 bp with reference to transcription start site region. This is in agreement with Halees [22] who reported that majority of motifs are located immediately upstream of a TSS. The candidate motifs were highly distributed in the positive strands than negative strands. The present analysis revealed that the best common motif, motif IV, bear resemblance with three transcription factor families: Zinc-finger family, SMAD family and E2A related factors; where majority (84.6%, 11/13) of them belong to Zinc-finger transcription family. This is in agreement with Samuel and Dinka's [17] finding who reported zinc finger family transcription factors are the main regulatory element for olfactory receptor in cattle. Adryan and Teichmann [23] showed that zinc finger transcription factors are strongly represented early in embryonic development and they are typically regulate gene expression by binding to specific DNA sequences via their DNA-binding zinc finger domains [24].
The current findings revealed that the observed SP1 and SP3 transcription factors have dual regulatory function and have major role in embryonic eye, placenta and skeletal system development. This is in close agreement with previous studies on the transcription factors Sp1 and Sp3 expression and regulatory functions in mammalian cells [25][26][27]. Similarly, findings from Uniprot database revealed that transcription factors KLF1, KLF5, TCF4 and EGR3 are transcriptionally activator and have role in different embryonic tissue development. This result is in agreement with Chen et al. [28] and Wang et al. [29] who reported that Krüppel-like factor families are important role in maintaining embryonic stem cells.
It has been reported that CGIs are highly involved in gene regulatory processes [9]. In this study, investigation of the CGIs indicated that MAGE genes encoding for embryonic development in cattle have slightly rich CGIs in their promoter and gene body regions. The in silico digestion results also revealed slightly rich in CpG islands in cattle MAGE genes encoding for embryonic development which is in agreement with the first method, Takai and Jones' algorithm. Similar findings are reported by Reik and Walter [30]. The author reported that the CpG islands associated with the MAGE genes have a CpG-rich region of 300-650 bp long at their 5'end. CpG islands are often associated with the promoters of most house-keeping genes and many tissue-specific genes, and thus have important regulatory functions and can be used as gene markers [31]. However, Samuel and Dinka [17] reported poor CGIs using MspI enzyme digestion for cattle olfactory receptor genes.
The present in silico study analyzed promoter and regulatory elements of MAGE genes in cattle using different algorithms. However, due to various physiological and biological functions as well as broad expression of MAGE genes in tissues, we are not sure to fully recommend the direct role of MAGE genes in embryonic development. Thus further in vitro or in vivo experiment should validate the findings. It is normal that validation is important for in silico study approach or other computational based approach. Thus the limitation of present study is that it is in silico analysis which requires confirmation by experimental validation.

Conclusions
Identification and characterization of promoter regions of MAGE genes encoding for embryonic development in cattle is essential for understanding the regulatory mechanisms that control its expression. The current finding showed that regulatory elements found in the promoter region of MAGE genes may play direct roles in the gametogenesis process and then in embryo development. The current results would assist animal scientists in boosting cattle reproduction efficiency. However, further experimental studies will be necessary to validate the role of identified transcription factors and their common binding sites in the regulation of MAGE genes encoding for embryonic development in cattle.

Selection/retrieval of MAGE gene from NCBI
Distinct coding sequences belonging to MAGE gene family were retrieved from NCBI database via web-server https:// www. ncbi. nlm. nih. gov. The MAGE genes of Angus*Brahman FI hybrid cattle breed were extracted from UOA_Brahman_1 genome assembly and they were further characterized using genomic resources UniProt (https:// www. unipr ot. org). Duplicate and nonfunctional sequences were discarded from analysis. In this analysis, from a total of twenty one (21), nineteen (19) representative functional protein coding genes, with single exons, that have ORF were considered. Multi-exon genes were excluded from analysis as they have variable promoter region and produce different protein isoforms at different promoters [32,33] that makes difficult to predict regulatory elements.

Determination of transcription start sites and promoter regions for MAGE genes
In order to determine TSSs of each gene, minimum of 1 kb upstream of the start codon were excised from each gene [34]. The retrieved segments were fitted to Neural Network Promoter Prediction (NNPP version 2.2) by setting the minimum standard predictive score (between 0 and 1) with a cut off value of 0.8 [35]. This tool helps us to locate the possible TSSs within the sequences upstream of the start codon. For sequences having multiple TSSs, the TSS with the highest prediction value was considered as statistically significant and accurate. The promoter regions were determined 1 kb region upstream of each TSS as previously described by Michaloski et al. [36] for mouse odorant and vomeronasal receptor (V1R) genes.

Identification of common candidate motifs and transcription factors (TFs)
The predicted promoter sequences of MAGE genes were analyzed using the MEME((Multiple Em for Motif Elicitation) version 5.3.3 searches [37] to discover common candidate motifs that serve for binding sites of transcription factors regulating expression of MAGE genes. The MEME output in HTML format, significant motif, was submitted to TOMTOM [38] for TF prediction. The TOMTOM compared one or more motifs against a database of known motifs and produce an alignment for each significant match and produced LOGOS with p-value and q-value [39].

Search for CpG islands
In order to identify CpG islands in the upstream of MAGE genes, 2 kb sequences upstream of the start codon were used from each gene. The body regions of MAGE genes were also analyzed. The CpG islands were studied using two algorithms. The first algorithm, Takai and Jones algorithm with GC content ≥ 55%, Observed CpG/Expected CpG ratio ≥ 0.65, and length ≥ 500 bp was used [40]. This analysis was done via CpG island searcher program (CpGi130) accessible at web link http:// dbcat. cgm. ntu. edu. tw/. Secondly, the offline tool, CLC Genomics Workbench version 5.5.2 (http:// clcbio. com, CLC Bio, Aarhus, Denmark) was used for searching the restriction enzyme MspI cutting sites (with fragment sizes between 40 and 220 bp parameters). Searching for MspI cutting sites is relevant for detection of CGIs and it recognizes CCGG sites [41].