Animal development starts from a single fertilised egg cell, which gives rise to different cells, tissues and organs. A Single cell develops into a complex multicellular organism in a remarkable process of development mediated by extensive cell division and differentiation into diverse cell types. The specification of unique cell types, their subsequent differentiation, and their different functions are determined by carefully orchestrated regulation of gene expression. Transcription factors (TFs) regulate gene expression by acting on associated DNA regulatory elements in the genome [1]. These cis-regulatory elements (CRMs) include promoters, repressors, insulators, and enhancers. Enhancers are cis-regulatory elements that enable precise spatiotemporal patterns of gene expression during development, able to function at large distance from their target genes and comprise binding sites for multiple transcription factors [2]. Enhancers have sequence-specific transcription factors bound to it and nucleosome region of open chromatin. Poised enhancers are primed enhancers containing repressive epigenetic chromatin mark a state [3]. DNA sequencing technology coupled to both chromatin immunoprecipitation (ChIP-Seq) and DNAse hypersensitivity assays (DNAse-Seq) can predict an enhancers on a genome wide scale [4,5]. Enhancers of a given cell or tissue type can now be easily identified throughout the genome using these assays according to their epigenetic features such as histone and chromatin modifications. Nucleosomes of enhancer elements are enriched for mono-methylation at lysine 4 of histone 3 (H3K4me1) while nucleosomes near the promoters tend to have tri-methylated H3K4 (H3K4me3) [6].

The model organism for this study is Drosophila, which is smaller in size compare to housefly. It can be cultured easily in a mass and has a short life time. The complete genome of fly was sequenced in 2000 contains approximately 13,600 genes [7]. It has 4 pairs of chromosomes: 3autosomal and one sex chromosome.

Rhomboid (rho) gene encodes a putative transmembrane receptor that is required for the differentiation of the ventral epidermis. It is initially expressed in embryo before the completion of cellularization within the presumptive neuroectoderm. Maternal morphogen Dorsal (dl) including other gene called twist (twi) activate expression of rho in both lateral and ventral regions. Expression is blocked in ventral regions (the presumptive mesoderm) by snail (sna) [8].

Enhancers are the DNA sequences that contain multiple binding sites for TFs known as transcription factor binding sites (TFBSs). Cluster of transcription factors is identified by using scoring matrix called position-weight matrix (PWM) [9]. In this study we have used ClusterDraw, which is one of the programs aimed to identify binding sites and binding site clusters for transcription factors. It filters overlapping binding sites by selecting those producing the best statistical scores [10]. It searches for the best clusters in the parameter space defined by the motif match P-value and the significance of the cluster. Many of the studies had been done using this tool for instance distribution of enhancer specific binding motifs was predicted responsible for anterior-posterior and dorsal-ventral patterning of Drosophila embryo [11]. Normal gene expression is produce by overlapping activities of multiple enhancers present in the gap gene, which was analysed by ChIP-chip method. Multiple distal and proximal enhancer of hunchback (hb) gene in combination to gap gene produces authentic expression pattern in Drosophila embryo [12].

There are multiple enhancers found in the activation of gene transcription. Primary enhancer contains multiple binding sites for transcription factors enhance the activity of gene expression. Secondary enhancer located at more remote position from the target gene mediates the expression of target gene by overlapping the primary enhancer. This secondary enhancer is known as shadow enhancer. One of the first instances of genes having multiple enhancers was reported in case of shavenbaby gene which directs the development of larval trichomes in Drosophila. Svb possesses three known enhancers- 7, E and A, located 50kb upstream of the gene [13]. Additional svb enhancer were located by constructing two reporter targets, resulting in identification of Z and DG2 which overlaps the expression of 7, E and A, and allow these 3 enhancers to develop larval trichomes in embryo under optimal temperature [14].

Sequence retrieval

Rhomboid gene and flanking sequences FASTA format, which is an online database. were obtained from Flybase (http://flybase.org/) version FB2015_02 in

Prediction of putative enhancer

Gene sequence along with flanking upstream and downstream sequences retrieved from flybase was uploaded to online server ClusterDraw (http://line.bioinfolab.net/webgate/submit.cgi) version 2.55 to predict binding sites and binding sites clusters. Sequence of binding motifs “Dorsal”, “Twist” and “Snail” were uploaded from the collection of “fly development” motifs (available at http://line.bioinfolab.net/webgate/help/dxp.htm) in FASTA format. For D. melanogaster ClusterDraw searches for minimum of one motif combination and 5 cluster significance. Uploaded gene and motifs sequence were submitted and result was visualized as a 2D graph, showing the location of gene, sequence of binding site. These results can be extracted in FASTA format and number of binding motifs are identified on submitted sequence.

Sequence alignment

Predicted enhancer sequence of D.melanogaster rhomboid gene from ClusterDraw was analysed with the sequence validated in Ip journal in online web server ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). It is software to visualize multiple sequence alignment. Parameters used in this software are gap penalties and weight matrix (PAM250 and BLOSUM62 matrix) [15]. It aligns the most closely related sequence from the phylogeny by calculating the number of k – tuple (combination of small words of certain size) matches. K-tuples value stands for the length of the word used to search for identity. Typical values of k-tuple are 1 or 2 for proteins and 3 forDNA. Both sequences were uploaded in the given sequence enter bar in the prescribed format. Default setting for alignment is slow type and sequences were submitted to obtain the results.

Identification of epigenetic features

Epigenetic features such as histone modifications can be visualized in UCSC genome browser (https://genome.ucsc.edu/). It provides reference sequence for a large collection of genomes. D. melanogaster genome was selected (Apr.2004 (BDGPR4/dm2)), rhomboid gene was entered in the search bar and details were submitted, result appeared in graphic form showing promoter, Dorsal, Twist and Snail binding predicted by ChIP-chip method at different stages of Drosophila embryo.

The sequence of rho gene was taken from Flybase (http://flybase.org/). The sequence of rho gene in D. melanogaster located is from 1463811-1468944 on 3L chromosome with its various regulatory elements. To predict putative enhancer for rho gene, 20 kb upstream and downstream sequence was analyzed. As previously described regulators for rho gene enhancer are Dorsal, Twistand Snail, cluster formation of these motifs at binding sites of gene enhancers was analysed using database ClusterDraw (http://line.bioinfolab.net/webgate/submit.cgi). Binding sites cluster was shown as colored peaks in which red color peak shows the highest cluster significance (Figure 1).

Figure 1: ClusterDraw output: 2D plot of cluster E-values using drosophila binding motifs Dorsal, Twist and Snail. X-axis shows the location of gene and 20kb flanking sequence and Y-axis defined the match P–value. Red peaks show the position of putative enhancer in all Drosophila melanogaster.

To analyse the results obtained from ClusterDraw, the predicted enhancer sequence of Drosophila melanogaster, were aligned with the validated enhancer of rhomboid gene of Drosophila melanogaster [16] by using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Result showed that validated enhancer sequence of rho gene lies within the sequence of putative enhancer sequence resulted from ClusterDraw (Figure 2).

Figure 2: Screenshot of pairwise sequence alignment of putative enhancer after ClusterDraw analysis of 20kb upstream and downstream Drosophila melanogaster ho gene with the rho NEE identified in Ip et al., 1992. Alignment was done by ClustalW.

Enhancer sequences are consisting of multiple binding sites for transcription binding factors. Online genome browser UCSC (https://genome.ucsc.edu/) is used for visualization of binding sites for polymerase, Dorsal, Twist and Snail in predicted putative enhancer sequences of rhomboid gene (Figure 3).

Figure 3: Screenshot of rho gene from UCSC browser visualizing binding sites for Polymerase, Dorsal, Twist and Snail at different embryonic stages.

Similarly, 20 kb upstream and downstream sequence of rhomboid gene of all other Drosophila species viz., D.sechellia, D.erecta, D.grimshawi, D.pseudoobscura, D.yakuba, D.willistoni, D.virilis, D.mojavensis, D.ananassae and D.persimilis were taken and cluster of binding sites of motifs were analysed. Clusters were seen as coloured peaks in which red peaks are more significant (Figure 4).

Figure 4: ClusterDraw output: 2D plot of cluster E-values using drosophila binding motifs Dorsal, Twist and Snail. X-axis shows the location of gene and 20 kb flanking sequence and Y-axis defined the match P – value. Red peaks show the position of putative enhancer in all Drosophila species.

Number of putative enhancers and binding sites ranges from 1-3 and 1-15, respectively (Table 1). D. ananassae, D. persimilis, D. yakuba have two putative enhancers. D. erecta have three pupative enhancer whereas other species viz., D. melanogaster, D. grimshawi, D. mojavensis and D. pseudoobscura have single putative enhancer element.

Table 1: Table shows the number of putative enhancer predicted from Cluster Draw in multiple species consisting of multiple Dorsal, Twist and Snail binding sites and distance of enhancer from rho gene.

From the analysis it is found that there are multiple binding sites for Dorsal, Twist and Snail and one or more than one enhancer are essential for the activation of expression of the gene and the estimated distance of enhancer shows that enhancer can be located upstream or downstream from the gene.

The rhomboid gene was first isolated from the follicle cell of the embryo and is activated initially at the early stage of the drosophila embryo (i.e. 0-4hr embryo) as regulatory elements required for the gene expression are found in follicle stage or regulatory elements which represses the gene expression found in late developmental stage of drosophila embryo in all the species [17] and expression is seen in the development of neuroectoderm in dorsal – ventral region. Dorsal, twist and snail together regulates the expression of gene.

In this study we have predicted enhancer elements binding motifs of the rhomboid gene. Analysis of clusters of binding sites of Dorsal, Twistand Snailin multiple species was done by using software ClusterDraw. It predicts the cluster of best binding sites. We have performed precition of these regulatory elements of the rhomboid gene among 11 species of Drosophila.

As a result of these predictions we reported that there are multiple binding sites for Dorsal, Twist and Snail motifs on the enhancer element of the genes. We also found that there are one or more than one enhancer elements are required for the activation of expression of the gene and the estimated distance of enhancer shows that enhancer can be located upstream or downstream from the gene.

Above discussed method predicts the binding sites of proteins on DNA segment at genomic level but to validate that a DNA segment on which transcription factors binds and regulates the expression of target gene requires various experimental techniques such as: in vitro assays and transgenic assays in which activity of enhancer is determined by using reporter gene which is detected by luciferase or beta-galactosidase [18]. Other method used for validating enhancer activity are enhancer- FACS seq (eFC) method [19] and sequencing of transcribed barcode using plasmid based systems.