Integrative analyses of conserved WNT clusters and their co-operative behaviour in human breast cancer

Abstract

In human, WNT gene clusters are highly conserved at specie level and associated with carcinogenesis. Among them, WNT-10A and WNT-6 genes clustered in chromosome 2q35 are homologous to WNT-10B and WNT-1 located in chromosome 12q13, respectively. In an attempt to study co-regulation, the coordinated expression of these genes was monitored in human breast cancer tissues. As compared to normal tissue, both WNT-10A and WNT-10B genes exhibited lower expression while WNT-6 and WNT-1 showed increased expression in breast cancer tissues. The co-expression pattern was elaborated by detailed phylogenetic and syntenic analyses. Moreover, the intergenic and intragenic regions for these gene clusters were analyzed for studying the transcriptional regulation. In this context, adequate conserved binding sites for SOX and TCF family of transcriptional factors were observed. We propose that SOX9 and TCF4 may compete for binding at the promoters of WNT family genes thus regulating the disease phenotype.

Keywords: SOX; TCF; WNT; breast cancer.

Figure 1

A comprehensive phylogenetic analysis of WNT family, displaying all 19 paralogs in Primates (Homo sapiens), Rodents (Rattus norvegicus), Fish (Tetradon nigroviridis) and Deuterostome (Drosophila melanogaster). An un-rooted tree is constructed using MEGA 5.0 and Neighbour-joining algorithm, boot strap values are shown at each cluster validating the clustering of genes and species. P-distance is used as a measure of evolutionary distance, which also includes the correction for hidden changes.

Figure 2

Elaborated phylogenetic history of WNT-6/-1 and WNT-10A/-10B clusters closely related to each other from Homo sapiens to Caenorhabditis elegans. Independent duplication is observed in case of Tetradon norvegicus WNT-10B. Both clusters illustrate the true reconciliation with species tree. Moreover, the tree clearly indicates the fact that there is an independent copy of WNT-1, WNT-10 and WNT-6 in Drosophila melanogaster, which depicts the ultra-conservation of this cluster in invertebrates along with its chromosomal orientation. Unrooted tree is constructed using MEGA 5.0 and one of distance based method (Neighbour Joining Algorithm). Bootstrap values are shown for individual branches to confirm the validity of clustering.

Figure 3

Chromosomal location of two clusters of WNT (WNT- 1/-6 and WNT-10A/-10B) and their surrounding syntenic regions in Human, Macaque and Mouse. (A) 105 MB region of chromosome 2 in Homo sapiens is conserved with Macaca mulatta (chromosome 12). 50 MB region of Mus musculus chromosome 1 has similar gene order syntenic with this 105 MB region. This conserved region in Homo sapiens, Mucaca mulatta and Mus musculus contains a cluster of WNT-10A and WNT-6 (B) Chromosomal location of WNT-1 and WNT-10B genes in Homo sapiens, Macaca mulatta and Mus musculus. The conserved region, approximately 17MB (in blue) contains the other two WNT genes under study in form of a cluster.

Figure 4

Synteny of a conserved locus (1 MB in size) in Homo sapiens, Macaca mulatta, Mus musculus and Danio rerio. (A) WNT-10A and WNT-6, seems to remain conserved in all species. (B) Shows the other homologous cluster of WNT-1 and WNT-10B. Interesting information here is the conserved regulatory region between WNT-10A and WNT-6 and WNT-10B and WNT-1. This region is almost 7KB in Human, Mouse and Macaque. However in case of Zebra fish it is of 38KB, which means that independent duplications have occurred in this particular intergenic sequence, which is strange and under study. (C) Chromosomal co-linearity of homologous WNT clusters in Homo sapiens. WNT10-A is homologous to WNT-10B, while WNT-6 is homologous to WNT-1 at human chromosomes 2 and 12, respectively. Along with these, there are also some other genes whose genetic paralogs exist on these locus including PRKAG3, IHH and DNAJB2, despite of the fact that their exact orientation is not same.

Figure 5

Alignment file of WNT-1 and WNT-6 showing the conservation of sequence between four species and also displaying conserved sequence motifs. (A) Sequence alignments of WNT-1 and WNT-6 illustrating the conserved sequence motifs. The conserved motifs including Palmitoylation site (IPR014788), WNT family signature (PS00246), Claudin-5 signature, S locus related glycoprotein 1 binding pollen coat and Somatostatin receptor signature (IPB001116A) are represented by colours as indicated. Motif enrichment analysis is performed by using PRINTS, PRODOM, Blocks, PFAM and InterProScan, respectively. (B) MSA of WNT-1 sequences and (C) MSA of WNT-6 derived from Homo sapiens, Macaca mullata, Mus musculus and Danio rerio. Black colour is indicating the level of conservation at amino acid level.

Figure 6

Multiple sequence alignment of WNT-10A and WNT- 10B. (A) Sequence alignments of WNT-10A and WNT-10B illustrating the conserved sequence motifs. The conserved motifs including Palmitoylation site (IPR014788), WNT family signature (PS00246), Claudin-5/-8 signature, Rhodopsin like GPCR family signature (IPR000276), HIV- TAT domain (IPR001831) and 4-disulphide core signature (IPR015874) are represented by colours as indicated. Motif enrichment analysis is performed by using PRINTS, PRODOM, BLOCKs, PFAM and InterProScan, respectively. (B) MSA of WNT-10A sequences and (C) MSA of WNT-10B derived from Homo sapiens, Macaca mullata, Mus musculus and Danio rerio. Black color indicates the level of conservation at amino acid level.

Figure 7

Reverse Transcriptase PCR analysis of WNT-1, WNT- 6, WNT-10A and WNT-10B. (A) Expression analysis of two homologs WNT-6 and WNT-10A, working in synergistic manner. WNT-6 showing up-regulation in tumour whereas WNT-10A in normal. (B) Expression analysis of WNT-1 and WNT-10B, also acting synergistically, WNT-1 up regulating in tumour just like its closest homolog WNT-6 and WNT-10B up regulating in normal tissue sample like WNT-10A. β-actin is shown as positive control.

Figure 8

Determination of novel cis-regulatory elements (CREs) at intergenic region of co-linear WNT genes (WNT-6 and WNT-10A) forming a cluster. (A) Intergenic region of 7000bp between WNT-6 and WNT-10A showing conserved motifs of TCF-4, SOX family, myc family, TBF and TBP. (B) 7000bp downstream region of WNT-10A. Transcription factor binding sites are present for Sox-9 and TCF-4. In case of mouse, there is also a conserved site of LEF-1 along with TCF-4. (C) 5'upstream regulatory region analysis of WNT-6. This analysis is showing the similar results as depicted in A and B. TFBSs of TCF-4 and SOX remained conserved in almost all orthologs, supporting the hypothesis.

Figure 9

(A)Novel cis-regulatory elements (CREs) at intergenic region of homologous WNT-1 and WNT-10B genes.(A) Intergenic region of 7KB between WNT-1 and WNT-10B showing conserved motifs of TCF-4, SOX family, myc family, TBF and TBP. Presence of multiple binding sites for TCF-4 and Sox-9 in human and macaque strongly provide the evidence supporting the hypothesis that these two might be the responsible regulatory elements acting as activator and suppressor. C-myc and n-myc binding sites are also shown to be conserved in vertebrates that might play role in tumorgenesis. (B) Downstream regulatory region of WNT-1 showing multiple sites for Sox-9 and TCF-4, depicting their strong conservation in case of both genes of WNT-1/WNT-10B cluster. Additionally, overlapping TFBSs were also analyzed using TrFAST tool (unpublished data) and are shown. TBP and TBF showing strong conservation in fish are DNA binding motifs; (C) Shows the similar results as elucidated in A and B. TFBSs of TCF-4 and SOX remain conserved in almost all orthologs.