210 DEFINING THE MINIMAL PROMOTER REGION OF BOVINE VASA HOMOLOGUE (Bvh) BY IN SILICO ANALYSIS

Abstract

The DEAD box polypeptide 4, DDX4 or VASA, is a highly conserved gene that encodes a putative RNA helicase with the motif DEAD (Asp-Glu-Ala-Asp). Although little is known about its role in germ cell genesis, VASA is one of the earliest, specialised markers of primordial germ cell (PGC) specification. Furthermore, this process of specification has been recapitulated to some degree in vitro using embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) in mice and humans, using VASA expression as one of the criteria for differentiation and sorting of the differentiated cells. In order to establish a system for isolation and tracking of bovine iPSC undergoing germ cell specification, we analysed all regulatory elements in the 5-kb upstream region [RC 5083564–5088564 Bos taurus (Hereford) chromosome 20 genomic scaffold: NW_003104511.1] of the bovine VASA homologue (Bvh) locus, which is thought to be the putative promoter region of Bvh, and in the in vivo validated promoter regions, for the corresponding homologous genes in human and mouse. We performed the analysis using 2 different approaches: at the sequence level, by orthologous promoter alignment of transcription factor (TF) binding sites (TFBS) using DiAling^®, and at the functional level, by functional unit analysis (complex model) using Frameworker^® (Genomatix, Munich, Germany). The initial DiAling^® analysis did not produce similarities between the 3 analysed species. In contrast, using the complex analysis of functional units, we identified 85 single elements common to all 3, and 795, 482, and 129 models composed of 2, 3, and 4 elements, respectively. The number of models was reduced to 3 [M1, M2, and M3 (P = 4.8 × 10^–11)] by increasing the number of TF (each model composed of 6 different elements). As a result, members of SOX/SRY-sex/testis determining and related HMG box factor family related with germ cell specification, pluripotent-related factors such as members of the octamer binding protein family, and TFs common to numerous vertebrate genes such as homeobox transcription factors were identified (Table 1). Based on these results, we determined a region of approximate 0.6 kb upstream of the Bvh gene, which encloses a core of TFBS conserved at the functional level between species. We propose that this sequence is the best candidate for driving the expression of reporter genes under Bvh promoter control.

Table 1.  Six element models