Identification and characterization of proteins binding to regulatory elements of the Hoxa-5 spatial-specific enhancer

Abstract

The Hox homeobox genes cooperate in providing positional information needed for spatial and temporal patteming of the vertebrate body axis. However, the biological mechanisms behind spatial Hox expression are largely unknown. In transgenic mice, gene fusions between Hoxa-5 (previously called Hox-13) 5' flanking regions and lacZ show tissueand time-specific expression in the brachial spinal cord in day 11-13 embryos. This spatiallyspecific expression is directed by a 604 bp regulatory region with enhancer properties. Finedetail mapping of this enhancer has identified several elements involved in region-specific expression. A brachial spinal cord element, is required for expression in the brachial spinal cord. Factors in embryonic day 12.5 nuclear extracts bind this element in electrophoretic mobility shift assays (EMS A) and protect three regions from DNasel digestion. All three sites contain an AAATAA sequence and mutations at these sites decrease or abolish binding. Furthermore, this element binds specific individual embryonic proteins on a protein blot. The binding appears as a gradient along the A-P axis with twoto three-fold higher levels observed in extracts from anterior regions than from posterior regions. In parallel with the EMS A, the proteins on the protein blot also show decreased binding to probes with mutations at the AAATAA sites. This brachial spinal cord element and its binding proteins are likely to be involved in spatial expression of Hoxa-5 during development INTRODUCTION The genetically defined cascade of homeobox gene regulation in Drosophila, which ultimately acts to define the components of the embryo, is an excellent model of how spatial expression of genes is regulated to result in segment identification. This cascade of gene regulation begins with the maternal and gap genes setting up gradients of positional information that regulate the pair-rule genes, which, in turn, act to establish the initial spatial expression of the homeobox-containing homeotic genes (reviewed in Lawrence and Morata, 1994). The homeobox is a 60 amino acid binding domain conserved in many regulatory genes, first discovered in Drosophila (McGinnis et al., 1984, and Scott and Weiner, 1984). Drosophila homeobox-containing genes are the most extensively characterized in terms of function.