Gilthorpe, Jonathan David; (1996) Regulation of mouse Hoxb-4 expression during embryogenesis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Previous work has defined the sequences sufficient to recapitulate the full expression pattern of the endogenous Hoxb-4 gene in transgenic mice. Several distinct regulatory regions have been identified which are responsible for particular aspects of Hoxb-4 expression. In this study I have begun a detailed analysis of the spatially-specific enhancer, region C, able to drive a major subset of Hoxb-4 expression in the mesoderm, central nervous system (CNS) and peripheral nervous system (PNS). Moreover, it is capable of imposing the correct anterior boundary of Hoxb-4 somitic expression on both the Hoxb-4 and hsp68 promoters. By a combination of sequence comparison and mutational analysis of a region C/hsp68-lacZ reporter gene, I have characterised two cw-regulatory elements that are critical for normal region C activity in transgenic mice. The first of these elements is located within an evolutionarily conserved region of the Hoxb-4 intron (CB1). Deletion of CB1 abolished lacZ reporter gene expression in the mesoderm, PNS and in the majority of the CNS of transgenic embryos. DNA electrophoretic-mobility shift assays revealed the presence of two overlapping binding sites for HoxTF and YY1 within CB1. Specific mutation of each binding-site showed that HoxTF is essential for efficient expression of Hoxb-4 in the embryo, whilst the role of YYl is unclear. UV crosslinking studies suggest that HoxTF binds to DNA as a heterodimer. Reporter gene analysis has shown that an isolated HoxTF binding element is capable of driving a consistent pattern of expression within the nervous system. These results show that the HoxTF element, though necessary, is not sufficient to drive Hoxb-4 mesodermal expression and requires the cooperation of other elements to achieve this. I have identified a second regulatory element, G5, required to achieve proper levels of region C enhancer activity. A transgenic reporter gene carrying an isolated fragment that contains the HoxTF/YY1 and G5 binding-sites drives a similar pattern of lacZ expression to that seen with the HoxTF site alone. This suggests that further elements are required to specify the Hoxb-4 pattern of mesodermal expression. 3' deletions have mapped these elements to a 269bp fragment that lies 3' to the HoxTF/YY1 site and within the 5' half of the intron. The 3' half of the intron is able to specify a limited pattern of expression in the PNS, implicating the role of a second HoxTF site that is involved in stabilising the activity of the region C enhancer.

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