The notochord is a defining feature of the chordate body plan. Experiments in 
ascidian, frog and mouse embryos have shown that co-expression of Brachyury and 
FoxA class transcription factors is required for notochord development. However, 
studies on the cis-regulatory sequences mediating the synergistic effects of 
these transcription factors are complicated by the limited knowledge of 
notochord genes and cis-regulatory modules (CRMs) that are directly targeted by 
both. We have identified an easily testable model for such investigations in a 
155-bp notochord-specific CRM from the ascidian Ciona intestinalis. This CRM 
contains functional binding sites for both Ciona Brachyury (Ci-Bra) and FoxA 
(Ci-FoxA-a). By combining point mutation analysis and misexpression experiments, 
we demonstrate that binding of both transcription factors to this CRM is 
necessary and sufficient to activate transcription. To gain insights into the 
cis-regulatory criteria controlling its activity, we investigated the 
organization of the transcription factor binding sites within the 155-bp CRM. 
The 155-bp sequence contains two Ci-Bra binding sites with identical core 
sequences but opposite orientations, only one of which is required for enhancer 
activity. Changes in both orientation and spacing of these sites substantially 
affect the activity of the CRM, as clusters of identical sites found in the 
Ciona genome with different arrangements are unable to activate transcription in 
notochord cells. This work presents the first evidence of a synergistic 
interaction between Brachyury and FoxA in the activation of an individual 
notochord CRM, and highlights the importance of transcription factor binding 
site arrangement for its function.