For gene products to function together, they often need to be expressed together. A new study that explores coexpression in Ciona urochordates reveals that the sequence elements involved are remarkably flexible in their architecture.

Brown and colleagues explored the basis of coexpression for 19 genes that function in muscle differentiation in two Ciona species. Cis-regulatory elements in all of these genes contain three classes of motif that bind different transcription factors and are responsible for directing muscle-specific expression. Within these motif classes there is variation — motifs of each type vary in sequence and, potentially, function.

The authors generated reporter constructs for regulatory elements involved in the expression of the 19 genes, and carried out extensive mutagenesis on them. After generating transgenic embryos expressing the mutagenized constructs, a quantitative whole-embryo gene expression assay was used to explore regulatory element function. This allowed the authors to define the elements that explain the expression of each gene. They then disrupted each of the putative transcription factor binding motifs in each element, either individually or in different combinations, allowing the relationship between the composition and function of the regulatory elements to be explored.

To analyse their results, the authors first needed to be able to determine the contribution of each motif to the expression that was driven by each element. A key question here was whether interactions between motifs are important for element function. This proved not to be the case — an additive model provided a good fit for the data, showing that motif interactions generally affect expression very little and providing a basis for further analysis.

Brown and colleagues then set about exploring how the architecture of different elements contributes to their function. Surprisingly, despite their shared expression pattern, there is a high level of flexibility among the elements that were investigated. First, these elements are diverse in terms of the number and types of motifs that they contain. Also, no rules related to motif spacing, order or orientation seem to explain their regulatory function. So, many different combinations and arrangements of motifs can be used to give rise to the same expression pattern.

Far less flexibility in the architecture of cis-regulatory elements is permitted over evolutionary time. When the authors compared orthologues of the coexpressed genes between the two Ciona species, they found a high degree of sequence constraint within the regulatory elements. Furthermore, measuring motif function in both species independently revealed that the functional contribution of each motif to the total regulatory activity is extraordinarily well conserved, despite the fact that the two species are diverged enough to have accumulated significant change at these motifs. This constraint seems to be relaxed when genes are duplicated: paralogous genes showed much more flexibility in terms of element use and composition.

As the authors point out, these findings have implications for understanding how genetic variation influences phenotype through polymorphisms in regulatory elements: the effect of alterations on both cis- and trans-acting factors is expected to vary widely between elements as a result of their diverse architectures.