Transcriptional regulatory networks play a central role in optimizing cell 
survival. How DNA binding domains and cis-regulatory DNA binding sequences have 
co-evolved to allow the expansion of transcriptional networks and how this 
contributes to cellular fitness remains unclear. Here we experimentally explore 
how the complex G1/S transcriptional network evolved in the budding yeast 
Saccharomyces cerevisiae by examining different chimeric transcription factor 
(TF) complexes. Over 200 G1/S genes are regulated by either one of the two TF 
complexes, SBF and MBF, which bind to specific DNA binding sequences, SCB and 
MCB, respectively. The difference in size and complexity of the G1/S 
transcriptional network across yeast species makes it well suited to investigate 
how TF paralogs (SBF and MBF) and DNA binding sequences (SCB and MCB) co-evolved 
after gene duplication to rewire and expand the network of G1/S target genes. 
Our data suggests that whilst SBF is the likely ancestral regulatory complex, 
the ancestral DNA binding element is more MCB-like. G1/S network expansion took 
place by both cis- and trans- co-evolutionary changes in closely related but 
distinct regulatory sequences. Replacement of the endogenous SBF DNA-binding 
domain (DBD) with that from more distantly related fungi leads to a contraction 
of the SBF-regulated G1/S network in budding yeast, which also correlates with 
increased defects in cell growth, cell size, and proliferation.