A conserved metabolic machinery forms the common basis of all cells; however, variation in the regulation of the genes that encode this machinery produces fundamental phenotypic differences between species. Recently, several groups have linked phenotypic traits to changes in the expression of conserved gene in diverse metazoans like Darwin finches, sticklebacks, and flies 
. At the transcriptional level, this differential gene expression can be obtained by varying the structure of cellular transcriptional regulatory networks (TRNs), and many types of modifications can drive changes in gene regulation. For example, the set of target genes of a transcription factor (TF) can evolve by cis
-regulatory sequence changes 
, as the appearance or disappearance of TF-binding motifs in genes or groups of genes allows their addition or removal from regulatory circuits. Changing the chromatin status of a gene by varying its nucleosome occupancy, its gene neighborhood, or its chromosome position can have impacts on its expression level 
. As well, trans
-acting factors and their interacting partners can be modified by the recruitment of chromatin modifying enzymes or by changes in properties such as their DNA-binding specificity, modular structure, trans
-activating potential, or combinatorial/cooperative binding characteristics 
. Furthermore, the regulation of a TF can be changed through it being connected to new regulatory circuits, and this would affect the expression of its targets 
. Recently, several studies have highlighted gene expression differences between species 
, but the flexibility of the regulatory network that drives these transcriptional changes still needs to be studied.
Ribosomal proteins (RPs) and rRNAs are among the most conserved components of the cell, and the transcriptional regulation required to produce their stoichiometric and condition-dependent expression is a central cellular process. In S. cerevisiae
, co-ordinate expression of RP subunit genes is brought about by a protein complex made of the essential factors Rap1, Hmo1, Fhl1, and Ifh1. Rap1 and Hmo1 recruit the nutrient-dependent Fhl1-Ifh1 complex exclusively to RP genes 
, although Rap1 separately also occupies telomeres, the mating type locus, and glycolytic gene promoters 
. The binding of Rap1, Fhl1, and Hmo1 is not modulated by stress or nutrient levels, but under conditions of rapid proliferation, Fhl1 recruits Ifh1 through a heterotypic interaction between their respective FHA and FHB domains. This recruitment activates RP gene transcription to maximal levels but is perturbed by stress, or by inhibition of TOR or PKA signaling pathways, resulting in Ifh1 being released from RP promoters and replaced by another FHB-containing protein, the Crf1 co-repressor 
. Therefore, in S. cerevisiae
, the regulation of RP genes depends on intricate interactions among four regulatory proteins, specific DNA elements, and signaling pathways.
Previous studies have proposed that RP regulation has a high level of flexibility during evolution 
. This is supported by our recent observation that the essential C. albicans
TF Tbf1 (assisted by Cbf1 at some loci) is the key DNA-binding regulator of RP genes and the rDNA locus in most fungal species 
. Therefore, a Tbf1-DNA interface prevails at RP genes and the rDNA locus of C. albicans
, while Rap1 governs the transcription of RP genes in S. cerevisiae
. But the means by which the C. albicans
Tbf1-dominated regulatory network performs the task of connecting ribosomal transcription with cellular signaling and the fate of the other S. cerevisiae
regulators remains unknown. Here, we have used chromatin immunoprecipitation followed by microarray analysis (ChIP-CHIP) with full-genome coverage to show that conserved orthologous TFs can be profoundly repositioned within the regulatory network during evolution. Specifically, their regulons, their connections with cellular functions, their hierarchical position within the regulatory network, their DNA-binding specificity, and their assembly into higher order complexes are shaped during evolution.