Proper control of gene expression is critical for the complex function of a living cell. Although gene expression can be regulated at multiple levels, one of the most important regulatory mechanisms is at the transcriptional level. The transcriptional program is regulated by binding of transcription factors (TFs) to the specific DNA sequences in regulatory regions of the genes. TFs are thus fundamental to the regulation of gene expression. However, several studies on specific TFs have observed that only a small fraction of genes that were bound by a TF were differentially expressed after that factor was knocked out or knocked down [1
]. Recently, a genome-wide study has carried out knockout experiments to determine knockout target genes (i.e. differentially expressed genes) for 269 budding yeast TFs under normal growth condition [5
]. Further analysis showed that there is only a low overlap (~3%) between the knockout targets and the binding targets identified by chromatin immunoprecipitation (ChIP)-chip [6
There are several possible explanations for the small overlap between knockout and binding targets. First, the large-scale ChIP-chip data set could be more likely to be affected by experimental noise [5
]. The overlap between Rap1 knockout and binding targets from a different high-quality ChIP-chip data set [7
] is as twice as that for the large-scale ChIP-chip data set [6
]. Second, binding and knockout experiments could capture distinct features of the transcriptional regulatory program [8
]. Binding experiments show a preference to uncover long-range interactions between telomeres and transcription factors, while knockout experiments tend to reveal downstream effects of interference with ribosome biogenesis. Third, mere TF binding is not sufficient to guarantee its regulation. The effect of bound TFs on target genes' expression might depend on the presence of other proteins. The location, orientation and spacing of transcription factor binding sites (TFBSs) also affect the regulatory function of TFs on their target genes [9
]. Finally, the knockout targets could include both direct and indirect targets. The overlap between the knockout and the binding targets indeed significantly improved after the elimination of the indirect targets [5
The small overlap between knockout and binding targets might be indicative of redundant TFs which mask the TF knockout effect. Most eukaryotic genes are regulated by a combination of TFs [11
]. Some TFs might work in a redundant fashion [14
], and they could compensate for each other's loss [15
]. The compensation among TFs should lead to the insensitivity of binding targets to the knockout of single TF. We referred to this insensitivity to the knockout of single TF as the robustness of transcriptional regulatory program. An interesting question arises concerning how the TFs involved in robust transcriptional regulatory program work in a redundant fashion. In addition, it is intuitive that this robustness should influence gene expression, but evidence for the effects remains to be elucidated.
In this study, we first distinguished between robust and fragile (unrobust) transcriptional regulatory programs according to their degrees of sensitivity to the knockout of TFs under normal growth condition. We referred to TFs involved in robust or fragile transcriptional regulatory program as robust or fragile TFs, and referred to genes having robust or fragile transcriptional regulatory program as robust or fragile genes. We found that robust cooperative TFs show significantly higher co-activity than fragile cooperative TFs. This result indicates that robust TFs regulate their common target genes in an activity-redundant fashion. We further revealed that fragile genes have high capacity to modulate gene expression upon changing conditions and have high evolutionary rates of genes expression. Paralogs provide backup mechanisms for expression variability of fragile genes. We compared the fragility of transcriptional regulatory program with other determinants of expression variability, and showed that the fragility is an important source of expression variability.