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Similar analyses using direct targets of HNF4A (Boj et?al., 2009) to explore TF function and TF binding intensity afforded similar results (data not shown). For additional methodological details, please see the section ��A Large Core Set of TF Binding Intensities Is Evolutionarily Stable across All Five Mouse Species but Is Decoupled from Functional Target Genes�� in the Extended Experimental Procedures. In summary, regions with stronger TF binding intensities involved more TFs and were less likely to be lost over evolutionary time. Within the conserved TF binding regions shared among all five mouse species, we observed more than 7,000 loci where the TF binding [https://en.wikipedia.org/wiki/Oxymatrine Oxymatrine] strength is constrained, and these loci, perhaps surprisingly, do not appear to be concentrated near functional target genes. We asked what effect genetic deletion of single component TFs would have on the stability of combinatorial TF binding and how the genetic stability is related to the evolutionary conservation of the TF binding within these clusters. We obtained livers from genetically engineered mice lacking either HNF4A or CEBPA. Although we cannot entirely rule out the influence of indirect effects, each TF knockout had minimal effect on the gene expression of the other liver-specific TFs (Kyrmizi et?al., 2006; data not shown). We then performed ChIP-seq experiments against HNF4A, CEBPA, and FOXA1. These experiments further confirmed that both genetic knockouts were successful [http://www.selleckchem.com/products/liraglutide.html click here] and that the targeted TF was largely absent from liver (Figure?6). We then asked what effect these genetic deletions have on 2TF and 3TF clusters that were consistently bound across all species of mice, expecting that these would be most robust to perturbations. We used two internal controls that should be unaffected by the deletion of a specific TF: (1) CTCF [http://www.selleckchem.com/products/verubecestat-mk-8931.html Verubecestat manufacturer] binding, which occurs in the genome independently of tissue-specific TF clusters (Faure et?al., 2012); and (2) the 2TF clusters not containing the deleted factor (Figure?6A). Our data confirmed that CTCF binding was unperturbed by knockout of the unrelated factor, as was TF binding in the 2TF clusters lacking the deleted regulator. The use of multiple internal controls afforded robustness to our analysis. We consistently found that deletion of HNF4A or CEBPA from a combinatorially bound region caused loss of cobound partner TFs (Figure?6). For instance, genetic deletion of HNF4A has no effect on the deeply shared CEBPA-FOXA1 2TF clusters (96% overlap with wild-type [WT]) but significantly destabilizes the CEBPA-HNF4A 2TF clusters (66% overlap with WT: p?