Distinguishing direct versus indirect transcription factor-DNA interactions

Abstract

Transcriptional regulation is largely enacted by transcription factors (TFs) binding DNA. Large numbers of TF binding motifs have been revealed by ChIP-chip experiments followed by computational DNA motif discovery. However, the success of motif discovery algorithms has been limited when applied to sequences bound in vivo (such as those identified by ChIP-chip) because the observed TF-DNA interactions are not necessarily direct: Some TFs predominantly associate with DNA indirectly through protein partners, while others exhibit both direct and indirect binding. Here, we present the first method for distinguishing between direct and indirect TF-DNA interactions, integrating in vivo TF binding data, in vivo nucleosome occupancy data, and motifs from in vitro protein binding microarray experiments. When applied to yeast ChIP-chip data, our method reveals that only 48% of the data sets can be readily explained by direct binding of the profiled TF, while 16% can be explained by indirect DNA binding. In the remaining 36%, none of the motifs used in our analysis was able to explain the ChIP-chip data, either because the data were too noisy or because the set of motifs was incomplete. As more in vitro TF DNA binding motifs become available, our method could be used to build a complete catalog of direct and indirect TF-DNA interactions. Our method is not restricted to yeast or to ChIP-chip data, but can be applied in any system for which both in vivo binding data and in vitro DNA binding motifs are available.

Figure 1.

Identification of highly enriched motifs in a ChIP-chip data set. We proceed in four steps: (A) For each TF with a PBM-derived motif (here, Gcn4) and each intergenic probe (here, iYER052c), we compute the probability that the TF binds that probe, as described in the Methods section. (B) For each TF (here, Gcn4) we rank all intergenic probes in decreasing order of the binding probability and then compute the enrichment of the motif in a ChIP-chip data set (here, Gcn4_SM) according to AUC. To calculate the AUC statistic, we defined the positive and negative sets to be the sets of intergenic regions with ChIP-chip P-values < 0.001 and >0.5, respectively, as calculated by Harbison et al. (2004). (C) For each ChIP-chip data set (here, Gcn4_SM), we ranked all TFs in decreasing order of their motif's AUC value. (D) We determine the significantly enriched motif(s) (here, Gcn4).

Figure 2.

High-scoring motifs in the Cbf1_YPD ChIP-chip data set. (A) AUC values for the 139 PBM-derived motifs in the Cbf1_YPD data set. The x-axis shows the TF ranks, computed as in Figure 1C. (B) The three motifs that exhibit high AUC values in this data set: Tye7, Cbf1, and Rtg3. (C) Venn diagram showing the overlap among the sets of probes bound by Tye7, Cbf1, and Rtg3 in rich medium (YPD). Given the high similarity among the three motifs and the small overlap among the probes bound by the three factors, we do not consider this a case of indirect DNA binding by Cbf1.

Figure 3.

High-scoring motifs in the Fkh2_H2O2Hi and Fkh2_H2O2Lo ChIP-chip data sets. (A,B) AUC values for the 139 PBM-derived motifs in the two data sets. The x-axes show the TF ranks, computed as in Figure 1C. (C) Motifs significantly enriched in the two data sets. The DNA binding motif of Fkh2 was correctly identified as one of the significantly enriched motifs. In addition to Fkh2, the Hcm1, Fkh1, and Mcm1 motifs are also highly enriched. The Hcm1 and Fkh1 motifs are similar to the Fkh2 motif. Mcm1 is known to bind cooperatively with Fkh2 (Hollenhorst et al. 2001). (D,E) Venn diagrams showing the overlaps between the sets of probes bound by Fhk2 and Mcm1 in different environmental conditions.

Figure 4.

An example of indirect DNA association by a TF. The Rap1 motif is the only significantly enriched motif in all three Fhl1 ChIP-chip data sets: Fhl1_RAPA, Fhl1_SM, and Fhl1_YPD. The Fhl1 motif has only moderate AUC values and associated P-values that do not pass our significance criteria. We infer that in such cases many sequences identified as “bound” in the ChIP-chip experiments are actually indirectly bound by the profiled factor (here, Fhl1) through an interacting factor (here, Rap1).

Figure 5.

Direct and indirect DNA binding by Ste12 and Tec1. Ste12 and Tec1 are both involved in two developmental processes: filamentation (induced by treatment with butanol, as in the BUT14 condition) and mating (induced by treatment with the alpha pheromone, as in the Alpha condition). (A) During filamentation, the Tec1-Ste12–Dig1 complex binds DNA through Tec1. Our method correctly identifies Tec1 as the only significantly enriched TF in the ChIP-chip experiments where filamentation occurs. (B) During mating, the Ste12–Dig1–Dig2 and Ste12–Tec1–Dig 1 complexes bind DNA through Ste12. Our method correctly identifies Ste12 as the only significantly enriched TF in the ChIP-chip experiments where mating occurs.