Identifying combinatorial regulation of transcription factors and binding motifs

Abstract

Background: Combinatorial interaction of transcription factors (TFs) is important for gene regulation. Although various genomic datasets are relevant to this issue, each dataset provides relatively weak evidence on its own. Developing methods that can integrate different sequence, expression and localization data have become important.

Results: Here we use a novel method that integrates chromatin immunoprecipitation (ChIP) data with microarray expression data and with combinatorial TF-motif analysis. We systematically identify combinations of transcription factors and of motifs. The various combinations of TFs involved multiple binding mechanisms. We reconstruct a new combinatorial regulatory map of the yeast cell cycle in which cell-cycle regulation can be drawn as a chain of extended TF modules. We find that the pairwise combination of a TF for an early cell-cycle phase and a TF for a later phase is often used to control gene expression at intermediate times. Thus the number of distinct times of gene expression is greater than the number of transcription factors. We also see that some TF modules control branch points (cell-cycle entry and exit), and in the presence of appropriate signals they can allow progress along alternative pathways.

Conclusions: Combining different data sources can increase statistical power as demonstrated by detecting TF interactions and composite TF-binding motifs. The original picture of a chain of simple cell-cycle regulators can be extended to a chain of composite regulatory modules: different modules may share a common TF component in the same pathway or a TF component cross-talking to other pathways.

Figure 1

Overview of the method. (a) Finding over-represented single motifs from ChIP data. Target promoters of each TF are determined from ChIP data, and these promoters are searched for over-represented motifs. (b) Finding over-represented motif combinations specific to each cell-cycle phase. For all over-represented motifs found in (a), all possible first (single)-, second (double)- and third (triple)-order combinations of motifs are searched against the promoters of the G1, S, S/G2, G2/M, M/G1 or 'All' groups of cell-cycle genes. (c) When an over-represented motif combination is found, we require that the genes possessing that combination have more coherent patterns of gene expression than the patterns for the phase-specific gene group.(d) From ChIP data, we find the set of TFs (over-represented TFs, see Materials and methods) binding to the promoters with a motif combination (from (b) and (c)), and the set of TFs that can bind over-represented motifs (from (a)) constituting a motif combination. For each motif combination and its component motifs, we take the intersection of TFs from these two sets. Such intersection TFs (in bold in the (d)) are assigned to the component motifs of a combination (see Materials and methods).

Figure 2

Over-represented motifs from ChIP data reflect three types of binding mechanisms. A solid arrow means that the TF binds directly to the motif. A dotted arrow means that a motif (for example, M1) is over-represented in TF1 ChIP data. This can occur (a) because of direct binding, or (b, c) because of two modes of indirect binding. (a) M1 is a direct-binding motif of TF1. (b) M1 is an indirect piggy-back binding motif of TF1 via TF2, where TF1 binds TF2, and TF2 binds the motif. For example, M1, TF1, and TF2 could correspond to GTAAACA (the Fkh2 motif), Ndd1, and Fkh2, respectively, as Ndd1 binds to Fkh2, which in turn binds to GTAAACA. (c) M1 is an indirect cross-binding motif of TF1 via TF2. For example, M1, TF1 and TF2 could correspond to GTAAACA (the Fkh2 motif), Mcm1, and Fkh2, respectively, since Mcm1 binds to its own motif (M2) on the same promoter as the Fkh2 motif.

Figure 3

Reconstructed transcriptional regulation model of the yeast cell cycle. Segments of the cycle contain motif combinations. The TF and motif combinations in black were known previously and are also confirmed here. Those in red are new combinations (or previously poorly characterized combinations). TFs and motifs in parentheses are those we could not detect (for example, because the TF was not included in the ChIP experiments) but whose presence is indicated by some other experimental work [25,46]. Almost all TFs appear as elements of a combination. {Swi5, Ste12, Dig1} shows a joint-process combination between cell cycle and pheromone response or filamentous growth (see Discussion).