DNA-dependent formation of transcription factor pairs alters their binding specificity

Abstract

PAPER ABSTRACTGene expression is regulated by transcription factors (TFs), proteins that recognize short DNA sequence motifs. Such sequences are very common in the human genome, and an important determinant of the specificity of gene expression is the cooperative binding of multiple TFs to closely located motifs. However, interactions between DNA-bound TFs have not been systematically characterized. To identify TF pairs that bind cooperatively to DNA, and to characterize their spacing and orientation preferences, we have performed consecutive affinity-purification systematic evolution of ligands by exponential enrichment (CAP-SELEX) analysis of 9,400 TF-TF-DNA interactions. This analysis revealed 315 TF-TF interactions recognizing 618 heterodimeric motifs, most of which have not been previously described. The observed cooperativity occurred promiscuously between TFs from diverse structural families. Structural analysis of the TF pairs, including a novel crystal structure of MEIS1 and DLX3 bound to their identified recognition site, revealed that the interactions between the TFs were predominantly mediated by DNA. Most TF pair sites identified involved a large overlap between individual TF recognition motifs, and resulted in recognition of composite sites that were markedly different from the individual TF's motifs. Together, our results indicate that the DNA molecule commonly plays an active role in cooperative interactions that define the gene regulatory lexicon. SUMMARYCharting transcription factor interactions. By using a new method, we discovered a large number of transcription factor (TF) pairs that bind cooperatively to DNA, and found that in many cases the TF pairs recognize a composite motif ('compound DNA word') that is markedly different from that expected from the individual TF motifs ('DNA words'). The problemIndividual TF recognition sequences (DNA words) are common in the human genome, so the specificity of gene expression depends on the cooperative binding of multiple TFs to sites close to each other. The critical role of particular TF combinations in cell-fate determination and development is well established 1,2 . The TFs in cultured cells bind to only a subset of their potential target sites, and many of the occupied sites do not contain high-affinity TF motifs, suggesting that cooperative interactions allow TFs to bind to low-affinity sites [3][4][5] . Research has shown many examples where two TFs bind DNA together as cooperative complexes that form as a result of protein-protein interactions 6 , DNA-facilitated protein-protein contacts, and interactions mediated by DNA [7][8][9] . Most cases have been discovered by studying individual TF pairs, however, so it has not been known how common such TF-TF interactions are. The lack of understanding of TF interactions is one reason why the gene regulatory code, which determines how DNA sequence defines geneexpression patterns, has remained poorly understood. The solutionTo systematically identify TF-TF interactions in the presence of DNA, we dev...

Complex Interdependence Regulates Heterotypic Transcription Factor Distribution and Coordinates Cardiogenesis

“…Besides the recognition of specific binding motifs, TF occupancy depends in high degree on the interaction with other TFs, giving rise to TFs complexes where strength of interaction is dictated by the combination of TF-TF and TF-DNA affinities. We have also observed permissive binding motif configurations, highlighting the role of DNA as an instructive partner for pairs of heterotypic TFs (Jolma et al, 2015). Our structural studies proved a biophysical basis for these interactions, and additionally suggest important TF-TF protein interactions away from the DNA-binding interface.…”

“…Despite the suggestion of combined function from occupancy or gain of function biochemical studies (eg. Jolma et al, 2015; Spitz and Furlong, 2012), the functional output of interactions between developmentally important TFs and their genomic and structural basis have not been explored on a broad scale or in sufficient detail. The nature and importance of heterotypic TF interactions are therefore incompletely understood.…”

Gene regulation at a distance: From remote enhancers to 3D regulatory ensembles

“…Recruitment of sequence-specific transcription factors (TFs) is an essential hallmark of enhancers. TFs usually bind specific motifs of 6-10 bp long, which are now known for most vertebrate TFs [3][4][5]. Computational prediction of enhancers as clusters of TF binding sites are possible, but since the short motifs that define most TF consensus binding sites are very frequent in large genomes, they remain difficult and prone to false positives (reviewed in [6,7]).…”

“…Moreover, an unknown proportion of enhancers can be missed by such approaches. For example, cooperativity between different TFs can allow them to bind to sequences that differ markedly from the preferred sites of the individual TFs [5]. Furthermore, recent studies emphasize that tissue-specific enhancers rely rather on combinations of low-affinity binding sites instead of using high-affinity consensus ones, as the latter ones tend to reduce expression specificity [8,9].…”

Decoding transcriptional enhancers: Evolving from annotation to functional interpretation

“…This knowledge gap renders prediction of functional enhancers and their mechanisms difficult. Additionally, predicting the functional effects of different sequence variants within enhancers is further complicated by the recently described phenomenon that pairs of factors cooperatively binding to a DNA segment influence motif preference and binding kinetics [24]. Furthermore,, although enhancer function is traditionally believed to be independent of DNA strand orientation, recent studies have challenged this notion [23, 25].…”