Protein−DNA binding in the absence of specific base-pair recognition

Abstract

Until now, it has been reasonably assumed that specific base-pair recognition is the only mechanism controlling the specificity of transcription factor (TF)−DNA binding. Contrary to this assumption, here we show that nonspecific DNA sequences possessing certain repeat symmetries, when present outside of specific TF binding sites (TFBSs), statistically control TF−DNA binding preferences. We used highthroughput protein−DNA binding assays to measure the binding levels and free energies of binding for several human TFs to tens of thousands of short DNA sequences with varying repeat symmetries. Based on statistical mechanics modeling, we identify a new protein−DNA binding mechanism induced by DNA sequence symmetry in the absence of specific base-pair recognition, and experimentally demonstrate that this mechanism indeed governs protein−DNA binding preferences. protein−DNA binding is an important biophysical mechanism operating in a living cell (1). This seminal work makes it possible to interpret experiments that measured how transcription factors (TFs) search for their specific target sites flanked by nonconsensus sequence elements (1-10). A specific consensus motif is a short DNA sequence, typically 6-20 base pairs (bp), that possesses an enhanced binding affinity for a particular TF. For example, the sequence CACGTG represents the specific consensus motif for the human protein Max used in this study (Fig. 1). The process of establishing specific, consensus protein−DNA binding requires the formation of precise geometrical fit between the protein and its consensus DNA motif, accompanied by the formation of specific hydrogen and electrostatic contacts at the protein−DNA binding interface (6, 7) ( Fig. 1). In addition to binding to their consensus DNA motifs, transcription factors can also bind, albeit with lower affinity, to DNA regions lacking any consensus motifs. The term "nonspecific protein−DNA binding" (6) is typically used to describe these weaker interactions. Von Hippel and Berg suggested classifying nonspecific protein−DNA binding into two related mechanisms (6). The first mechanism includes protein binding to its mutated specific motifs that retain some residual, reduced specificity. The second mechanism is largely DNA sequence independent, and it involves electrostatic binding modulated by the overall DNA geometry (6). Despite significant experimental progress, molecular mechanisms responsible for these two types of nonspecific binding remain poorly understood, and the free energy of nonspecific protein−DNA binding has not been systematically characterized (11)(12)(13)(14). The interplay between consensus and nonconsensus DNA sequence elements emerges as a dominant factor that governs protein−DNA binding preferences. However, this interplay is also poorly understood (15, 16). Until now, it has been reasonably assumed that specific (consensus) base-pair recognition must control the genome-wide specificity of TF−DNA binding.Contrary to this assumption, here we identify a general mechanism for protein−DNA bi...

A widespread role of the motif environment in transcription factor binding across diverse protein families

“…We found that TF sequence preferences reach far beyond the core motifs and their direct flanks, which were previously shown to contribute to TF binding preferences (Gordân et al 2013;Afek et al 2014;Levo et al 2015). An intriguing question is how the protein can identify the unique environment so far beyond its binding site.…”

“…However, selective binding of motifs by TFs has also been observed in a variety of in vitro experiments (Noyes et al 2008;Badis et al 2009;Berger and Bulyk 2009;Zhao et al 2009;Slattery et al 2011;Enuameh et al 2013;Gordân et al 2013;Jolma et al 2013;Afek et al 2014;Weirauch et al 2014;Abe et al 2015;Levo et al 2015). These in vitro studies show that TFs can bind to different sequences containing a similar motif with a large range of different affinities, which suggests that TF-DNA binding specificity is influenced by the DNA context surrounding the motif.…”

“…These in vitro studies show that TFs can bind to different sequences containing a similar motif with a large range of different affinities, which suggests that TF-DNA binding specificity is influenced by the DNA context surrounding the motif. Indeed, the contribution of the regions directly flanking the motif to binding specificity in vitro has been demonstrated for a small number of TFs (Gordân et al 2013;Afek et al 2014;Yang et al 2014;Levo et al 2015). The sequence environment of a motif has also been shown to contribute to transcriptional regulation by the TF Cone-rod homeobox (CRX) (White et al 2013).…”

Relationship between histone modifications and transcription factor binding is protein family specific

“…DNA sequence and shape preferences at flanking regions of core motifs play important roles in achieving binding specificity for TFs from certain protein families, both in vitro (Gordân et al 2013;Afek et al 2014) and in vivo . However, these DNA properties alone are insufficient to explain the different in vivo TF binding preferences observed across distinct cell types (The ENCODE Project Consortium.…”

Busca Alvo de Fatores de Transcrição em Cadeias de ADN

“…Os resultados de estudos nesta linha podem impactar a compreensão dos princípios moleculares biofísicos de regulação da transcrição e melhorar significativamente a nossa capacidade de prever como as variações nas sequências de ADN, tais como mutações ou polimorfismos e concentrações de proteína, influenciam programas de expressão gênica em células vivas [1]. Apesar de ser largamente aceito, a forma geral da difusão facilitada, a princípio, apresentaria um problema conhecido como paradoxo velocidade-estabilidade.…”