High-throughput data and modeling reveal insights into the mechanisms of cooperative DNA-binding by transcription factor proteins.

Cooperative DNA-binding by transcription factor (TF) proteins is critical for eukaryotic gene regulation. In the human genome, many regulatory regions contain TF-binding sites in close proximity to each other, which can facilitate cooperative interactions. However, binding site proximity does not necessarily imply cooperative binding, as TFs can also bind independently to each of their neighboring target sites. Currently, the rules that drive cooperative TF binding are not well understood. In addition, it is oftentimes difficult to infer direct TF-TF cooperativity from existing DNA-binding data. Here, we show that in vitro binding assays using DNA libraries of a few thousand genomic sequences with putative cooperative TF-binding events can be used to develop accurate models of cooperativity and to gain insights into cooperative binding mechanisms. Using factors ETS1 and RUNX1 as our case study, we show that the distance and orientation between ETS1 sites are critical determinants of cooperative ETS1-ETS1 binding, while cooperative ETS1-RUNX1 interactions show more flexibility in distance and orientation and can be accurately predicted based on the affinity and sequence/shape features of the binding sites. The approach described here, combining custom experimental design with machine-learning modeling, can be easily applied to study the cooperative DNA-binding patterns of any TFs.

QBiC-Pred: quantitative predictions of transcription factor binding changes due to sequence variants.

Non-coding genetic variants/mutations can play functional roles in the cell by disrupting regulatory interactions between transcription factors (TFs) and their genomic target sites. For most human TFs, a myriad of DNA-binding models are available and could be used to predict the effects of DNA mutations on TF binding. However, information on the quality of these models is scarce, making it hard to evaluate the statistical significance of predicted binding changes. Here, we present QBiC-Pred, a web server for predicting quantitative TF binding changes due to nucleotide variants. QBiC-Pred uses regression models of TF binding specificity trained on high-throughput in vitro data. The training is done using ordinary least squares (OLS), and we leverage distributional results associated with OLS estimation to compute, for each predicted change in TF binding, a P-value reflecting our confidence in the predicted effect. We show that OLS models are accurate in predicting the effects of mutations on TF binding in vitro and in vivo, outperforming widely-used PWM models as well as recently developed deep learning models of specificity. QBiC-Pred takes as input mutation datasets in several formats, and it allows post-processing of the results through a user-friendly web interface. QBiC-Pred is freely available at http://qbic.genome.duke.edu.

Transcription Factor-Centric Approach to Identify Non-Recurring Putative Regulatory Drivers in Cancer.

Recent efforts to sequence the genomes of thousands of matched normal-tumor samples have led to the identification of millions of somatic mutations, the majority of which are non-coding. Most of these mutations are believed to be passengers, but a small number of non-coding mutations could contribute to tumor initiation or progression, e.g. by leading to dysregulation of gene expression. Efforts to identify putative regulatory drivers rely primarily on information about the recurrence of mutations across tumor samples. However, in regulatory regions of the genome, individual mutations are rarely seen in more than one donor. Instead of using recurrence information, here we present a method to identify putative regulatory driver mutations based on the magnitude of their effects on transcription factor-DNA binding. For each gene, we integrate the effects of mutations across all its regulatory regions, and we ask whether these effects are larger than expected by chance, given the mutation spectra observed in regulatory DNA in the cohort of interest. We applied our approach to analyze mutations in a liver cancer data set with ample somatic mutation and gene expression data available. By combining the effects of mutations across all regulatory regions of each gene, we identified dozens of genes whose regulation in tumor cells is likely to be significantly perturbed by non-coding mutations. Overall, our results show that focusing on the functional effects of non-coding mutations, rather than their recurrence, has the potential to identify putative regulatory drivers and the genes they dysregulate in tumor cells.