Inherent limitations of probabilistic models for protein-DNA binding specificity

The specificities of transcription factors are most commonly represented with probabilistic models. These models provide a probability for each base occurring at each position within the binding site and the positions are assumed to contribute independently. The model is simple and intuitive and is the basis for many motif discovery algorithms. However, the model also has inherent limitations that prevent it from accurately representing true binding probabilities, especially for the highest affinity sites under conditions of high protein concentration. The limitations are not due to the assumption of independence between positions but rather are caused by the non-linear relationship between binding affinity and binding probability and the fact that independent normalization at each position skews the site probabilities. Generally probabilistic models are reasonably good approximations, but new high-throughput methods allow for biophysical models with increased accuracy that should be used whenever possible

Quantitative profiling of BATF family proteins/JUNB/IRF hetero-trimers using Spec-seq

Additional file 6. Half site analysis for BATFx-JUNB-IRFx. (A) Single variants from half sites of these oligos in the library were used to generate energy logos. Bolded positions represent the half sites generated in B. (B) Energy logos from Spec-seq results of BATFx-JUNB-IRFx. The Y-axis is negative energy so the preferred sequence is on the top

Combining SELEX with quantitative assays to rapidly obtain accurate models of protein–DNA interactions

Models for the specificity of DNA-binding transcription factors are often based on small amounts of qualitative data and therefore have limited accuracy. In this study we demonstrate a simple and efficient method of affinity chromatography-SELEX followed by a quantitative binding (QuMFRA) assay to rapidly collect the data necessary for more accurate models. Using the zinc finger protein EGR as an e.g. we show that many bindings sites can be obtained efficiently with affinity chromatography-SELEX, but those sequences alone provide a weight matrix model with limited accuracy. Using a QuMFRA assay to determine the quantitative relative affinity for only a subset of the sequences obtained by SELEX leads to a much more accurate model. Application of this method to variants of a transcription factor would allow us to generate a large collection of quantitative data for modeling protein–DNA interactions that could facilitate the determination of recognition codes for different transcription factor families

Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

BACKGROUND: Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. RESULTS: Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. CONCLUSION: The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data

Assessing the effects of symmetry on motif disovery and modeling

BACKGROUND: Identifying the DNA binding sites for transcription factors is a key task in modeling the gene regulatory network of a cell. Predicting DNA binding sites computationally suffers from high false positives and false negatives due to various contributing factors, including the inaccurate models for transcription factor specificity. One source of inaccuracy in the specificity models is the assumption of asymmetry for symmetric models. METHODOLOGY/PRINCIPAL FINDINGS: Using simulation studies, so that the correct binding site model is known and various parameters of the process can be systematically controlled, we test different motif finding algorithms on both symmetric and asymmetric binding site data. We show that if the true binding site is asymmetric the results are unambiguous and the asymmetric model is clearly superior to the symmetric model. But if the true binding specificity is symmetric commonly used methods can infer, incorrectly, that the motif is asymmetric. The resulting inaccurate motifs lead to lower sensitivity and specificity than would the correct, symmetric models. We also show how the correct model can be obtained by the use of appropriate measures of statistical significance. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the most commonly used motif-finding approaches usually model symmetric motifs incorrectly, which leads to higher than necessary false prediction errors. It also demonstrates how alternative motif-finding methods can correct the problem, providing more accurate motif models and reducing the errors. Furthermore, it provides criteria for determining whether a symmetric or asymmetric model is the most appropriate for any experimental dataset

Finding motifs using DNA images derived from sparse representations

MOTIVATION: Motifs play a crucial role in computational biology, as they provide valuable information about the binding specificity of proteins. However, conventional motif discovery methods typically rely on simple combinatoric or probabilistic approaches, which can be biased by heuristics such as substring-masking for multiple motif discovery. In recent years, deep neural networks have become increasingly popular for motif discovery, as they are capable of capturing complex patterns in data. Nonetheless, inferring motifs from neural networks remains a challenging problem, both from a modeling and computational standpoint, despite the success of these networks in supervised learning tasks. RESULTS: We present a principled representation learning approach based on a hierarchical sparse representation for motif discovery. Our method effectively discovers gapped, long, or overlapping motifs that we show to commonly exist in next-generation sequencing datasets, in addition to the short and enriched primary binding sites. Our model is fully interpretable, fast, and capable of capturing motifs in a large number of DNA strings. A key concept emerged from our approach-enumerating at the image level-effectively overcomes the k-mers paradigm, enabling modest computational resources for capturing the long and varied but conserved patterns, in addition to capturing the primary binding sites. AVAILABILITY AND IMPLEMENTATION: Our method is available as a Julia package under the MIT license at https://github.com/kchu25/MOTIFs.jl, and the results on experimental data can be found at https://zenodo.org/record/7783033

Discovering structural cis-regulatory elements by modeling the behaviors of mRNAs

Gene expression is regulated at each step from chromatin remodeling through translation and degradation. Several known RNA-binding regulatory proteins interact with specific RNA secondary structures in addition to specific nucleotides. To provide a more comprehensive understanding of the regulation of gene expression, we developed an integrative computational approach that leverages functional genomics data and nucleotide sequences to discover RNA secondary structure-defined cis-regulatory elements (SCREs). We applied our structural cis-regulatory element detector (StructRED) to microarray and mRNA sequence data from Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens. We recovered the known specificities of Vts1p in yeast and Smaug in flies. In addition, we discovered six putative SCREs in flies and three in humans. We characterized the SCREs based on their condition-specific regulatory influences, the annotation of the transcripts that contain them, and their locations within transcripts. Overall, we show that modeling functional genomics data in terms of combined RNA structure and sequence motifs is an effective method for discovering the specificities and regulatory roles of RNA-binding proteins

Folding Free Energies of 5′-UTRs Impact Post-Transcriptional Regulation on a Genomic Scale in Yeast

Using high-throughput technologies, abundances and other features of genes and proteins have been measured on a genome-wide scale in Saccharomyces cerevisiae. In contrast, secondary structure in 5′–untranslated regions (UTRs) of mRNA has only been investigated for a limited number of genes. Here, the aim is to study genome-wide regulatory effects of mRNA 5′-UTR folding free energies. We performed computations of secondary structures in 5′-UTRs and their folding free energies for all verified genes in S. cerevisiae. We found significant correlations between folding free energies of 5′-UTRs and various transcript features measured in genome-wide studies of yeast. In particular, mRNAs with weakly folded 5′-UTRs have higher translation rates, higher abundances of the corresponding proteins, longer half-lives, and higher numbers of transcripts, and are upregulated after heat shock. Furthermore, 5′-UTRs have significantly higher folding free energies than other genomic regions and randomized sequences. We also found a positive correlation between transcript half-life and ribosome occupancy that is more pronounced for short-lived transcripts, which supports a picture of competition between translation and degradation. Among the genes with strongly folded 5′-UTRs, there is a huge overrepresentation of uncharacterized open reading frames. Based on our analysis, we conclude that (i) there is a widespread bias for 5′-UTRs to be weakly folded, (ii) folding free energies of 5′-UTRs are correlated with mRNA translation and turnover on a genomic scale, and (iii) transcripts with strongly folded 5′-UTRs are often rare and hard to find experimentally