Short tandem repeats (STRs) are enriched in eukaryotic cis -regulatory elements and alter gene expression, yet how they regulate transcription remains unknown. We found that STRs modulate transcription factor (TF)–DNA affinities and apparent on-rates by about 70-fold by directly binding TF DNA-binding domains, with energetic impacts exceeding many consensus motif mutations. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density, with impacts well predicted by statistical mechanics. Confirming that STRs also affect TF binding in cells, neural networks trained only on in vivo occupancies predicted effects identical to those observed in vitro. Approximately 90% of TFs preferentially bound STRs that need not resemble known motifs, providing a cis -regulatory mechanism to target TFs to genomic sites.

Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hypermutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein–DNA binding specificity. We applied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all the TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs, we identified a surprising but reproducible effect at certain nonconsensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and nonconsensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.

The chemical synthesis of site‐specifically modified transcription factors (TFs) is a powerful method to investigate how post‐translational modifications (PTMs) influence TF‐DNA interactions and impact gene expression. Among these TFs, Max plays a pivotal role in controlling the expression of 15 % of the genome. The activity of Max is regulated by PTMs; Ser‐phosphorylation at the N‐terminus is considered one of the key regulatory mechanisms. In this study, we developed a practical synthetic strategy to prepare homogeneous full‐length Max for the first time, to explore the impact of Max phosphorylation. We prepared a focused library of eight Max variants, with distinct modification patterns, including mono‐phosphorylated, and doubly phosphorylated analogues at Ser2/Ser11 as well as fluorescently labeled variants through native chemical ligation. Through comprehensive DNA binding analyses, we discovered that the phosphorylation position plays a crucial role in the DNA‐binding activity of Max. Furthermore, in vitro high‐throughput analysis using DNA microarrays revealed that the N‐terminus phosphorylation pattern does not interfere with the DNA sequence specificity of Max. Our work provides insights into the regulatory role of Max′s phosphorylation on the DNA interactions and sequence specificity, shedding light on how PTMs influence TF function.

Chemical protein synthesis provides a powerful means to prepare novel modified proteins with precision down to the atomic level, enabling an unprecedented opportunity to understand fundamental biological processes. Of particular interest is the process of gene expression, orchestrated through the interactions between transcription factors (TFs) and DNA. Here, we combined chemical protein synthesis and high-throughput screening technology to decipher the role of post-translational modifications (PTMs), e.g., Lys-acetylation on the DNA binding activity of Max TF. We synthesized a focused library of singly, doubly, and triply modified Max variants including site-specifically acetylated and fluorescently tagged analogs. The resulting synthetic analogs were employed to decipher the molecular role of Lys-acetylation on the DNA binding activity and sequence specificity of Max. We provide evidence that the acetylation sites at Lys-31 and Lys-57 significantly inhibit the DNA binding activity of Max. Furthermore, by utilizing high-throughput binding measurements, we assessed the binding activities of the modified Max variants across diverse DNA sequences. Our results indicate that acetylation marks can alter the binding specificities of Max toward certain sequences flanking its consensus binding sites. Our work provides insight into the hidden molecular code of PTM-TFs and DNA interactions, paving the way to interpret gene expression regulation programs.

Transcription factors (TFs) intricately navigate the vast genomic landscape to locate and bind specific DNA sequences for the regulation of gene expression programs. These interactions occur within a dynamic cellular environment, where both DNA and TF proteins experience continual chemical and structural perturbations, including epigenetic modifications, DNA damage, mechanical stress, and post-translational modifications (PTMs). While many of these factors impact TF-DNA binding interactions, understanding their effects remains challenging and incomplete. This review explores the existing literature on these dynamic changes and their potential impact on TF-DNA interactions.