Biology subjects

Afek, A.

Publications and source records attributed to Afek, A..

10 recordsLinked to original sources

bioRxiv2026

EpiBinder: a multimodal framework for cell-type-specific prediction and interpretation of transcription factor binding

Transcription factor (TF) occupancy in vivo depends not only on the underlying DNA sequence but also on the local epigenetic environment, which varies across cell types and strongly influences whether sequence-encoded binding potential becomes functional. Here we present EpiBinder, a multimodal deep-learning framework for cell-type-specific prediction of TF binding that jointly models DNA sequence with base-resolution epigenetic information, including cytosine methylation from whole-genome bisulfite sequencing and chromatin accessibility from DNase I hypersensitivity data. Across multiple human cell lines, EpiBinder consistently outperforms strong sequence-only baselines, improving TF-binding prediction by up to 10% in area under the precision-recall curve. Beyond predictive performance, EpiBinder provides base-level attribution maps that enable systematic interrogation of regulatory context, including candidate methylation-sensitive loci, contextual motif dependencies, and putative TF-TF interactions. These results position EpiBinder as a practical framework for modeling and exploring the local regulatory grammar underlying cell-type-specific TF occupancy.

genomics↗

bioRxiv2025

IRF1 Tunes Basal Immunity and Antiviral Readiness in a Context-Dependent Manner

Interferon Regulatory Factor 1 (IRF1) plays a pivotal role in interferon (IFN) signaling, yet its context-dependent regulatory functions remain incompletely understood. Here, we dissect the impact of IRF1 on gene regulation in HeLa cells, by targeted knockout (KO) or overexpression (OE) of IRF1. IRF1 KO did not impair interferon stimulated gene (ISG) expression regulation upon IFN-{beta} stimulation, but partially diminished IFN-{psi} induced gene regulation. IRF1 KO did show a homeostatic role in basal gene abundance, including increasing the abundance of some antiviral genes. RNA-seq analysis showed altered expression of both ISGs and immune signaling genes, implicating IRF1 as a dual regulator that fine-tunes gene abundance through both activation and repression. IRF1 OE induced potent antiviral protection in the absence of exogenous IFN, mediated by type I IFN secretion, particularly of IFN- subtypes. This paracrine effect was confirmed by transcriptomics, cytokine profiling, and mass spectrometry, and was functional even in JAK1-deficient or Ruxolitinib-treated cells but not type I IFN receptor KO cells, suggesting the involvement of non-canonical signaling pathways. Hierarchical clustering of RNA-seq data revealed distinct IFN-independent gene clusters activated or repressed by IRF1, including pathways related to adaptive immunity and T cell function. Using protein-binding microarrays and predictive modeling, we mapped IRF1 binding across promoters and validated functional motifs in the IFIT2 gene promoter by a reporter assay. Our integrative approach establishes IRF1 as a central regulator of antiviral immunity, capable of shaping gene expression both through cytokine signaling and direct promoter binding.

biochemistry↗

bioRxiv2025

Systematic DNA nicking reveals the structural logic of protein recognition

Transcription factors (TFs) bind to specific genomic sites to regulate gene expression1,2. These interactions almost universally require DNA deformation and the accumulation of local mechanical strain within the double helix. As a result, TF-DNA recognition is determined not only by the linear base sequence but also by the spatial alignment of bases and phosphates, as well as their ability to adopt and retain structural deformations3. However, the sequence-centric focus of existing studies makes it challenging to directly probe DNA structural determinants and to decouple their impact from alterations in base sequences, limiting our ability to unravel the key factors influencing binding beyond the sequence identity and leaving significant gaps in our understanding of the principles governing TF-DNA recognition. Here, we introduce a high-throughput strategy to perturb TF binding sites without altering their base sequence, enabling systematic investigation of the structural features of DNA that govern TF binding. Our method, PIC-NIC, introduces single-strand breaks (SSBs) at every position within the binding site, selectively disrupting backbone continuity while preserving nucleotide identity, with the resulting effects on TF binding measured quantitatively. Applied to 15 human TFs spanning eight structural classes, and supported by seven high-resolution TF-DNA crystal structures and molecular dynamics simulations, PIC-NIC uncovers discrete backbone positions serving as structural anchor points where nicks can abolish binding, rewire sequence preferences, or even enhance affinity. By decoupling structural and chemical contributions, we demonstrate that DNA mechanics--encoded in backbone geometry and continuity--can independently shape binding specificity beyond the linear code of base identity. These findings shift the paradigm of TF-DNA recognition, establishing the backbone not as a passive scaffold, but as a functional determinant capable of directing regulatory mechanisms through its physical architecture.

biophysics↗

bioRxiv2025

TMPRSS2-ERG confers resistance to antiandrogens: mechanism and therapeutic implications

Approximately 50% of prostate cancer (PCa) patients harbor fusions involving the TMPRSS2 and ERG genes. Despite this, tailored therapies targeting the fused gene, tERG, remain undeveloped. Our study analyzed biopsy samples from two clinical trials assessing the efficacies of androgen receptor (AR) signaling inhibitors (ARSIs). The results revealed that tERG promotes resistance to ARSIs and is associated with elevated levels of the glucocorticoid receptor (GR). Subsequent assays showed that GR directly interacts with tERG, alleviates allosteric autoinhibition and prevents chemotherapy-induced tERG degradation. In PCa models, either inhibiting GR or lowering cortisol levels suppressed tumor growth in tERG-positive models, but not in fusion-negative models. In addition, patient-derived fusion-positive xenografts displayed enhanced sensitivity to combined GR and AR inhibitors. Collectively, these findings highlight TMPRSS2-ERG as a new biomarker and propose that simultaneous inhibition of GR and AR may specifically benefit tERG-positine patients. However, GR stimulatory corticosteroid therapies may not be advisable for this patient subgroup.

cancer biology↗

bioRxiv2022

UV irradiation remodels the specificity landscape of transcription factors

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 hyper-mutation 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 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 non-consensus 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 non-consensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.

biophysics↗

bioRxiv2022

Short tandem repeats bind transcription factors to tune eukaryotic gene expression

Short tandem repeats (STRs) are enriched in eukaryotic cis-regulatory elements and their polymorphisms alter gene expression, yet how they regulate transcription remains unknown. We find that STRs can modulate transcription factor (TF)-DNA affinities and on rates by up to 70-fold by directly binding TF DNA-binding domains, with energetic impacts approaching or exceeding mutations to consensus sites. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density near motifs to speed target search. Confirming that STRs also impact TF binding in cells, neural networks trained only on in vivo occupancies predict identical effects to those observed in vitro. Approximately 90% of TFs preferentially bind STRs that need not resemble known motifs, providing a novel cis-regulatory mechanism to target TFs to cognate sites.

biophysics↗

bioRxiv2020

Reconfiguring Okazaki fragment start sites on a genome by using a data driven approach

DNA-protein interactions are essential in all aspects of every living cell. Understanding of how features embedded in the DNA sequence affect specific interactions with proteins is challenging but important, since it may contribute to finding the means to regulate metabolic pathways involving DNA-protein interactions. Using a massive experimental benchmark dataset of binding scores for DNA sequences and a machine learning workflow, we describe the binding to DNA of T7 primase, as a model system for specific DNA-protein interactions. Effective binding of T7 primase to its specific DNA recognition sequences triggers the formation of RNA primers that serve as Okazaki fragment start sites during DNA replication.

biochemistry↗

bioRxiv2019

DNA mismatches reveal widespread conformational penalties in protein-DNA recognition

Transcription-factor (TF) proteins recognize specific genomic sequences, despite an overwhelming excess of non-specific DNA, to regulate complex gene expression programs1-3. While there have been significant advances in understanding how DNA sequence and shape contribute to recognition, some fundamental aspects of protein-DNA binding remain poorly understood2,3. Many DNA-binding proteins induce changes in the DNA structure outside the intrinsic B-DNA envelope. How the energetic cost associated with distorting DNA contributes to recognition has proven difficult to study and measure experimentally because the distorted DNA structures exist as low-abundance conformations in the naked B-DNA ensemble4-10. Here, we use a novel high-throughput assay called SaMBA (Saturation Mismatch-Binding Assay) to investigate the role of DNA conformational penalties in TF-DNA recognition. The approach introduces mismatched base-pairs (i.e. mispairs) within TF binding sites to pre-induce a variety of DNA structural distortions much larger than those induced by changes in Watson-Crick sequence. Strikingly, while most mismatches either weakened TF binding (~70%) or had negligible effects (~20%), approximately 10% of mismatches increased binding and at least one mismatch was found that increased the binding affinity for each of 21 examined TFs. Mismatches also converted sites from the non-specific affinity range into specific sites, and high-affinity sites into \"super-sites\" stronger than any known canonical binding site. These findings reveal a complex binding landscape that cannot be explained based on DNA sequence alone. Analysis of crystal structures together with NMR and molecular dynamics simulations revealed that many of the mismatches that increase binding induce distortions similar to those induced by TF binding, thus pre-paying some of the energetic cost to deform the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit TFs and thus modulate replication and repair activities in the cell11,12.

biophysics↗

bioRxiv2019

Unexpected implications of STAT3 acetylation revealed by genetic encoding of acetyl-lysine

The signal transducer and activator of transcription 3 (STAT3) protein is activated by phosphorylation of a specific tyrosine residue (Tyr705) in response to various extracellular signals. STAT3 activity was also found to be regulated by acetylation of Lys685. However, the molecular mechanism by which Lys685 acetylation affects the transcriptional activity of STAT3 remains elusive. By genetically encoding the co-translational incorporation of acetyl-lysine into position Lys685 and co-expression with the Elk receptor tyrosine kinase, we were able to biochemically characterize site-specifically acetylated, and simultaneously acetylated and phosphorylated STAT3. We measured the effect of acetylation on the crystal structure, and DNA binding affinity and specificity of Tyr705-phosphorylated and non-phosphorylated STAT3. In addition, we monitored the deacetylation of acetylated Lys685 by reconstituting the mammalian enzymatic deacetylation reaction in live bacteria. Surprisingly, we found that acetylation, per se, had no effect on the crystal structure, and DNA binding affinity or specificity of STAT3, implying that the previously observed acetylation-dependent transcriptional activity of STAT3 involves an additional cellular component. In addition, we discovered that Tyr705-phosphorylation protects Lys685 from deacetylation in bacteria, providing a new possible explanation for the observed correlation between STAT3 activity and Lys685 acetylation.

biochemistry↗

bioRxiv2018

DNA sequence context controls the binding and processivity of T7 DNA primase

Primases are key enzymes involved in DNA replication. They act on single-stranded DNA, and catalyze the synthesis of short RNA primers used by DNA polymerases. Here, we investigate the DNA-binding and activity of the bacteriophage T7 primase using a new workflow called High-Throughput Primase Profiling (HTPP). Using a unique combination of high-throughput binding assays and biochemical analyses, HTPP reveals a complex landscape of binding specificity and functional activity for the T7 primase, determined by sequences flanking the primase recognition site. We identified specific features, such as G/T-rich flanks, which increase primase-DNA binding up to 10-fold and, surprisingly, also increase the length of newly formed RNA (up to 3-fold). To our knowledge, variability in primer length has not been reported for this primase. We expect that applying HTPP to additional enzymes will reveal new insights into the effects of DNA sequence composition on the DNA recognition and functional activity of primases.

biophysics↗