DNA-Based Electrodes and Computational Approaches on the Intercalation Study of Antitumoral Drugs

The binding between anticancer drugs and double-stranded DNA (dsDNA) is a key issue to understand their mechanism of action, and many chemical methods have been explored on this task. Molecular docking techniques successfully predict the affinity of small molecules into the DNA binding sites. In turn, various DNA-targeted drugs are electroactive; in this regard, their electrochemical behavior may change according to the nature and strength of interaction with DNA. A carbon paste electrode (CPE) modified with calf thymus ds-DNA (CPDE) and computational methods were used to evaluate the drug–DNA intercalation of doxorubicin (DOX), daunorubicin (DAU), idarubicin (IDA), dacarbazine (DAR), mitoxantrone (MIT), and methotrexate (MTX), aiming to evaluate eventual correlations. CPE and CPDE were immersed in pH 7 0.1 mM solutions of each drug with different incubation times. As expected, the CPDE response for all DNA-targeted drugs was higher than that of CPE, evidencing the drug–DNA interaction. A peak current increase of up to 10-fold was observed; the lowest increase was seen for MTX, and the highest increase for MIT. Although this increase in the sensitivity is certainly tied to preconcentration effects of DNA, the data did not agree entirely with docking studies, evidencing the participation of other factors, such as viscosity, interfacial electrostatic interactions, and coefficient of diffusion.

Analysing the Protein-DNA Binding Sites in Arabidopsis thaliana from ChIP-seq Experiments

Computational genomics aim at supporting the discovery of how the functionality of the genome of the organism under study is affected both by its own sequence and structure, and by the network of interaction between this genome and different biological or physical factors. In this work, we focus on the analysis of ChIP-seq data, for which many methods have been proposed in the recent years. However, to the best of our knowledge, those methods lack an appropriate mathematical formalism. We have developed a method based on multivariate models for the analysis of the set of peaks obtained from a ChIP-seq experiment. This method can be used to characterize an individual experiment and to compare different experiments regardless of where and when they were conducted. The method is based on a multivariate hypergeometric distribution, which fits the complexity of the biological data and is better suited to deal with the uncertainty generated in this type of experiments than the dichotomous models used by the state of the art methods. We have validated this method with Arabidopsis thaliana datasets obtained from the Remap2020 database, obtaining results in accordance with the original study of these samples. Our work shows a novel way for analyzing ChIP-seq data.

Quantitative-enhancer-FACS-seq (QeFS) reveals epistatic interactions among motifs within transcriptional enhancers in developing Drosophila tissue

Understanding the contributions of transcription factor DNA binding sites to transcriptional enhancers is a significant challenge. We developed Quantitative enhancer-FACS-Seq for highly parallel quantification of enhancer activities from a genomically integrated reporter in Drosophila melanogaster embryos. We investigate the contributions of the DNA binding motifs of four poorly characterized TFs to the activities of twelve embryonic mesodermal enhancers. We measure quantitative changes in enhancer activity and discover a range of epistatic interactions among the motifs, both synergistic and alleviating. We find that understanding the regulatory consequences of TF binding motifs requires that they be investigated in combination across enhancer contexts.

Menin inhibition suppresses castration-resistant prostate cancer and enhances chemosensitivity

Disease progression and therapeutic resistance of prostate cancer (PC) are linked to multiple molecular events that promote survival and plasticity. We previously showed that heat shock protein 27 (HSP27) acted as a driver of castration-resistant phenotype (CRPC) and developed an oligonucleotides antisense (ASO) against HSP27 with evidence of anti-cancer activity in men with CRPC. Here, we show that the tumor suppressor Menin (MEN1) is highly regulated by HSP27. Menin is overexpressed in high-grade PC and CRPC. High MEN1 mRNA expression is associated with decreased biochemical relapse-free and overall survival. Silencing Menin with ASO technology inhibits CRPC cell proliferation, tumor growth, and restores chemotherapeutic sensitivity. ChIP-seq analysis revealed differential DNA binding sites of Menin in various prostatic cells, suggesting a switch from tumor suppressor to oncogenic functions in CRPC. These data support the evaluation of ASO against Menin for CRPC.

Structural and functional characterization of DdrC, a novel DNA damage-induced nucleoid associated protein involved in DNA compaction

Deinococcus radiodurans is a spherical bacterium well-known for its outstanding resistance to DNA-damaging agents. Exposure to such agents leads to drastic changes in the transcriptome of D. radiodurans. In particular, four Deinococcus-specific genes, known as DNA Damage Response genes, are strongly up-regulated and have been shown to contribute to the resistance phenotype of D. radiodurans. One of these, DdrC, is expressed shortly after exposure to γ-radiation and is rapidly recruited to the nucleoid. In vitro, DdrC has been shown to compact circular DNA, circularize linear DNA, anneal complementary DNA strands and protect DNA from nucleases. To shed light on the possible functions of DdrC in D. radiodurans, we determined the crystal structure of the domain-swapped DdrC dimer at a resolution of 2.2 Å and further characterized its DNA binding and compaction properties. Notably, we show that DdrC bears two asymmetric DNA binding sites located on either side of the dimer and can modulate the topology and level of compaction of circular DNA. These findings suggest that DdrC may be a DNA damage-induced nucleoid-associated protein that enhances nucleoid compaction to limit the dispersion of the fragmented genome and facilitate DNA repair after exposure to severe DNA damaging conditions.

Rescuing Biologically Relevant Consensus Regions Across Replicated Samples

Motivation Protein-DNA binding sites of ChIP-seq experiments are identified where the binding affinity is significant based on a given threshold. The choice of the threshold is a trade-off between conservative region identification and discarding weak, but true binding sites. Results We argue the biological relevance of weak binding sites and the information they add when rescued. The sites are rescued using MSPC, which exploits replicates to lower the threshold required to identify a binding site while keeping a low false-positive rate. We extend MSPC to call consensus regions across any number of replicated samples, accounting for differences between biological and technical replicates. We observed several master transcription regulators (e.g., SP1 and GATA3) and HDAC2-GATA1 regulatory networks on rescued regions. Availability and implementation An implementation of the proposed method and the scripts to reproduce the performed analysis are freely available at https://genometric.github.io/MSPC/, MSPC is distributed as a command-line application, an R package available from Bioconductor (https://doi.org/doi:10.18129/B9.bioc.rmspc), and a C# library.

Systematic identification of non-canonical transcription factor motifs

Sequence-specific transcription factors (TFs) recognize motifs of related nucleotide sequences at their DNA binding sites. Upon binding at these sites, TFs regulate critical molecular processes such as gene expression. It is widely assumed that a TF recognizes a single “canonical” motif, although recent studies have identified additional “non-canonical” motifs for some TFs. A comprehensive approach to identify non-canonical DNA binding motifs and the functional importance of those motifs’ matches in the human genome is necessary for fully understanding the mechanisms of TF-regulated molecular processes in human cells. To address this need, we developed a statistical pipeline for in vitro HT-SELEX data that identifies and characterizes the distributions of non-canonical TF motifs in a stringent manner. Analyzing ~170 human TFs’ HT-SELEX data, we found non-canonical motifs for 19 TFs (11%). These non-canonical motifs occur independently of the TFs’ canonical motifs. Non-canonical motif occurrences in the human genome show similar evolutionary conservation to canonical motif occurrences, explain TF binding in locations without canonical motifs, and occur within gene promoters and epigenetically marked regulatory sequences in human cell lines and tissues. Our approach and collection of non-canonical motifs expand current understanding of functionally relevant DNA binding sites for human TFs.

AlphaFold-aware prediction of protein-DNA binding sites using graph transformer

MotivationProtein-DNA interactions play crucial roles in the biological systems, and identifying protein-DNA binding sites is the first step for mechanistic understanding of various biological activities (such as transcription and repair) and designing novel drugs. How to accurately identify DNA-binding residues from only protein sequence remains a challenging task. Currently, most existing sequence-based methods only consider contextual features of the sequential neighbors, which are limited to capture spatial information.ResultsBased on the recent breakthrough in protein structure prediction by AlphaFold2, we propose an accurate predictor, GraphSite, for identifying DNA-binding residues based on the structural models predicted by AlphaFold2. Here, we convert the binding site prediction problem into a graph node classification task and employ a transformerbased variant model to take the protein structural information into account. By leveraging predicted protein structures and graph transformer, GraphSite substantially improves over the latest sequence-based and structure-based methods. The algorithm was further confirmed on the independent test set of 196 proteins, where GraphSite surpasses the state-of-the-art structure-based method by 12.3% in AUPR and 9.3% in MCC, [email protected]

Primate-specific cis- and trans-regulators shape transcriptional networks during human development

The human genome contains more than 4.5 million inserts derived from transposable elements (TE), the result of recurrent waves of invasion and internal propagation throughout evolution. For new TE copies to be inherited, they must become integrated in the genome of the germline or preimplantation embryo, which requires that their source TE be expressed at these stages. Accordingly, many TEs harbor DNA binding sites for the pluripotency factors OCT4, NANOG, SOX2, KLFs and are transiently expressed during embryonic genome activation. Here, we describe how many primate-restricted TEs have additional binding sites for lineage-specific transcription factors driving their expression during human gastrulation and later steps of fetal development. These TE integrants serve as lineage-specific enhancers fostering the transcription, amongst other targets, of KRAB-zinc finger proteins of similar evolutionary age, which in turn corral the activity of TE-embedded regulatory sequences in an equally lineage-restricted fashion. Thus, TEs and their KZFP controllers play broad roles in shaping transcriptional networks during early human development.

A micro-evolutionary change in target binding sites as key determinant of Ultrabithorax function in Drosophila.

All Hox proteins are known to recognize, in vitro, similar DNA-binding sites containing a TAAT core sequence. This poor DNA-binding specificity is in sharp contrast with their specific functions in vivo. Here we report a new binding motif with TAAAT core sequence to which the Hox protein Ultrabithorax (Ubx) binds with higher affinity and specificity. Using transgenic and luciferase assays, we show that this new motif is critical for Ubx-mediated regulation of a target gene in Drosophila melanogaster. Interestingly, this new motif with TAAAT core sequences is not associated with the targets of Ubx in the honeybee, Apis mellifera, wherein hindwings are nearly identical to the forewings. We show that introduction of TAAAT motif in the place of TAAT motif is sufficient to bring an enhancer of a wing-promoting gene of A. mellifera under the regulation of Ubx. Our results, thus, suggest that binding motifs with a TAAAT core sequence may help identify functionally relevant direct targets of Ubx in D. melanogaster and the emergence of these binding sites may be crucial for Hox-mediated morphological changes during insect evolution.