Biology subjects

Gralak, A. J.

Publications and source records attributed to Gralak, A. J..

5 recordsLinked to original sources

bioRxiv2024

Cross-platform DNA motif discovery and benchmarking to explore binding specificities of poorly studied human transcription factors

A DNA sequence pattern, or "motif", is an essential representation of DNA-binding specificity of a transcription factor (TF). Any particular motif model has potential flaws due to shortcomings of the underlying experimental data and computational motif discovery algorithm. As a part of the Codebook/GRECO-BIT initiative, here we evaluated at large scale the cross-platform recognition performance of positional weight matrices (PWMs), which remain popular motif models in many practical applications. We applied ten different DNA motif discovery tools to generate PWMs from the "Codebook" data comprised of 4,237 experiments from five different platforms profiling the DNA-binding specificity of 394 human proteins, focusing on understudied transcription factors of different structural families. For many of the proteins, there was no prior knowledge of a genuine motif. By benchmarking-supported human curation, we constructed an approved subset of experiments comprising about 30% of all experiments and 50% of tested TFs which displayed consistent motifs across platforms and replicates. We present the Codebook Motif Explorer (https://mex.autosome.org), a detailed online catalog of DNA motifs, including the top-ranked PWMs, and the underlying source and benchmarking data. We demonstrate that in the case of high-quality experimental data, most of the popular motif discovery tools detect valid motifs and generate PWMs, which perform well both on genomic and synthetic data. Yet, for each of the algorithms, there were problematic combinations of proteins and platforms, and the basic motif properties such as nucleotide composition and information content offered little help in detecting such pitfalls. By combining multiple PMWs in decision trees, we demonstrate how our setup can be readily adapted to train and test binding specificity models more complex than PWMs. Overall, our study provides a rich motif catalog as a solid baseline for advanced models and highlights the power of the multi-platform multi-tool approach for reliable mapping of DNA binding specificities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/619379v2_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@79561forg.highwire.dtl.DTLVardef@54c0aorg.highwire.dtl.DTLVardef@1c33f34org.highwire.dtl.DTLVardef@16a93ba_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

bioinformatics↗

bioRxiv2024

Identification of methylation-sensitive human transcription factors using meSMiLE-seq

Transcription factors (TFs) are key players in eukaryotic gene regulation, but the DNA binding specificity of many TFs remains unknown. Here, we assayed 284 mostly poorly characterized, putative human TFs using selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq), revealing 72 new DNA binding motifs. To investigate whether some of the 158 TFs for which we did not find motifs preferably bind epigenetically modified DNA (i.e. methylated CG dinucleotides), we developed methylation-sensitive SMiLE-seq (meSMiLE-seq). This microfluidic assay simultaneously probes the affinity of a protein to methylated and unmethylated DNA, augmenting the capabilities of the original method to infer methylation-aware binding sites. We assayed 114 TFs with meSMiLE-seq and identified DNA-binding models for 48 proteins, including the known methylation-sensitive binding modes for POU5F1 and RFX5. For 11 TFs, binding to methylated DNA was preferred or resulted in the discovery of alternative, methylation-dependent motifs (e.g. PRDM13), while aversion towards methylated sequences was found for 13 TFs (e.g. USF3). Finally, we uncovered a potential role for ZHX2 as a putative binder of Z-DNA, a left-handed helical DNA structure which is adopted more frequently upon CpG methylation. Altogether, our study significantly expands the human TF codebook by identifying DNA binding motifs for 98 TFs, while providing a versatile platform to quantitatively assay the impact of DNA modifications on TF binding.

genomics↗

bioRxiv2024

Perspectives on Codebook: sequence specificity of uncharacterized human transcription factors

Gene expression is regulated by transcription factors (TFs), which recognize specific DNA sequence motifs. Several hundred putative human TFs, identified mainly by an apparent DNA-binding domain, lack known binding motifs1, and even for well-characterized TFs, it remains controversial to what degree motifs accurately reflect binding sites in living cells2,3. Here, we describe a systematic effort ("Codebook") to determine the sequence specificity of 332 putative and poorly characterized human TFs. Over 4,000 independent experiments, encompassing multiple in vitro and in vivo assays, produced motifs for just over half (177, or 53%), of which most are unique to a single protein, thereby extending the vocabulary of sequence recognition encoded by human TFs by [~]100 distinct motifs. Moreover, binding motifs identified in vitro are strongly enriched within cellular binding sites. Collectively, the data reveal tens of thousands of previously unknown, conserved, and direct TF binding sites across the human genome. These sites are concentrated in promoter regions, and are predictive of gene expression, illustrating that this new data atlas provides an important step forward in decoding the human genome.

genomics↗

bioRxiv2020

Bioorthogonal red and far-red fluorogenic probes for wash-free live-cell and super-resolution microscopy

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labelling via bioorthogonal tetrazine chemistry confers minimal label size and rapid labelling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine-dye conjugates with minimal distance between tetrazine and fluorophore. Two synthetic routes give access to a series of cell permeable and impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as dominant excited state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multi-colour and super-resolution STED and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

cell biology↗

bioRxiv2020

A chemical strategy to control protein networks in vivo

Direct control of protein interaction by chemically induced protein proximity (CIPP) holds great potential for cell- and synthetic biology as well as therapeutic applications. However, toxicity, low cell-permeability and lack of orthogonality currently limit the use of available chemical inducers of proximity (CIP). We present Mandi, a novel CIP and demonstrate its applicability in cell culture systems as well as living organisms for protein translocation, protein network shuttling and manipulation of endogenous proteins.

synthetic biology↗