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1. Design and PCR Synthesis of Infrared Fluorophore-Labeled Modular DNA Probes

   https://doi.org/10.17504/protocols.io.wfjfbkn  

   Van Dyke, Michael (December 2018, Protocols.io)

   R fluorophore-modified DNAs and IR fluorescence imaging provide a safe and effective alternative for investigating ligand-DNA interactions than those involving radioactivity. These DNAs can be synthesized by conventional phosphoramidite chemistries and are commercially available. However, they are relatively expensive, which can be prohibitive if many different DNA probes are required. Described here is the design of modular DNA probes that can be synthesized by PCR using conventional templates and a defined set of IR fluorophore-modified primers. These have been found effective for investigating protein-DNA interactions in a variety of assays, including Electrophoretic Mobility Shift Assays (EMSA), Restriction Endonuclease Protection Assays (REPA), and the iterative selection method Restriction Endonuclease Protection, Selection, and Amplification (REPSA). 

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2. Electrophoretic Mobility Shift Assays using Infrared-Fluorescent DNA Probes

   https://doi.org/10.17504/protocols.io.mbdc2i6  

   Van Dyke, Michael; Cox, James (December 2018, Protocols.io)

   Protein-DNA binding interactions are critical in several biological processes, especially the regulation of gene expression at the level of transcription initiation. An important technique for studying these interactions is the electrophoretic mobility shift assay (EMSA), whereby protein-DNA complexes are resolved on the basis of their mass:charge ratio using native polyacrylamide gel electrophoresis (nPAGE). Here we describe EMSA using PCR-generated, near infrared-fluorescent DNA probes, and IR fluorescence imaging to qualitatively and quantitatively study the interaction of transcriptional regulatory proteins from thermophilic organisms with different DNAs. Direct imaging of IR fluorophore-labeled DNA probes is advantageous because it provides high sensitivity (subnanomolar) without the need for intermediate staining steps or costly and problematic radiolabeled probes, thereby providing a more affordable and sensitive option to image protein-DNA on polyacrylamide gels by techniques such as EMSA. 

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3. Prediction accuracy of regulatory elements from sequence varies by functional sequencing technique

   https://doi.org/10.3389/fcimb.2023.1182567  

   Nowling, Ronald J.; Njoya, Kimani; Peters, John G.; Riehle, Michelle M. (August 2023, Frontiers in Cellular and Infection Microbiology)

   Introduction Various sequencing based approaches are used to identify and characterize the activities of cis -regulatory elements in a genome-wide fashion. Some of these techniques rely on indirect markers such as histone modifications (ChIP-seq with histone antibodies) or chromatin accessibility (ATAC-seq, DNase-seq, FAIRE-seq), while other techniques use direct measures such as episomal assays measuring the enhancer properties of DNA sequences (STARR-seq) and direct measurement of the binding of transcription factors (ChIP-seq with transcription factor-specific antibodies). The activities of cis -regulatory elements such as enhancers, promoters, and repressors are determined by their sequence and secondary processes such as chromatin accessibility, DNA methylation, and bound histone markers. Methods Here, machine learning models are employed to evaluate the accuracy with which cis -regulatory elements identified by various commonly used sequencing techniques can be predicted by their underlying sequence alone to distinguish between cis -regulatory activity that is reflective of sequence content versus secondary processes. Results and discussion Models trained and evaluated on D. melanogaster sequences identified through DNase-seq and STARR-seq are significantly more accurate than models trained on sequences identified by H3K4me1, H3K4me3, and H3K27ac ChIP-seq, FAIRE-seq, and ATAC-seq. These results suggest that the activity detected by DNase-seq and STARR-seq can be largely explained by underlying DNA sequence, independent of secondary processes. Experimentally, a subset of DNase-seq and H3K4me1 ChIP-seq sequences were tested for enhancer activity using luciferase assays and compared with previous tests performed on STARR-seq sequences. The experimental data indicated that STARR-seq sequences are substantially enriched for enhancer-specific activity, while the DNase-seq and H3K4me1 ChIP-seq sequences are not. Taken together, these results indicate that the DNase-seq approach identifies a broad class of regulatory elements of which enhancers are a subset and the associated data are appropriate for training models for detecting regulatory activity from sequence alone, STARR-seq data are best for training enhancer-specific sequence models, and H3K4me1 ChIP-seq data are not well suited for training and evaluating sequence-based models for cis -regulatory element prediction. 

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4. Using Restriction Endonuclease, Protection, Selection, and Amplification to Identify Preferred DNA-Binding Sequences of Microbial Transcription Factors

   https://doi.org/10.1128/spectrum.04397-22  

   Barrows, John K.; Van Dyke, Michael W. (February 2023, Microbiology Spectrum)

   Polen, Tino (Ed.)

   ABSTRACT Regulation of gene expression is a vital component of cellular biology. Transcription factor proteins often bind regulatory DNA sequences upstream of transcription start sites to facilitate the activation or repression of RNA polymerase. Research laboratories have devoted many projects to understanding the transcription regulatory networks for transcription factors, as these regulated genes provide critical insight into the biology of the host organism. Various in vivo and in vitro assays have been developed to elucidate transcription regulatory networks. Several assays, including SELEX-seq and ChIP-seq, capture DNA-bound transcription factors to determine the preferred DNA-binding sequences, which can then be mapped to the host organism’s genome to identify candidate regulatory genes. In this protocol, we describe an alternative in vitro , iterative selection approach to ascertaining DNA-binding sequences of a transcription factor of interest using restriction endonuclease, protection, selection, and amplification (REPSA). Contrary to traditional antibody-based capture methods, REPSA selects for transcription factor-bound DNA sequences by challenging binding reactions with a type IIS restriction endonuclease. Cleavage-resistant DNA species are amplified by PCR and then used as inputs for the next round of REPSA. This process is repeated until a protected DNA species is observed by gel electrophoresis, which is an indication of a successful REPSA experiment. Subsequent high-throughput sequencing of REPSA-selected DNAs accompanied by motif discovery and scanning analyses can be used for determining transcription factor consensus binding sequences and potential regulated genes, providing critical first steps in determining organisms’ transcription regulatory networks. IMPORTANCE Transcription regulatory proteins are an essential class of proteins that help maintain cellular homeostasis by adapting the transcriptome based on environmental cues. Dysregulation of transcription factors can lead to diseases such as cancer, and many eukaryotic and prokaryotic transcription factors have become enticing therapeutic targets. Additionally, in many understudied organisms, the transcription regulatory networks for uncharacterized transcription factors remain unknown. As such, the need for experimental techniques to establish transcription regulatory networks is paramount. Here, we describe a step-by-step protocol for REPSA, an inexpensive, iterative selection technique to identify transcription factor-binding sequences without the need for antibody-based capture methods. 

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5. Arsenite oxidation regulator AioR regulates bacterial chemotaxis towards arsenite in Agrobacterium tumefaciens GW4

   Article | OPEN | Published: 03 March 2017 Shi, K; Fan, X; Qiao, Z; Han, Y; McDermott, TR; Wang, Q; Wang, G. (March 2017, Scientific reports)

   Some arsenite [As(III)]-oxidizing bacteria exhibit positive chemotaxis towards As(III), however, the related As(III) chemoreceptor and regulatory mechanism remain unknown. The As(III)-oxidizing bacterium Agrobacterium tumefaciens GW4 displays positive chemotaxis towards 0.5–2 mM As(III). Genomic analyses revealed a putative chemoreceptor-encoding gene, mcp, located in the arsenic gene island and having a predicted promoter binding site for the As(III) oxidation regulator AioR. Expression of mcp and other chemotaxis related genes (cheA, cheY2 and fliG) was inducible by As(III), but not in the aioR mutant. Using capillary assays and intrinsic tryptophan fluorescence spectra analysis, Mcp was confirmed to be responsible for chemotaxis towards As(III) and to bind As(III) (but not As(V) nor phosphate) as part of the sensing mechanism. A bacterial one-hybrid system technique and electrophoretic mobility shift assays showed that AioR interacts with the mcp regulatory region in vivo and in vitro, and the precise AioR binding site was confirmed using DNase I foot-printing. Taken together, these results indicate that this Mcp is responsible for the chemotactic response towards As(III) and is regulated by AioR. Additionally, disrupting the mcp gene affected bacterial As(III) oxidation and growth, inferring that Mcp may exert some sort of functional connection between As(III) oxidation and As(III) chemotaxis. 

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