Biology subjects

Peters, J. P.

Publications and source records attributed to Peters, J. P..

3 recordsLinked to original sources

bioRxiv2026

LOOP-TAG: massively-parallel measurement of length-dependent protein-mediated DNA looping probabilities by tagmentation in vitro

We are interested in DNA stiffness in vitro and in vivo. Classical approaches for measuring this parameter in vitro involve tedious T4 DNA ligase-mediated cyclization kinetics experiments performed for one DNA fragment length at a time to establish effective local end-end concentrations (J-factors). Protein-mediated DNA looping is a more relevant biological reaction. An efficient approach to determine length-dependent protein-mediated DNA J-loop values in vitro has been lacking. We describe LOOP-TAG, a massively-parallel method. We leverage intramolecular DNA looping and tagmentation catalyzed by Tn5 transposase (Tnp) tethered to the terminus of a ~1,000-bp bead-bound duplex DNA. We detected loops terminating with Tnp. Counts of the resulting DNA looping-dependent bead-bound tagmentation products from deep sequencing determine DNA loop length probabilities at single-base pair resolution for all bead-bound fragments. When normalized to DNA fragment length probabilities from tagmentation using known concentrations of free Tnp, length-dependent J-loop values are directly determined. We demonstrate LOOP-TAG for Tnp tethered in two ways and in the presence of DNA-kinking architectural DNA binding protein Nhp6A. Intermediate length J-loop values are consistent with expectations of wormlike chain theory after accounting for strong sequence-dependence of Tnp. DNA loops are shifted to smaller sizes in the presence of Nhp6A, as predicted.

biophysics↗

bioRxiv2026

Defining the DNA Binding Specificity of GRHL2

Grainyhead-like 2 (GRHL2) is an epithelial transcription factor with context-dependent regulatory roles, yet the sequence rules governing its DNA recognition remain incompletely defined. In this study, a high-density genomic Specificity and Affinity for Protein (SNAP) DNA-binding array containing 772,732 tiled probes derived from GRHL2 ChIP-seq regions was used to resolve GRHL2 binding specificity at 6 base pair resolution across genomic sequences. From high-affinity probes, de novo motif analysis recovered the canonical 5-AACCGGTT-3 motif. Sequence specificity landscapes revealed a stepwise reduction in binding as mismatches were introduced, with the strongest effects at the C (position 3) and G (position 6) within the motif, greater tolerance at the central CG dinucleotide, and intermediate tolerance at the A/T bases at the motif edges. This analysis also demonstrated the influence of nearby flanking sequences. Extended motif and spacing analyses indicated dimeric binding at paired motifs, with periodic helical spacing consistent with interactions on the same face of the DNA helix. Integration of SNAP array binding with ChIP-seq data distinguished direct, motif-encoded GRHL2 occupancy from indirect, cofactor-mediated recruitment at genomic sites. These results define the sequence specificity of GRHL2 interactions with variations in the DNA consensus motif and flanking sequences within an endogenous genomic context. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/719077v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@237363org.highwire.dtl.DTLVardef@16c97d7org.highwire.dtl.DTLVardef@64b251org.highwire.dtl.DTLVardef@f72090_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Designed architectural proteins that tune DNA looping in bacteria

Architectural proteins alter the shape of DNA, often by distorting the double helix and introducing sharp kinks that relieve strain in tightly-bent DNA structures. Here we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from E. coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

biochemistry↗