Teif lab

@teiflab.bsky.social

3032 Followers 1098 Following

Teif lab at the University of Essex. We work on gene regulation in chromatin and applications to liquid biopsies, using approaches of genomics, biophysics, bioinformatics & AI. Our focus is nucleosomics, TF binding, CTCF, cfDNA. https://generegulation.org

How transcription factors (TFs) and their binding sites organize and engage nucleosomes at natural genomic locations remains poorly understood. Here, we develop Benzonase-seq to measure the rotational phasing of nucleosomes in human cells and enhance chromatin immunoprecipitation (ChIP)-exo (v6) to measure rotational phasing on the same DNA molecule bound by a TF. Unbound CTCF sites were found to be rotationally accessible on nucleosomes, and this rotational accessibility is encoded by classical dinucleotide periodicities. CTCF binding results in nucleosome displacement to adjacent DNA phasing sequences. Upon examining 40 TF classes, unbound sites were found to be phased either inward or outward or to lack phasing. In all examined cases, TF binding (e.g., NFIA and FoxA) results in adjacent rotational and translational phasing, which is not dinucleotide encoded. Benzonase-seq also more robustly maps nucleosome and subnucleosome positions in hard-to-map CpG islands. These findings provide a clearer view of how TFs engage and position nucleosomes to shape the natural chromatin landscape.
The consensus mode for linker histone (H1) association coincides with ‘on-dyad’ binding to an individual nucleosome, making it challenging to rationalize the chromatin dynamics and compacting activities of H1 in the context of a highly heterogeneous structural scaffold. Here, we investigate the activity of the somatic H1 variants by conducting crystallographic analysis of nucleosomal assemblies and characterization of nucleosome array condensates, which recapitulate long-range nucleosome fiber interactions in chromatin. H1 is observed to associate variant-dependently with nucleosomes through a diversity of binding modes that include linking multiple nucleosomes/fibers together. Binding versatility is facilitated by the proclivity of the H1 globular domain to recognize DNA structural motifs, which are similar between an individual nucleosome and specific niches within clusters of nucleosomes. We propose that linker histones support a structurally and functionally complex repertoire for chromatin regulation by assuming a variety of context-and variant-dependent DNA binding modes.
Figure 1.(A) Classical gel electrophoresis experiments showing mono-, di-, tri-, tetra-, and further multinucleosome bands upon chromatin digestion. (B) The nucleosome repeat length (NRL) is defined as the genomic distance between the centres of two neighbouring nucleosomes.
The molecular details governing transcription factor (TF) binding and the formation of accessible chromatin are not yet quantitatively understood—including how sequence context modulates affinity, how TFs search DNA, the kinetics of TF occupancy, and how motif grammars coordinate binding. To resolve these questions for a human TF, erythroid Krüppel-like factor (eKLF/KLF1), we quantitatively compare, in high throughput, in vitro TF binding rates and affinities with in vivo single-molecule TF and nucleosome occupancies and in vivo-derived deep learning models. We find that 40-fold flanking sequence effects on affinity are consistent with distal flanks tuning TF search parameters and captured by a linear energy model. Motif recognition probability, rather than time in the bound state, drives affinity changes, and in vitro and in nuclei measurements exhibit consistent, minutes-long TF residence times. Finally, in vitro biophysical parameters predict in vivo sequence preferences and single-molecule chromatin states for unseen motif grammars.
Fig. 1: Schematic of the human telomeric chromatin architecture. The human telomere is made up of TTAGGG repeats arranged into nucleosomes, bound by a six-subunit shelterin complex (comprising TRF1, TRF2, RAP1, TIN2, TPP1 and POT1). Some complexes are depicted without TPP1 and POT1, which it has been suggested are less abundant than the other subunits. Recent in vitro structural analyses of telomeric chromatin revealed a columnar stacking of histones on TTAGGG repeats, characterized by face-to-face nucleosome arrangements, as well as an alternative open configuration where two histone octamers are oriented at a nearly 90° angle. These in vitro columnar arrays exhibit an NRL of approximately 130 bp. In vivo, however, the telomeric NRL is 157 bp, longer than the NRL of the proposed columnar histone arrangement, but shorter than the ~197-bp NRL of bulk chromatin. To reconcile this, we depict here a model in which shelterin remodels the telomeric chromatin to integrate features of a columnar nucleosome organization with the nucleosome periodicity observed in vivo. At the telomere terminus, a 3′ single-stranded overhang of TTAGGG repeats invades the duplex telomeric DNA to form a lariat structure known as the T-loop. This loop structure serves a protective function by preventing the telomere from being recognized as damaged DNA. The size of the T-loop is variable and may differ between telomeres, contributing to the structural heterogeneity of the telomere. The extent to which nucleosomes are present within the T-loop remains unclear.
Four-panel black-and-white stick-figure cartoon comparing work styles. Panel 1, “Boss”: one person sits on a cart full of books and points forward while three others pull the cart. Panel 2, “Leader”: four people pull the cart together, with the front person pointing the direction while still pulling. Panel 3, “Introvert”: a single person pulls the cart alone. Panel 4, “Introvert with AI”: the same person walks easily while a small robot pulls two book-filled carts behind them.
Wishing you a happy, healthy, successful and science-filled new year!
Various analytes of liquid biopsies. Cancer cells shed various analytes into the bloodstream, all of which can be analyzed in liquid biopsies. (A) Extracellular vesicles contain various molecules, including DNA, RNA, and proteins representing the biology of the tumor. (B) Several types of cell-free RNA (cfRNA) molecules can be found in plasma, including noncoding micro-RNA (miRNA), long noncoding RNA (lncRNA), messenger RNA (mRNA), and tumor-derived RNA fusions. (C) Circulating tumor cells (CTCs) from primary tumors or metastatic sites directly represent cancer biology at the site of origin. CTCs can group together with healthy cells, creating CTC clusters. (D) Many features of cell-free DNA (cfDNA) provide great insights into tumor biology. These features include fragmentomics focusing on cfDNA fragment lengths and fragment end motifs (FEMs), 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) patterns and post-translational modifications (PTMs) of circulating histones. In addition, somatic variants, such as single-nucleotide variants (SNVs), insertions and deletions (indels), and copy number alterations (CNAs) are detectable in liquid biopsies.
Schematic of the IceQream (IQ) workflow: (i) Single-cell ATAC raw counts are transformed into estimated access probabilities (AP). (ii) The IQ model incorporates transcription factor (TF) models, epigenomic context variables, and pairwise interactions of TF models. Each TF model integrates contributions from strong and weak affinity sequences, weighted by spatial preferences around the accessible hotspot, which are transformed into dose-response-like spatial binding preference curves using pre-defined non-linear functions. (iii) Model initialization involves scanning candidate TF models from PSSM (position-specific scoring matrix) databases and de novo motif regression. (iv) An integrated IQ model predicts differential AP (dAP) across a selected manifold trajectory. (v) IQ models from multiple trajectories are fused to create a manifold-wide set of common TF motif models.
Oscillatory pattern of chain extension and nucleosome self-association with variation in protein-free DNA linker length in simulated nucleosome-decorated DNA chains. Regular structures bearing 15 nucleosomes constructed from the mean rigid-body parameters between successive base pairs; nucleosomal cores depicted as wedge-shaped objects and color-coded to highlight the two- and three-start organization of the structures. Nucleosomal DNA constrained to the 147-bp pathway found in the best-resolved core particle structure, pdb_id 1kx5
Transcription factors create accessibility through distinct mechanisms involving nucleosome sliding and eviction. A) Estimation of nucleosome-free (NFR) and nucleosomal fragment probabilities using ATAC-seq fragment size distributions. B) Example genomic locus showing ATAC insertion profile, nucleosome occupancy scores, and dyad positions inferred from NucleoATAC.
cFOOT-seq maps chromatin accessibility, nucleosome occupancy, and TF footprints A. Schematic of the cFOOT-seq workflow. After cell permeabilization, dsDNA deaminases (blue) convert cytosine to uracil in accessible chromatin DNA (blue lines), but DNA occupied by nucleosomes or transcription factors (TFs) (gray lines) remains unconverted. The profile of DNA conversion rate defines nucleosome occupancy and TF footprints.
The formation of condensed heterochromatin is critical for establishing cell-specific transcriptional programs. To reveal structural transitions underlying heterochromatin formation in maturing mouse rod photoreceptors, we apply cryo-EM tomography, AI-assisted deep denoising, and molecular modeling. We find that chromatin isolated from immature retina cells contains many closely apposed nucleosomes with extremely short or absent nucleosome linkers, which are inconsistent with the typical two-start zigzag chromatin folding. In mature retina cells, the fraction of short-linker nucleosomes is much lower, supporting stronger chromatin compaction. By Cryo-EM-assisted nucleosome interaction capture we observe that chromatin in immature retina is enriched with i±1 interactions while chromatin in mature retina contains predominantly i±2 interactions typical of the two-start zigzag. By mesoscale modeling and computational simulation, we clarify that the unusually short linkers typical of immature retina are sufficient to inhibit the two-start zigzag and chromatin compaction by the interference of very short linkers with linker DNA stems. We propose that this short linker composition renders nucleosome arrays more open in immature retina and that, as the linker DNA length increases in mature retina, chromatin fibers become globally condensed via tight zigzag folding. This mechanism may be broadly utilized to introduce higher chromatin folding entropy for epigenomic plasticity.
Microsatellites are essential genomic components increasingly linked to transcriptional regulation. FoxP3, a transcription factor critical for regulatory T cell (Treg) development, recognizes TTTG repeat microsatellites by forming multimers along DNA. However, FoxP3 also binds a broader range of TnG repeats (n = 2–5), often at the edges of accessible chromatin regions. This raises questions about how FoxP3 adapts to sequence variability and the potential role of nucleosomes. Using cryoelectron microscopy and single-molecule analyses, we show that murine FoxP3 assembles into various distinct supramolecular structures, depending on DNA sequence. This structural plasticity enables FoxP3 to bridge 2–4 DNA duplexes, forming ultrastable structures that coordinate multiple genomic loci. Nucleosomes further facilitate FoxP3 assembly by inducing local DNA bending, creating a nucleus that recruits distal DNA elements through multiway bridging. Our findings thus reveal FoxP3’s unusual ability to shapeshift to accommodate evolutionarily dynamic microsatellites and its potential to reinforce chromatin boundaries and three-dimensional genomic architecture.