Biology subjects

Boettiger, A.

Publications and source records attributed to Boettiger, A..

8 recordsLinked to original sources

bioRxiv2026

Loop Extrusion Accelerates Long-Range Enhancer-Promoter Searches in Living Embryos

Long-range gene regulation underlies a variety of human developmental disorders including Cornelia de Lange syndrome1 and polydactyly2. Cohesin-mediated loop extrusion and tether-like elements are two major mechanisms implicated in fostering long-range enhancer-promoter (E-P) contacts3. However, our understanding of the contributions of these mechanisms to the kinetics of E-P interactions is limited. Here we employ a combination of quantitative single-cell imaging, genetic manipulations and polymer simulations to examine the interplay of cohesin-mediated loop extrusion and tethering elements4,5 in the timely activation of gene expression in living Drosophila embryos. Depletion of NIPBL or deletion of an enhancer-proximal CTCF anchor element reduced expression without changing the duration of individual transcriptional bursts. Genetic epistasis experiments recapitulated polymer simulations predicting complementation of tether deletions by augmenting the stability of cohesin via reduced levels of WAPL6. We propose a "scan and snag" model whereby directional cohesin-driven enhancer scanning promotes diffusion-mediated tethering interactions to produce successful E-P contacts and transcriptional activation. We discuss how modulating cohesin stability and the "stickiness" of looping factors7-10 can fine-tune the levels and timing of gene expression in mammalian developmental and disease processes.

molecular biology↗

bioRxiv2025

Kinetic organization of the genome revealed by ultra-resolution, multiscale live imaging

In the last decade, sequencing methods like Hi-C have made it clear the genome is intricately folded, and that this organization contributes significantly to the control of gene expression and thence cell fate and behavior. Single-cell DNA tracing microscopy and polymer physics-based simulations of genome folding have proposed these population-scale patterns arise from motor- driven, heterogeneous movement, rather than stable 3D genomic architecture, implying that motion, rather than structure, is key to understanding genome function. However, tools to directly observe this motion in vivo have been limited in coverage and resolution. Here we describe TRansposon Assisted Chromatin Kinetic Imaging Technology (TRACK-IT), which combines a suite of imaging and labeling improvements to achieve ultra-resolution in space and time, with self-mapping transposons to distribute labels across the chromosome, uncovering dynamic behaviors across four orders of magnitude of genomic separation. We find that sequences separated by sub-megabase distances, typically 200-500 nm of nanometers apart, can transition to close proximity in tens of seconds - faster than previously hypothesized. This rapid motion is dependent upon cohesin and is exhibited only within certain genomic domains. Domain borders act as kinetic impediments to this search process, substantially slowing the rate and frequency of the transition to proximity. The genomic separation-dependent scaling of the search time for cis-interactions within a domain violates predictions of diffusion, suggesting motor driven folding. This distinctive scaling is lost following cohesin depletion, replaced with a behavior consistent with diffusion. Finally, we found cohesin containing cells exhibited rare, processive movements, not seen in cohesin depleted cells. These processive trajectories exhibit extrusion rates of [~]2.7 kb/s across three distinct genomic intervals, faster than recent in vitro measurements and prior estimates from in vivo data. Taken together, these results reveal a genome in motion across multiple genomic and temporal scales, where motor-dependent extrusion divides the sequence, not into spatially separate domains, but into kinetically separated domains that experience accelerated local search.

biophysics↗

bioRxiv2024

Long range regulation of transcription scales with genomic distance in a gene specific manner

While critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here we bypass the need for sequence-specific transcription factors and recruit activators directly using CARGO-VPR, an approach for targeting dCas9-VPR using a multiplexed array of RNA guides. We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible by single dCas9. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mESCs, where neither gene is expressed. We demonstrate that while activator recruitment to any tested region results in transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence or presence of the repressive chromatin marks at the locus.

molecular biology↗

bioRxiv2022

SnapFISH: a computational pipeline to identify chromatin loops from multiplexed DNA FISH data

Multiplexed DNA fluorescence in situ hybridization (FISH) imaging technologies have been developed to map the folding of chromatin fibers at tens of nanometer and tens of kilobase resolution in single cells. However, computational methods to reliably identify chromatin loops from such imaging datasets are still lacking. Here we present a Single-Nucleus Analysis Pipeline for multiplexed DNA FISH (SnapFISH), to process the multiplexed DNA FISH data and identify chromatin loops. SnapFISH can identify known chromatin loops from mouse embryonic stem cells with high sensitivity and accuracy. In addition, SnapFISH obtained comparable results of chromatin loops across datasets generated from diverse imaging technologies.

bioinformatics↗

bioRxiv2022

Polycomb repression of Hox genes involves spatial feedback but not domain compaction or demixing

Polycomb group (PcG) proteins modulate higher-order genome folding and play a critical role in silencing transcription during development. It is commonly proposed that PcG dependent changes in genome folding, which compact chromatin, contribute directly to repression by blocking binding of activating complexes and demixing repressed targets from non-repressed chromatin. To test this model we utilized Optical Reconstruction of Chromatin Architecture (ORCA) to trace the 3-dimensional folding of the Hoxa gene cluster, a canonical Polycomb target, allowing us to analyze thousands of DNA traces in single cells. In cell types ranging from embryonic stem cells to brain tissue, we find that PcG-bound chromatin frequently explores decompact states and partial mixing with neighboring chromatin, while remaining uniformly repressed, challenging the repression-by-compaction model. Using polymer physics simulations, we show that the flexible ensembles we observe can be explained by dynamic contacts mediated by multivalent interactions that are too weak to induce phase separation. Instead, these transient contacts contribute to accurate propagation of the epigenetic state without ectopic spreading or gradual erosion. We propose that the distinctive 3D organization of Polycomb chromatin, reflects a mechanism of "spatial feedback" required for stable repression.

molecular biology↗

bioRxiv2020

How subtle changes in 3D structure can create large changes in transcription

Animal genomes are organized into topologically associated domains (TADs), which exhibit more intra-domain than inter-domain contact. However, the absolute difference in contact is usually no more than twofold, even though disruptions to TAD boundaries can change gene expression by 8-10 fold. Existing models fail to explain this superlinear transcriptional response to changes in genomic contact. Here, we propose a futile cycle model where an enzyme stimulated by association with its products can exhibit bistability and hysteresis, allowing a small increase in enhancer-promoter contact to produce a large change in expression without obvious correlation between E-P contact and promoter activity. Through mathematical analysis and stochastic simulation, we show that this system can create an illusion of enhancer-promoter specificity and explain the importance of weak TAD boundaries. It also offers a mechanism to reconcile recent global cohesin loop disruption and TAD boundary deletion experiments. We discuss the model in the context of these recent controversial experiments. Together, these analyses advance our interpretation and understanding of cis-regulatory contacts in controlling gene expression, and suggest new experimental directions.

biophysics↗

bioRxiv2020

Polycomb-mediated Genome Architecture Enables Long-range Spreading of H3K27 methylation

Polycomb-group proteins play critical roles in gene silencing through the deposition of histone H3 lysine 27 trimethylation (H3K27me3) and chromatin compaction1-5. This process is essential for embryonic stem cell (ESCs) pluripotency, differentiation, and development. Polycomb repressive complex 2 (PRC2) can both read and write H3K27me3, enabling progressive spread of H3K27me3 on the linear genome6. Long-range Polycomb-associated DNA contacts have also been described, but their regulation and role in gene silencing remains unclear7-10. Here, we apply H3K27me3 HiChIP11-13, a protein-directed chromosome conformation method, and optical reconstruction of chromatin architecture14 to profile long-range Polycomb-associated DNA loops that span tens to hundreds of megabases across multiple topological associated domains in mouse ESCs and human induced pluripotent stem cells7-10. We find that H3K27me3 loop anchors are enriched for Polycomb nucleation points and coincide with key developmental genes, such as Hmx1, Wnt6 and Hoxa. Genetic deletion of H3K27me3 loop anchors revealed a coupling of Polycomb-associated genome architecture and H3K27me3 deposition evidenced by disruption of spatial contact between distant loci and altered H3K27me3 in cis, both locally and megabases away on the same chromosome. Further, we find that global alterations in PRC2 occupancy resulting from an EZH2 mutant15 selectively deficient in RNA binding is accompanied by loss of Polycomb-associated DNA looping. Together, these results suggest PRC2 acts as a "genomic wormhole", using RNA binding to enhance long range chromosome folding and H3K27me3 spreading. Additionally, developmental gene loci have novel roles in Polycomb spreading, emerging as important architectural elements of the epigenome.

genetics↗

bioRxiv2019

Quantitative Cytogenetics Reveals the Longitudinal Architecture of Meiotic Chromosome Axes

During meiosis, chromosomes adopt a specialized organization involving assembly of a cohesin-based axis along their lengths, with DNA loops emanating from this axis. We applied novel, quantitative and widely applicable cytogenetic strategies to elucidate the molecular bases of this organization using C. elegans. Analyses of WT chromosomes and de novo circular mini-chromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with two functionally-distinct cohesin complexes (REC-8 and COH-3/4) in defined stoichiometry. We further found that REC-8 cohesins, which load during S phase and mediate sister chromatid cohesion, usually occur as individual complexes, supporting a model wherein sister cohesion is mediated locally by a single cohesin ring. REC-8 complexes are interspersed in an alternating pattern with cohorts of axis-organizing COH-3/4 complexes (averaging three per cohort), which are insufficient to confer cohesion but can bind to individual chromatids, suggesting a mechanism to enable formation of asymmetric sister chromatid loops. Indeed, immuno-FISH assays demonstrate frequent asymmetry in genomic content between the loops formed on sister chromatids. We discuss how features of chromosome axis/loop architecture inferred from our data can help to explain enigmatic, yet essential, aspects of the meiotic program.

cell biology↗