Alistair Boettiger is an Assistant Professor in the Department of Developmental Biology at the Stanford University School of Medicine and is a member of Bio-X and the Biophysics Program. As an undergraduate he studied Physics and Molecular Biology at Princeton University, where he had the opportunity to experience lab research under the mentorship of Stas Shvartsman, who introduced him to his abiding interest in quantitative imaging and animal development. He conducted his Ph.D. research under the guidance of Michael Levine at UC Berkeley, where he studied cis-regulatory sequences that modulate the precision and robustness of gene expression (in particular shadow enhancers and paused promoters). As a postdoc in Xiaowei Zhuang's single-molecule imaging group at Harvard University, he studied gene regulation through super-resolution microscopy, multiplexed, error-correcting imaging, and deep sequencing. Dr. Boettiger started his lab at Stanford in 2016.
Honors & Awards
New Innovator Award, NIH (2018-2023)
Packard Fellowship, Packard Foundation (2018-2023)
Beckman Young Investigator, Beckman Foundation (2018-2022)
Kavli Fellow, NAS/Kavli Frontiers of Science (2018)
Searle Scholars Award, Chicago Community Trust (2017-2020)
Career Award at the Scientific Interface (CASI), Burroughs Wellcome Fund (2016-2021)
Dale F. Frey Award for Breakthrough Scientists, Damon Runyon Cancer Research Foundation (2016-2018)
Damon Runyon Fellowship Award, Damon Runyon Cancer Research Foundation (2012-2016)
Graduate Research Fellowship, National Science Foundation (2009-2011)
Postdoc, Harvard University, Single-molecule Imaging (2016)
Ph.D., UC Berkeley, Biophysics (2011)
A.B., Princeton University, Physics (2007)
Current Research and Scholarly Interests
My research aims to improve our understanding of how cells with the same genome can develop dramatically different behaviors. For example, consider the mechanical abilities of a muscle cell compared to the electrical excitability of a neuron, or the industrious activity of bone building cells in a youthful person compared to an elderly one. Each of these cells, (if taken from the same individual) has an identical genome — and yet each is “reading” a very distinct subset of that genome and consequently carrying out very different behaviors. The choice of what to read and what to hide away is made during development. An increasing body of data suggests this is accomplished by modifying the genome both in the nature of the proteins bound to different sequences and in the spatial organization of those sequences relative to each other. The spatial organization or folding of the genome may be particularly important in complex multicellular organisms, since many of the sequences known to interact based on genetic data are nonetheless substantially separated from each other along the linear genome. By regulating the folding of this linear sequence into a higher order structure, a cell might change which regulatory sequences have access to which genes, and achieve different behavioral states.
So far we have little imaging data on how the genome is folded within a cell on the length scale of individual genes, or whether this folding is regulated in any way relevant to the behavior of the cell. Our limited knowledge stems largely from want of a method that has both the resolution and specificity to visualize such genomic substructure. Conventional fluorescent microscopy has developed excellent tools for coloring specific regions of DNA and particular DNA-associated proteins with uniquely colored dyes — but lacks the resolution to turn these colored blurs into structures. Electron-microscopy has substantially greater resolution but lacks compatibility with specific labeling techniques to tell different gene clusters or different protein types apart. Super-resolution imaging approaches promise to address this balance by allowing the use of fluorescent labels while simultaneously resolving structures on the nano-scale. I have been adapting this approach to uncover the nano-scale structure of chromatin and determine to how this structure changes when bound by different types of nuclear proteins. While individual gene clusters appear as quite diverse structures, there appear to be a few general features, for example, linking structure with the epigenetic state.
- Developmental Biology
DBIO 210 (Spr)
Independent Studies (6)
- Directed Reading in Biophysics
BIOPHYS 399 (Sum)
- Directed Reading in Developmental Biology
DBIO 299 (Aut, Win, Spr, Sum)
- Graduate Research
BIOPHYS 300 (Aut, Win, Spr, Sum)
- Graduate Research
DBIO 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
DBIO 370 (Aut, Win, Spr, Sum)
- Undergraduate Research
DBIO 199 (Aut, Win, Spr, Sum)
- Directed Reading in Biophysics
- Prior Year Courses
Single-cell chromatin state transitions during epigenetic memory formation.
bioRxiv : the preprint server for biology
Repressive chromatin modifications are thought to compact chromatin to silence transcription. However, it is unclear how chromatin structure changes during silencing and epigenetic memory formation. We measured gene expression and chromatin structure in single cells after recruitment and release of repressors at a reporter gene. Chromatin structure is heterogeneous, with open and compact conformations present in both active and silent states. Recruitment of repressors associated with epigenetic memory produces chromatin compaction across 10-20 kilobases, while reversible silencing does not cause compaction at this scale. Chromatin compaction is inherited, but changes molecularly over time from histone methylation (H3K9me3) to DNA methylation. The level of compaction at the end of silencing quantitatively predicts epigenetic memory weeks later. Similarly, chromatin compaction at the Nanog locus predicts the degree of stem-cell fate commitment. These findings suggest that the chromatin state across tens of kilobases, beyond the gene itself, is important for epigenetic memory formation.
View details for DOI 10.1101/2023.10.03.560616
View details for PubMedID 37873344
View details for PubMedCentralID PMC10592931
SnapFISH: a computational pipeline to identify chromatin loops from multiplexed DNA FISH data.
2023; 14 (1): 4873
Multiplexed DNA fluorescence in situ hybridization (FISH) imaging technologies have been developed to map the folding of chromatin fibers at tens of nanometers and up to several kilobases in 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 obtains comparable results of chromatin loops across datasets generated from diverse imaging technologies. SnapFISH is freely available at https://github.com/HuMingLab/SnapFISH .
View details for DOI 10.1038/s41467-023-40658-3
View details for PubMedID 37573342
View details for PubMedCentralID 5763923
Leveraging polymer modeling to reconstruct chromatin connectivity from live images.
Chromosomal dynamics plays a central role in a number of critical biological processes, such as transcriptional regulation, genetic recombination, and DNA replication. However, visualization of chromatin is generally limited to live imaging of a few fluorescently labeled chromosomal loci or high-resolution reconstruction of multiple loci from a single time frame. To aid in mapping the underlying chromosomal structure based on parsimonious experimental measurements, we present an exact analytical expression for the evolution of the polymer configuration based on a flexible-polymer model, and we propose an algorithm that tracks the polymer configuration from live images of chromatin marked with several fluorescent marks. Our theory identifies the resolution of microscopy needed to achieve high-accuracy tracking for a given spacing of markers, establishing the statistical confidence in the assignment of genome identity to the visualized marks. We then leverage experimental data of locus-tracking measurements to demonstrate the validity of our modeling approach and to establish a basis for the design of experiments with a desired resolution. Altogether, this work provides a computational approach founded on polymer physics that vastly improves the interpretation of in vivo measurements of biopolymer dynamics.
View details for DOI 10.1016/j.bpj.2023.08.001
View details for PubMedID 37542372
Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project.
The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.
View details for DOI 10.1016/j.molcel.2023.06.018
View details for PubMedID 37419111
Extreme long-range gene regulatory perturbation drives a human craniofacial disorder
SPRINGERNATURE. 2023: 14
View details for Web of Science ID 001050507000024
Loop stacking organizes genome folding from TADs to chromosomes.
2023; 83 (9): 1377
Although population-level analyses revealed significant roles for CTCF and cohesin in mammalian genome organization, their contributions at the single-cell level remain incompletely understood. Here, we used a super-resolution microscopy approach to measure the effects of removal of CTCF or cohesin in mouse embryonic stem cells. Single-chromosome traces revealed cohesin-dependent loops, frequently stacked at their loop anchors forming multi-way contacts (hubs), bridging across TAD boundaries. Despite these bridging interactions, chromatin in intervening TADs was not intermixed, remaining separated in distinct loops around the hub. At the multi-TAD scale, steric effects from loop stacking insulated local chromatin from ultra-long range (>4 Mb) contacts. Upon cohesin removal, the chromosomes were more disordered and increased cell-cell variability in gene expression. Our data revise the TAD-centric understanding of CTCF and cohesin and provide a multi-scale, structural picture of how they organize the genome on the single-cell level through distinct contributions to loop stacking.
View details for DOI 10.1016/j.molcel.2023.04.008
View details for PubMedID 37146570
Structural elements promote architectural stripe formation and facilitate ultra-long-range gene regulation at a human disease locus.
Enhancer clusters overlapping disease-associated mutations in Pierre Robin sequence (PRS) patients regulate SOX9 expression at genomic distances over 1.25 Mb. We applied optical reconstruction of chromatin architecture (ORCA) imaging to trace 3D locus topology during PRS-enhancer activation. We observed pronounced changes in locus topology between cell types. Subsequent analysis of single-chromatin fiber traces revealed that these ensemble-average differences arise through changes in the frequency of commonly sampled topologies. We further identified two CTCF-bound elements, internal to the SOX9 topologically associating domain, which promote stripe formation, are positioned near the domain's 3D geometric center, and bridge enhancer-promoter contacts in a series of chromatin loops. Ablation of these elements results in diminished SOX9 expression and altered domain-wide contacts. Polymer models with uniform loading across the domain and frequent cohesin collisions recapitulate this multi-loop, centrally clustered geometry. Together, we provide mechanistic insights into architectural stripe formation and gene regulation over ultra-long genomic ranges.
View details for DOI 10.1016/j.molcel.2023.03.009
View details for PubMedID 36996812
Recent progress and challenges in single-cell imaging of enhancer-promoter interaction.
Current opinion in genetics & development
2023; 79: 102023
In the past two years, approaches relying on high-resolution microscopy and live-cell imaging have increasingly contributed to our understanding of the 3D genome organization and its importance for transcriptional control. Here, we describe recent progress that has highlighted how flexible and heterogeneous 3D chromatin structure is, on the length scales relevant to transcriptional control. We describe work that has investigated how robust transcriptional outcomes may be derived from such flexible organization without the need for clearly distinct structures in active and silent cells. We survey the latest state of the art in directly observing the dynamics of chromatin interactions, and suggest how some recent, apparently contradictory conclusions may be reconciled.
View details for DOI 10.1016/j.gde.2023.102023
View details for PubMedID 36854248
The spatial organization of transcriptional control.
Nature reviews. Genetics
In animals, the sequences for controlling gene expression do not concentrate just at the transcription start site of genes, but are frequently thousands to millions of base pairs distal to it. The interaction of these sequences with one another and their transcription start sites is regulated by factors that shape the three-dimensional (3D) organization of the genome within the nucleus. Over the past decade, indirect tools exploiting high-throughput DNA sequencing have helped to map this 3D organization, have identified multiple key regulators of its structure and, in the process, have substantially reshaped our view of how 3D genome architecture regulates transcription. Now, new tools for high-throughput super-resolution imaging of chromatin have directly visualized the 3D chromatin organization, settling some debates left unresolved by earlier indirect methods, challenging some earlier models of regulatory specificity and creating hypotheses about the role of chromatin structure in transcriptional regulation.
View details for DOI 10.1038/s41576-022-00526-0
View details for PubMedID 36104547
Polycomb-mediated genome architecture enables long-range spreading of H3K27 methylation.
Proceedings of the National Academy of Sciences of the United States of America
2022; 119 (22): e2201883119
SignificanceThe relationship between long-range Polycomb-associated chromatin contacts and the linear propagation of histone H3 lysine 27 trimethylation (H3K27me3) by Polycomb repressive complex 2 (PRC2) is not well-characterized. Here, we nominate a role for developmental loci as genomic architectural elements that enable long-range spreading of H3K27me3. Polycomb-associated loops are disrupted upon loss of PRC2 binding and deletion of loop anchors results in alterations of H3K27me3 deposition and ectopic gene expression. These results suggest that Polycomb-mediated genome architecture is important for gene repression during embryonic development.
View details for DOI 10.1073/pnas.2201883119
View details for PubMedID 35617427
Domain stacking enables a limb enhancer to act across multiple TAD boundaries.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
2022; 36 Suppl 1
Many developmentally important genes have their promoter and enhancers within different TADs. Although there are hypotheses about molecular mechanisms enabling such cross-TAD interactions between these cis-elements, but they remain to be assessed. To test these hypotheses, we use Optical Reconstruction of Chromatin Architecture (ORCA) to characterize the conformations of the Pitx1 locus on thousands of single chromosomes in developing mouse limbs. Our data supports a model in which cis-elements adjacent to TAD boundaries are collected in transient hub-like structures when neighboring boundaries are stacked with each other as a result of loop-extrusion. Through molecular dynamics simulations, we further propose that increasing boundary strengths (eg. by increasing CTCF occupancy) facilitates the formation of the stacked boundary conformation. This work provides a revised view of the TAD borders' function both facilitating as well as preventing cis-regulatory interactions and introduces a framework to distinguish border-crossing from border-respecting enhancer-promoter pairs.
View details for DOI 10.1096/fasebj.2022.36.S1.0I198
View details for PubMedID 35551698
A leukemia-protective germline variant mediates chromatin module formation via transcription factor nucleation.
2022; 13 (1): 2042
Non-coding variants coordinate transcription factor (TF) binding and chromatin mark enrichment changes over regions spanning >100kb. These molecularly coordinated regions are named "variable chromatin modules" (VCMs), providing a conceptual framework of how regulatory variation might shape complex traits. To better understand the molecular mechanisms underlying VCM formation, here, we mechanistically dissect a VCM-modulating noncoding variant that is associated with reduced chronic lymphocytic leukemia (CLL) predisposition and disease progression. This common, germline variant constitutes a 5-bp indel that controls the activity of an AXIN2 gene-linked VCM by creating a MEF2 binding site, which, upon binding, activates a super-enhancer-like regulatory element. This triggers a large change in TF binding activity and chromatin state at an enhancer cluster spanning >150kb, coinciding with subtle, long-range chromatin compaction and robust AXIN2 up-regulation. Our results support a model in which the indel acts as an AXIN2 VCM-activating TF nucleation event, which modulates CLL pathology.
View details for DOI 10.1038/s41467-022-29625-6
View details for PubMedID 35440565
How subtle changes in 3D structure can create large changes in transcription.
Animal genomes are organized into topologically associated domains (TADs). TADs are thought to contribute to gene regulation by facilitating enhancer-promoter (E-P) contacts within a TAD preventing these contacts across TAD borders. However, the absolute difference in contact frequency across TAD boundaries is usually less than two-fold, even though disruptions of TAD borders can change gene expression by ten-fold. Existing models fail to explain this hypersensitive response. Here, we propose a futile cycle model of enhancer-mediated regulation that can exhibit hypersensitivity through bistability and hysteresis. Consistent with recent experiments, this regulation does not exhibit strong correlation between enhancer-promoter contact and promoter activity, even though regulation occurs through contact. Through mathematical analysis and stochastic simulation, we show that this system can create an illusion of enhancer-promoter biochemical specificity and explain the importance of weak TAD boundaries. It also offers a mechanism to reconcile apparently contradictory results from recent global TAD disruption with local TAD boundary deletion experiments. Together, these analyses advance our understanding of cis-regulatory contacts in controlling gene expression, and suggest new experimental directions.
View details for DOI 10.7554/eLife.64320
View details for PubMedID 34240703
mSWI/SNF promotes Polycomb repression both directly and through genome-wide redistribution.
Nature structural & molecular biology
2021; 28 (6): 501-511
The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.
View details for DOI 10.1038/s41594-021-00604-7
View details for PubMedID 34117481
- Voices of biotech research. Nature biotechnology 2021
Tracing DNA paths and RNA profiles in cultured cells and tissues with ORCA.
Chromatin conformation capture (3C) methods and fluorescent in situ hybridization (FISH) microscopy have been used to investigate the spatial organization of the genome. Although powerful, both techniques have limitations. Hi-C is challenging for low cell numbers and requires very deep sequencing to achieve its high resolution. In contrast, FISH can be done on small cell numbers and capture rare cell populations, but typically targets pairs of loci at a lower resolution. Here we detail a protocol for optical reconstruction of chromatin architecture (ORCA), a microscopy approach to trace the 3D DNA path within the nuclei of fixed tissues and cultured cells with a genomic resolution as fine as 2 kb and a throughput of ~10,000 cells per experiment. ORCA can identify structural features with comparable resolution to Hi-C while providing single-cell resolution and multimodal measurements characteristic of microscopy. We describe how to use this DNA labeling in parallel with multiplexed labeling of dozens of RNAs to relate chromatin structure and gene expression in the same cells. Oligopaint probe design, primary probe making, sample collection, cryosectioning and RNA/DNA primary probe hybridization can be completed in 1.5 weeks, while automated RNA/DNA barcode hybridization and RNA/DNA imaging typically takes 2-6 d for data collection and 2-7 d for the automated steps of image analysis.
View details for DOI 10.1038/s41596-020-00478-x
View details for PubMedID 33619390
Deep learning connects DNA traces to transcription to reveal predictive features beyond enhancer-promoter contact.
2021; 12 (1): 3423
Chromatin architecture plays an important role in gene regulation. Recent advances in super-resolution microscopy have made it possible to measure chromatin 3D structure and transcription in thousands of single cells. However, leveraging these complex data sets with a computationally unbiased method has been challenging. Here, we present a deep learning-based approach to better understand to what degree chromatin structure relates to transcriptional state of individual cells. Furthermore, we explore methods to "unpack the black box" to determine in an unbiased manner which structural features of chromatin regulation are most important for gene expression state. We apply this approach to an Optical Reconstruction of Chromatin Architecture dataset of the Bithorax gene cluster in Drosophila and show it outperforms previous contact-focused methods in predicting expression state from 3D structure. We find the structural information is distributed across the domain, overlapping and extending beyond domains identified by prior genetic analyses. Individual enhancer-promoter interactions are a minor contributor to predictions of activity.
View details for DOI 10.1038/s41467-021-23831-4
View details for PubMedID 34103507
Advances in Chromatin Imaging at Kilobase-Scale Resolution.
Trends in genetics : TIG
It is now widely appreciated that the spatial organization of the genome is nonrandom, and its complex 3D folding has important consequences for many genome processes. Recent developments in multiplexed, super-resolution microscopy have enabled an unprecedented view of the polymeric structure of chromatin - from the loose folds of whole chromosomes to the detailed loops of cis-regulatory elements that regulate gene expression. Facilitated by the use of robotics, microfluidics, and improved approaches to super-resolution, thousands to hundreds of thousands of individual cells can now be analyzed in an individual experiment. This has led to new insights into the nature of genomic structural features identified by sequencing, such as topologically associated domains (TADs), and the nature of enhancer-promoter interactions underlying transcriptional regulation. We review these recent improvements.
View details for DOI 10.1016/j.tig.2019.12.010
View details for PubMedID 32007290
Quantitative cytogenetics reveals molecular stoichiometry and longitudinal organization of meiotic chromosome axes and loops.
2020; 18 (8): e3000817
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 Caenorhabditis elegans. Analyses of wild-type (WT) chromosomes and de novo circular minichromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with 2 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 3 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, immunofluorescence fluorescence in situ hybridization (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.
View details for DOI 10.1371/journal.pbio.3000817
View details for PubMedID 32813728
Atlas of Subcellular RNA Localization Revealed by APEX-Seq.
We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.
View details for DOI 10.1016/j.cell.2019.05.027
View details for PubMedID 31230715
Visualizing DNA folding and RNA in embryos at single-cell resolution.
The establishment of cell types during development requires precise interactions between genes and distal regulatory sequences. We have a limited understanding of how these interactions look in three dimensions, vary across cell types in complex tissue, and relate to transcription. Here we describe optical reconstruction of chromatin architecture (ORCA), a method that can trace the DNA path in single cells with nanoscale accuracy and genomic resolution reaching two kilobases. We used ORCA to study a Hox gene cluster in cryosectioned Drosophila embryos and labelled around 30 RNA species in parallel. We identified cell-type-specific physical borders between active and Polycomb-repressed DNA, and unexpected Polycomb-independent borders. Deletion of Polycomb-independent borders led to ectopic enhancer-promoter contacts, aberrant gene expression, and developmental defects. Together, these results illustrate an approach for high-resolution, single-cell DNA domain analysis in vivo, identify domain structures that change with cell identity, and show that border elements contribute to the formation of physical domains in Drosophila.
View details for PubMedID 30886393
Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells.
Science (New York, N.Y.)
2018; 362 (6413)
The spatial organization of chromatin is pivotal for regulating genome functions. We report an imaging method for tracing chromatin organization with kilobase- and nanometer-scale resolution, unveiling chromatin conformation across topologically associating domains (TADs) in thousands of individual cells. Our imaging data revealed TAD-like structures with globular conformation and sharp domain boundaries in single cells. The boundaries varied from cell to cell, occurring with nonzero probabilities at all genomic positions but preferentially at CCCTC-binding factor (CTCF)- and cohesin-binding sites. Notably, cohesin depletion, which abolished TADs at the population-average level, did not diminish TAD-like structures in single cells but eliminated preferential domain boundary positions. Moreover, we observed widespread, cooperative, multiway chromatin interactions, which remained after cohesin depletion. These results provide critical insight into the mechanisms underlying chromatin domain and hub formation.
View details for PubMedID 30361340
In Situ Super-Resolution Imaging of Genomic DNA with OligoSTORM and OligoDNA-PAINT
SUPER-RESOLUTION MICROSCOPY: METHODS AND PROTOCOLS
2017; 1663: 231–52
OligoSTORM and OligoDNA-PAINT meld the Oligopaint technology for fluorescent in situ hybridization (FISH) with, respectively, Stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) to enable in situ single-molecule super-resolution imaging of nucleic acids. Both strategies enable ≤20 nm resolution and are appropriate for imaging nanoscale features of the genomes of a wide range of species, including human, mouse, and fruit fly (Drosophila).
View details for PubMedID 28924672
View details for PubMedCentralID PMC5919218
Spatial organization shapes the turnover of a bacterial transcriptome
Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Time-resolved RNA-sequencing revealed that degradation rates of inner-membrane-protein mRNAs are on average greater that those of the other mRNAs and that this selective destabilization of inner-membrane-protein mRNAs is abolished by dissociating the RNA degradosome from the membrane. Together, these results demonstrate that the bacterial transcriptome is spatially organized and suggest that this organization shapes the post-transcriptional dynamics of mRNAs.
View details for DOI 10.7554/eLife.13065
View details for Web of Science ID 000376881500001
View details for PubMedID 27198188
View details for PubMedCentralID PMC4874777
Super-resolution imaging reveals distinct chromatin folding for different epigenetic states
2016; 529 (7586): 418-?
Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression. Here we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive or Polycomb-repressed states, and observed distinct chromatin organizations for each state. All three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed domains show the densest packing and most intriguing chromatin folding behaviour, in which chromatin packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins play an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly relevant to genome regulation.
View details for DOI 10.1038/nature16496
View details for Web of Science ID 000368354800051
View details for PubMedID 26760202
View details for PubMedCentralID PMC4905822
Chromatin topology is coupled to Polycomb group protein subnuclear organization.
2016; 7: 10291-?
The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, here we show that PcG proteins are organized into hundreds of nanoscale protein clusters. We manipulated PcG clusters by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions.
View details for DOI 10.1038/ncomms10291
View details for PubMedID 26759081
View details for PubMedCentralID PMC4735512
Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes
Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.
View details for DOI 10.1038/ncomms8147
View details for Web of Science ID 000355533700006
View details for PubMedID 25962338
View details for PubMedCentralID PMC4430122
RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells.
Science (New York, N.Y.)
2015; 348 (6233)
Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here, we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 to 1000 distinct RNA species in hundreds of individual cells. Correlation analysis of the ~10(4) to 10(6) pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins.
View details for DOI 10.1126/science.aaa6090
View details for PubMedID 25858977
View details for PubMedCentralID PMC4662681
Analytic Approaches to Stochastic Gene Expression in Multicellular Systems
2013; 105 (12): 2629-2640
Deterministic thermodynamic models of the complex systems, which control gene expression in metazoa, are helping researchers identify fundamental themes in the regulation of transcription. However, quantitative single cell studies are increasingly identifying regulatory mechanisms that control variability in expression. Such behaviors cannot be captured by deterministic models and are poorly suited to contemporary stochastic approaches that rely on continuum approximations, such as Langevin methods. Fortunately, theoretical advances in the modeling of transcription have assembled some general results that can be readily applied to systems being explored only through a deterministic approach. Here, I review some of the recent experimental evidence for the importance of genetically regulating stochastic effects during embryonic development and discuss key results from Markov theory that can be used to model this regulation. I then discuss several pairs of regulatory mechanisms recently investigated through a Markov approach. In each case, a deterministic treatment predicts no difference between the mechanisms, but the statistical treatment reveals the potential for substantially different distributions of transcriptional activity. In this light, features of gene regulation that seemed needlessly complex evolutionary baggage may be appreciated for their key contributions to reliability and precision of gene expression.
View details for DOI 10.1016/j.bpj.2013.10.033
View details for Web of Science ID 000328597400008
View details for PubMedID 24359735
View details for PubMedCentralID PMC3909422
Rapid Transcription Fosters Coordinate snail Expression in the Drosophila Embryo
2013; 3 (1): 8-15
Transcription is commonly held to be a highly stochastic process, resulting in considerable heterogeneity of gene expression among the different cells in a population. Here, we employ quantitative in situ hybridization methods coupled with high-resolution imaging assays to measure the expression of snail, a developmental patterning gene necessary for coordinating the invagination of the mesoderm during gastrulation of the Drosophila embryo. Our measurements of steady-state mRNAs suggest that there is very little variation in snail expression across the different cells that make up the mesoderm and that synthesis approaches the kinetic limits of Pol II processivity. We propose that rapid transcription kinetics and negative autoregulation are responsible for the remarkable homogeneity of snail expression and the coordination of mesoderm invagination.
View details for DOI 10.1016/j.celrep.2012.12.015
View details for Web of Science ID 000321891400002
View details for PubMedID 23352665
View details for PubMedCentralID PMC4257496
Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (33): 13570-13575
Segmentation of the Drosophila embryo begins with the establishment of spatially restricted gap gene-expression patterns in response to broad gradients of maternal transcription factors, such as Bicoid. Numerous studies have documented the fidelity of these expression patterns, even when embryos are subjected to genetic or environmental stress, but the underlying mechanisms for this transcriptional precision are uncertain. Here we present evidence that every gap gene contains multiple enhancers with overlapping activities to produce authentic patterns of gene expression. For example, a recently identified hunchback (hb) enhancer (located 5-kb upstream of the classic enhancer) ensures repression at the anterior pole. The combination of intronic and 5' knirps (kni) enhancers produces a faithful expression pattern, even though the intronic enhancer alone directs an abnormally broad expression pattern. We present different models for "enhancer synergy," whereby two enhancers with overlapping activities produce authentic patterns of gene expression.
View details for DOI 10.1073/pnas.1109873108
View details for Web of Science ID 000293895100046
View details for PubMedID 21825127
View details for PubMedCentralID PMC3158186
Inferring ecological and behavioral drivers of African elephant movement using a linear filtering approach
2011; 92 (8): 1648-1657
Understanding the environmental factors influencing animal movements is fundamental to theoretical and applied research in the field of movement ecology. Studies relating fine-scale movement paths to spatiotemporally structured landscape data, such as vegetation productivity or human activity, are particularly lacking despite the obvious importance of such information to understanding drivers of animal movement. In part, this may be because few approaches provide the sophistication to characterize the complexity of movement behavior and relate it to diverse, varying environmental stimuli. We overcame this hurdle by applying, for the first time to an ecological question, a finite impulse-response signal-filtering approach to identify human and natural environmental drivers of movements of 13 free-ranging African elephants (Loxodonta africana) from distinct social groups collected over seven years. A minimum mean-square error (MMSE) estimation criterion allowed comparison of the predictive power of landscape and ecological model inputs. We showed that a filter combining vegetation dynamics, human and physical landscape features, and previous movement outperformed simpler filter structures, indicating the importance of both dynamic and static landscape features, as well as habit, on movement decisions taken by elephants. Elephant responses to vegetation productivity indices were not uniform in time or space, indicating that elephant foraging strategies are more complex than simply gravitation toward areas of high productivity. Predictions were most frequently inaccurate outside protected area boundaries near human settlements, suggesting that human activity disrupts typical elephant movement behavior. Successful management strategies at the human-elephant interface, therefore, are likely to be context specific and dynamic. Signal processing provides a promising approach for elucidating environmental factors that drive animal movements over large time and spatial scales.
View details for Web of Science ID 000293459100011
View details for PubMedID 21905431
Transcriptional Regulation: Effects of Promoter Proximal Pausing on Speed, Synchrony and Reliability
PLOS COMPUTATIONAL BIOLOGY
2011; 7 (5)
Recent whole genome polymerase binding assays in the Drosophila embryo have shown that a substantial proportion of uninduced genes have pre-assembled RNA polymerase-II transcription initiation complex (PIC) bound to their promoters. These constitute a subset of promoter proximally paused genes for which mRNA elongation instead of promoter access is regulated. This difference can be described as a rearrangement of the regulatory topology to control the downstream transcriptional process of elongation rather than the upstream transcriptional initiation event. It has been shown experimentally that genes with the former mode of regulation tend to induce faster and more synchronously, and that promoter-proximal pausing is observed mainly in metazoans, in accord with a posited impact on synchrony. However, it has not been shown whether or not it is the change in the regulated step per se that is causal. We investigate this question by proposing and analyzing a continuous-time Markov chain model of PIC assembly regulated at one of two steps: initial polymerase association with DNA, or release from a paused, transcribing state. Our analysis demonstrates that, over a wide range of physical parameters, increased speed and synchrony are functional consequences of elongation control. Further, we make new predictions about the effect of elongation regulation on the consistent control of total transcript number between cells. We also identify which elements in the transcription induction pathway are most sensitive to molecular noise and thus possibly the most evolutionarily constrained. Our methods produce symbolic expressions for quantities of interest with reasonable computational effort and they can be used to explore the interplay between interaction topology and molecular noise in a broader class of biochemical networks. We provide general-purpose code implementing these methods.
View details for DOI 10.1371/journal.pcbi.1001136
View details for Web of Science ID 000291015800010
View details for PubMedID 21589887
View details for PubMedCentralID PMC3093350
Shadow Enhancers Foster Robustness of Drosophila Gastrulation
2010; 20 (17): 1562-1567
Critical developmental control genes sometimes contain "shadow" enhancers that can be located in remote positions, including the introns of neighboring genes . They nonetheless produce patterns of gene expression that are the same as or similar to those produced by more proximal primary enhancers. It was suggested that shadow enhancers help foster robustness in gene expression in response to environmental or genetic perturbations [2, 3]. We critically tested this hypothesis by employing a combination of bacterial artificial chromosome (BAC) recombineering and quantitative confocal imaging methods [2, 4]. Evidence is presented that the snail gene is regulated by a distal shadow enhancer located within a neighboring locus. Removal of the proximal primary enhancer does not significantly perturb snail function, including the repression of neurogenic genes and formation of the ventral furrow during gastrulation at normal temperatures. However, at elevated temperatures, there is sporadic loss of snail expression and coincident disruptions in gastrulation. Similar defects are observed at normal temperatures upon reductions in the levels of Dorsal, a key activator of snail expression (reviewed in ). These results suggest that shadow enhancers represent a novel mechanism of canalization whereby complex developmental processes "bring about one definite end-result regardless of minor variations in conditions" .
View details for DOI 10.1016/j.cub.2010.07.043
View details for Web of Science ID 000281941100034
View details for PubMedID 20797865
View details for PubMedCentralID PMC4257487
Morphogen Gradients: Limits to Signaling or Limits to Measurement?
2010; 20 (5): R232-R234
In Drosophila embryos, a concentration gradient of nuclear Dorsal protein controls pattern formation along the dorsal-ventral axis. Recent quantitative studies agree on the temporal dynamics of the gradient, but disagree on its spatial limits.
View details for DOI 10.1016/j.cub.2010.01.040
View details for Web of Science ID 000275476900008
View details for PubMedID 20219171
Emergent complexity in simple neural systems.
Communicative & integrative biology
2009; 2 (6): 467-470
The ornate and diverse patterns of seashells testify to the complexity of living systems. Provocative computational explorations have shown that similarly complex patterns may arise from the collective interaction of a small number of rules. This suggests that, although a system may appear complex, it may still be understood in terms of simple principles. It is still debatable whether shell patterns emerge from some undiscovered simple principles, or are the consequence of an irreducibly complex interaction of many effects. Recent work by Boettiger, Ermentrout and Oster on the biological mechanisms of shell patterning has provided compelling evidence that, at least for this system, simplicity produces diversity and complexity.
View details for PubMedID 20195452
View details for PubMedCentralID PMC2829821
Synchronous and Stochastic Patterns of Gene Activation in the Drosophila Embryo
2009; 325 (5939): 471-473
Drosophila embryogenesis is characterized by rapid transitions in gene activity, whereby crudely distributed gradients of regulatory proteins give way to precise on/off patterns of gene expression. To explore the underlying mechanisms, a partially automated, quantitative in situ hybridization method was used to visualize expression profiles of 14 developmental control genes in hundreds of embryos. These studies revealed two distinct patterns of gene activation: synchronous and stochastic. Synchronous genes display essentially uniform expression of nascent transcripts in all cells of an embryonic tissue, whereas stochastic genes display erratic patterns of de novo activation. RNA polymerase II is "pre-loaded" (stalled) in the promoter regions of synchronous genes, but not stochastic genes. Transcriptional synchrony might ensure the orderly deployment of the complex gene regulatory networks that control embryogenesis.
View details for DOI 10.1126/science.1173976
View details for Web of Science ID 000268255100055
View details for PubMedID 19628867
View details for PubMedCentralID PMC4280267
The neural origins of shell structure and pattern in aquatic mollusks
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (16): 6837-6842
We present a model to explain how the neurosecretory system of aquatic mollusks generates their diversity of shell structures and pigmentation patterns. The anatomical and physiological basis of this model sets it apart from other models used to explain shape and pattern. The model reproduces most known shell shapes and patterns and accurately predicts how the pattern alters in response to environmental disruption and subsequent repair. Finally, we connect the model to a larger class of neural models.
View details for DOI 10.1073/pnas.0810311106
View details for Web of Science ID 000265506800074
View details for PubMedID 19351900
View details for PubMedCentralID PMC2672551
Evolution of insect dorsoventral patterning mechanisms.
Cold Spring Harbor symposia on quantitative biology
2009; 74: 275-279
The dorsoventral (DV) patterning of the early Drosophila embryo depends on Dorsal, a maternal sequence-specific transcription factor related to mammalian NF-kappaB. Dorsal controls DV patterning through the differential regulation of approximately 50 target genes in a concentration-dependent manner. Whole-genome methods, including ChIP-chip and ChIP-seq assays, have identified approximately 100 Dorsal target enhancers, and more than one-third of these have been experimentally confirmed via transgenic embryo assays. Despite differences in DV patterning among divergent insects, a number of the Dorsal target enhancers are located in conserved positions relative to the associated transcription units. Thus, the evolution of novel patterns of gene expression might depend on the modification of old enhancers, rather than the invention of new ones. As many as half of all Dorsal target genes appear to contain "shadow" enhancers: a second enhancer that directs the same or similar expression pattern as the primary enhancer. Preliminary studies suggest that shadow enhancers might help to ensure resilience of gene expression in response to environmental and genetic perturbations. Finally, most Dorsal target genes appear to contain RNA polymerase II (pol II) prior to their activation. Stalled pol II fosters synchronous patterns of gene activation in the early embryo. In contrast, DV patterning genes lacking stalled pol II are initially activated in an erratic or stochastic fashion. It is possible that stalled pol II confers fitness to a population by ensuring coordinate deployment of the gene networks controlling embryogenesis.
View details for DOI 10.1101/sqb.2009.74.021
View details for PubMedID 19843594
View details for PubMedCentralID PMC4280271
Nuclear trapping shapes the terminal gradient in the Drosophila embryo
2008; 18 (12): 915-919
Patterning of the terminal regions of the Drosophila embryo relies on the gradient of phosphorylated ERK/MAPK (dpERK), which is controlled by the localized activation of the Torso receptor tyrosine kinase [1-4]. This model is supported by a large amount of data, but the gradient itself has never been quantified. We present the first measurements of the dpERK gradient and establish a new intracellular layer of its regulation. Based on the quantitative analysis of the spatial pattern of dpERK in mutants with different levels of Torso as well as the dynamics of the wild-type dpERK pattern, we propose that the terminal-patterning gradient is controlled by a cascade of diffusion-trapping modules. A ligand-trapping mechanism establishes a sharply localized pattern of the Torso receptor occupancy on the surface of the embryo. Inside the syncytial embryo, nuclei play the role of traps that localize diffusible dpERK. We argue that the length scale of the terminal-patterning gradient is determined mainly by the intracellular module.
View details for DOI 10.1016/j.cub.2008.05.034
View details for Web of Science ID 000257151900032
View details for PubMedID 18571412
View details for PubMedCentralID PMC2500156
Modeling the bicold gradient: Diffusion and reversible nuclear trapping of a stable protein
2007; 312 (2): 623-630
The Bicoid gradient in the Drosophila embryo provided the first example of a morphogen gradient studied at the molecular level. The exponential shape of the Bicoid gradient had always been interpreted within the framework of the localized production, diffusion, and degradation model. We propose an alternative mechanism, which assumes no Bicoid degradation. The medium where the Bicoid gradient is formed and interpreted is very dynamic. Most notably, the number of nuclei changes over three orders of magnitude from fertilization, when Bicoid synthesis is initiated, to nuclear cycle 14 when most of the measurements were taken. We demonstrate that a model based on Bicoid diffusion and nucleocytoplasmic shuttling in the presence of the growing number of nuclei can account for most of the properties of the Bicoid concentration profile. Consistent with experimental observations, the Bicoid gradient in our model is established before nuclei migrate to the periphery of the embryo and remains stable during subsequent nuclear divisions.
View details for DOI 10.1016/j.ydbio.2007.09.058
View details for Web of Science ID 000251734300013
View details for PubMedID 18001703
View details for PubMedCentralID PMC2789574
Role of boundary conditions in an experimental model of epithelial wound healing
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
2006; 291 (1): C68-C75
Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis, but our understanding of the mechanisms that coordinate the behavior of multiple cells in these processes is far from complete. Recent experiments with Madin-Darby canine kidney epithelial monolayers revealed a wave-like pattern of injury-induced MAPK activation and showed that it is essential for collective cell migration after wounding. To investigate the effects of the different aspects of wounding on cell sheet migration, we engineered a system that allowed us to dissect the classic wound healing assay. We studied Madin-Darby canine kidney sheet migration under three different conditions: 1) the classic wound healing assay, 2) empty space induction, where a confluent monolayer is grown adjacent to a slab of polydimethylsiloxane and the monolayer is not injured but allowed to migrate upon removal of the slab, and 3) injury via polydimethylsiloxane membrane peel-off, where an injured monolayer migrates onto plain tissue culture surface, as in the case of empty space induction allowing for direct comparison. By tracking the motion of individual cells within the sheet under these three conditions, we show how the dynamics of the individual cells' motion is responsible for the coordinated migration of the sheet and is coordinated with the activation of ERK1/2 MAPK. In addition, we demonstrate that the propagation of the waves of MAPK activation depends on the generation of reactive oxygen species at the wound edge.
View details for DOI 10.1152/ajpcell.00411.2005
View details for Web of Science ID 000238287500009
View details for PubMedID 16495370