Enhancer-promoter interactions are reconfigured through the formation of long-range multiway hubs as mouse ES cells exit pluripotency.

Enhancers bind transcription factors, chromatin regulators, and non-coding transcripts to modulate the expression of target genes. Here, we report 3D genome structures of single mouse ES cells as they are induced to exit pluripotency and transition through a formative stage prior to undergoing neuroectodermal differentiation. We find that there is a remarkable reorganization of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5-8 distant chromosomal sites that are often separated by many Mb from each other. In the formative state, genes important for pluripotency exit establish contacts with emerging enhancers within these multiway hubs, suggesting that the structural changes we have observed may play an important role in modulating transcription and establishing new cell identities.

Unorthodox PCNA Binding by Chromatin Assembly Factor 1.

The eukaryotic DNA replication fork is a hub of enzymes that continuously act to synthesize DNA, propagate DNA methylation and other epigenetic marks, perform quality control, repair nascent DNA, and package this DNA into chromatin. Many of the enzymes involved in these spatiotemporally correlated processes perform their functions by binding to proliferating cell nuclear antigen (PCNA). A long-standing question has been how the plethora of PCNA-binding enzymes exert their activities without interfering with each other. As a first step towards deciphering this complex regulation, we studied how Chromatin Assembly Factor 1 (CAF-1) binds to PCNA. We demonstrate that CAF-1 binds to PCNA in a heretofore uncharacterized manner that depends upon a cation-pi (π) interaction. An arginine residue, conserved among CAF-1 homologs but absent from other PCNA-binding proteins, inserts into the hydrophobic pocket normally occupied by proteins that contain canonical PCNA interaction peptides (PIPs). Mutation of this arginine disrupts the ability of CAF-1 to bind PCNA and to assemble chromatin. The PIP of the CAF-1 p150 subunit resides at the extreme C-terminus of an apparent long α-helix (119 amino acids) that has been reported to bind DNA. The length of that helix and the presence of a PIP at the C-terminus are evolutionarily conserved among numerous species, ranging from yeast to humans. This arrangement of a very long DNA-binding coiled-coil that terminates in PIPs may serve to coordinate DNA and PCNA binding by CAF-1.

Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3.

In mammalian genomes, differentially methylated regions (DMRs) and histone marks including trimethylation of histone 3 lysine 27 (H3K27me3) at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. However, neither parent-of-origin-specific transcription nor imprints have been comprehensively mapped at the blastocyst stage of preimplantation development. Here, we address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos. We find that seventy-one genes exhibit previously unreported parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expressed). Uniparental expression of nBiX genes disappears soon after implantation. Micro-whole-genome bisulfite sequencing (µWGBS) of individual uniparental blastocysts detects 859 DMRs. We further find that 16% of nBiX genes are associated with a DMR, whereas most are associated with parentally-biased H3K27me3, suggesting a role for Polycomb-mediated imprinting in blastocysts. nBiX genes are clustered: five clusters contained at least one published imprinted gene, and five clusters exclusively contained nBiX genes. These data suggest that early development undergoes a complex program of stage-specific imprinting involving different tiers of regulation.

Citrullination of HP1γ chromodomain affects association with chromatin.

BACKGROUND: Stem cell differentiation involves major chromatin reorganisation, heterochromatin formation and genomic relocalisation of structural proteins, including heterochromatin protein 1 gamma (HP1γ). As the principal reader of the repressive histone marks H3K9me2/3, HP1 plays a key role in numerous processes including heterochromatin formation and maintenance. RESULTS: We find that HP1γ is citrullinated in mouse embryonic stem cells (mESCs) and this diminishes when cells differentiate, indicating that it is a dynamically regulated post-translational modification during stem cell differentiation. Peptidylarginine deiminase 4, a known regulator of pluripotency, citrullinates HP1γ in vitro. This requires R38 and R39 within the HP1γ chromodomain, and the catalytic activity is enhanced by trimethylated H3K9 (H3K9me3) peptides. Mutation of R38 and R39, designed to mimic citrullination, affects HP1γ binding to H3K9me3-containing peptides. Using live-cell single-particle tracking, we demonstrate that R38 and R39 are important for HP1γ binding to chromatin in vivo. Furthermore, their mutation reduces the residence time of HP1γ on chromatin in differentiating mESCs. CONCLUSION: Citrullination is a novel post-translational modification of the structural heterochromatin protein HP1γ in mESCs that is dynamically regulated during mESC differentiation. The citrullinated residues lie within the HP1γ chromodomain and are important for H3K9me3 binding in vitro and chromatin association in vivo.

FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells.

A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.

Combining fluorescence imaging with Hi-C to study 3D genome architecture of the same single cell.

Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000-100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.

Calculation of 3D genome structures for comparison of chromosome conformation capture experiments with microscopy: An evaluation of single-cell Hi-C protocols.

Single-cell chromosome conformation capture approaches are revealing the extent of cell-to-cell variability in the organization and packaging of genomes. These single-cell methods, unlike their multi-cell counterparts, allow straightforward computation of realistic chromosome conformations that may be compared and combined with other, independent, techniques to study 3D structure. Here we discuss how single-cell Hi-C and subsequent 3D genome structure determination allows comparison with data from microscopy. We then carry out a systematic evaluation of recently published single-cell Hi-C datasets to establish a computational approach for the evaluation of single-cell Hi-C protocols. We show that the calculation of genome structures provides a useful tool for assessing the quality of single-cell Hi-C data because it requires a self-consistent network of interactions, relating to the underlying 3D conformation, with few errors, as well as sufficient longer-range cis- and trans-chromosomal contacts.

Sensitive light-sheet microscopy in multiwell plates using an AFM cantilever.

We present a sensitive inverted light sheet microscope, capable of single-molecule fluorescence imaging of cells in 96-well plates. Light sheet microscope designs are often complex and costly, requiring custom-made sample chambers that are incompatible with standard cell culture samples. To overcome this limitation, we have developed single-objective cantilever selective plane illumination microscopy (socSPIM), which introduces a light sheet through the objective lens of an inverted microscope using an AFM tip. We demonstrate the effectiveness of this setup by performing 3D imaging of nuclear pore complexes, as well as live whole-cell 3D imaging of lysosomes and super-resolution imaging of the T-cell membrane. The unique advantage offered by socSPIM is the minimal footprint of the cantilever, which allowed us to perform super-resolution reflected light-sheet microscopy by PAINT in 96-well plates, paving the way for high-throughput studies.

Architecture of TAF11/TAF13/TBP complex suggests novel regulation properties of general transcription factor TFIID.

General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-interaction domain (CTID) in TAF13, which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.

Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function.

Single-molecule localization microscopy, typically based on total internal reflection illumination, has taken our understanding of protein organization and dynamics in cells beyond the diffraction limit. However, biological systems exist in a complicated three-dimensional environment, which has required the development of new techniques, including the double-helix point spread function (DHPSF), to accurately visualize biological processes. The application of the DHPSF approach has so far been limited to the study of relatively small prokaryotic cells. By matching the refractive index of the objective lens immersion liquid to that of the sample media, we demonstrate DHPSF imaging of up to 15-µm-thick whole eukaryotic cell volumes in three to five imaging planes. We illustrate the capabilities of the DHPSF by exploring large-scale membrane reorganization in human T cells after receptor triggering, and by using single-particle tracking to image several mammalian proteins, including membrane, cytoplasmic, and nuclear proteins in T cells and embryonic stem cells.

3D structures of individual mammalian genomes studied by single-cell Hi-C.

The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.

Are There Knots in Chromosomes?

Recent developments have for the first time allowed the determination of three-dimensional structures of individual chromosomes and genomes in nuclei of single haploid mouse embryonic stem (ES) cells based on Hi⁻C chromosome conformation contact data. Although these first structures have a relatively low resolution, they provide the first experimental data that can be used to study chromosome and intact genome folding. Here we further analyze these structures and provide the first evidence that G1 phase chromosomes are knotted, consistent with the fact that plots of contact probability vs sequence separation show a power law dependence that is intermediate between that of a fractal globule and an equilibrium structure.

A microfluidic platform for trapping, releasing and super-resolution imaging of single cells.

A multi-layer device, combining hydrodynamic trapping with microfluidic valving techniques, has been developed for on-chip manipulation and imaging of single cells and particles. Such a device contains a flow layer with trapping channels to capture single particles or cells and a control layer with valve channels to selectively control the trap and release processes. Particles and cells have been successfully trapped and released using the proposed device. The device enables the trapping of single particles with a trapping efficiency of greater than 95%, and allows for single particles and cells to be trapped, released and manipulated by simply controlling corresponding valves. Moreover, the trap and release processes are found to be compatible with biological samples like cells. Our device allows stable immobilisation of large numbers of single cells in a few minutes, significantly easing the experiment setup for single-cell characterisation and offering a stable platform for both single-molecule and super-resolution imaging. Proof-of-concept super- resolution imaging experiments with mouse embryonic stem cells (mESCs) have been conducted by exploiting super-resolution photoactivated localisation microscopy (PALM). Cells and nuclei were stably trapped and imaged. Centromeres of ∼200 nm size could be identified with a localisation precision of <15 nm.

Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localisation Microscopy.

Single-molecule localisation microscopy (SMLM) allows the super-resolved imaging of proteins within mammalian nuclei at spatial resolutions comparable to that of a nucleosome itself (~20 nm). The technique is therefore well suited to the study of chromatin structure. Fixed-cell SMLM has already allowed temporal 'snapshots' of how proteins are arranged on chromatin within mammalian nuclei. In this chapter, we focus on how recent developments, for example in selective plane illumination and protein labelling, have led to a range of live-cell SMLM studies. We describe how to carry out single-particle tracking (SPT) of single proteins and, by analysing their diffusion parameters, how to determine whether proteins interact with chromatin, diffuse freely or do both. We can study the numbers of proteins that interact with chromatin and also determine their residence time on chromatin. We can determine whether these proteins form functional clusters within the nucleus as well as whether they form specific nuclear structures.

Dynamic monitoring of single cell lysis in an impedance-based microfluidic device.

A microfluidic device that is capable of trapping and sensing dynamic variations in the electrical properties of individual cells is demonstrated. The device is applied to the real-time recording of impedance measurements of mouse embryonic stem cells (mESCs) during the process of membrane lysis, with the resulting changes in the electrical properties of cells during this process being quantitatively tracked over time. It is observed that the impedance magnitude decreases dramatically after cell membrane lysis. A significant shift in the phase spectrum is also observed during the time course of this process. By fitting experimental data to physical models, the electrical parameters of cells can be extracted and parameter variations quantified during the process. In the cell lysis experiments, the equivalent conductivity of the cell membrane is found to increase significantly due to pore formation in the membrane during lysis. An increase in the specific capacitance of the membrane is also observed. On the other hand, the conductivity of the cytoplasm is observed to decrease, which may be explained the fact that excess water enters the cell through the gradual permeabilization of the membrane during lysis. Cells can be trapped in the device for periods up to several days, and their electrical response can be monitored by real-time impedance measurements in a label-free and non-invasive manner. Furthermore, due to the highly efficient single cell trapping capacity of the device, a number of cells can be trapped and held in separate wells for concurrent parallel experiments, allowing for the possibility of stepped parametric experiments and studying cell heterogeneity by combining measurements across the array.

Single cell studies of mouse embryonic stem cell (mESC) differentiation by electrical impedance measurements in a microfluidic device.

Biological populations of cells show considerable cell-to-cell variability. Study of single cells and analysis of cell heterogeneity are considered to be critical in understanding biological processes such as stem cell differentiation and cancer development. Recent advances in lab-on-a-chip techniques have allowed single-cell capture in microfluidic channels with the possibility of precise environmental control and high throughput of experiments with minimal usage of samples and reagents. In recent years, label-free techniques such as electrical impedance spectroscopy have emerged as a non-invasive approach to studying cell properties. In this study, we have designed and fabricated a microfluidic device that combines hydrodynamic trapping of single cells in pre-defined locations with the capability of running electrical impedance measurements within the same device. We have measured mouse embryonic stem cells (mESCs) at different states during differentiation (t=0h, 24h and 48h) and quantitatively analysed the changes in electrical parameters of cells during differentiation. A marked increase in the magnitude of the cell impedance is found during cell differentiation, which can be attributed to an increase in cell size. The analysis of the measurements shows that the nucleus-to-cytoplasm ratio decreases during this process. The degree of cell heterogeneity is observed to be the highest when the cells are at the transition state (24h), compare with cells at undifferentiated (0h) and fully differentiated (48h) states. The device enables highly efficient single cell trapping and provides sensitive, label-free electrical impedance measurements of individual cells, enabling the possibility of quantitatively analysing their physical state as well as studying the associated heterogeneity of a cell population.

Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.

The pentameric nucleoplasmin fold is present in Drosophila FKBP39 and a large number of chromatin-related proteins.

Nucleoplasmin is a histone chaperone that consists of a pentameric N-terminal domain and an unstructured C-terminal tail. The pentameric core domain, a doughnut-like structure with a central pore, is only found in the nucleoplasmin family. Here, we report the first structure of a nucleoplasmin-like domain (NPL) from the unrelated Drosophila protein, FKBP39, and we present evidence that this protein associates with chromatin. Furthermore, we show that two other chromatin proteins, Arabidopsis thaliana histone deacetylase type 2 (HD2) and Saccharomyces cerevisiae Fpr4, share the NPL fold and form pentamers, or a dimer of pentamers in the case of HD2. Thus, we propose a new family of proteins that share the pentameric nucleoplasmin-like NPL domain and are found in protists, fungi, plants and animals.

Structure calculation, refinement and validation using CcpNmr Analysis.

CcpNmr Analysis provides a streamlined pipeline for both NMR chemical shift assignment and structure determination of biological macromolecules. In addition, it encompasses tools to analyse the many additional experiments that make NMR such a pivotal technique for research into complex biological questions. This report describes how CcpNmr Analysis can seamlessly link together all of the tasks in the NMR structure-determination process. It details each of the stages from generating NMR restraints [distance, dihedral, hydrogen bonds and residual dipolar couplings (RDCs)], exporting these to and subsequently re-importing them from structure-calculation software (such as the programs CYANA or ARIA) and analysing and validating the results obtained from the structure calculation to, ultimately, the streamlined deposition of the completed assignments and the refined ensemble of structures into the PDBe repository. Until recently, such solution-structure determination by NMR has been quite a laborious task, requiring multiple stages and programs. However, with the new enhancements to CcpNmr Analysis described here, this process is now much more intuitive and efficient and less error-prone.

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.