Nucleosome spacing controls chromatin spatial structure and accessibility.

Recent research highlights the significance of the three-dimensional structure of chromatin in regulating various cellular processes, particularly transcription. This is achieved through dynamic chromatin structures that facilitate long-range contacts and control spatial accessibility. Chromatin consists of DNA and a variety of proteins, of which histones play an essential structural role by forming nucleosomes. Extensive experimental and theoretical research in recent decades has yielded conflicting results about key factors that regulate the spatial structure of chromatin, which remains enigmatic. By using a computer model that allows us to simulate chromatin volumes containing physiological nucleosome concentrations, we investigated whether nucleosome spacing or nucleosome density is fundamental for three-dimensional chromatin accessibility. Unexpectedly, the regularity of the nucleosome spacing is crucial for determining the accessibility of the chromatin network to diffusive processes, whereas variation in nucleosome concentrations has only minor effects. Using only the basic physical properties of DNA and nucleosomes was sufficient to generate chromatin structures consistent with published electron microscopy data. Contrary to other work, we found that nucleosome density did not substantially alter the properties of chromatin fibers or contact probabilities of genomic loci. No breakup of fiber-like structures was observed at high molar density. These findings challenge previous assumptions and highlight the importance of nucleosome spacing as a key driver of chromatin organization. These results identified changes in nucleosome spacing as a tentative mechanism for altering the spatial chromatin structure and thus genomic functions.

Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor.

Cellular senescence is acknowledged as a key contributor to organismal ageing and late-life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro-inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent-like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity.

Regulatory evolution tuning pigmentation intensity quantitatively in Drosophila.

Quantitative variation in attributes such as color, texture, or stiffness dominates morphological diversification. It results from combinations of alleles at many Mendelian loci. Here, we identify an additional source of quantitative variation among species, continuous evolution in a gene regulatory region. Specifically, we examined the modulation of wing pigmentation in a group of fly species and showed that inter-species variation correlated with the quantitative expression of the pigmentation gene yellow. This variation results from an enhancer of yellow determining darkness through species-specific activity. We mapped the divergent activities between two sister species and found the changes to be broadly distributed along the enhancer. Our results demonstrate that enhancers can act as dials fueling quantitative morphological diversification by modulating trait properties.

IKKß primes inflammasome formation by recruiting NLRP3 to the trans-Golgi network.

The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: the first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited. Here, we show that IKKß, a kinase activated during priming, induces recruitment of NLRP3 to phosphatidylinositol-4-phosphate (PI4P), a phospholipid enriched on the trans-Golgi network. NEK7, a mitotic spindle kinase that had previously been thought to be indispensable for NLRP3 activation, was redundant for inflammasome formation when IKKß recruited NLRP3 to PI4P. Studying iPSC-derived human macrophages revealed that the IKKß-mediated NEK7-independent pathway constitutes the predominant NLRP3 priming mechanism in human myeloid cells. Our results suggest that PI4P binding represents a primed state into which NLRP3 is brought by IKKß activity.

Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling.

Many life-science techniques and assays rely on selective labeling of biological target structures with commercial fluorophores that have specific yet invariant properties. Consequently, a fluorophore (or dye) is only useful for a limited range of applications, e.g., as a label for cellular compartments, super-resolution imaging, DNA sequencing or for a specific biomedical assay. Modifications of fluorophores with the goal to alter their bioconjugation chemistry, photophysical or functional properties typically require complex synthesis schemes. We here introduce a general strategy that allows to customize these properties during biolabelling with the goal to introduce the fluorophore in the last step of biolabelling. For this, we present the design and synthesis of 'linker' compounds, that bridge biotarget, fluorophore and a functional moiety via well-established labeling protocols. Linker molecules were synthesized via the Ugi four-component reaction (Ugi-4CR) which facilitates a modular design of linkers with diverse functional properties and bioconjugation- and fluorophore attachment moieties. To demonstrate the possibilities of different linkers experimentally, we characterized the ability of commercial fluorophores from the classes of cyanines, rhodamines, carbopyronines and silicon-rhodamines to become functional labels on different biological targets in vitro and in vivo via thiol-maleimide chemistry. With our strategy, we showed that the same commercial dye can become a photostable self-healing dye or a sensor for bivalent ions subject to the linker used. Finally, we quantified the photophysical performance of different self-healing linker-fluorophore conjugates and demonstrated their applications in super-resolution imaging and single-molecule spectroscopy.

Differences in nanoscale organization of regulatory active and inactive human chromatin.

Methodological advances in conformation capture techniques have fundamentally changed our understanding of chromatin architecture. However, the nanoscale organization of chromatin and its cell-to-cell variance are less studied. Analyzing genome-wide data from 733 human cell and tissue samples, we identified 2 prototypical regions that exhibit high or absent hypersensitivity to deoxyribonuclease I, respectively. These regulatory active or inactive regions were examined in the lymphoblast cell line K562 by using high-throughput super-resolution microscopy. In both regions, we systematically measured the physical distance of 2 fluorescence in situ hybridization spots spaced by only 5 kb of DNA. Unexpectedly, the resulting distance distributions range from very compact to almost elongated configurations of more than 200-nm length for both the active and inactive regions. Monte Carlo simulations of a coarse-grained model of these chromatin regions based on published data of nucleosome occupancy in K562 cells were performed to understand the underlying mechanisms. There was no parameter set for the simulation model that can explain the microscopically measured distance distributions. Obviously, the chromatin state given by the strength of internucleosomal interaction, nucleosome occupancy, or amount of histone H1 differs from cell to cell, which results in the observed broad distance distributions. This large variability was not expected, especially in inactive regions. The results for the mechanisms for different distance distributions on this scale are important for understanding the contacts that mediate gene regulation. Microscopic measurements show that the inactive region investigated here is expected to be embedded in a more compact chromatin environment. The simulation results of this region require an increase in the strength of internucleosomal interactions. It may be speculated that the higher density of chromatin is caused by the increased internucleosomal interaction strength.

Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy.

Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site-specific modification of proteins. Antibody-oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence-encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub-tag® technology in combination with Cu-catalyzed azide-alkyne cycloaddition for the site-specific conjugation of single DNA and PNA strands to an eGFP-binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence-specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP-tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody-oligonucleotides for reversible immunofluorescence imaging.

Cohesin depleted cells rebuild functional nuclear compartments after endomitosis.

Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.

Regulatory encoding of quantitative variation in spatial activity of a Drosophila enhancer.

Developmental enhancers control the expression of genes prefiguring morphological patterns. The activity of an enhancer varies among cells of a tissue, but collectively, expression levels in individual cells constitute a spatial pattern of gene expression. How the spatial and quantitative regulatory information is encoded in an enhancer sequence is elusive. To link spatial pattern and activity levels of an enhancer, we used systematic mutations of the yellow spot enhancer, active in developing Drosophila wings, and tested their effect in a reporter assay. Moreover, we developed an analytic framework based on the comprehensive quantification of spatial reporter activity. We show that the quantitative enhancer activity results from densely packed regulatory information along the sequence, and that a complex interplay between activators and multiple tiers of repressors carves the spatial pattern. Our results shed light on how an enhancer reads and integrates trans-regulatory landscape information to encode a spatial quantitative pattern.

BigStitcher: reconstructing high-resolution image datasets of cleared and expanded samples.

Light-sheet imaging of cleared and expanded samples creates terabyte-sized datasets that consist of many unaligned three-dimensional image tiles, which must be reconstructed before analysis. We developed the BigStitcher software to address this challenge. BigStitcher enables interactive visualization, fast and precise alignment, spatially resolved quality estimation, real-time fusion and deconvolution of dual-illumination, multitile, multiview datasets. The software also compensates for optical effects, thereby improving accuracy and enabling subsequent biological analysis.

Nanoparticle mediated delivery and small molecule triggered activation of proteins in the nucleus.

Protein transfection is a versatile tool to study or manipulate cellular processes and also shows great therapeutic potential. However, the repertoire of cost effective techniques for efficient and minimally cytotoxic delivery remains limited. Mesoporous silica nanoparticles (MSNs) are multifunctional nanocarriers for cellular delivery of a wide range of molecules, they are simple and economical to synthesize and have shown great promise for protein delivery. In this work we present a general strategy to optimize the delivery of active protein to the nucleus. We generated a bimolecular Venus based optical sensor that exclusively detects active and bioavailable protein for the performance of multi-parameter optimization of protein delivery. In conjunction with cell viability tests we maximized MSN protein delivery and biocompatibility and achieved highly efficient protein transfection rates of 80%. Using the sensor to measure live-cell protein delivery kinetics, we observed heterogeneous timings within cell populations which could have a confounding effect on function studies. To address this problem we fused a split or dimerization dependent protein of interest to chemically induced dimerization (CID) components, permitting control over its activity following cellular delivery. Using the split Venus protein we directly show that addition of a small molecule dimerizer causes synchronous activation of the delivered protein across the entire cell population. This combination of cellular delivery and triggered activation provides a defined starting point for functional studies and could be applied to other protein transfection methods.

Data-Driven Modeling of Intracellular Auxin Fluxes Indicates a Dominant Role of the ER in Controlling Nuclear Auxin Uptake.

In plants, the phytohormone auxin acts as a master regulator of developmental processes and environmental responses. The best characterized process in the auxin regulatory network occurs at the subcellular scale, wherein auxin mediates signal transduction into transcriptional programs by triggering the degradation of Aux/IAA transcriptional repressor proteins in the nucleus. However, whether and how auxin movement between the nucleus and the surrounding compartments is regulated remain elusive. Using a fluorescent auxin analog, we show that its diffusion into the nucleus is restricted. By combining mathematical modeling with time course assays on auxin-mediated nuclear signaling and quantitative phenotyping in single plant cell systems, we show that ER-to-nucleus auxin flux represents a major subcellular pathway to directly control nuclear auxin levels. Our findings propose that the homeostatically regulated auxin pool in the ER and ER-to-nucleus auxin fluxes underpin auxin-mediated downstream responses in plant cells.

In vitro and in vivo delivery of siRNA via VIPER polymer system to lung cells.

The block copolymer VIPER (virus-inspired polymer for endosomal release) has been reported to be a promising novel delivery system of DNA plasmids both in vitro and in vivo. VIPER is comprised of a polycation segment for condensation of nucleic acids as well as a pH-sensitive segment that exposes the membrane lytic peptide melittin in acidic environments to facilitate endosomal escape. The objective of this study was to investigate VIPER/siRNA polyplex characteristics, and compare their in vitro and in vivo performance with commercially available transfection reagents and a control version of VIPER lacking melittin. VIPER/siRNA polyplexes were formulated and characterized at various charge ratios and shown to be efficiently internalized in cultured cells. Target mRNA knockdown was confirmed by both flow cytometry and qRT-PCR and the kinetics of knockdown was monitored by live cell spinning disk microscopy, revealing knockdown starting by 4 h post-delivery. Intratracheal instillation of VIPER particles formulated with sequence specific siRNA to the lung of mice resulted in a significantly more efficient knockdown of GAPDH compared to treatment with VIPER particles formulated with scrambled sequence siRNA. We also demonstrated using pH-sensitive labels that VIPER particles experience less acidic environments compared to control polyplexes. In summary, VIPER/siRNA polyplexes efficiently deliver siRNA in vivo resulting in robust gene silencing (>75% knockdown) within the lung.

Desmoglein 2 regulates the intestinal epithelial barrier via p38 mitogen-activated protein kinase.

Intestinal epithelial barrier properties are maintained by a junctional complex consisting of tight junctions (TJ), adherens junctions (AJ) and desmosomes. Desmoglein 2 (Dsg2), an adhesion molecule of desmosomes and the only Dsg isoform expressed in enterocytes, is required for epithelial barrier properties and may contribute to barrier defects in Crohn's disease. Here, we identified extradesmosomal Dsg2 on the surface of polarized enterocytes by Triton extraction, confocal microscopy, SIM and STED. Atomic force microscopy (AFM) revealed Dsg2-specific binding events along the cell border on the surface of enterocytes with a mean unbinding force of around 30pN. Binding events were blocked by an inhibitory antibody targeting Dsg2 which under same conditions activated p38MAPK but did not reduce cell cohesion. In enterocytes deficient for Dsg2, p38MAPK activity was reduced and both barrier integrity and reformation were impaired. Dsc2 rescue did not restore p38MAPK activity indicating that Dsg2 is required. Accordingly, direct activation of p38MAPK in Dsg2-deficient cells enhanced barrier reformation demonstrating that Dsg2-mediated activation of p38MAPK is crucial for barrier function. Collectively, our data show that Dsg2, beside its adhesion function, regulates intestinal barrier function via p38MAPK signalling. This is in contrast to keratinocytes and points towards tissue-specific signalling functions of desmosomal cadherins.

Identification of the elementary structural units of the DNA damage response.

Histone H2AX phosphorylation is an early signalling event triggered by DNA double-strand breaks (DSBs). To elucidate the elementary units of phospho-H2AX-labelled chromatin, we integrate super-resolution microscopy of phospho-H2AX during DNA repair in human cells with genome-wide sequencing analyses. Here we identify phospho-H2AX chromatin domains in the nanometre range with median length of ∼75 kb. Correlation analysis with over 60 genomic features shows a time-dependent euchromatin-to-heterochromatin repair trend. After X-ray or CRISPR-Cas9-mediated DSBs, phospho-H2AX-labelled heterochromatin exhibits DNA decondensation while retaining heterochromatic histone marks, indicating that chromatin structural and molecular determinants are uncoupled during repair. The phospho-H2AX nano-domains arrange into higher-order clustered structures of discontinuously phosphorylated chromatin, flanked by CTCF. CTCF knockdown impairs spreading of the phosphorylation throughout the 3D-looped nano-domains. Co-staining of phospho-H2AX with phospho-Ku70 and TUNEL reveals that clusters rather than nano-foci represent single DSBs. Hence, each chromatin loop is a nano-focus, whose clusters correspond to previously known phospho-H2AX foci.

Chimeric PD-1:28 Receptor Upgrades Low-Avidity T cells and Restores Effector Function of Tumor-Infiltrating Lymphocytes for Adoptive Cell Therapy.

Inherent intermediate- to low-affinity T-cell receptors (TCR) that develop during the natural course of immune responses may not allow sufficient activation for tumor elimination, making the majority of T cells suboptimal for adoptive T-cell therapy (ATT). TCR affinity enhancement has been implemented to provide stronger T-cell activity but carries the risk of creating undesired cross-reactivity leading to potential serious adverse effects in clinical application. We demonstrate here that engineering of low-avidity T cells recognizing a naturally processed and presented tumor-associated antigen with a chimeric PD-1:28 receptor increases effector function to levels seen with high-avidity T cells of identical specificity. Upgrading the function of low-avidity T cells without changing the TCR affinity will allow a large arsenal of low-avidity T cells previously thought to be therapeutically inefficient to be considered for ATT. PD-1:28 engineering reinstated Th1 function in tumor-infiltrating lymphocytes that had been functionally disabled in the human renal cell carcinoma environment without unleashing undesired Th2 cytokines or IL10. Involved mechanisms may be correlated to restoration of ERK and AKT signaling pathways. In mouse tumor models of ATT, PD-1:28 engineering enabled low-avidity T cells to proliferate stronger and prevented PD-L1 upregulation and Th2 polarization in the tumor milieu. Engineered T cells combined with checkpoint blockade secreted significantly more IFNγ compared with T cells without PD-1:28, suggesting a beneficial combination with checkpoint blockade therapy or other therapeutic strategies. Altogether, the supportive effects of PD-1:28 engineering on T-cell function make it an attractive tool for ATT. Cancer Res; 77(13); 3577-90. ©2017 AACR.

Universal Super-Resolution Multiplexing by DNA Exchange.

Super-resolution microscopy allows optical imaging below the classical diffraction limit of light with currently up to 20× higher spatial resolution. However, the detection of multiple targets (multiplexing) is still hard to implement and time-consuming to conduct. Here, we report a straightforward sequential multiplexing approach based on the fast exchange of DNA probes which enables efficient and rapid multiplexed target detection with common super-resolution techniques such as (d)STORM, STED, and SIM. We assay our approach using DNA origami nanostructures to quantitatively assess labeling, imaging, and washing efficiency. We furthermore demonstrate the applicability of our approach by imaging multiple protein targets in fixed cells.

Calcium spikes, waves and oscillations in a large, patterned epithelial tissue.

While calcium signaling in excitable cells, such as muscle or neurons, is extensively characterized, calcium signaling in epithelial tissues is little understood. Specifically, the range of intercellular calcium signaling patterns elicited by tightly coupled epithelial cells and their function in the regulation of epithelial characteristics are little explored. We found that in Drosophila imaginal discs, a widely studied epithelial model organ, complex spatiotemporal calcium dynamics occur. We describe patterns that include intercellular waves traversing large tissue domains in striking oscillatory patterns as well as spikes confined to local domains of neighboring cells. The spatiotemporal characteristics of intercellular waves and oscillations arise as emergent properties of calcium mobilization within a sheet of gap-junction coupled cells and are influenced by cell size and environmental history. While the in vivo function of spikes, waves and oscillations requires further characterization, our genetic experiments suggest that core calcium signaling components guide actomyosin organization. Our study thus suggests a possible role for calcium signaling in epithelia but importantly, introduces a model epithelium enabling the dissection of cellular mechanisms supporting the initiation, transmission and regeneration of long-range intercellular calcium waves and the emergence of oscillations in a highly coupled multicellular sheet.

Interface Contractility between Differently Fated Cells Drives Cell Elimination and Cyst Formation.

Although cellular tumor-suppression mechanisms are widely studied, little is known about mechanisms that act at the level of tissues to suppress the occurrence of aberrant cells in epithelia. We find that ectopic expression of transcription factors that specify cell fates causes abnormal epithelial cysts in Drosophila imaginal discs. Cysts do not form cell autonomously but result from the juxtaposition of two cell populations with divergent fates. Juxtaposition of wild-type and aberrantly specified cells induces enrichment of actomyosin at their entire shared interface, both at adherens junctions as well as along basolateral interfaces. Experimental validation of 3D vertex model simulations demonstrates that enhanced interface contractility is sufficient to explain many morphogenetic behaviors, which depend on cell cluster size. These range from cyst formation by intermediate-sized clusters to segregation of large cell populations by formation of smooth boundaries or apical constriction in small groups of cells. In addition, we find that single cells experiencing lateral interface contractility are eliminated from tissues by apoptosis. Cysts, which disrupt epithelial continuity, form when elimination of single, aberrantly specified cells fails and cells proliferate to intermediate cell cluster sizes. Thus, increased interface contractility functions as error correction mechanism eliminating single aberrant cells from tissues, but failure leads to the formation of large, potentially disease-promoting cysts. Our results provide a novel perspective on morphogenetic mechanisms, which arise from cell-fate heterogeneities within tissues and maintain or disrupt epithelial homeostasis.

DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination.

DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.