The relationship between nanoscale genome organization and gene expression in mouse embryonic stem cells during pluripotency transition.

During early development, gene expression is tightly regulated. However, how genome organization controls gene expression during the transition from naïve embryonic stem cells to epiblast stem cells is still poorly understood. Using single-molecule microscopy approaches to reach nanoscale resolution, we show that genome remodeling affects gene transcription during pluripotency transition. Specifically, after exit from the naïve pluripotency state, chromatin becomes less compacted, and the OCT4 transcription factor has lower mobility and is more bound to its cognate sites. In epiblast cells, the active transcription hallmark, H3K9ac, decreases within the Oct4 locus, correlating with reduced accessibility of OCT4 and, in turn, with reduced expression of Oct4 nascent RNAs. Despite the high variability in the distances between active pluripotency genes, distances between Nodal and Oct4 decrease during epiblast specification. In particular, highly expressed Oct4 alleles are closer to nuclear speckles during all stages of the pluripotency transition, while only a distinct group of highly expressed Nodal alleles are in close proximity to Oct4 when associated with a nuclear speckle in epiblast cells. Overall, our results provide new insights into the role of the spatiotemporal genome remodeling during mouse pluripotency transition and its correlation with the expression of key pluripotency genes.

Transcription-mediated supercoiling regulates genome folding and loop formation.

The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.

Distinct properties and functions of CTCF revealed by a rapidly inducible degron system.

CCCTC-binding factor (CTCF) is a conserved zinc finger transcription factor implicated in a wide range of functions, including genome organization, transcription activation, and elongation. To explore the basis for CTCF functional diversity, we coupled an auxin-induced degron system with precision nuclear run-on. Unexpectedly, oriented CTCF motifs in gene bodies are associated with transcriptional stalling in a manner independent of bound CTCF. Moreover, CTCF at different binding sites (CBSs) displays highly variable resistance to degradation. Motif sequence does not significantly predict degradation behavior, but location at chromatin boundaries and chromatin loop anchors, as well as co-occupancy with cohesin, are associated with delayed degradation. Single-molecule tracking experiments link chromatin residence time to CTCF degradation kinetics, which has ramifications regarding architectural CTCF functions. Our study highlights the heterogeneity of CBSs, uncovers properties specific to architecturally important CBSs, and provides insights into the basic processes of genome organization and transcription regulation.

Mesoscale Modeling and Single-Nucleosome Tracking Reveal Remodeling of Clutch Folding and Dynamics in Stem Cell Differentiation.

Nucleosomes form heterogeneous groups in vivo, named clutches. Clutches are smaller and less dense in mouse embryonic stem cells (ESCs) compared to neural progenitor cells (NPCs). Using coarse-grained modeling of the pluripotency Pou5f1 gene, we show that the genome-wide clutch differences between ESCs and NPCs can be reproduced at a single gene locus. Larger clutch formation in NPCs is associated with changes in the compaction and internucleosome contact probability of the Pou5f1 fiber. Using single-molecule tracking (SMT), we further show that the core histone protein H2B is dynamic, and its local mobility relates to the structural features of the chromatin fiber. H2B is less stable and explores larger areas in ESCs compared to NPCs. The amount of linker histone H1 critically affects local H2B dynamics. Our results have important implications for how nucleosome organization and H2B dynamics contribute to regulate gene activity and cell identity.

Two-parameter single-molecule analysis for measurement of chromatin mobility.

This protocol provides a two-parameter analysis of single-molecule tracking (SMT) trajectories of Halo-tagged histones in living adherent cell lines and unveils a chromatin mobility landscape composed of five chromatin types, ranging from low to high mobility. When the analysis is applied to Halo-tagged, chromatin-binding proteins, it associates chromatin interaction properties with known functions in a way that previously used SMT parameters did not. For complete information on the use and execution of this protocol, please refer to Lerner et al. (2020).

Two-Parameter Mobility Assessments Discriminate Diverse Regulatory Factor Behaviors in Chromatin.

Enzymatic probes of chromatin structure reveal accessible versus inaccessible chromatin states, while super-resolution microscopy reveals a continuum of chromatin compaction states. Characterizing histone H2B movements by single-molecule tracking (SMT), we resolved chromatin domains ranging from low to high mobility and displaying different subnuclear localizations patterns. Heterochromatin constituents correlated with the lowest mobility chromatin, whereas transcription factors varied widely with regard to their respective mobility with low- or high-mobility chromatin. Pioneer transcription factors, which bind nucleosomes, can access the low-mobility chromatin domains, whereas weak or non-nucleosome binding factors are excluded from the domains and enriched in higher mobility domains. Nonspecific DNA and nucleosome binding accounted for most of the low mobility of strong nucleosome interactor FOXA1. Our analysis shows how the parameters of the mobility of chromatin-bound factors, but not their diffusion behaviors or SMT-residence times within chromatin, distinguish functional characteristics of different chromatin-interacting proteins.

Super-resolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo.

Chromatin organization is crucial for regulating gene expression. Previously, we showed that nucleosomes form groups, termed clutches. Clutch size correlated with the pluripotency grade of mouse embryonic stem cells and human induced pluripotent stem cells. Recently, it was also shown that regions of the chromatin containing activating epigenetic marks were composed of small and dispersed chromatin nanodomains with lower DNA density compared to the larger silenced domains. Overall, these results suggest that clutch size may regulate DNA packing density and gene activity. To directly test this model, we carried out 3D, two-color super-resolution microscopy of histones and DNA with and without increased histone tail acetylation. Our results showed that lower percentage of DNA was associated with nucleosome clutches in hyperacetylated cells. We further showed that the radius and compaction level of clutch-associated DNA decreased in hyperacetylated cells, especially in regions containing several neighboring clutches. Importantly, this change was independent of clutch size but dependent on the acetylation state of the clutch. Our results directly link the epigenetic state of nucleosome clutches to their DNA packing density. Our results further provide in vivo support to previous in vitro models that showed a disruption of nucleosome-DNA interactions upon hyperacetylation.

Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT.

Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore's response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as "frequency multiplexed DNA-PAINT," or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.

(Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes.

CRISPR/dCas9-based labeling has allowed direct visualization of genomic regions in living cells. However, poor labeling efficiency and signal-to-background ratio have limited its application to visualize genome organization using super-resolution microscopy. We developed (Po)STAC (Polycistronic SunTAg modified CRISPR) by combining CRISPR/dCas9 with SunTag labeling and polycistronic vectors. (Po)STAC enhances both labeling efficiency and fluorescence signal detected from labeled loci enabling live cell imaging as well as super-resolution fixed-cell imaging of multiple genes with high spatiotemporal resolution.

Fluorescence analysis of a tumor model in the chorioallantoic membrane used for the evaluation of different photosensitizers for photodynamic therapy.

The development of a tumor in the chicken chorioallantoic membrane (CAM) enables a more individualized understanding of the dynamics of the photosensitizer (PS) interaction with the tumor blood vessels and cells. Photogem® and 5-aminolevulinic acid (ALA), a protoporphyrin IX (PpIX) precursor, were used as PS and their red fluorescence enabled the monitoring of PS dynamic distribution in the vessels and in the tumor. With a tumor model in CAM and fluorescence assessment, the aim of this study was to evaluate the PDT parameters comparing different photosensitezers. In this model, the topical application was chosen as the best way of drug delivery and widefield fluorescence images were at every 30min. The images were processed in a MATLAB® routine for a semi-quantitative analysis of the red fluorescence. PpIX formation in the blood vessels and in the tumor region was observed after 3h and 1.5h, respectively, whereas Photogem® was accumulated in the tumor region after 2h. The illumination was performed by a diode laser with emission centered at 635nm and irradiance of 80mW/cm2 for 10min. After PDT irradiation, the photobleaching for both compounds was observed. Photogem® showed a reduced photobleaching, however, both PS induced a destruction of the tumor mass and vascular network in the treated area.