Angiogenesis is uncoupled from osteogenesis during calvarial bone regeneration.

Bone regeneration requires a well-orchestrated cellular and molecular response including robust vascularization and recruitment of mesenchymal and osteogenic cells. In femoral fractures, angiogenesis and osteogenesis are closely coupled during the complex healing process. Here, we show with advanced longitudinal intravital multiphoton microscopy that early vascular sprouting is not directly coupled to osteoprogenitor invasion during calvarial bone regeneration. Early osteoprogenitors emerging from the periosteum give rise to bone-forming osteoblasts at the injured calvarial bone edge. Microvessels growing inside the lesions are not associated with osteoprogenitors. Subsequently, osteogenic cells collectively invade the vascularized and perfused lesion as a multicellular layer, thereby advancing regenerative ossification. Vascular sprouting and remodeling result in dynamic blood flow alterations to accommodate the growing bone. Single cell profiling of injured calvarial bones demonstrates mesenchymal stromal cell heterogeneity comparable to femoral fractures with increase in cell types promoting bone regeneration. Expression of angiogenesis and hypoxia-related genes are slightly elevated reflecting ossification of a vascularized lesion site. Endothelial Notch and VEGF signaling alter vascular growth in calvarial bone repair without affecting the ossification progress. Our findings may have clinical implications for bone regeneration and bioengineering approaches.

Eph-ephrin signaling couples endothelial cell sorting and arterial specification.

Cell segregation allows the compartmentalization of cells with similar fates during morphogenesis, which can be enhanced by cell fate plasticity in response to local molecular and biomechanical cues. Endothelial tip cells in the growing retina, which lead vessel sprouts, give rise to arterial endothelial cells and thereby mediate arterial growth. Here, we have combined cell type-specific and inducible mouse genetics, flow experiments in vitro, single-cell RNA sequencing and biochemistry to show that the balance between ephrin-B2 and its receptor EphB4 is critical for arterial specification, cell sorting and arteriovenous patterning. At the molecular level, elevated ephrin-B2 function after loss of EphB4 enhances signaling responses by the Notch pathway, VEGF and the transcription factor Dach1, which is influenced by endothelial shear stress. Our findings reveal how Eph-ephrin interactions integrate cell segregation and arteriovenous specification in the vasculature, which has potential relevance for human vascular malformations caused by EPHB4 mutations.

Single-cell transcriptomics reveals functionally specialized vascular endothelium in brain.

The blood-brain barrier (BBB) limits the entry of leukocytes and potentially harmful substances from the circulation into the central nervous system (CNS). While BBB defects are a hallmark of many neurological disorders, the cellular heterogeneity at the neurovascular interface, and the mechanisms governing neuroinflammation are not fully understood.Through single-cell RNA sequencing of non-neuronal cell populations of the murine cerebral cortex during development, adulthood, ageing, and neuroinflammation, we identify reactive endothelial venules, a compartment of specialized postcapillary endothelial cells that are characterized by consistent expression of cell adhesion molecules, preferential leukocyte transmigration, association with perivascular macrophage populations, and endothelial activation initiating CNS immune responses. Our results provide novel insights into the heterogeneity of the cerebral vasculature and a useful resource for the molecular alterations associated with neuroinflammation and ageing.

Induction of osteogenesis by bone-targeted Notch activation.

Declining bone mass is associated with aging and osteoporosis, a disease characterized by progressive weakening of the skeleton and increased fracture incidence. Growth and lifelong homeostasis of bone rely on interactions between different cell types including vascular cells and mesenchymal stromal cells (MSCs). As these interactions involve Notch signaling, we have explored whether treatment with secreted Notch ligand proteins can enhance osteogenesis in adult mice. We show that a bone-targeting, high affinity version of the ligand Delta-like 4, termed Dll4(E12), induces bone formation in male mice without causing adverse effects in other organs, which are known to rely on intact Notch signaling. Due to lower bone surface and thereby reduced retention of Dll4(E12), the same approach failed to promote osteogenesis in female and ovariectomized mice but strongly enhanced trabecular bone formation in combination with parathyroid hormone. Single cell analysis of stromal cells indicates that Dll4(E12) primarily acts on MSCs and has comparably minor effects on osteoblasts, endothelial cells, or chondrocytes. We propose that activation of Notch signaling by bone-targeted fusion proteins might be therapeutically useful and can avoid detrimental effects in Notch-dependent processes in other organs.

FAN-C: a feature-rich framework for the analysis and visualisation of chromosome conformation capture data.

Chromosome conformation capture data, particularly from high-throughput approaches such as Hi-C, are typically very complex to analyse. Existing analysis tools are often single-purpose, or limited in compatibility to a small number of data formats, frequently making Hi-C analyses tedious and time-consuming. Here, we present FAN-C, an easy-to-use command-line tool and powerful Python API with a broad feature set covering matrix generation, analysis, and visualisation for C-like data ( https://github.com/vaquerizaslab/fanc ). Due to its compatibility with the most prevalent Hi-C storage formats, FAN-C can be used in combination with a large number of existing analysis tools, thus greatly simplifying Hi-C matrix analysis.

CHESS enables quantitative comparison of chromatin contact data and automatic feature extraction.

Dynamic changes in the three-dimensional (3D) organization of chromatin are associated with central biological processes, such as transcription, replication and development. Therefore, the comprehensive identification and quantification of these changes is fundamental to understanding of evolutionary and regulatory mechanisms. Here, we present Comparison of Hi-C Experiments using Structural Similarity (CHESS), an algorithm for the comparison of chromatin contact maps and automatic differential feature extraction. We demonstrate the robustness of CHESS to experimental variability and showcase its biological applications on (1) interspecies comparisons of syntenic regions in human and mouse models; (2) intraspecies identification of conformational changes in Zelda-depleted Drosophila embryos; (3) patient-specific aberrant chromatin conformation in a diffuse large B-cell lymphoma sample; and (4) the systematic identification of chromatin contact differences in high-resolution Capture-C data. In summary, CHESS is a computationally efficient method for the comparison and classification of changes in chromatin contact data.

Heterochromatin establishment during early mammalian development is regulated by pericentromeric RNA and characterized by non-repressive H3K9me3.

Following fertilization in mammals, the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodelling of constitutive heterochromatin, although the functional relevance of this is unknown. Here, we show that heterochromatin establishment relies on the stepwise expression and regulated activity of SUV39H enzymes. Enforcing precocious acquisition of constitutive heterochromatin results in compromised development and epigenetic reprogramming, which demonstrates that heterochromatin remodelling is essential for natural reprogramming at fertilization. We find that de novo H3K9 trimethylation (H3K9me3) in the paternal pronucleus after fertilization is catalysed by SUV39H2 and that pericentromeric RNAs inhibit SUV39H2 activity and reduce H3K9me3. De novo H3K9me3 is initially non-repressive for gene expression, but instead bookmarks promoters for compaction. Overall, we uncover the functional importance for the restricted transmission of constitutive heterochromatin during reprogramming and a non-repressive role for H3K9me3.

Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells.

How chromosome organization is related to genome function remains poorly understood. Cohesin, loop extrusion, and CCCTC-binding factor (CTCF) have been proposed to create topologically associating domains (TADs) to regulate gene expression. Here, we examine chromosome conformation in embryonic stem cells lacking cohesin and find, as in other cell types, that cohesin is required to create TADs and regulate A/B compartmentalization. However, in the absence of cohesin, we identify a series of long-range chromosomal interactions that persist. These correspond to regions of the genome occupied by the polycomb repressive system and are dependent on PRC1. Importantly, we discover that cohesin counteracts these polycomb-dependent interactions, but not interactions between super-enhancers. This disruptive activity is independent of CTCF and insulation and appears to modulate gene repression by the polycomb system. Therefore, we discover that cohesin disrupts polycomb-dependent chromosome interactions to modulate gene expression in embryonic stem cells.

Pump-probe laser system at the FXE and SPB/SFX instruments of the European X-ray Free-Electron Laser Facility.

User operation at the European X-ray Free-Electron Laser Facility started at the SASE1 undulator beamline in fall 2017. The majority of the experiments utilize optical lasers (mostly ultrafast) for pump-probe-type measurements in combination with X-ray pulses. This manuscript describes the purpose-developed pump-probe laser system as installed at SASE1, implemented features and plans for further upgrades.

Chromatin conformation analysis of primary patient tissue using a low input Hi-C method.

Chromatin conformation constitutes a fundamental level of eukaryotic genome regulation. However, our ability to examine its biological function and role in disease is limited by the large amounts of starting material required to perform current experimental approaches. Here, we present Low-C, a Hi-C method for low amounts of input material. By systematically comparing Hi-C libraries made with decreasing amounts of starting material we show that Low-C is highly reproducible and robust to experimental noise. To demonstrate the suitability of Low-C to analyse rare cell populations, we produce Low-C maps from primary B-cells of a diffuse large B-cell lymphoma patient. We detect a common reciprocal translocation t(3;14)(q27;q32) affecting the BCL6 and IGH loci and abundant local structural variation between the patient and healthy B-cells. The ability to study chromatin conformation in primary tissue will be fundamental to fully understand the molecular pathogenesis of diseases and to eventually guide personalised therapeutic strategies.

A molecular roadmap for the emergence of early-embryonic-like cells in culture.

Unlike pluripotent cells, which generate only embryonic tissues, totipotent cells can generate a full organism, including extra-embryonic tissues. A rare population of cells resembling 2-cell-stage embryos arises in pluripotent embryonic stem (ES) cell cultures. These 2-cell-like cells display molecular features of totipotency and broader developmental plasticity. However, their specific nature and the process through which they arise remain outstanding questions. Here we identified intermediate cellular states and molecular determinants during the emergence of 2-cell-like cells. By deploying a quantitative single-cell expression approach, we identified an intermediate population characterized by expression of the transcription factor ZSCAN4 as a precursor of 2-cell-like cells. By using a small interfering RNA (siRNA) screen, we identified epigenetic regulators of 2-cell-like cell emergence, including the non-canonical PRC1 complex PRC1.6 and the EP400-TIP60 complex. Our data shed light on the mechanisms that underlie exit from the ES cell state toward the formation of early-embryonic-like cells in culture and identify key epigenetic pathways that promote this transition.

Chromatin Architecture Emerges during Zygotic Genome Activation Independent of Transcription.

Chromatin architecture is fundamental in regulating gene expression. To investigate when spatial genome organization is first established during development, we examined chromatin conformation during Drosophila embryogenesis and observed the emergence of chromatin architecture within a tight time window that coincides with the onset of transcription activation in the zygote. Prior to zygotic genome activation, the genome is mostly unstructured. Early expressed genes serve as nucleation sites for topologically associating domain (TAD) boundaries. Activation of gene expression coincides with the establishment of TADs throughout the genome and co-localization of housekeeping gene clusters, which remain stable in subsequent stages of development. However, the appearance of TAD boundaries is independent of transcription and requires the transcription factor Zelda for locus-specific TAD boundary insulation. These results offer insight into when spatial organization of the genome emerges and identify a key factor that helps trigger this architecture.

TADtool: visual parameter identification for TAD-calling algorithms.

Eukaryotic genomes are hierarchically organized into topologically associating domains (TADs). The computational identification of these domains and their associated properties critically depends on the choice of suitable parameters of TAD-calling algorithms. To reduce the element of trial-and-error in parameter selection, we have developed TADtool: an interactive plot to find robust TAD-calling parameters with immediate visual feedback. TADtool allows the direct export of TADs called with a chosen set of parameters for two of the most common TAD calling algorithms: directionality and insulation index. It can be used as an intuitive, standalone application or as a Python package for maximum flexibility. AVAILABILITY AND IMPLEMENTATION: TADtool is available as a Python package from GitHub (https://github.com/vaquerizaslab/tadtool) or can be installed directly via PyPI, the Python package index (tadtool). CONTACT: kai.kruse@mpi-muenster.mpg.de, jmv@mpi-muenster.mpg.deSupplementary information: Supplementary data are available at Bioinformatics online.

Intrinsically disordered segments affect protein half-life in the cell and during evolution.

Precise control of protein turnover is essential for cellular homeostasis. The ubiquitin-proteasome system is well established as a major regulator of protein degradation, but an understanding of how inherent structural features influence the lifetimes of proteins is lacking. We report that yeast, mouse, and human proteins with terminal or internal intrinsically disordered segments have significantly shorter half-lives than proteins without these features. The lengths of the disordered segments that affect protein half-life are compatible with the structure of the proteasome. Divergence in terminal and internal disordered segments in yeast proteins originating from gene duplication leads to significantly altered half-life. Many paralogs that are affected by such changes participate in signaling, where altered protein half-life will directly impact cellular processes and function. Thus, natural variation in the length and position of disordered segments may affect protein half-life and could serve as an underappreciated source of genetic variation with important phenotypic consequences.

A complex network framework for unbiased statistical analyses of DNA-DNA contact maps.

Experimental techniques for the investigation of three-dimensional (3D) genome organization are being developed at a fast pace. Currently, the associated computational methods are mostly specific to the individual experimental approach. Here we present a general statistical framework that is widely applicable to the analysis of genomic contact maps, irrespective of the data acquisition and normalization processes. Within this framework DNA-DNA contact data are represented as a complex network, for which a broad number of directly applicable methods already exist. In such a network representation, DNA segments and contacts between them are denoted as nodes and edges, respectively. Furthermore, we present a robust method for generating randomized contact networks that explicitly take into account the inherent 3D nature of the genome and serve as realistic null-models for unbiased statistical analyses. By integrating a variety of large-scale genome-wide datasets we demonstrate that meiotic crossover sites display enriched genomic contacts and that cohesin-bound genes are significantly colocalized in the yeast nucleus. We anticipate that the complex network framework in conjunction with the randomization of DNA-DNA contact networks will become a widely used tool in the study of nuclear architecture.

Comparing flux balance analysis to network expansion: producibility, sustainability and the scope of compounds.

The producibility of metabolites from available resources is investigated systematically using flux balance analysis (FBA) and network expansion. Calculations are performed for the genome-scale metabolic networks of Escherichia coli and Methanosarcina barkeri. Strict biological interpretation of the results obtained with FBA leads to the concept of sustainability, which reduces the set of producible metabolites by assuming a growing and dividing cell. A systematic comparison showed that applying network expansion in many cases results in exactly the set of all sustainable metabolites. The purely heuristic approach of allowing for certain cofactors to facilitate reactions during the process of network expansion dramatically helps to improve agreement of the results from the two different approaches. In conclusion, we state that network expansion, due to its enormous advantages in computational speed, is a valuable alternative to determining producible metabolites with FBA.