Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions

Even odder after twenty-three years : the superconducting order parameter puzzle of Sr2RuO4

Funding: Max Planck Society.In this short review, we aim to provide a topical update on the status of efforts to understand the superconductivity of Sr2RuO4. We concentrate on the quest to identify a superconducting order parameter symmetry that is compatible with all the major pieces of experimental knowledge of the material, and highlight some major discrepancies that have become even clearer in recent years. As the pun in the title suggests, we have tried to start the discussion from scratch, making no assumptions even about fundamental issues such as the parity of the superconducting state. We conclude that no consensus is currently achievable in Sr2RuO4, and that the reasons for this go to the heart of how well some of the key probes of unconventional superconductivity are really understood. This is therefore a puzzle that merits continued in-depth study.Publisher PDFPeer reviewe

Hydrodynamic electron flow and Hall viscosity

The authors acknowledge support from the Emergent Phenomena in Quantum Systems initiative of the Gordon and Betty Moore Foundation (T. S.) and NSF DMR-1507141 and a Simons Investigatorship (J. E. M.). We also acknowledge the support of the Max Planck Society and the UK Engineering and Physical Sciences Research Council under Grant No. EP/I032487/1.In metallic samples of small enough size and sufficiently strong momentum-conserving scattering, the viscosity of the electron gas can become the dominant process governing transport. In this regime, momentum is a long-lived quantity whose evolution is described by an emergent hydrodynamical theory. Furthermore, breaking time-reversal symmetry leads to the appearance of an odd component to the viscosity called the Hall viscosity, which has attracted considerable attention recently due to its quantized nature in gapped systems but still eludes experimental confirmation. Based on microscopic calculations, we discuss how to measure the effects of both the even and odd components of the viscosity using hydrodynamic electronic transport in mesoscopic samples under applied magnetic fields.PostprintPeer reviewe

Strong peak in Tc of Sr2RuO4 under uniaxial pressure

Sr2RuO4 is an unconventional superconductor that has attracted widespread study because of its high purity and the possibility that its superconducting order parameter has odd parity. We study the dependence of its superconductivity on anisotropic strain. Applying uniaxial pressures of up to ~1 gigapascals along a 〈100〉 direction (a axis) of the crystal lattice results in the transition temperature (Tc) increasing from 1.5 kelvin in the unstrained material to 3.4 kelvin at compression by ≈0.6%, and then falling steeply. Calculations give evidence that the observed maximum Tc occurs at or near a Lifshitz transition when the Fermi level passes through a Van Hove singularity, and open the possibility that the highly strained, Tc = 3.4 K Sr2RuO4 has an even-parity, rather than an odd-parity, order parameter.PostprintPeer reviewe

The linker histone H1.0 generates epigenetic and functional intratumor heterogeneity

Tumors comprise functionally diverse subpopulations of cells with distinct proliferative potential. Here, we show that dynamic epigenetic states defined by the linker histone H1.0 determine which cells within a tumor can sustain the long-term cancer growth. Numerous cancer types exhibit high inter- and intratumor heterogeneity of H1.0, with H1.0 levels correlating with tumor differentiation status, patient survival, and, at the single-cell level, cancer stem cell markers. Silencing of H1.0 promotes maintenance of self-renewing cells by inducing derepression of megabase-sized gene domains harboring downstream effectors of oncogenic pathways. Self-renewing epigenetic states are not stable, and reexpression of H1.0 in subsets of tumor cells establishes transcriptional programs that restrict cancer cells’ long-term proliferative potential and drive their differentiation. Our results uncover epigenetic determinants of tumor-maintaining cells

Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria

Lysosome-mediated processing of chromatin in senescence

Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C–negative, but strongly γ-H2AX–positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression

Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions

Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von hippel-lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities

E3 ubiquitin ligases are attractive targets in the ubiquitin-proteasome system, however, the development of small-molecule ligands has been rewarded with limited success. The von Hippel-Lindau protein (pVHL) is the substrate recognition subunit of the VHL E3 ligase that targets HIF-1α for degradation. We recently reported inhibitors of the pVHL:HIF-1α interaction, however they exhibited moderate potency. Herein, we report the design and optimization, guided by X-ray crystal structures, of a ligand series with nanomolar binding affinities

Epigenetics and Cancer Stem Cells: Unleashing, Hijacking, and Restricting Cellular Plasticity

Epigenetic regulators are one of the most commonly mutated classes of genes in cancer. During cancer initiation, mutated epigenetic regulators lead to oncogenic cellular reprogramming and promote the acquisition of uncontrolled self-renewal. The emergence of CSCs requires elaborate reorganization of the epigenome. During cancer growth, epigenetic mechanisms integrate the effect of cell-intrinsic (i.e., subclonal mutations) and cell-extrinsic (i.e., signaling from the microenvironment) changes and establish intratumoral heterogeneity, either promoting or inhibiting the CSC state. ‘Loose’ epigenetic constraints in cancer cells enhance cellular plasticity and allow reversible transitions between different phenotypic states. Enhanced cellular plasticity favors cancer cell adaptability and resistance to therapy. Modulation of epigenetic processes allows targeting of the most downstream determinants of the CSC state