WNT signalling control by KDM5C during development affects cognition.

Although KDM5C is one of the most frequently mutated genes in X-linked intellectual disability1, the exact mechanisms that lead to cognitive impairment remain unknown. Here we use human patient-derived induced pluripotent stem cells and Kdm5c knockout mice to conduct cellular, transcriptomic, chromatin and behavioural studies. KDM5C is identified as a safeguard to ensure that neurodevelopment occurs at an appropriate timescale, the disruption of which leads to intellectual disability. Specifically, there is a developmental window during which KDM5C directly controls WNT output to regulate the timely transition of primary to intermediate progenitor cells and consequently neurogenesis. Treatment with WNT signalling modulators at specific times reveal that only a transient alteration of the canonical WNT signalling pathway is sufficient to rescue the transcriptomic and chromatin landscapes in patient-derived cells and to induce these changes in wild-type cells. Notably, WNT inhibition during this developmental period also rescues behavioural changes of Kdm5c knockout mice. Conversely, a single injection of WNT3A into the brains of wild-type embryonic mice cause anxiety and memory alterations. Our work identifies KDM5C as a crucial sentinel for neurodevelopment and sheds new light on KDM5C mutation-associated intellectual disability. The results also increase our general understanding of memory and anxiety formation, with the identification of WNT functioning in a transient nature to affect long-lasting cognitive function.

High-throughput Oligopaint screen identifies druggable 3D genome regulators.

The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.

Cancer epigenetics: promises and pitfalls for cancer therapy.

Over the past few decades, epigenetic regulators have emerged as major players in cellular processes that drive cancer initiation and progression, and subsequently modulate the responsiveness of cancers to therapeutic agents. This Special Issue of The FEBS Journal, Cancer Epigenetics, features an exciting collection of review articles that focus on the functions of a broad spectrum of epigenetic modulators in cancer. The diverse topics explored herein range from the roles of transposable elements and chromatin architecture in cancer and the most recent research advances on cancer-associated histone variants (oncohistones), to the effects of altered epigenetics on transcription and advanced cancer cell phenotypes. Moreover, the prospective key function of cancer metabolism in linking epigenetics and transcriptional regulation, and the potential of epigenetics for targeted cancer therapeutics is discussed. We hope that this collection of articles will give readers an enlightening overview of the most recent advances in the fast-moving field of cancer epigenetics.

Progesterone Inhibits the Establishment of Activation-Associated Chromatin During TH1 Differentiation.

TH1-mediated diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA) improve during pregnancy, coinciding with increasing levels of the pregnancy hormone progesterone (P4), highlighting P4 as a potential mediator of this immunomodulation. Here, we performed detailed characterization of how P4 affects the chromatin and transcriptomic landscape during early human TH1 differentiation, utilizing both ATAC-seq and RNA-seq. Time series analysis of the earlier events (0.5-24 hrs) during TH1 differentiation revealed that P4 counteracted many of the changes induced during normal differentiation, mainly by downregulating key regulatory genes and their upstream transcription factors (TFs) involved in the initial T-cell activation. Members of the AP-1 complex such as FOSL1, FOSL2, JUN and JUNB were particularly affected, in both in promoters and in distal regulatory elements. Moreover, the changes induced by P4 were significantly enriched for disease-associated changes related to both MS and RA, revealing several shared upstream TFs, where again JUN was highlighted to be of central importance. Our findings support an immune regulatory role for P4 during pregnancy by impeding T-cell activation, a crucial checkpoint during pregnancy and in T-cell mediated diseases, and a central event prior to T-cell lineage commitment. Indeed, P4 is emerging as a likely candidate involved in disease modulation during pregnancy and further studies evaluating P4 as a potential treatment option are needed.

PARP inhibitor BMN-673 induced apoptosis by trapping PARP-1 and inhibiting base excision repair via modulation of pol-ß in chromatin of breast cancer cells.

PARP inhibitors emerged as clinically effective anti-tumor agents in combination with DNA damaging agents but the toxicity of DNA damaging agents and their off-target effects caused serious problems in cancer therapy. They confer cytotoxicity in cancer cells both by catalytic inhibition and trapping of PARP-1 at the DNA damage site. There is a lack of direct evidence to quantitatively determine the trapped PARP-1 in cellular DNA. Here, we have precisely evaluated the mechanism of PARP trapping mediated anti-cancer action of Quinacrine (QC), BMN-673, and their combination (QC + BMN-673) in breast cancer cells. We introduced a strategy to measure the cellular PARP trapping potentiality of BMN-673 in QC pretreated cells using a fluorescence-based assay system. It was found that QC+ BMN-673 induced apoptosis by triggering DNA damage in breast cancer cells. Treatment with QC + BMN-673 stimulated the expression of PARP-1 in the chromatin compared to that of PARP-2 and PARP-3. QC + BMN-673 treatment also caused a dose-dependent and time-dependent accumulation of PARP-1 and inhibition of PARylation in the chromatin. Upregulation of BER components (pol-ß and FEN-1), an unchanged HR and NHEJ pathway proteins, and reduction of luciferase activity of the cells transfected with R-p21-P (LP-BER) were noted in combined drug-treated cells. Interestingly, silencing of pol-ß resulted in unchanged PARP-1 trapping and PAR activity in the chromatin with increasing time after QC + BMN-673 treatment without altering APC and FEN-1 expression. Thus, our data suggested that the QC + BMN-673 augmented breast cancer cell death by pol-ß mediated repair inhibition primarily through trapping of PARP-1 besides PARP-1 catalytic inhibition.

Therapeutic targeting of chromatin: status and opportunities.

The molecular characterization of mechanisms underlying transcriptional control and epigenetic inheritance since the 1990s has paved the way for the development of targeted therapies that modulate these pathways. In the past two decades, cancer genome sequencing approaches have uncovered a plethora of mutations in chromatin modifying enzymes across tumor types, and systematic genetic screens have identified many of these proteins as specific vulnerabilities in certain cancers. Now is the time when many of these basic and translational efforts start to bear fruit and more and more chromatin-targeting drugs are entering the clinic. At the same time, novel pharmacological approaches harbor the potential to modulate chromatin in unprecedented fashion, thus generating entirely novel opportunities. Here, we review the current status of chromatin targets in oncology and describe a vision for the epigenome-modulating drugs of the future.

Oncohistones: a roadmap to stalled development.

Since the discovery of recurrent mutations in histone H3 variants in paediatric brain tumours, so-called 'oncohistones' have been identified in various cancers. While their mechanism of action remains under active investigation, several studies have shed light on how they promote genome-wide epigenetic perturbations. These findings converge on altered post-translational modifications on two key lysine (K) residues of the H3 tail, K27 and K36, which regulate several cellular processes, including those linked to cell differentiation during development. We will review how these oncohistones affect the methylation of cognate residues, but also disrupt the distribution of opposing chromatin marks, creating genome-wide epigenetic changes which participate in the oncogenic process. Ultimately, tumorigenesis is promoted through the maintenance of a progenitor state at the expense of differentiation in defined cellular and developmental contexts. As these epigenetic disruptions are reversible, improved understanding of oncohistone pathogenicity can result in needed alternative therapies.

Clinical advances in targeting epigenetics for cancer therapy.

The appropriate coordination between epigenetic regulators is essential for spatial and temporal regulation of gene expression and maintenance of cell identity. Cancer is a disease driven by both genetic and epigenetic alterations. The widespread dysregulation and reversible nature of epigenetic alterations confer cancer cells with vulnerabilities for therapeutic interventions. Over the past decades, remarkable progress has been made in developing drugs that target epigenetic regulators, with many drugs under evaluation in clinical trials. Here, we summarize the epigenetic drugs currently in clinical investigations and highlight the potentials and challenges in their implication to treat cancer. We also discuss the preclinical and clinical results of combination therapies with epigenetic drugs and other therapies such as targeted and immune-based therapies.

Early-life midazolam exposure persistently changes chromatin accessibility to impair adult hippocampal neurogenesis and cognition.

Linkage between early-life exposure to anesthesia and subsequent learning disabilities is of great concern to children and their families. Here we show that early-life exposure to midazolam (MDZ), a widely used drug in pediatric anesthesia, persistently alters chromatin accessibility and the expression of quiescence-associated genes in neural stem cells (NSCs) in the mouse hippocampus. The alterations led to a sustained restriction of NSC proliferation toward adulthood, resulting in a reduction of neurogenesis that was associated with the impairment of hippocampal-dependent memory functions. Moreover, we found that voluntary exercise restored hippocampal neurogenesis, normalized the MDZ-perturbed transcriptome, and ameliorated cognitive ability in MDZ-exposed mice. Our findings thus explain how pediatric anesthesia provokes long-term adverse effects on brain function and provide a possible therapeutic strategy for countering them.

A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress.

Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.

Quantifying the phase separation property of chromatin-associated proteins under physiological conditions using an anti-1,6-hexanediol index.

BACKGROUND: Liquid-liquid phase separation (LLPS) is an important organizing principle for biomolecular condensation and chromosome compartmentalization. However, while many proteins have been reported to undergo LLPS, quantitative and global analysis of chromatin LLPS property remains absent. RESULTS: Here, by combining chromatin-associated protein pull-down, quantitative proteomics and 1,6-hexanediol (1,6-HD) treatment, we develop Hi-MS and define an anti-1,6-HD index of chromatin-associated proteins (AICAP) to quantify 1,6-HD sensitivity of chromatin-associated proteins under physiological conditions. Compared with known physicochemical properties involved in phase separation, we find that proteins with lower AICAP are associated with higher content of disordered regions, higher hydrophobic residue preference, higher mobility and higher predicted LLPS potential. We also construct BL-Hi-C libraries following 1,6-HD treatment to study the sensitivity of chromatin conformation to 1,6-HD treatment. We find that the active chromatin and high-order structures, as well as the proteins enriched in corresponding regions, are more sensitive to 1,6-HD treatment. CONCLUSIONS: Our work provides a global quantitative measurement of LLPS properties of chromatin-associated proteins and higher-order chromatin structure. Hi-MS and AICAP data provide an experimental tool and quantitative resources valuable for future studies of biomolecular condensates.

Pharmacological Inhibition of Core Regulatory Circuitry Liquid-liquid Phase Separation Suppresses Metastasis and Chemoresistance in Osteosarcoma.

Liquid-liquid phase-separated (LLPS) transcriptional factor assemblies at super-enhancers (SEs) provide a conceptual framework for underlying transcriptional control in mammal cells. However, the mechanistic understanding of LLPS in aberrant transcription driven by dysregulation of SEs in human malignancies is still elusive. By integrating SE profiling and core regulatory circuitry (CRC) calling algorithm, the CRC of metastatic and chemo-resistant osteosarcoma is delineated. CRC components, HOXB8 and FOSL1, produce dense and dynamic phase-separated droplets in vitro and liquid-like puncta in cell nuclei. Disruption of CRC phase separation decreases the chromatin accessibility in SE regions and inhibits the release of RNA polymerase II from the promoter of SE-driven genes. Importantly, absence of CRC key component causes a reduction in osteosarcoma tumor growth and metastasis. Moreover, it is shown that CRC condensates can be specifically attenuated by the H3K27 demethylase inhibitor, GSK-J4. Pharmacological inhibition of the CRC phase separation results in metastasis suppression and re-sensitivity to chemotherapy drugs in patient-derived xenograft model. Taken together, this study reveals a previously unknown mechanism that CRC factors formed LLPS condensates, and provides a phase separation-based pharmacological strategy to target undruggable CRC components for the treatment of metastatic and chemo-resistant osteosarcoma.

Transcriptome and chromatin landscape changes associated with trastuzumab resistance in HER2+ breast cancer cells.

We set out to uncover transcriptome and chromatin landscape changes that occur in HER2 + breast cancer (BC) cells upon acquiring resistance to trastuzumab. RNA-seq analysis was applied to two independently-derived BC cell lines with acquired resistance to trastuzumab (SKBr3.HerR and BT-474HerR) and their parental drug-sensitive cell lines (SKBr3 and BT-474). Chromatin landscape analysis indicated that the most significant increase in accessibility in resistant cells occurs in PPP1R1B within a segment spanning introns 1b through intron 3. Footprint analysis of this segment revealed that FoxJ3 (within intron 2) and Pou5A1/Sox2 (within inton 3) transcription factor motifs are protected in resistant cells. Overall, 344 shared genes were upregulated in both resistant cell lines relative to their parental counterparts and 453 shared genes were downregulated in both resistant cell lines relative to their parental counterparts. In resistant cells, genes associated with autophagy and mitochondria organization are upregulated and genes associated with ribosome assembly and cell cycle are downregulated relative to parental cells. The five top upregulated genes in drug-resistant breast cancer cells are APOD, AZGP1, ETV5, ALPP, and PPP1R1B. This is the first report of increased chromatin accessibility within PPP1R1B associated with its t-Darpp transcript increase, and points to a possible mechanism for its activation in trastuzumab-resistant cells.

Hexavalent chromium disrupts chromatin architecture.

Accessibility of DNA elements and the orchestration of spatiotemporal chromatin-chromatin interactions are critical mechanisms in the regulation of gene transcription. Thus, in an ever-changing milieu, cells mount an adaptive response to environmental stimuli by modulating gene expression that is orchestrated by coordinated changes in chromatin architecture. Correspondingly, agents that alter chromatin structure directly impact transcriptional programs in cells. Heavy metals, including hexavalent chromium (Cr(VI)), are of special interest because of their ability to interact directly with cellular protein, DNA and other macromolecules, resulting in general damage or altered function. In this review we highlight the chromium-mediated mechanisms that promote disruption of chromatin architecture and how these processes are integral to its carcinogenic properties. Emerging evidence shows that Cr(VI) targets nucleosomal architecture in euchromatin, particularly in genomic locations flanking binding sites of the essential transcription factors CTCF and AP1. Ultimately, these changes contribute to an altered chromatin state in critical gene regulatory regions, which disrupts gene transcription in functionally relevant biological processes.

New Insights into Alterations in PL Proteins Affecting Their Binding to DNA after Exposure of Mytilus galloprovincialis to Mercury-A Possible Risk to Sperm Chromatin Structure?

Mercury (Hg) is a highly toxic and widespread pollutant. We previously reported that the exposure of Mytilus galloprovincialis for 24 h to doses of HgCl2 similar to those found in seawater (range 1-100 pM) produced alterations in the properties of protamine-like (PL) proteins that rendered them unable to bind and protect DNA from oxidative damage. In the present work, to deepen our studies, we analyzed PL proteins by turbidimetry and fluorescence spectroscopy and performed salt-induced release analyses of these proteins from sperm nuclei after the exposure of mussels to HgCl2 at the same doses. Turbidity assays indicated that mercury, at these doses, induced PL protein aggregates, whereas fluorescence spectroscopy measurements showed mercury-induced conformational changes. Indeed, the mobility of the PLII band changed in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, particularly after exposure to 10-pM HgCl2, confirming the mercury-induced structural rearrangement. Finally, exposure to HgCl2 at all doses produced alterations in PL-DNA binding, detectable by DNA absorption spectra after the PL protein addition and by a decreased release of PLII and PLIII from the sperm nuclei. In conclusion, in this paper, we reported Hg-induced PL protein alterations that could adversely affect mussel reproductive activity, providing an insight into the molecular mechanism of Hg-related infertility.

Bivalent chromatin as a therapeutic target in cancer: An in silico predictive approach for combining epigenetic drugs.

Tumour cell heterogeneity is a major barrier for efficient design of targeted anti-cancer therapies. A diverse distribution of phenotypically distinct tumour-cell subpopulations prior to drug treatment predisposes to non-uniform responses, leading to the elimination of sensitive cancer cells whilst leaving resistant subpopulations unharmed. Few strategies have been proposed for quantifying the variability associated to individual cancer-cell heterogeneity and minimizing its undesirable impact on clinical outcomes. Here, we report a computational approach that allows the rational design of combinatorial therapies involving epigenetic drugs against chromatin modifiers. We have formulated a stochastic model of a bivalent transcription factor that allows us to characterise three different qualitative behaviours, namely: bistable, high- and low-gene expression. Comparison between analytical results and experimental data determined that the so-called bistable and high-gene expression behaviours can be identified with undifferentiated and differentiated cell types, respectively. Since undifferentiated cells with an aberrant self-renewing potential might exhibit a cancer/metastasis-initiating phenotype, we analysed the efficiency of combining epigenetic drugs against the background of heterogeneity within the bistable sub-ensemble. Whereas single-targeted approaches mostly failed to circumvent the therapeutic problems represented by tumour heterogeneity, combinatorial strategies fared much better. Specifically, the more successful combinations were predicted to involve modulators of the histone H3K4 and H3K27 demethylases KDM5 and KDM6A/UTX. Those strategies involving the H3K4 and H3K27 methyltransferases MLL2 and EZH2, however, were predicted to be less effective. Our theoretical framework provides a coherent basis for the development of an in silico platform capable of identifying the epigenetic drugs combinations best-suited to therapeutically manage non-uniform responses of heterogenous cancer cell populations.

Differential responses of neurons, astrocytes, and microglia to G-quadruplex stabilization.

The G-quadruplex (G4-DNA or G4) is a secondary DNA structure formed by DNA sequences containing multiple runs of guanines. While it is now firmly established that stabilized G4s lead to enhanced genomic instability in cancer cells, whether and how G4s contribute to genomic instability in brain cells is still not clear. We previously showed that, in cultured primary neurons, small-molecule G4 stabilizers promote formation of DNA double-strand breaks (DSBs) and downregulate the Brca1 gene. Here, we determined if G4-dependent Brca1 downregulation is unique to neurons or if the effects in neurons also occur in astrocytes and microglia. We show that primary neurons, astrocytes and microglia basally exhibit different G4 landscapes. Stabilizing G4-DNA with the G4 ligand pyridostatin (PDS) differentially modifies chromatin structure in these cell types. Intriguingly, PDS promotes DNA DSBs in neurons, astrocytes and microglial cells, but fails to downregulate Brca1 in astrocytes and microglia, indicating differences in DNA damage and repair pathways between brain cell types. Taken together, our findings suggest that stabilized G4-DNA contribute to genomic instability in the brain and may represent a novel senescence pathway in brain aging.

Chromatin-mediated epigenetic regulation of HSV-1 transcription as a potential target in antiviral therapy.

The ability to establish, and reactivate from, latent infections is central to the biology and pathogenesis of HSV-1. It also poses a strong challenge to antiviral therapy, as latent HSV-1 genomes do not replicate or express any protein to be targeted. Although the processes regulating the establishment and maintenance of, and reactivation from, latency are not fully elucidated, the current general consensus is that epigenetics play a major role. A unifying model postulates that whereas HSV-1 avoids or counteracts chromatin silencing in lytic infections, it becomes silenced during latency, silencing which is somewhat disrupted during reactivation. Many years of work by different groups using a variety of approaches have also shown that the lytic HSV-1 chromatin is distinct and has unique biophysical properties not shared with most cellular chromatin. Nonetheless, the lytic and latent viral chromatins are typically enriched in post translational modifications or histone variants characteristic of active or repressed transcription, respectively. Moreover, a variety of small molecule epigenetic modulators inhibit viral replication and reactivation from latency. Despite these successes in culture and animal models, it is not obvious how epigenetic modulation would be used in antiviral therapy if the same epigenetic mechanisms governed viral and cellular gene expression. Recent work has highlighted several important differences between the viral and cellular chromatins, which appear to be of consequence to their respective epigenetic regulations. In this review, we will discuss the distinctiveness of the viral chromatin, and explore whether it is regulated by mechanisms unique enough to be exploited in antiviral therapy.

Dual Targeting of Chromatin Stability By The Curaxin CBL0137 and Histone Deacetylase Inhibitor Panobinostat Shows Significant Preclinical Efficacy in Neuroblastoma.

PURPOSE: We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma. EXPERIMENTAL DESIGN: The effects of the drug combination on cancer growth were examined in vitro and in animal models of MYCN-amplified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction. RESULTS: The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137/panobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination. CONCLUSIONS: The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies.

[Impact of the use of systemic corticosteroid therapy on the effectiveness of immune checkpoint inhibitors]. / Impact de l'utilisation de la corticothérapie systémique sur l'efficacité des inhibiteurs de points de contrôle immunitaire.

Immunotherapy, which consists in using molecules targeting the immune system, has existed for many years in oncology (vaccines, interleukins, monoclonal antibodies) but has recently expanded due to the development of immune checkpoint inhibitors. These monoclonal antibodies help to restore the immunity against cancer by specifically targeting some immune checkpoints such as CTLA-4, PD-1 and PD-L1. Furthermore, in oncology, it is common to use systemic corticosteroids in the management of symptoms linked to the natural history of the disease (pain, spinal cord compression, cerebral edema) and toxicities linked to anticancer treatment. The impact of corticosteroids on the efficacy of immune checkpoint inhibitors is still poorly understood and they should be used cautiously. According to previously published studies, there seems to be a deleterious effect of corticosteroid therapy on the efficacy of immune checkpoint inhibitors when administered before or at the initiation of immunotherapy, while this effect does not seem present when corticosteroids are administered to patients already undergoing immunotherapy. The aim of this work is to analyze the existing data evaluating the impact of corticosteroid use of on the efficacy of immune checkpoint inhibitors.