CRISPR screens in sister chromatid cohesion defective cells reveal PAXIP1-PAGR1 as regulator of chromatin association of cohesin.

The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.

Late Toxicity and Health-Related Quality of Life Following Definitive Chemoradiotherapy for Esophageal Cancer: A Systematic Review and Meta-analysis.

PURPOSE: Definitive chemoradiotherapy (dCRT) is a treatment option with curative intent for patients with esophageal cancer that could result in late toxicities and affect health-related quality of life (HRQoL). This study aimed to review the literature and perform a meta-analysis to investigate the effect of dCRT on late toxicities and HRQoL in esophageal cancer. METHODS AND MATERIALS: A systematic search was performed in MEDLINE, EMBASE, and PsychINFO. Prospective phase II and III clinical trials, population-based studies, and retrospective chart reviews investigating late toxicity or HRQoL after dCRT (≥50 Gy) were included. The HRQoL outcomes were analyzed using linear mixed-effect models with restricted cubic spline transformation. Any HRQoL changes of ≥10 points were considered clinically relevant. The risk of toxicities was calculated using the number of events and the total study population. RESULTS: Among 41 included studies, 10 assessed HRQoL and 31 late toxicity. Global health status remained stable over time and improved after 36 months compared with baseline (mean change, +11). Several tumor-specific symptoms, including dysphagia, eating restrictions, and pain, improved after 6 months compared with baseline. Compared with baseline, dyspnea worsened after 6 months (mean change, +16 points). The risk of any late toxicity was 48% (95% CI, 33%-64%). Late toxicity risk of any grade for the esophagus was 17% (95% CI, 12%-21%), pulmonary 21% (95% CI, 11%-31%), cardiac 12% (95% CI, 6%-17%), and any other organ 24% (95% CI, 2%-45%). CONCLUSIONS: Global health status remained stable over time, and tumor-specific symptoms improved within 6 months after dCRT compared with baseline, with the exception of dyspnea. In addition, substantial risks of late toxicity were observed.

Predictive Value of SLCO1B1 c.521T>C Polymorphism on Observed Changes in the Treatment of 1136 Statin-Users.

Pharmacogenomic testing is a method to prevent adverse drug reactions. Pharmacogenomics could be relevant to optimize statin treatment, by identifying patients at high risk for adverse drug reactions. We aim to investigate the clinical validity and utility of pre-emptive pharmacogenomics screening in primary care, with SLCO1B1 c.521T>C as a risk factor for statin-induced adverse drug reactions. The focus was on changes in therapy as a proxy for adverse drug reactions observed in statin-users in a population-based Dutch cohort. In total, 1136 statin users were retrospectively genotyped for the SLCO1B1 c.521T>C polymorphism (rs4149056) and information on their statin dispensing was evaluated as cross-sectional research. Approximately half of the included participants discontinued or switched their statin treatment within three years. In our analyses, we could not confirm an association between the SLCO1B1 c.521T>C genotype and any change in statin therapy or arriving at a stable dose sooner in primary care. To be able to evaluate the predictive values of SLCO1B1 c.521T>C genotype on adverse drug reactions from statins, prospective data collection of actual adverse drug reactions and reasons to change statin treatment should be facilitated.

The histone methyltransferase SETD2 negatively regulates cell size.

Cell size varies between cell types but is tightly regulated by cell intrinsic and extrinsic mechanisms. Cell size control is important for cell function, and changes in cell size are frequently observed in cancer. Here, we uncover a role for SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Using multiple independent perturbation strategies, we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that overexpression of the H3K36 demethylase KDM4A or the oncohistone H3.3K36M also increase cell size. In addition, ectopic overexpression of a decoy SRI domain increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development.

SETD2: from chromatin modifier to multipronged regulator of the genome and beyond.

Histone modifying enzymes play critical roles in many key cellular processes and are appealing proteins for targeting by small molecules in disease. However, while the functions of histone modifying enzymes are often linked to epigenetic regulation of the genome, an emerging theme is that these enzymes often also act by non-catalytic and/or non-epigenetic mechanisms. SETD2 (Set2 in yeast) is best known for associating with the transcription machinery and methylating histone H3 on lysine 36 (H3K36) during transcription. This well-characterized molecular function of SETD2 plays a role in fine-tuning transcription, maintaining chromatin integrity, and mRNA processing. Here we give an overview of the various molecular functions and mechanisms of regulation of H3K36 methylation by Set2/SETD2. These fundamental insights are important to understand SETD2's role in disease, most notably in cancer in which SETD2 is frequently inactivated. SETD2 also methylates non-histone substrates such as α-tubulin which may promote genome stability and contribute to the tumor-suppressor function of SETD2. Thus, to understand its role in disease, it is important to understand and dissect the multiple roles of SETD2 within the cell. In this review we discuss how histone methylation by Set2/SETD2 has led the way in connecting histone modifications in active regions of the genome to chromatin functions and how SETD2 is leading the way to showing that we also have to look beyond histones to truly understand the physiological role of an 'epigenetic' writer enzyme in normal cells and in disease.

MRI and pathology correlations in the medulla in sudden unexpected death in epilepsy (SUDEP): a postmortem study.

AIMS: Sudden unexpected death in epilepsy (SUDEP) likely arises as a result of autonomic dysfunction around the time of a seizure. In vivo MRI studies report volume reduction in the medulla and other brainstem autonomic regions. Our aim, in a pathology series, is to correlate regional quantitative features on 9.4T MRI with pathology measures in medullary regions. METHODS: Forty-seven medullae from 18 SUDEP, 18 nonepilepsy controls and 11 epilepsy controls were studied. In 16 cases, representing all three groups, ex vivo 9.4T MRI of the brainstem was carried out. Five regions of interest (ROI) were delineated, including the reticular formation zone (RtZ), and actual and relative volumes (RV), as well as T1, T2, T2* and magnetization transfer ratio (MTR) measurements were evaluated on MRI. On serial sections, actual and RV estimates using Cavalieri stereological method and immunolabelling indices for myelin basic protein, synaptophysin and Microtubule associated protein 2 (MAP2) were carried out in similar ROI. RESULTS: Lower relative RtZ volumes in the rostral medulla but higher actual volumes in the caudal medulla were observed in SUDEP (P < 0.05). No differences between groups for T1, T2, T2* and MTR values in any region was seen but a positive correlation between T1 values and MAP2 labelling index in RtZ (P < 0.05). Significantly lower MAP2 LI were noted in the rostral medulla RtZ in epilepsy cases (P < 0.05). CONCLUSIONS: Rostro-caudal alterations of medullary volume in SUDEP localize with regions containing respiratory regulatory nuclei. They may represent seizure-related alterations, relevant to the pathophysiology of SUDEP.

Application of Recombination -Induced Tag Exchange (RITE) to study histone dynamics in human cells.

In eukaryotes, nucleosomes form a barrier to DNA templated reactions and must be dynamically disrupted to provide access to the genome. During nucleosome (re)assembly, histones can be replaced by new histones, erasing post-translational modifications. Measuring histone turnover in mammalian cells has mostly relied on inducible overexpression of histones, which may influence and distort natural histone deposition rates. We have previously used recombination-induced tag exchange (RITE) to study histone dynamics in budding yeast. RITE is a method to follow protein turnover by genetic switching of epitope tags using Cre recombinase and does not rely on inducible overexpression. Here, we applied RITE to study the dynamics of the replication-independent histone variant H3.3 in human cells. Epitope tag-switching could be readily detected upon induction of Cre-recombinase, enabling the monitoring old and new H3.3 in the same pool of cells. However, the rate of tag-switching was lower than in yeast cells. Analysis of histone H3.3 incorporation by chromatin immunoprecipitation did not recapitulate previously reported aspects of H3.3 dynamics such as high turnover rates in active promoters and enhancers. We hypothesize that asynchronous Cre-mediated DNA recombination in the cell population leads to a low time resolution of the H3.3-RITE system in human cells. We conclude that RITE enables the detection of old and new proteins in human cells and that the time-scale of tag-switching prevents the capture of high turnover events in a population of cells. Instead, RITE might be more suited for tracking long-lived histone proteins in human cells.

Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L-dose dependency in HDAC1-deficient thymic lymphoma.

DOT1L methylates histone H3K79 and is aberrantly regulated in MLL-rearranged leukemia. Inhibitors have been developed to target DOT1L activity in leukemia, but cellular mechanisms that regulate DOT1L are still poorly understood. We have identified the histone deacetylase Rpd3 as a negative regulator of budding yeast Dot1. At its target genes, the transcriptional repressor Rpd3 restricts H3K79 methylation, explaining the absence of H3K79me3 at a subset of genes in the yeast genome. Similar to the crosstalk in yeast, inactivation of the murine Rpd3 homolog HDAC1 in thymocytes led to an increase in H3K79 methylation. Thymic lymphomas that arise upon genetic deletion of Hdac1 retained the increased H3K79 methylation and were sensitive to reduced DOT1L dosage. Furthermore, cell lines derived from Hdac1Δ/Δ thymic lymphomas were sensitive to a DOT1L inhibitor, which induced apoptosis. In summary, we identified an evolutionarily conserved crosstalk between HDAC1 and DOT1L with impact in murine thymic lymphoma development.

Spatiotemporal dynamics of PDGFRß expression in pericytes and glial scar formation in penetrating brain injuries in adults.

AIMS: Understanding the spatiotemporal dynamics of reactive cell types following brain injury is important for future therapeutic interventions. We have previously used penetrating cortical injuries following intracranial recordings as a brain repair model to study scar-forming nestin-expressing cells. We now explore the relationship between nestin-expressing cells, PDGFRß+ pericytes and Olig2+ glia, including their proliferation and functional maturation. METHODS: In 32 cases, ranging from 3 to 461 days post injury (dpi), immunohistochemistry for PDGFRß, nestin, GFAP, Olig2, MCM2, Aquaporin 4 (Aq4), Glutamine Synthetase (GS) and Connexin 43 (Cx43) was quantified for cell densities, labelling index (LI) and cellular co-expression at the injury site compared to control regions. RESULTS: PDGFRß labelling highlighted both pericytes and multipolar parenchymal cells. PDGFRß LI and PDGFRß+ /MCM2+ cells significantly increased in injury Zones at 10-13 dpi with migration of pericytes away from vessels with increased co-localization of PDGRFß with nestin compared to control regions (P < 0.005). Olig2+ /MCM2+ cell populations peaked at 13 dpi with significantly higher cell densities at injury sites than in control regions (P < 0.01) and decreasing with dpi (P < 0.05). Cx43 LI was reduced in acute injuries but increased with dpi (P < 0.05) showing significant cellular co-localization with nestin and GFAP (P < 0.005 and P < 0.0001) but not PDGFRß. CONCLUSIONS: These findings indicate that PDGFRß+ and Olig2+ cells contribute to the proliferative fraction following penetrating brain injuries, with evidence of pericyte migration. Dynamic changes in Cx43 in glial cell types with dpi suggest functional alterations during temporal stages of brain repair.

Review: Challenges in the histopathological classification of ganglioglioma and DNT: microscopic agreement studies and a preliminary genotype-phenotype analysis.

Low-grade epilepsy-associated brain tumours (LEAT) are the second most common cause for drug-resistant, focal epilepsy, that is ganglioglioma (GG) and dysembryoplastic neuroepithelial tumours (DNT). However, molecular pathogenesis, risk factors for malignant progression and their frequent association with drug-resistant focal seizures remain poorly understood. This contrasts recent progress in understanding the molecular-genetic basis and targeted treatment options in diffuse gliomas. The Neuropathology Task Force of the International League Against Epilepsy examined available literature to identify common obstacles in diagnosis and research of LEAT. Analysis of 10 published tumour series from epilepsy surgery pointed to poor inter-rater agreement for the histopathology diagnosis. The Task Force tested this hypothesis using a web-based microscopy agreement study. In a series of 30 LEAT, 25 raters from 18 countries agreed in only 40% of cases. Highest discordance in microscopic diagnosis occurred between GG and DNT variants, when oligodendroglial-like cell patterns prevail, or ganglion cells were difficult to discriminate from pre-existing neurons. Suggesting new terminology or major histopathological criteria did not satisfactorily increase the yield of histopathology agreement in four consecutive trials. To this end, the Task Force applied the WHO 2016 strategy of integrating phenotype analysis with molecular-genetic data obtained from panel sequencing and 450k methylation arrays. This strategy was helpful to distinguish DNT from GG variants in all cases. The Task Force recommends, therefore, to further develop diagnostic panels for the integration of phenotype-genotype analysis in order to reliably classify the spectrum of LEAT, carefully characterize clinically meaningful entities and make better use of published literature.

Dot1 promotes H2B ubiquitination by a methyltransferase-independent mechanism.

The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions of Dot1, we generated genetic interaction maps of increased-dosage alleles of DOT1. We identified a functional relationship between increased Dot1 dosage and loss of the DUB module of the SAGA co-activator complex, which deubiquitinates H2Bub1 and thereby negatively regulates H3K79 methylation. Increased Dot1 dosage was found to promote H2Bub1 in a dose-dependent manner and this was exacerbated by the loss of SAGA-DUB activity, which also caused a negative genetic interaction. The stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of methyltransferase activity. Our findings show that Dot1 and H2Bub1 are subject to bi-directional crosstalk and that Dot1 possesses chromatin regulatory functions that are independent of its methyltransferase activity.

Review: The past, present and future challenges in epilepsy-related and sudden deaths and biobanking.

Awareness and research on epilepsy-related deaths (ERD), in particular Sudden Unexpected Death in Epilepsy (SUDEP), have exponentially increased over the last two decades. Most publications have focused on guidelines that inform clinicians dealing with these deaths, educating patients, potential risk factors and mechanisms. There is a relative paucity of information available for pathologists who conduct these autopsies regarding appropriate post mortem practice and investigations. As we move from recognizing SUDEP as the most common form of ERD toward in-depth investigations into its causes and prevention, health professionals involved with these autopsies and post mortem procedure must remain fully informed. Systematizing a more comprehensive and consistent practice of examining these cases will facilitate (i) more precise determination of cause of death, (ii) identification of SUDEP for improved epidemiological surveillance (the first step for an intervention study), and (iii) biobanking and cell-based research. This article reviews how pathologists and healthcare professionals have approached ERD, current practices, logistical problems and areas to improve and harmonize. The main neuropathology, cardiac and genetic findings in SUDEP are outlined, providing a framework for best practices, integration of clinical, pathological and molecular genetic investigations in SUDEP, and ultimately prevention.

Review: Neurodegenerative processes in temporal lobe epilepsy with hippocampal sclerosis: Clinical, pathological and neuroimaging evidence.

Cognitive decline is increasingly described as a co-morbidity of temporal lobe epilepsy (TLE). Mechanisms underlying cognitive impairment are not fully understood despite examining clinical factors, such as seizure frequency, and cellular mechanisms of excitotoxicity. We review the neuropsychometry evidence for progressive cognitive decline and examine the pathology and neuroimaging evidence supporting a neurodegenerative process in hippocampal sclerosis (HS)-related TLE. Accelerated cognitive decline is described in groups of adult HS-related TLE patients. Large childhood studies show early onset of seizures result in poor development of verbal memory and a hindrance in achieving cognitive potential. We discuss HS classification according to different patterns of neuronal loss and correlation to post-temporal lobectomy cognitive outcomes in refractory TLE patients. Factors such as lateralization of HS pathology, neuronal density and subtype have correlated to cognitive outcomes with varying significance between different studies. Furthermore, alterations in neuronal maturity, regenerative capacity and aberrant connectivity appear to affect cognitive performance post-operatively suggesting a complex multifactorial process. More recent studies have identified tau pathology being present in HS-related TLE and correlated to post-operative cognitive decline in some patients. A traumatic head injury-related or novel tauopathy has been hypothesized as an underlying process. We discuss the value of prospective and cross-sectional imaging in assessing cognition and review volumetric magnetic resonance studies with progressive ipsilateral hippocampal atrophy identified to correlate with seizure frequency. Finally, we consider the use of positron emission tomography biomarkers, such as tau tracers, and connectivity studies that may examine in vivo pathways and further explore cognitive decline in TLE.

Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1.

Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features.

Expression of neurodegenerative disease-related proteins and caspase-3 in glioneuronal tumours.

AIMS: Recent evidence supports the activation of mechanisms underlying cellular ageing and neurodegeneration in developmental lesions associated with epilepsy. The present study examined the ongoing cell injury and vulnerability to neuronal degeneration in glioneuronal tumours (GNT). METHODS: We evaluated a series of GNT (n = 31 gangliogliomas, GG and n = 30 dysembryoplastic neuroepithelial tumours, DNT). Sections were processed for immunohistochemistry using markers for the evaluation of caspase-3 and neurodegeneration-related proteins/pathways and their expression was correlated with the tumour features and the clinical history of epilepsy. RESULTS: Both GG and DNT specimens contained caspase-3-positive cells. In GG, expression of activated caspase-3 was negatively correlated the with the BRAF V600E mutation status. We also observed an abnormal expression of death receptor-6 and ß-amyloid precursor protein (APP). Moreover, dysplastic neurones expressed p62, phosphorylated (p)TDP43 and pTau. Double labelling experiments showed colocalization of phosphorylated S6 (marker of mammalian target of rapamycin, mTOR, pathway activation) with pTau and p62. In GG, neuronal p62 expression was positively correlated with pS6. The immunoreactivity score (IRS) of caspase-3, APP, DR6, p62 and pTDP43 were found to be significantly higher in GG than in DNT. Expression of APP, DR6, pTau (in GG and DNT) and caspase-3 (in GG) positively correlated with duration of epilepsy. In GG, the expression of neuronal caspase-3, DR6 and glial p62 was associated with a worse postoperative seizure outcome. CONCLUSIONS: Our observations in GNT provide evidence of premature activation of mechanisms of neurodegeneration which are associated with the clinical course of epilepsy in patient with GG.