The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells.

Invariant natural killer T cells (iNKT cells) are innate-like lymphocytes that protect against infection, autoimmune disease and cancer. However, little is known about the epigenetic regulation of iNKT cell development. Here we found that the H3K27me3 histone demethylase UTX was an essential cell-intrinsic factor that controlled an iNKT-cell lineage-specific gene-expression program and epigenetic landscape in a demethylase-activity-dependent manner. UTX-deficient iNKT cells exhibited impaired expression of iNKT cell signature genes due to a decrease in activation-associated H3K4me3 marks and an increase in repressive H3K27me3 marks within the promoters occupied by UTX. We found that JunB regulated iNKT cell development and that the expression of genes that were targets of both JunB and the iNKT cell master transcription factor PLZF was UTX dependent. We identified iNKT cell super-enhancers and demonstrated that UTX-mediated regulation of super-enhancer accessibility was a key mechanism for commitment to the iNKT cell lineage. Our findings reveal how UTX regulates the development of iNKT cells through multiple epigenetic mechanisms.

Reprogramming roadmap reveals route to human induced trophoblast stem cells.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development1-6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

Histone H3.3 phosphorylation promotes heterochromatin formation by inhibiting H3K9/K36 histone demethylase.

Histone H3.3 is an H3 variant which differs from the canonical H3.1/2 at four residues, including a serine residue at position 31 which is evolutionarily conserved. The H3.3 S31 residue is phosphorylated (H3.3 S31Ph) at heterochromatin regions including telomeres and pericentric repeats. However, the role of H3.3 S31Ph in these regions remains unknown. In this study, we find that H3.3 S31Ph regulates heterochromatin accessibility at telomeres during replication through regulation of H3K9/K36 histone demethylase KDM4B. In mouse embryonic stem (ES) cells, substitution of S31 with an alanine residue (H3.3 A31 -phosphorylation null mutant) results in increased KDM4B activity that removes H3K9me3 from telomeres. In contrast, substitution with a glutamic acid (H3.3 E31, mimics S31 phosphorylation) inhibits KDM4B, leading to increased H3K9me3 and DNA damage at telomeres. H3.3 E31 expression also increases damage at other heterochromatin regions including the pericentric heterochromatin and Y chromosome-specific satellite DNA repeats. We propose that H3.3 S31Ph regulation of KDM4B is required to control heterochromatin accessibility of repetitive DNA and preserve chromatin integrity.

ABCA12 regulates insulin secretion from ß-cells.

Dysregulation of lipid homeostasis is intimately associated with defects in insulin secretion, a key feature of type 2 diabetes. Here, we explore the role of the putative lipid transporter ABCA12 in regulating insulin secretion from ß-cells. Mice with ß-cell-specific deletion of Abca12 display impaired glucose-stimulated insulin secretion and eventual islet inflammation and ß-cell death. ABCA12's action in the pancreas is independent of changes in the abundance of two other cholesterol transporters, ABCA1 and ABCG1, or of changes in cellular cholesterol or ceramide content. Instead, loss of ABCA12 results in defects in the genesis and fusion of insulin secretory granules and increases in the abundance of lipid rafts at the cell membrane. These changes are associated with dysregulation of the small GTPase CDC42 and with decreased actin polymerisation. Our findings establish a new, pleiotropic role for ABCA12 in regulating pancreatic lipid homeostasis and insulin secretion.

Functional Proteomic Analysis of Repressive Histone Methyltransferase Complexes Reveals ZNF518B as a G9A Regulator.

Cell-type specific gene silencing by histone H3 lysine 27 and lysine 9 methyltransferase complexes PRC2 and G9A-GLP is crucial both during development and to maintain cell identity. Although studying their interaction partners has yielded valuable insight into their functions, how these factors are regulated on a network level remains incompletely understood. Here, we present a new approach that combines quantitative interaction proteomics with global chromatin profiling to functionally characterize repressive chromatin modifying protein complexes in embryonic stem cells. We define binding stoichiometries of 9 new and 12 known interaction partners of PRC2 and 10 known and 29 new interaction partners of G9A-GLP, respectively. We demonstrate that PRC2 and G9A-GLP interact physically and share several interaction partners, including the zinc finger proteins ZNF518A and ZNF518B. Using global chromatin profiling by targeted mass spectrometry, we discover that even sub-stoichiometric binding partners such as ZNF518B can positively regulate global H3K9me2 levels. Biochemical analysis reveals that ZNF518B directly interacts with EZH2 and G9A. Our systematic analysis suggests that ZNF518B may mediate the structural association between PRC2 and G9A-GLP histone methyltransferases and additionally regulates the activity of G9A-GLP.

Pattern Center and Distortion Determined from Faint, Diffuse Electron Diffraction Rings from Amorphous Materials.

Diffuse rings from amorphous materials sit on a steep background resulting in a monotonically decreasing intensity over scattering vector length, frequently with no clear local maximum that could be used to determine the center of the ring. The novelty of the method reported here is that it successful processes such weak patterns. It is based on separating the angular dependence of the positions of the maxima on the azimuthal angle in the measured two-dimensional pattern for a manually preselected peak. Both pattern center and elliptical distortion are simultaneously refined from this angular dependence. Both steps are based on nonlinear least square fitting, using the Levenberg-Marquardt method. It can be successfully applied to any amorphous patterns provided they were recorded with experimental conditions that facilitate dividing them into sectors with acceptable statistics. Patterns with the center shifted to the camera corner (recording a quadrant of a ring) can also be reliably evaluated, keeping precalibrated values of the elliptical distortion fixed during the fit. Finally, the limited number of counts in any pattern is overcome by cumulating many patterns (from equivalent areas) into a single pattern. Eliminating false effects is facilitated by masking out unwanted parts of any recorded pattern from processing.

Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline.

The Piwi proteins of the Argonaute superfamily are required for normal germline development in Drosophila, zebrafish, and mice and associate with 24-30 nucleotide RNAs termed piRNAs. We identify a class of 21 nucleotide RNAs, previously named 21U-RNAs, as the piRNAs of C. elegans. Piwi and piRNA expression is restricted to the male and female germline and independent of many proteins in other small-RNA pathways, including DCR-1. We show that Piwi is specifically required to silence Tc3, but not other Tc/mariner DNA transposons. Tc3 excision rates in the germline are increased at least 100-fold in piwi mutants as compared to wild-type. We find no evidence for a Ping-Pong model for piRNA amplification in C. elegans. Instead, we demonstrate that Piwi acts upstream of an endogenous siRNA pathway in Tc3 silencing. These data might suggest a link between piRNA and siRNA function.

Urban residential road traffic noise and hypertension: a cross-sectional study of adult population.

Results from studies involving exposure to road traffic noise and risk of hypertension are diverse and have seldom reached statistical significance. This study was designed with the aim of investigating whether there is any association between road traffic noise and prevalence of hypertension in an urban adult population. Similar studies have never been reported from India. A cross-sectional study was performed on 909 adults (533 female and 376 male) aged 18-80 years residing in close proximity to roadways in Asansol City. Time-weighted equivalent noise level (L den) was estimated using a standard modeling platform. Odds for hypertension in relation to traffic noise exposure were estimated by univariate and multifactorial logistic regression. The adjusted odds ratio (OR) for self-reported hypertension was 1.99 (95 % confidence interval (CI) 1.66-2.39) per 5 dB(A) increase of L den (range 55.1-77.9). A gender-related risk difference was observed among the male (OR 1.81 (1.42-2.31)) and female (OR 2.18 (1.66-2.88)) respondents. For increase in 9 years of age, the odds of hypertension risk increased by 60 % (OR 1.66 (1.43-1.91) among those exposed above L den 60 dB(A). Vulnerable subgroups were female aged 35-54 years and male aged 45-54 years. The study suggests that a threshold exposure to road traffic noise at L den > 65 dB(A) for men and L den > 60 dB(A) in women may be associated with the occurrence of hypertension.

Post-caesarean Section Headache: A Case Report of Post-dural Puncture Headache and Cerebral Venous Thrombosis Following Epidural Anaesthesia.

Post-dural puncture headache (PDPH) is a common complication of epidural and spinal anaesthesia in obstetric medicine. In rare cases, PDPH can be associated with complications such as cerebral venous thrombosis (CVT) as well. We discuss a recent case of a young female who developed PDPH and CVT concurrently after undergoing epidural anaesthesia for initially uncomplicated labour and delivered via an emergency caesarean section. She developed an orthostatic headache a few hours post administration of the epidural anaesthetic, which was initially treated as a suspected PDPH by giving simple analgesia and caffeine. Her symptoms did not improve and she underwent further neuroimaging, which revealed the development of a CVT. Despite the prompt administration of enoxaparin, the headache persisted and did not respond to increased doses of analgesia. After deliberation and inter-departmental discussion, an epidural blood patch was performed, leading to the prompt resolution of the headache. This report highlights a rare concurrence of PDPH and CVT, causing a diagnostic dilemma that resulted in treatment delays for the patient. Treating both conditions raises difficult practical questions, especially regarding the use of an epidural blood patch as opposed to anticoagulation. Given the risk of fatal complications such as venous cerebral infarction, seizures, and subdural hematoma, prompt treatment of both PDPH and CVT is strongly recommended. The multifactorial mechanism by which CVT develops with intracranial hypotension and PDPH also makes it essential for clinicians to keep an open mind when managing post-caesarean headaches, requiring inter-departmental cooperation to ensure optimal patient outcomes.

Simple ePDF: A Pair Distribution Function Method Based on Electron Diffraction Patterns to Reveal the Local Structure of Amorphous and Nanocrystalline Materials.

Amorphous, glassy or disordered materials play important roles in developing structural materials from metals or ceramics, devices from semiconductors or medicines from organic compounds. Their local structure is frequently similar to crystalline ones. A computer program is presented here that runs under the Windows operating system on a PC to extract pair distribution function (PDF) from electron diffraction in a transmission electron microscope (TEM). A polynomial correction reduces small systematic deviations from the expected average Q-dependence of scattering. Neighbor distance and coordination number measurements are supplemented by either measurement or enforcement of number density. Quantification of similarity is supported by calculation of Pearson's correlation coefficient and fingerprinting. A rough estimate of fractions in a mixture is computed by multiple least-square fitting using the PDFs from components of the mixture. PDF is also simulated from crystalline structural models (in addition to measured ones) to be used in libraries for fingerprinting or fraction estimation. Crystalline structure models for simulations are obtained from CIF files or str files of ProcessDiffraction. Data from inorganic samples exemplify usage. In contrast to previous free ePDF programs, our stand-alone program does not need a special software environment, which is a novelty. The program is available from the author upon request.

Influence of surface scattering on the anomalous conductance plateaus in an asymmetrically biased InAs/In(0.52)Al(0.48)As quantum point contact.

We study of the appearance and evolution of several anomalous (i.e., G < G(0) D 2e(2)/h) conductance plateaus in an In(0.52)Al(0.48)As/InAs quantum point contact (QPC). This work was performed at T = 4:2 K as a function of the offset bias ΔV(G) between the two in-plane gates of the QPC. The number and location of the anomalous conductance plateaus strongly depend on the polarity of the offset bias. The anomalous plateaus appear only over an intermediate range of offset bias of several volts. They are quite robust, being observed over a maximum range of nearly 1 V for the common sweep voltage applied to the two gates. These results are interpreted as evidence for the sensitivity of the QPC spin polarization to defects (surface roughness and impurity (dangling bond) scattering) generated during the etching process that forms the QPC side walls. This assertion is supported by non-equilibrium Green function simulations of the conductance of a single QPC in the presence of dangling bonds on its walls. Our simulations show that a spin conductance polarization as high as 98% can be achieved despite the presence of dangling bonds. The maximum in is not necessarily reached where the conductance of the channel is equal to 0:5G(0).

TINC- A Method to Dissect Regulatory Complexes at Single-Locus Resolution- Reveals an Extensive Protein Complex at the Nanog Promoter.

Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general.

Genetic unmasking of an epigenetically silenced microRNA in human cancer cells.

The mechanisms underlying microRNA (miRNA) disruption in human disease are poorly understood. In cancer cells, the transcriptional silencing of tumor suppressor genes by CpG island promoter hypermethylation has emerged as a common hallmark. We wondered if the same epigenetic disruption can "hit" miRNAs in transformed cells. To address this issue, we have used cancer cells genetically deficient for the DNA methyltransferase enzymes in combination with a miRNA expression profiling. We have observed that DNA hypomethylation induces a release of miRNA silencing in cancer cells. One of the main targets is miRNA-124a, which undergoes transcriptional inactivation by CpG island hypermethylation in human tumors from different cell types. Interestingly, we functionally link the epigenetic loss of miRNA-124a with the activation of cyclin D kinase 6, a bona fide oncogenic factor, and the phosphorylation of the retinoblastoma, a tumor suppressor gene.

Novel TEM Microscopy and Electron Diffraction Techniques to Characterize Cultural Heritage Materials: From Ancient Greek Artefacts to Maya Mural Paintings.

To understand in-depth material properties, manufacturing, and conservation in cultural heritage artefacts, there is a strong need for advanced characterization tools that enable analysis down to the nanometric scale. Transmission electron microscopy (TEM) and electron diffraction (ED) techniques, like 3D precession electron diffraction tomography and ASTAR phase/orientation mapping, are proposed to study cultural heritage materials at nanoscale. In this work, we show how electron crystallography in TEM helps to determine precise structural information and phase/orientation distribution of various pigments in cultural heritage materials from various historical periods like Greek amphorisks, Roman glass tesserae, and pre-Hispanic Maya mural paintings. Such TEM-based methods can be an alternative to synchrotron techniques and can allow distinguishing accurately different crystalline phases even in cases of identical or very close chemical compositions at the nanometric scale.

Inhibition of a K9/K36 demethylase by an H3.3 point mutation found in paediatric glioblastoma.

An array of oncogenic histone point mutations have been identified across a number of different cancer studies. It has been suggested that some of these mutant histones can exert their effects by inhibiting epigenetic writers. Here, we report that the H3.3 G34R (glycine to arginine) substitution mutation, found in paediatric gliomas, causes widespread changes in H3K9me3 and H3K36me3 by interfering with the KDM4 family of K9/K36 demethylases. Expression of a targeted single-copy of H3.3 G34R at endogenous levels induced chromatin alterations that were comparable to a KDM4 A/B/C triple-knockout. We find that H3.3 G34R preferentially binds KDM4 while simultaneously inhibiting its enzymatic activity, demonstrating that histone mutations can act through inhibition of epigenetic erasers. These results suggest that histone point mutations can exert their effects through interactions with a range of epigenetic readers, writers and erasers.

Chronic Myelogenous Leukemia- Initiating Cells Require Polycomb Group Protein EZH2.

Tyrosine kinase inhibitors (TKI) have revolutionized chronic myelogenous leukemia (CML) management. Disease eradication, however, is hampered by innate resistance of leukemia-initiating cells (LIC) to TKI-induced killing, which also provides the basis for subsequent emergence of TKI-resistant mutants. We report that EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), is overexpressed in CML LICs and required for colony formation and survival and cell-cycle progression of CML cell lines. A critical role for EZH2 is supported by genetic studies in a mouse CML model. Inactivation of Ezh2 in conventional conditional mice and through CRISPR/Cas9-mediated gene editing prevents initiation and maintenance of disease and survival of LICs, irrespective of BCR-ABL1 mutational status, and extends survival. Expression of the EZH2 homolog EZH1 is reduced in EZH2-deficient CML LICs, creating a scenario resembling complete loss of PRC2. EZH2 dependence of CML LICs raises prospects for improved therapy of TKI-resistant CML and/or eradication of disease by addition of EZH2 inhibitors. SIGNIFICANCE: This work defines EZH2 as a selective vulnerability for CML cells and their LICs, regardless of BCR-ABL1 mutational status. Our findings provide an experimental rationale for improving disease eradication through judicious use of EZH2 inhibitors within the context of standard-of-care TKI therapy. Cancer Discov; 6(11); 1237-47. ©2016 AACR.See related article by Scott et al., p. 1248This article is highlighted in the In This Issue feature, p. 1197.

Flow-induced protein kinase A-CREB pathway acts via BMP signaling to promote HSC emergence.

Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)-cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA-CREB and BMP pathways in isolated AGM VE-cadherin(+) cells from mid-gestation embryos, we demonstrate that PKA-CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA-CREB-BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.

Zolpidem withdrawal delirium.

The Z-category hypnotics are promoted for their relative safety. However, this view is challenged by the emerging clinical evidence in the form of zolpidem related intoxication delirium and seizures, and dependence and complicated withdrawal. We report the case of a zolpidem-naive alcohol-dependent inpatient that, while undergoing alcohol de-addiction, was prescribed zolpidem for insomnia and developed delirium during taper-off. He was successfully detoxified for alcohol, treated for delirium and put on disulfiram prophylaxis. The case highlights the need for being cautious while using zolpidem for insomnia in alcohol dependent subjects.

MicroRNA biogenesis is required for mouse primordial germ cell development and spermatogenesis.

BACKGROUND: MicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. Apart from miRNAs, mouse germ cells express another type of small RNA, piwi-interacting RNAs (piRNAs). Although it has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we first revealed the expression pattern of miRNAs by using a real-time PCR-based 220-plex miRNA expression profiling method. During development of germ cells, miR-17-92 cluster, which is thought to promote cell cycling, and the ES cell-specific cluster encoding miR-290 to -295 (miR-290-295 cluster) were highly expressed in primordial germ cells (PGCs) and spermatogonia. A set of miRNAs was developmentally regulated. We next analysed function of miRNA biogenesis in germ cell development by using conditional Dicer-knockout mice in which Dicer gene was deleted specifically in the germ cells. Dicer-deleted PGCs and spermatogonia exhibited poor proliferation. Retrotransposon activity was unexpectedly suppressed in Dicer-deleted PGCs, but not affected in the spermatogonia. In Dicer-deleted testis, spermatogenesis was retarded at an early stage when proliferation and/or early differentiation. Additionally, we analysed spermatogenesis in conditional Argonaute2-deficient mice. In contrast to Dicer-deficient testis, spermatogenesis in Argonaute2-deficient testis was indistinguishable from that in wild type. CONCLUSION/SIGNIFICANCE: These results illustrate that miRNAs are important for the proliferation of PGCs and spermatogonia, but dispensable for the repression of retrotransposons in developing germ cells. Consistently, miRNAs promoting cell cycling are highly expressed in PGCs and spermatogonia. Furthermore, based on normal spermatogenesis in Argonaute2-deficient testis, the critical function of Dicer in spermatogenesis is independent of Argonaute2.

Requirement of bic/microRNA-155 for normal immune function.

MicroRNAs are a class of small RNAs that are increasingly being recognized as important regulators of gene expression. Although hundreds of microRNAs are present in the mammalian genome, genetic studies addressing their physiological roles are at an early stage. We have shown that mice deficient for bic/microRNA-155 are immunodeficient and display increased lung airway remodeling. We demonstrate a requirement of bic/microRNA-155 for the function of B and T lymphocytes and dendritic cells. Transcriptome analysis of bic/microRNA-155-deficient CD4+ T cells identified a wide spectrum of microRNA-155-regulated genes, including cytokines, chemokines, and transcription factors. Our work suggests that bic/microRNA-155 plays a key role in the homeostasis and function of the immune system.