Bivalent State Research Articles

Regulation of Pluripotency and Self- Renewal of ESCs through Epigenetic- Threshold Modulation and mRNA Pruning

Embryonic stem cell (ESC) pluripotency requires bivalent epigenetic modifications of key developmental genes regulated by various transcription factors and chromatin-modifying enzymes. How these factors coordinate with one another to maintain the bivalent chromatin state so that ESCs can undergo rapid self-renewal while retaining pluripotency is poorly understood. We report that Utf1,a target of Oct4 and Sox2, is a bivalent chromatin component that buffers poised states of bivalent genes. By limiting PRC2 loading and histone 3 lysine-27 trimethylation, Utf1 sets proper activation thresholds for bivalent genes. It also promotes nuclear tagging of messenger RNAs (mRNAs) transcribed from insufficiently silenced bivalent genes for cytoplasmic degradation through mRNA decapping. These opposing functions of Utf1 promote coordinated differentiation. The mRNA degradation function also ensures rapid cell proliferation by blocking the Myc-Arf feedback control. Thus, Utf1 couples the core pluripotency factors with Myc and PRC2 networks to promote the pluripotency and proliferation of ESCs.

Epigenetic control of CCR5 transcript levels in immune cells and modulation by small molecules inhibitors

Previously, we have shown that CCR5 transcription is regulated by CREB-1. However, the ubiquitous pattern of CREB-1 expression suggests the involvement of an additional level of transcriptional control in the cell type–specific expression of CCR5. In this study, we show that epigenetic changes (i.e. DNA methylation and histone modifications) within the context of the CCR5 P1 promoter region correlate with transcript levels of CCR5 in healthy and in malignant CD4+ T lymphocytes as well as in CD14+ monocytes. In normal naïve T cells and CD14+ monocytes the CCR5 P1 promoter resembles a bivalent chromatin state, with both repressive and permissive histone methylation and acetylation marks. The CCR5-expressing CD14+ monocytes however show much higher levels of acetylated histone H3 (AcH3) compared to the non–CCR5-expressing naïve T cells. Combined with a highly methylated promoter in CD14+ monocytes, this indicates a dominant role for AcH3 in CCR5 transcription. We also show that pharmacological interference in the epigenetic repressive mechanisms that account for the lack of CCR5 transcription in T leukaemic cell lines results in an increase in CREB-1 association with CCR5 P1 chromatin. Furthermore, RNA polymerase II was also recruited into CCR5 P1 chromatin resulting in CCR5 re-expression. Together, these data indicate that epigenetic modifications of DNA, and of histones, contribute to the control of CCR5 transcription in immune effector cells.

The broadband NIR emission properties of Bi doped La2O3–Al2O3–SiO2 glass

Bi2O3 doped 65SiO2–20Al2O3–15La2O3 (in mole%) glasses were prepared by the traditional melting–quenching method. The spectroscopic properties and mechanism of NIR broadband emission in these glasses were investigated in this work. Three excitation wavelengths of 500, 700 and 800nm were used to test emission spectra. The emission band under 500nm excitation can be regarded as combination of emission bands under 700 and 800nm excitation. 2.0mole% is found to be the optimal Bi2O3 doping level in this glass. Under 500nm excitation its emission peak, FWHM and lifetime of emission band are 1160nm, 300nm and 569μs, respectively. The longest fluorescent lifetime reaches 620μs under 700nm excitation. The valence state of Bi in these glasses is suggested to be lower than +3 by X-ray photoelectron spectroscopy. With the help of energy matching, we infer that both Bi0 and Bi+ centers are responsible for the NIR fluorescence of Bi2O3 doped 65SiO2–20Al2O3–15La2O3 glass.

Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules

The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.

Polycomb-Repressed Genes Have Permissive Enhancers that Initiate Reprogramming

Key regulatory genes, suppressed by Polycomb and H3K27me3, become active during normal differentiation and induced reprogramming. Using the well-characterized enhancer/promoter pair of MYOD1 as a model, we have identified a critical role for enhancers in reprogramming. We observed an unexpected nucleosome-depleted region (NDR) at the H3K4me1-enriched enhancer at which transcriptional regulators initially bind, leading to subsequent changes in the chromatin at the cognate promoter. Exogenous Myod1 activates its own transcription by binding first at the enhancer, leading to an NDR and transcription-permissive chromatin at the associated MYOD1 promoter. Exogenous OCT4 also binds first to the permissive MYOD1 enhancer but has a different effect on the cognate promoter, where the monovalent H3K27me3 marks are converted to the bivalent state characteristic of stem cells. Genome-wide, a high percentage of Polycomb targets are associated with putative enhancers in permissive states, suggesting that they may provide a widespread avenue for the initiation of cell-fate reprogramming.

Formation of a stable bivalent state of Yb ions in Na4Y6F22:Ce3+, Yb3+ crystal under intense UV irradiation

The evolution dynamics of absorption spectra induced in samples of Na4Y6F22:Ce3+, Yb3+ crystal by radiation resonant with 4f-5d transitions of Ce3+ ions was studied and analyzed. It was found that at least two types of color centers with different life times are induced in the studied crystal. It is established that the group of absorption bands in the UV spectral range that demonstrate long-term stability after excitation is caused by the 4f 13–4f 125d transitions of bivalent ytterbium ions. The sequence of processes that lead to the reduction of ytterbium ions from the trivalent to the bivalent state is proposed.

Dynamics of bivalent chromatin domains upon drug induced reactivation and resilencing in cancer cells

Epigenetic deregulation revealed by altered profiles of DNA methylation and histone modifications is a frequent event in cancer cells and results in abnormal patterns of gene expression. Cancer silenced genes constitute prime therapeutic targets and considerable progress has been made in the epigenetic characterization of the chromatin scenarios associated with their inactivation and drug induced reactivation. Despite these advances, the mechanisms involved in the maintenance or resetting of epigenetic states in both physiological and pharmacological situations are poorly known. To get insights into the dynamics of chromatin regulation upon drug-induced reactivation, we have investigated the epigenetic profiles of two chromosomal regions undergoing long range epigenetic silencing in colon cancer cells in time-course settings after exposure of cells to chromatin reactivating agents. The DNA methylation states and the balance between histone H3K4 methylation and H3K27 methylation marks clearly define groups of genes with alternative responses to therapy. We show that the expected epigenetic remodeling induced by the reactivating drugs, just achieves a transient disruption of the bivalent states, which overcome the treatment and restore the transcriptional silencing approximately four weeks after drug exposure. The interplay between DNA methylation and bivalent histone marks appears to configure a plastic but stable chromatin scenario that is fully restored in silenced genes after drug withdrawal. These data suggest that improvement of epigenetic therapies may be achieved by designing strategies with long lasting effects.

MnTUD-1: synthesis, characterization and catalytic behavior in liquid-phase oxidation of cyclohexane

Manganese containing three dimensional sponge-like amorphous mesoporous silicate MnTUD-1 with Si/Mn ratio of 115, 44 and 25 was prepared by hydrothermal method using tetraethylene glycol as the template. Nitrogen physisorption indicates that MnTUD-1 is a mesoporous material possessing surface area of 430–490 m2/g with mesopore volume of 0.61–0.71 cm3/g and an average adsorption pore diameter of 4.1–4.7 nm. The framework incorporation of manganese was confirmed by FT-IR, EPR and DRS UV–Vis studies. Manganese exists in bivalent and trivalent states in all the materials. MnTUD-1 showed good catalytic activity (13.5% cyclohexane conversion) and high selectivity (84.5%) in the liquid phase oxidation of cyclohexane with tert-butyl hydroperoxide as the oxidant under mild solvent-less reaction conditions.

Influence of Na on the structure of Bi 4Ti 3O 12 films deposited by liquid-delivery spin MOCVD

Thin Na-substituted Bi 4Ti 3O 12 films were grown by the liquid-delivery spin metal-organic chemical vapor deposition (MOCVD) method with different concentrations of sodium bis(trimethylsilyl)amide [Na(TMSA)] as Na precursor. At a substrate temperature of 600 °C the original Aurivillius structure was preserved, however high resolution x-ray diffraction (HRXRD) studies indicate that the Na-substituted phase exhibits a slightly smaller lattice parameter compared to the pure Bi 4Ti 3O 12 phase. From additional x-ray photoemission spectroscopy (XPS) results, we have concluded that monovalent Na + ions have been incorporated on Bi 3+ sites in the perovskite units. The proposed charge compensation for this aliovalent substitution is explained by a shift of the valence state of Bi 3+ ions in the vicinity of the incorporated Na + ions from 3+ to 5+. Due to the small ionic radius of the Bi 5+ ions, the incorporation efficiency amounts to a few atomic percent only.

Differences in the epigenetic regulation of MT-3 gene expression between parental and Cd+2 or As+3 transformed human urothelial cells

BackgroundStudies have shown that metallothionein 3 (MT-3) is not expressed in normal urothelium or in the UROtsa cell line, but is expressed in urothelial cancer and in tumors generated from the UROtsa cells that have been transformed by cadmium (Cd+2) or arsenite (As+3).The present study had two major goals. One, to determine if epigenetic modifications control urothelial MT-3 gene expression and if regulation is altered by malignant transformation by Cd+2 or As+3. Two, to determine if MT-3 expression might translate clinically as a biomarker for malignant urothelial cells released into the urine.ResultsThe histone deacetylase inhibitor MS-275 induced MT-3 mRNA expression in both parental UROtsa cells and their transformed counterparts. The demethylating agent, 5-Aza-2'-deoxycytidine (5-AZC) had no effect on MT-3 mRNA expression. ChIP analysis showed that metal-responsive transformation factor-1 (MTF-1) binding to metal response elements (MRE) elements of the MT-3 promoter was restricted in parental UROtsa cells, but MTF-1 binding to the MREs was unrestricted in the transformed cell lines. Histone modifications at acetyl H4, trimethyl H3K4, trimethyl H3K27, and trimethyl H3K9 were compared between the parental and transformed cell lines in the presence and absence of MS-275. The pattern of histone modifications suggested that the MT-3 promoter in the Cd+2 and As+3 transformed cells has gained bivalent chromatin structure, having elements of being "transcriptionally repressed" and "transcription ready", when compared to parental cells. An analysis of MT-3 staining in urinary cytologies showed that a subset of both active and non-active patients with urothelial cancer shed positive cells in their urine, but that control patients only rarely shed MT-3 positive cells.ConclusionThe MT-3 gene is silenced in non-transformed urothelial cells by a mechanism involving histone modification of the MT-3 promoter. In contrast, transformation of the urothelial cells with either Cd+2 or As+3 modified the chromatin of the MT-3 promoter to a bivalent state of promoter readiness. Urinary cytology for MT-3 positive cells would not improve the diagnosis of urothelial cancer, but might have potential as a biomarker for tumor progression.

Cu related doublets green band emission in ZnO:Cu thin films

Cu-doped ZnO (ZnO:Cu) thin films were grown on Si (111) substrate by low-pressure metal-organic chemical vapor deposition equipment. The crystal structures and optical properties of as-grown sample were examined. X-ray diffraction patterns indicated a lattice relaxation after the Cu doping. The incorporation of Cu atoms into ZnO film and its existence in a bivalent state were demonstrated by x-ray photoelectron spectroscopy measurements. Low-temperature photoluminescence was carried out at temperature of 11.4 K for both unintentionally doped and Cu-doped ZnO films. A characteristic green-luminescence with fine structure consisted of doublets emission peak was observed, which was believed to be associated with Cu doping. A theoretical model based on hydrogen analog has been proposed to explain this phenomenon. It provides new information about the detailed role of Cu in ZnO thin films.

Preparation and characterization of antibacterial Zn2+-exchanged montmorillonites

Zn-montmorillonites(Zn-MMTs) as antibacterial compounds were prepared by an ion-exchange reaction. The reaction time, initial pH value and molar ratios of CEC influencing zinc content in Zn-MMTs were investigated, and Zn-MMTs were characterized by means of EDX, XRD, XPS, and SEM. The results of bacterial growth tests were confirmed by determination of the minimum inhibition concentrations (MICs) and minimum bactericidal concentrations (MBCs). The experimental results show that the zinc is confirmed as bivalent zinc state, the d001 basal spacing of Zn-MMTs is enlarged with the enhancement of the zinc content, and the particles of Zn-MMTs are formed with irregular shape. Moreover, the antibacterial activity of Zn-MMTs increases with increasing the zinc content, and Zn-MMT-3 containing 6.76 mass% of zinc exhibits optimum antibacterial activity against Escherichia coli and Staphylococcus aureus. The intercalated zinc ions act as very effective antibacterial substances in the long term.

Facile fabrication of copper-supported ordered mesoporous carbon for antibacterial behavior

Copper-supported ordered mesoporous carbon (Cu/CMK-3) was prepared by impregnating ordered mesoporous carbon (CMK-3) with CuCl 2 aqueous solution. CMK-3 was served as a carrier for the continuous immobilization of Cu. The supported copper was observed to be the bivalence state, indicating that the Cu 2+ ion was not reduced into cuprous species or metallic copper in the CMK-3. The BET surface area and pore volume of Cu/CMK-3 were 728 m 2/g and 0.95 cm 3/g, respectively. The antibacterial activities of Cu/CMK-3 were tested by means of minimal inhibitory concentration (MIC) and viable cell counting method. The results show that Cu/CMK-3 presents a good antibacterial performance against Escherichia coli ( E. coli) and Staphylococcus aureus ( S. aureus), which indicates its potential applications as antibacterial material for microbiocides.

Abstract 2310: Snail-induced epigenetic regulation and its implication in plasticity of cellular morphology

Abstract Epithelial and mesenchymal cells exhibit distinct phenotypic and functional characteristics, yet the two cell types possess substantial plasticity and are interconvertable. Bidirectional transitions between epithelial and mesenchymal cells play crucial roles in physiological and pathological processes. Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are fundamental to embryonic development. In human cancer, EMT confers migratory and invasive properties on carcinoma cells, thereby promoting tumor invasion and dissemination. Moreover, EMT may endow cells with stem cell-like traits. After migrating tumor cells reach ectopic organ sites, conversion of mesenchymal tumor cells to epithelial cells through MET (including differentiation of tumor stem cells) may facilitate macrometastatic growth. However, despite its critical relevance in normal development and cancer metastasis, the molecular basis underpinning the interconvertibility between epithelial and mesenchymal states remains incompletely understood. The zinc-finger transcription factor Snail induces EMT by directly repressing E-cadherin, a defining marker of epithelial cells. Here we show that Snail physically interacts with and recruits the histone demethylase LSD1 to the E-cadherin promoter to remove dimethylation of lysine 4 on histone H3 (H3K4m2), a histone modification associated with active chromatin. Furthermore, demethylation of H3K4m2 by LSD1 is essential for Snail-mediated transcriptional repression. In contrast to H3K4me2, the active trimethylated H3K4 (H3K4m3) mark is not a substrate for LSD1 and it remains at high levels at the inactive E-cadherin promoter despite Snail expression. In addition, Snail induces H3K27 trimethylation (H3K27m3), a repressive chromatin mark. As a consequence, the Snail-repressed E-cadherin promoter simultaneously possesses opposite H3K4m3 and H3K27m3 marks, a pattern characteristic of bivalent chromatin modifications. Bivalent chromatin domains were initially identified and are enriched in stem cells. Bivalent promoters are inactive but are poised for activation. Although it awaits to be determined whether Snail-induced chromatin bivalency might contribute to cellular stemness, this observation suggests that the E-cadherin gene repressed by Snail remains in a transcriptionally ready state for re-activation, which may be critical for MET. These results exemplify how a bivalent chromatin state is established and provide new insights into epigenetic regulation of E-cadherin transcription and the plasticity of cellular morphology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2310.

Genome wide mapping of histone methylation reveals a distinct epigenomic signature in human pluripotent stem cells

In human pluripotent stem cell cultures, differentiation‐associated genes are silenced but “poised” for expression by bivalent chromatin marks: the concurrent presence of activating (H3K4me3) and repressing (H3K27me3) histone modifications. Here we sought to map this bivalent state across the genome of a subset of heterogeneous cultured human embryonic stem cells; the fraction that expresses the highest amounts of pluripotency‐associated factors (hpESCs). To this end, ChIP‐Seq was performed for H3K4me3 and H3K27me3 in hpESCs and the heterogeneous culture from which they were purified. Consistent with previous reports, both populations displayed high levels of H3K27me3 at differentiation‐associated genes. While H3K4me3 was also abundant at differentiation‐associated genes in the heterogeneous culture, this histone modification was generally absent at these genes in hpESCs. Therefore, our findings indicate that hpESCs possess a distinct epigenomic signature that typically lacks bivalent domains and that the repression of differentiation‐associated genes is regulated primarily by Polycomb. Collectively, our results strongly suggest that bivalency represents a transitory state between pluripotent and lineage committed cells.

Epigenetic chromatin states uniquely define the developmental plasticity of murine hematopoietic stem cells

AbstractHeritable epigenetic signatures are proposed to serve as an important regulatory mechanism in lineage fate determination. To investigate this, we profiled chromatin modifications in murine hematopoietic stem cells, lineage-restricted progenitors, and CD4+ T cells using modified genome-scale mini-chromatin immunoprecipitation technology. We show that genes involved in mature hematopoietic cell function associate with distinct chromatin states in stem and progenitor cells, before their activation or silencing upon cellular maturation. Many lineage-restricted promoters are associated with bivalent histone methylation and highly combinatorial histone modification patterns, which may determine their selective priming of gene expression during lineage commitment. These bivalent chromatin states are conserved in mammalian evolution, with a particular overrepresentation of promoters encoding key regulators of hematopoiesis. After differentiation into progenitors and T cells, activating histone modifications persist at transcriptionally repressed promoters, suggesting that these transcriptional programs might be reactivated after lineage restriction. Collectively, our data reveal the epigenetic framework that underlies the cell fate options of hematopoietic stem cells.

Pretreatment of well water with high level of iron before RO plant

Two methods of pretreatment of well water for reverse osmosis plant operating at the one of the Ukrainian breweries are considered. A new technology of pretreatment of well water with high level of iron has been suggested instead of traditional one based on oxidation of iron followed by filtration through quartz sand and active carbon. The new technology is based on keeping iron in the bivalent state followed by adsorption on carboxylic cationite. The well water pretreatment plant based on suggested technology with capacity of 160 m 3/h has been operated since November 2006. It is shown that application of the new technology causes both simplifying of technology and lowering of operational expenses by 35% and capital expenses by 40% in comparison with traditional one. Such results were achieved due to higher flow rates of filtration and improvement of pretreated water quality which causes cutback of consumption of chemicals for RO elements cleaning.

Room‐temperature ferromagnetism of Cu‐doped ZnO films deposited by helicon magnetron sputtering

AbstractWurtzite structure ZnO films doped with 0.5 and 1.7 at% Cu were deposited by helicon magnetron sputtering. The prepared films exhibited room‐temperature (RT) ferromagnetism (FM). Maximum RT saturation magnetization of 2 emu/cm3 (∼0.3μB/Cu) was observed for ZnO film with 1.7 at% Cu. Cu ions were in a bivalent state as identified by X‐ray photoelectron spectroscopy (XPS). In photoluminescence spectra, the green emission peak increased and redshifted due to the incorporation of Cu or defects induced by Cu incorporation. Since Cu and Cu‐related oxides are not RT ferromagnetic, and no trace of ferromagnetic contamination was detected in XPS results, the observed FM is considered to be an intrinsic property of Cu‐doped ZnO films. The FM was thought to originate from defect‐related mechanisms. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Properties and structure of (As2Se3)1 − z (SnSe2) z − x (Tl2Se) x and (As2Se3)1 − z (SnSe) z − x (Tl2Se) x glasses

Tin is stabilized in the bivalent and tetravalent states in the structure of (As2Se3)1 − z (SnSe2) z − x (Tl2Se) x and (As2Se3)1 − z (SnSe) z − x (Tl2Se) x glasses. The presence of bivalent tin in the structural network of a glass does not give rise to extrinsic conductivity. Dependences of density, microhardness, and the glass-transition temperature on the composition of the glasses are interpreted using a model according to which the structure of the glasses is composed of structural units that correspond to As2Se3, AsSe, TlAsSe2, Tl2Se, SnSe, and SnSe2 compounds.

Structure, magnetic and transport properties of MnxGe1−x films

Abstract Ge 1− x Mn x ( x =0.05, 0.07, 0.11, 0.15, 0.19, 0.23, 0.26, 0.29) thin films were prepared by magnetron sputtering. All the films had a Ge cubic structure, and no indication of a secondary phase was found in any sample using X-ray diffraction (XRD). The crystal lattice constant increases with the Mn concentration, in accordance with Vegard's law. No films show any clear magnetic domain structure under a magnetic force microscope (MFM). Atom force microscope (AFM) measurements show that all films have a uniform particle size distribution, and a columnar growth pattern. X-ray photoelectron spectroscopy (XPS) measurements indicate that the Mn atoms are not solely in a bivalent state. Electrical transport properties show that the resistance of the films increases with increasing Mn concentration, suggesting that the Mn ions are in deep-level acceptor states, while resistance decreases with increasing temperature, which is a typical semiconductor property. The ferromagnetism is intrinsic and long-ranged, mediated mainly by hole carriers through s, p–d exchange coupling.