Molecular templating of layered halide perovskite nanowires.

Layered metal-halide perovskites, or two-dimensional perovskites, can be synthesized in solution, and their optical and electronic properties can be tuned by changing their composition. We report a molecular templating method that restricted crystal growth along all crystallographic directions except for [110] and promoted one-dimensional growth. Our approach is widely applicable to synthesize a range of high-quality layered perovskite nanowires with large aspect ratios and tunable organic-inorganic chemical compositions. These nanowires form exceptionally well-defined and flexible cavities that exhibited a wide range of unusual optical properties beyond those of conventional perovskite nanowires. We observed anisotropic emission polarization, low-loss waveguiding (below 3 decibels per millimeter), and efficient low-threshold light amplification (below 20 microjoules per square centimeter).

A Role for Monomethylation of Histone H3-K27 in Gene Activity in Drosophila.

Polycomb repressive complex 2 (PRC2) is a conserved chromatin-modifying enzyme that methylates histone H3 on lysine-27 (K27). PRC2 can add one, two, or three methyl groups and the fully methylated product, H3-K27me3, is a hallmark of Polycomb-silenced chromatin. Less is known about functions of K27me1 and K27me2 and the dynamics of flux through these states. These modifications could serve mainly as intermediates to produce K27me3 or they could each convey distinct epigenetic information. To investigate this, we engineered a variant of Drosophila melanogaster PRC2 which is converted into a monomethyltransferase. A single substitution, F738Y, in the lysine-substrate binding pocket of the catalytic subunit, E(Z), creates an enzyme that retains robust K27 monomethylation but dramatically reduced di- and trimethylation. Overexpression of E(Z)-F738Y in fly cells triggers desilencing of Polycomb target genes significantly more than comparable overexpression of catalytically deficient E(Z), suggesting that H3-K27me1 contributes positively to gene activity. Consistent with this, normal genomic distribution of H3-K27me1 is enriched on actively transcribed Drosophila genes, with localization overlapping the active H3-K36me2/3 chromatin marks. Thus, distinct K27 methylation states link to either repression or activation depending upon the number of added methyl groups. If so, then H3-K27me1 deposition may involve alternative methyltransferases beyond PRC2, which is primarily repressive. Indeed, assays on fly embryos with PRC2 genetically inactivated, and on fly cells with PRC2 chemically inhibited, show that substantial H3-K27me1 accumulates independently of PRC2. These findings imply distinct roles for K27me1 vs. K27me3 in transcriptional control and an expanded machinery for methylating H3-K27.

Phase 3 efficacy and safety trial of gadobutrol, a 1.0 molar macrocyclic MR imaging contrast agent, in patients referred for contrast-enhanced MR imaging of the central nervous system.

PURPOSE: Gadobutrol is a 1.0 M macrocyclic magnetic resonance imaging (MRI) contrast agent. A study was performed to evaluate the efficacy and safety of gadobutrol-enhanced versus unenhanced imaging for central nervous system (CNS) lesion visualization and detection. MATERIALS AND METHODS: An international, multicenter, open-label, Phase III clinical trial. Patients underwent unenhanced and gadobutrol 1.0 M-enhanced (0.1 mmol/kg BW) MR imaging using a standardized protocol. Unenhanced and combined unenhanced/gadobutrol-enhanced images were scored by three independent, blinded readers for degree of lesion enhancement, border delineation, internal morphology, and total number of lesions detected (primary efficacy variables). Exact match of the MR diagnoses with the final clinical diagnosis, detection of malignant CNS lesions, and confidence in diagnosis were secondary efficacy variables. RESULTS: Of 343 enrolled patients, 321 were evaluated for efficacy. All primary efficacy endpoints were met: superiority was demonstrated for gadobutrol-enhanced versus unenhanced MR images (P < 0.0001 in all cases) for lesion enhancement, border delineation, and internal morphology. Noninferiority was met for mean number of lesions detected. There were improvements in the sensitivity of malignant lesion detection, without a loss in specificity, exact-match diagnostic accuracy, and reader confidence. Treatment-related adverse events were reported in 4.1% (n = 14); all were nonserious. CONCLUSION: Gadobutrol 1.0M is an effective and well-tolerated contrast agent for CNS MRI.

A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element.

Polycomb/Trithorax response elements (PRE/TREs) can switch their function reversibly between silencing and activation by mechanisms that are poorly understood. Here we show that a switch in forward and reverse noncoding transcription from the Drosophila melanogaster vestigial (vg) PRE/TRE switches the status of the element between silencing (induced by the forward strand) and activation (induced by the reverse strand). In vitro, both noncoding RNAs inhibit PRC2 histone methyltransferase activity, but, in vivo, only the reverse strand binds PRC2. Overexpression of the reverse strand evicts PRC2 from chromatin and inhibits its enzymatic activity. We propose that the interaction of RNAs with PRC2 is differentially regulated in vivo, allowing regulated inhibition of local PRC2 activity. Genome-wide analysis shows that strand switching of noncoding RNAs occurs at several hundred Polycomb-binding sites in fly and vertebrate genomes. This work identifies a previously unreported and potentially widespread class of PRE/TREs that switch function by switching the direction of noncoding RNA transcription.

Elements of the polycomb repressor SU(Z)12 needed for histone H3-K27 methylation, the interface with E(Z), and in vivo function.

Polycomb repressive complex 2 (PRC2) is an essential chromatin-modifying enzyme that implements gene silencing. PRC2 methylates histone H3 on lysine-27 and is conserved from plants to flies to humans. In Drosophila melanogaster, PRC2 contains four core subunits: E(Z), SU(Z)12, ESC, and NURF55. E(Z) bears a SET domain that houses the enzyme active site. However, PRC2 activity depends upon critical inputs from SU(Z)12 and ESC. The stimulatory mechanisms are not understood. We present here functional dissection of the SU(Z)12 subunit. SU(Z)12 contains two highly conserved domains: an ∼140-amino-acid VEFS domain and a Cys2-His2 zinc finger (ZnF). Analysis of recombinant PRC2 bearing VEFS domain alterations, including some modeled after leukemia mutations, identifies distinct elements needed for SU(Z)12 assembly with E(Z) and stimulation of histone methyltransferase. The results define an extensive VEFS subdomain that organizes the SU(Z)12-E(Z) interface. Although the SU(Z)12 ZnF is not needed for methyltransferase in vitro, genetic rescue assays show that the ZnF is required in vivo. Chromatin immunoprecipitations reveal that this ZnF facilitates PRC2 binding to a genomic target. This study defines functionally critical SU(Z)12 elements, including key determinants of SU(Z)12-E(Z) communication. Together with recent findings, this illuminates PRC2 modulation by conserved inputs from its noncatalytic subunits.

Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put.

Chromatin modification by Polycomb proteins provides an essential strategy for gene silencing in higher eukaryotes. Polycomb repressive complexes (PRCs) silence key developmental regulators and are centrally integrated in the transcriptional circuitry of stem cells. PRC2 trimethylates histone H3 on lysine 27 (H3K27me3), and PRC1-type complexes ubiquitylate histone H2A and compact polynucleosomes. How PRCs are deployed to select and silence genomic targets is the subject of intense investigation. We review advances on targeting, modulation, and functions of PRC1 and PRC2 and progress on defining the transcriptional steps they impact. Recent findings emphasize PRC1 targeting independent of H3K27me3, nonenzymatic PRC1-mediated compaction, and connections between PRCs and noncoding RNAs. Systematic analyses of Polycomb complexes and associated histone modifications during DNA replication and mitosis have also emerged. The stage is now set to reveal fundamental epigenetic mechanisms that determine how Polycomb target genes are silenced and how Polycomb silence is preserved through cell-cycle progression.

Inner workings and regulatory inputs that control Polycomb repressive complex 2.

Polycomb repressive complex 2 (PRC2) is a conserved multisubunit enzyme that methylates histone H3 on lysine-27. This chromatin modification is a hallmark of target genes transcriptionally silenced by the Polycomb system. At its core, PRC2 activity depends upon the SET domain active site of its catalytic subunit, EZH2, as well as critical stimulatory inputs from noncatalytic subunits, especially EED and SU(Z)12. We review recent progress on this core PRC2 machinery, including key features of the active site, control mechanisms that operate via EZH2 phosphorylation, and subunit elements and architectures that influence PRC2 function. Among these, we highlight work identifying an EED regulatory site that enables PRC2 to bind pre-existing methylated H3-K27 and stimulate enzyme output. These advances illuminate basic inner workings of PRC2 and also provide insights that could aid design of PRC2 inhibitors. The chromatin landscape that PRC2 encounters in vivo is decorated with many histone modifications that accompany active transcription, such as H3-K4 methylation. It has long been assumed that these "active" modifications oppose PRC2 at some level but, until recently, mechanisms of this antagonistic cross-talk have been elusive. We discuss new findings that illuminate how H3-K4 and H3-K36 methylation, H3-K27 acetylation, and H3-S28 phosphorylation each exert a negative impact on PRC2 function. The emerging picture presents PRC2 as a cooperative multipart machine, intricately outfitted to sense and respond to the local chromatin environment and other cues. This PRC2 design ensures flexibility and fine tuning of its fundamental gene silencing roles in diverse biological contexts.

Small ubiquitin-like modifier (SUMO) conjugation impedes transcriptional silencing by the polycomb group repressor Sex Comb on Midleg.

The Drosophila protein Sex Comb on Midleg (Scm) is a member of the Polycomb group (PcG), a set of transcriptional repressors that maintain silencing of homeotic genes during development. Recent findings have identified PcG proteins both as targets for modification by the small ubiquitin-like modifier (SUMO) protein and as catalytic components of the SUMO conjugation pathway. We have found that the SUMO-conjugating enzyme Ubc9 binds to Scm and that this interaction, which requires the Scm C-terminal sterile α motif (SAM) domain, is crucial for the efficient sumoylation of Scm. Scm is associated with the major Polycomb response element (PRE) of the homeotic gene Ultrabithorax (Ubx), and efficient PRE recruitment requires an intact Scm SAM domain. Global reduction of sumoylation augments binding of Scm to the PRE. This is likely to be a direct effect of Scm sumoylation because mutations in the SUMO acceptor sites in Scm enhance its recruitment to the PRE, whereas translational fusion of SUMO to the Scm N terminus interferes with this recruitment. In the metathorax, Ubx expression promotes haltere formation and suppresses wing development. When SUMO levels are reduced, we observe decreased expression of Ubx and partial haltere-to-wing transformation phenotypes. These observations suggest that SUMO negatively regulates Scm function by impeding its recruitment to the Ubx major PRE.

Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2.

The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression. Here, we demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing.

Chromatin compaction at Hox loci: a polycomb tale beyond histone tails.

A new study in this issue of Molecular Cell (Eskeland et al., 2010) implicates Polycomb repressive complex 1 (PRC1) in compacting Hox gene chromatin in mouse embryonic stem cells and suggests that compaction, rather than histone tail ubiquitylation, confers Hox gene silencing.

Comparative analysis of chromatin binding by Sex Comb on Midleg (SCM) and other polycomb group repressors at a Drosophila Hox gene.

Sex Comb on Midleg (SCM) is a transcriptional repressor in the Polycomb group (PcG), but its molecular role in PcG silencing is not known. Although SCM can interact with Polycomb repressive complex 1 (PRC1) in vitro, biochemical studies have indicated that SCM is not a core constituent of PRC1 or PRC2. Nevertheless, SCM is just as critical for Drosophila Hox gene silencing as canonical subunits of these well-characterized PcG complexes. To address functional relationships between SCM and other PcG components, we have performed chromatin immunoprecipitation studies using cultured Drosophila Schneider line 2 (S2) cells and larval imaginal discs. We find that SCM associates with a Polycomb response element (PRE) upstream of the Ubx gene which also binds PRC1, PRC2, and the DNA-binding PcG protein Pleiohomeotic (PHO). However, SCM is retained at this Ubx PRE despite genetic disruption or knockdown of PHO, PRC1, or PRC2, suggesting that SCM chromatin targeting does not require prior association of these other PcG components. Chromatin immunoprecipitations (IPs) to test the consequences of SCM genetic disruption or knockdown revealed that PHO association is unaffected, but reduced levels of PRE-bound PRC2 and PRC1 were observed. We discuss these results in light of current models for recruitment of PcG complexes to chromatin targets.

Mechanisms of polycomb gene silencing: knowns and unknowns.

Polycomb proteins form chromatin-modifying complexes that implement transcriptional silencing in higher eukaryotes. Hundreds of genes are silenced by Polycomb proteins, including dozens of genes that encode crucial developmental regulators in organisms ranging from plants to humans. Two main families of complexes, called Polycomb repressive complex 1 (PRC1) and PRC2, are targeted to repressed regions. Recent studies have advanced our understanding of these complexes, including their potential mechanisms of gene silencing, the roles of chromatin modifications, their means of delivery to target genes and the functional distinctions among variant complexes. Emerging concepts include the existence of a Polycomb barrier to transcription elongation and the involvement of non-coding RNAs in the targeting of Polycomb complexes. These findings have an impact on the epigenetic programming of gene expression in many biological systems.

Roles of the EZH2 histone methyltransferase in cancer epigenetics.

EZH2 is the catalytic subunit of Polycomb repressive complex 2 (PRC2), which is a highly conserved histone methyltransferase that targets lysine-27 of histone H3. This methylated H3-K27 chromatin mark is commonly associated with silencing of differentiation genes in organisms ranging from plants to flies to humans. Studies on human tumors show that EZH2 is frequently over-expressed in a wide variety of cancerous tissue types, including prostate and breast. Although the mechanistic contributions of EZH2 to cancer progression are not yet determined, functional links between EZH2-mediated histone methylation and DNA methylation suggest partnership with the gene silencing machinery implicated in tumor suppressor loss. Here we review the basic molecular biology of EZH2 and the findings that implicate EZH2 in different cancers. We also discuss EZH2 connections to other silencing enzymes, such as DNA methyltransferases and histone deacetylases, and we consider progress on deciphering mechanistic consequences of EZH2 overabundance and its potential roles in tumorigenesis. Finally, we review recent findings that link EZH2 roles in stem cells and cancer, and we consider prospects for integrating EZH2 blockade into strategies for developing epigenetic therapies.

Dominant alleles identify SET domain residues required for histone methyltransferase of Polycomb repressive complex 2.

Polycomb gene silencing requires histone methyltransferase activity of Polycomb repressive complex 2 (PRC2), which methylates lysine 27 of histone H3. Information on how PRC2 works is limited by lack of structural data on the catalytic subunit, Enhancer of zeste (E(Z)), and the paucity of E(z) mutant alleles that alter its SET domain. Here we analyze missense alleles of Drosophila E(z), selected for molecular study because of their dominant genetic effects. Four missense alleles identify key E(Z) SET domain residues, and a fifth is located in the adjacent CXC domain. Analysis of mutant PRC2 complexes in vitro, and H3-K27 methylation in vivo, shows that each SET domain mutation disrupts PRC2 histone methyltransferase. Based on known SET domain structures, the mutations likely affect either the lysine-substrate binding pocket, the binding site for the adenosylmethionine methyl donor, or a critical tyrosine predicted to interact with the substrate lysine epsilon-amino group. In contrast, the CXC mutant retains catalytic activity, Lys-27 specificity, and trimethylation capacity. Deletion analysis also reveals a functional requirement for a conserved E(Z) domain N-terminal to CXC and SET. These results identify critical SET domain residues needed for PRC2 enzyme function, and they also emphasize functional inputs from outside the SET domain.

Drosophila histone deacetylase-3 controls imaginal disc size through suppression of apoptosis.

Histone deacetylases (HDACs) execute biological regulation through post-translational modification of chromatin and other cellular substrates. In humans, there are eleven HDACs, organized into three distinct subfamilies. This large number of HDACs raises questions about functional overlap and division of labor among paralogs. In vivo roles are simpler to address in Drosophila, where there are only five HDAC family members and only two are implicated in transcriptional control. Of these two, HDAC1 has been characterized genetically, but its most closely related paralog, HDAC3, has not. Here we describe the isolation and phenotypic characterization of hdac3 mutations. We find that both hdac3 and hdac1 mutations are dominant suppressors of position effect variegation, suggesting functional overlap in heterochromatin regulation. However, all five hdac3 loss-of-function alleles are recessive lethal during larval/pupal stages, indicating that HDAC3 is essential on its own for Drosophila development. The mutant larvae display small imaginal discs, which result from abnormally elevated levels of apoptosis. This cell death occurs as a cell-autonomous response to HDAC3 loss and is accompanied by increased expression of the pro-apoptotic gene, hid. In contrast, although HDAC1 mutants also display small imaginal discs, this appears to result from reduced proliferation rather than from elevated apoptosis. The connection between HDAC loss and apoptosis is important since HDAC inhibitors show anticancer activities in animal models through mechanisms involving apoptotic induction. However, the specific HDACs implicated in tumor cell killing have not been identified. Our results indicate that protein deacetylation by HDAC3 plays a key role in suppression of apoptosis in Drosophila imaginal tissue.

Estimates of serotonin and norepinephrine transporter inhibition in depressed patients treated with paroxetine or venlafaxine.

Paroxetine and venlafaxine are potent serotonin transporter (SERT) antagonists and weaker norepinephrine transporter (NET) antagonists. However, the relative magnitude of effect at each of these sites during treatment is unknown. Using a novel blood assay that estimates CNS transporter occupancy we estimated the relative SERT and NET occupancy of paroxetine and venlafaxine in human subjects to assess the relative magnitude of SERT and NET inhibition. Outpatient subjects (N=86) meeting criteria for major depression were enrolled in a multicenter, 8 week, randomized, double-blind, parallel group, antidepressant treatment study. Subjects were treated by forced-titration of paroxetine CR (12.5-75 mg/day) or venlafaxine XR (75-375 mg/day) over 8 weeks. Blood samples were collected weekly to estimate transporter inhibition. Both medications produced dose-dependent inhibition of the SERT and NET. Maximal SERT inhibition at week 8 for paroxetine and venlafaxine was 90% (SD 7) and 85% (SD 10), respectively. Maximal NET inhibition for paroxetine and venlafaxine at week 8 was 36% (SD 19) and 60% (SD 13), respectively. The adjusted mean change from baseline (mean 28.6) at week 8 LOCF in MADRS total score was -16.7 (SE 8.59) and -17.3 (SE 8.99) for the paroxetine and venlafaxine-treated patients, respectively. The magnitudes of the antidepressant effects were not significantly different from each other (95%CI -3.42, 4.54, p=0.784). The results clearly demonstrate that paroxetine and venlafaxine are potent SERT antagonists and less potent NET antagonists in vivo. NET antagonism has been posited to contribute to the antidepressant effects of these compounds. The clinical significance of the magnitude of NET antagonism by both medications remains unclear at present.

The efficacy and safety of aripiprazole as adjunctive therapy in major depressive disorder: a second multicenter, randomized, double-blind, placebo-controlled study.

Nonresponse to one or more antidepressants is common and an important public health problem. This study evaluated the efficacy and safety of adjunctive aripiprazole or placebo to standard antidepressant therapy (ADT) in patients with major depressive disorder who showed an inadequate response to at least 1 and up to 3 historical and 1 additional prospective ADT. The study comprised a 7-28-day screening, an 8-week prospective treatment, and a 6-week randomization phase. During prospective treatment, patients experiencing a major depressive episode (17-item Hamilton Rating Scale for Depression total score > or = 18) received single-blind adjunctive placebo plus clinicians' choice of ADT (escitalopram, fluoxetine, paroxetine controlled-release, sertraline, or venlafaxine extended-release). Subjects with inadequate response were randomized to adjunctive placebo (n = 190) or adjunctive aripiprazole (n = 191) (starting dose 5 mg/d, dose adjustments 2-20 mg/d, mean end-point dose of 11.0 mg/d). The primary efficacy endpoint was the mean change in Montgomery-Asberg Depression Rating Scale total score from end of prospective treatment phase to end of randomized treatment phase (last observation carried forward). Mean change in Montgomery-Asberg Depression Rating Scale total score was significantly greater with adjunctive aripiprazole than placebo (-8.5 vs -5.7; P = 0.001). Remission rates were significantly greater with adjunctive aripiprazole than placebo (25.4% vs 15.2%; P = 0.016) as were response rates (32.4% vs 17.4%; P < 0.001). Adverse events occurring in 10% of patients or more with adjunctive placebo or aripiprazole were akathisia (4.2% vs 25.9%), headache (10.5% vs 9.0%), and fatigue (3.7% vs 10.1%). Incidence of adverse events leading to discontinuation was low (adjunctive placebo [1.1%] vs adjunctive aripiprazole [3.7%]). Aripiprazole is an effective and safe adjunctive therapy as demonstrated in this short-term study for patients who are nonresponsive to standard ADT.