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.

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.

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.

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.

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).

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.

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.

Alternative ESC and ESC-like subunits of a polycomb group histone methyltransferase complex are differentially deployed during Drosophila development.

The Extra sex combs (ESC) protein is a Polycomb group (PcG) repressor that is a key noncatalytic subunit in the ESC-Enhancer of zeste [E(Z)] histone methyltransferase complex. Survival of esc homozygotes to adulthood based solely on maternal product and peak ESC expression during embryonic stages indicate that ESC is most critical during early development. In contrast, two other PcG repressors in the same complex, E(Z) and Suppressor of zeste-12 [SU(Z)12], are required throughout development for viability and Hox gene repression. Here we describe a novel fly PcG repressor, called ESC-Like (ESCL), whose biochemical, molecular, and genetic properties can explain the long-standing paradox of ESC dispensability during postembryonic times. Developmental Western blots show that ESCL, which is 60% identical to ESC, is expressed with peak abundance during postembryonic stages. Recombinant complexes containing ESCL in place of ESC can methylate histone H3 with activity levels, and lysine specificity for K27, similar to that of the ESC-containing complex. Coimmunoprecipitations show that ESCL associates with E(Z) in postembryonic cells and chromatin immunoprecipitations show that ESCL tracks closely with E(Z) on Ubx regulatory DNA in wing discs. Furthermore, reduced escl+ dosage enhances esc loss-of-function phenotypes and double RNA interference knockdown of ESC/ESCL in wing disc-derived cells causes Ubx derepression. These results suggest that ESCL and ESC have similar functions in E(Z) methyltransferase complexes but are differentially deployed as development proceeds.

Programming off and on states in chromatin: mechanisms of Polycomb and trithorax group complexes.

Polycomb and trithorax group proteins are evolutionarily conserved chromatin components that maintain stable states of gene expression. Recent studies have identified and characterized several multiprotein complexes containing these transcriptional regulators. Advances in understanding molecular activities of these complexes in vitro, and functional domains present in their subunits, suggest that they control transcription through multistep mechanisms that involve nucleosome modification, chromatin remodeling, and interaction with general transcription factors.

Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes.

The ESC-E(Z) complex of Drosophila melanogaster Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55. The four-subunit complex is >1,000-fold more active than E(Z) alone. Here we show that ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. We also show that loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. We describe missense mutations in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. We also find that a recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set.

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.

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.

Purification and functional characterization of SET8, a nucleosomal histone H4-lysine 20-specific methyltransferase.

BACKGROUND: Covalent modifications of histone N-terminal tails play fundamental roles in regulating chromatin structure and function. Extensive studies have established that acetylation of specific lysine residues in the histone tails plays an important role in transcriptional regulation. Besides acetylation, recent studies have revealed that histone methylation also has significant effects on heterochromatin formation and transcriptional regulation. Histone methylation occurs on specific arginine and lysine residues of histones H3 and H4. Thus far, only 2 residues on histone H4 are known to be methylated. While H4-arginine 3 (H4-R3) methylation is mediated by PRMT1, the enzyme(s) responsible for H4-lysine 20 (H4-K20) methylation is not known. RESULTS: To gain insight into the function of H4-K20 methylation, we set out to identify the enzyme responsible for this modification. We purified and cloned a novel human SET domain-containing protein, named SET8, which specifically methylates H4 at K20. SET8 is a single subunit enzyme and prefers nucleosomal substrates. We find that H4-K20 methylation occurs in a wide range of higher eukaryotic organisms and that SET8 homologs exist in C. elegans and Drosophila. We demonstrate that the Drosophila SET8 homolog has the same substrate specificity as its human counterpart. Importantly, disruption of SET8 in Drosophila reduces levels of H4-K20 methylation in vivo and results in lethality. Although H4-K20 methylation does not correlate with gene activity, it appears to be regulated during the cell cycle. CONCLUSIONS: We identified and characterized an evolutionarily conserved nucleosomal H4-K20-specific methyltransferase and demonstrated its essential role in Drosophila development.

Developing a bioterrorism preparedness campaign for veterans: Using focus groups to inform materials development.

In the context of a global war on terrorism experts have focused on the potential for a bioterrorist incident to cause widespread health and psychological consequences. Preparation is critical to improving the U.S. response to future bioterrorist incidents and educating the public is recognized as a vital part of this preparedness effort. Under a grant from the U.S. Veterans Health Administration (VHA), researchers from a network of VA health care and research facilities initiated a program to develop and evaluate educational materials for veterans--including those with mental illness. This article describes the results of a series of focus groups with three veteran subpopulations of interest to characterize their concerns and information needs and summarizes the insights gained that helped guide materials development.

Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression.

The Drosophila Sex Comb on Midleg (SCM) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish SCM as a crucial PcG member, its molecular role is not known. To investigate how SCM might link to PcG complexes, we analyzed the in vivo role of a conserved protein interaction module, the SPM domain. This domain is found in SCM and in another PcG protein, Polyhomeotic (PH), which is a core component of Polycomb repressive complex 1 (PRC1). SCM-PH interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize >30 missense mutations in the SPM domain of SCM. Genetic rescue assays showed that SCM repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of SCM with a reconstituted PRC1 core complex shows that SCM can partner with PRC1. However, gel filtration chromatography showed that the bulk of SCM is biochemically separable from PH in embryo nuclear extracts. These results suggest that SCM, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing.

Aripiprazole augmentation of antidepressants for the treatment of partially responding and nonresponding patients with major depressive disorder.

OBJECTIVE: To determine the efficacy and tolerability of aripiprazole, a dopamine D2 and 5-HT1A receptor partial agonist, as augmentation of antidepressant treatment of partially responding and nonresponding patients with major depressive disorder. METHOD: Fifteen patients with major depressive disorder (diagnosed with a site-generated form described in the text) and an incomplete response or no response to > or = 8 weeks of antidepressant (selective serotonin reuptake inhibitor, venlafaxine, or bupropion) monotherapy were treated with aripiprazole augmentation in an 8-week, open-label study. Data were gathered from July 2003 to March 2004. RESULTS: The mean duration of antidepressant monotherapy at baseline was 43.1 weeks. At baseline, mean Clinical Global Impressions-Severity of Illness scale and Hamilton Rating Scale for Depression (HAM-D) scores were 4.3 and 18.9, respectively. After initiation of aripiprazole augmentation, 6 of 15 patients achieved remission (HAM-D score < or = 7) at week 1, and 9 of 15 patients remitted by week 2. All 8 completers achieved remission by study endpoint. Akathisia in 2 patients who withdrew prematurely prompted a reduction in the starting dose of aripiprazole from 10 mg/day to 2.5 mg/day, resulting in a 50% reduction in attrition due to akathisia (2/7 withdrew due to akathisia with the 10-mg starting dose, 1/8 withdrew due to akathisia with the 2.5-mg starting dose). Discontinuation rates after 4 weeks of treatment were lower for the 2.5-mg starting dose (1/8 patients) than for the 10-mg starting dose (3/7 patients). Overall discontinuation rates at endpoint were lower for the 2.5-mg dose (3/8 patients) than the 10-mg dose (4/7 patients). Response to aripiprazole augmentation did not appear to be related to the antidepressant used at study initiation. CONCLUSION: Aripiprazole is an effective augmentation strategy for improving therapeutic response in patients with treatment-resistant major depressive disorder when administered in combination with standard antidepressant therapy. Based on this clinical signal, a double-blind, placebo-controlled trial is warranted.

Extended-release venlafaxine in relapse prevention for patients with major depressive disorder.

Many studies have demonstrated that venlafaxine is an efficacious and safe treatment for major depressive disorder (MDD). This double-blind, placebo-controlled study was performed to evaluate the efficacy of venlafaxine extended-release (XR) (75-225 mg/day) in the prevention of relapse of depression. Patients with MDD who responded to an 8-week course of venlafaxine XR treatment, i.e., had a score < or = 3 on the Clinical Global Impressions scale-Severity of Illness item (CGI-S) and a 21-item Hamilton Rating Scale for Depression (HAM-D(21)) score < or = 10, were randomly assigned to receive continuation treatment (up to 6 months) with venlafaxine XR (n=161) or placebo (n=157). The main efficacy outcome measure was the number of patients who experienced a relapse of depression. Relapse was defined by either a combination of a patient meeting Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria for MDD and a CGI-S score > or = 4, two consecutive CGI-S scores > or = 4, or a final CGI-S score > or = 4 for a patient who withdrew from the study. The cumulative probability of relapse was calculated using the Kaplan-Meier method of survival analysis. During the 6-month evaluation period, significantly more patients in the placebo group had a relapse of MDD than did patients who continued treatment with venlafaxine XR. Cumulative relapse rates at 3 and 6 months were 19 and 28%, respectively, for venlafaxine XR, and 44 and 52%, respectively, for placebo. This study demonstrates that venlafaxine XR is an effective and safe continuation therapy.

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.