PcG Proteins, DNA Methylation, and Gene Repression by Chromatin Looping

Many DNA hypermethylated and epigenetically silenced genes in adult cancers are Polycomb group (PcG) marked in embryonic stem (ES) cells. We show that a large region upstream (∼30 kb) of and extending ∼60 kb around one such gene, GATA-4, is organized—in Tera-2 undifferentiated embryonic carcinoma (EC) cells—in a topologically complex multi-loop conformation that is formed by multiple internal long-range contact regions near areas enriched for EZH2, other PcG proteins, and the signature PcG histone mark, H3K27me3. Small interfering RNA (siRNA)–mediated depletion of EZH2 in undifferentiated Tera-2 cells leads to a significant reduction in the frequency of long-range associations at the GATA-4 locus, seemingly dependent on affecting the H3K27me3 enrichments around those chromatin regions, accompanied by a modest increase in GATA-4 transcription. The chromatin loops completely dissolve, accompanied by loss of PcG proteins and H3K27me3 marks, when Tera-2 cells receive differentiation signals which induce a ∼60-fold increase in GATA-4 expression. In colon cancer cells, however, the frequency of the long-range interactions are increased in a setting where GATA-4 has no basal transcription and the loops encompass multiple, abnormally DNA hypermethylated CpG islands, and the methyl-cytosine binding protein MBD2 is localized to these CpG islands, including ones near the gene promoter. Removing DNA methylation through genetic disruption of DNA methyltransferases (DKO cells) leads to loss of MBD2 occupancy and to a decrease in the frequency of long-range contacts, such that these now more resemble those in undifferentiated Tera-2 cells. Our findings reveal unexpected similarities in higher order chromatin conformation between stem/precursor cells and adult cancers. We also provide novel insight that PcG-occupied and H3K27me3-enriched regions can form chromatin loops and physically interact in cis around a single gene in mammalian cells. The loops associate with a poised, low transcription state in EC cells and, with the addition of DNA methylation, completely repressed transcription in adult cancer cells

Inhibition of SIRT1 Reactivates Silenced Cancer Genes without Loss of Promoter DNA Hypermethylation

The class III histone deactylase (HDAC), SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs) in which 5′ CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively), had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA)–mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression

Ευρετικές προσεγγίσεις του μοναδιάστατου προβλήματος πακετοποίησης

Article 59.1, of the International Code of Nomenclature for Algae, Fungi, and Plants (ICN; Melbourne Code), which addresses the nomenclature of pleomorphic fungi, became effective from 30 July 2011. Since that date, each fungal species can have one nomenclaturally correct name in a particular classification. All other previously used names for this species will be considered as synonyms. The older generic epithet takes priority over the younger name. Any widely used younger names proposed for use, must comply with Art. 57.2 and their usage should be approved by the Nomenclature Committee for Fungi (NCF). In this paper, we list all genera currently accepted by us in Dothideomycetes (belonging to 23 orders and 110 families), including pleomorphic and non-pleomorphic genera. In the case of pleomorphic genera, we follow the rulings of the current ICN and propose single generic names for future usage. The taxonomic placements of 1261 genera are listed as an outline. Protected names and suppressed names for 34 pleomorphic genera are listed separately. Notes and justifications are provided for possible proposed names after the list of genera. Notes are also provided on recent advances in our understanding of asexual and sexual morph linkages in Dothideomycetes. A phylogenetic tree based on four gene analyses supported 23 orders and 75 families, while 35 families still lack molecular data

A novel 6C assay uncovers Polycomb-mediated higher order chromatin conformations

We describe construction of a novel modification, “6C,” of chromatin looping assays that allows specific proteins that may mediate long-range chromatin interactions to be defined. This approach combines the standard looping approaches previously defined with an immunoprecipitation step to investigate involvement of the specific protein. The efficacy of this approach is demonstrated by using a Polycomb group (PcG) protein, Enhancer of Zeste (EZH2), as an example of how our assay might be used. EZH2, as a protein of the PcG complex, PRC2, has an important role in the propagation of epigenetic memory through deposition of the repressive mark, histone H3, lysine 27, tri-methylation (H3K27me3). Using our new 6C assay, we show how EZH2 is a direct mediator of long-range intra- and interchromosomal interactions that can regulate transcriptional down-regulation of multiple genes by facilitating physical proximities between distant chromatin regions, thus targeting sites within to PcG machinery

A DNA hypermethylation module for the stem/progenitor cell signature of cancer

Many DNA-hypermethylated cancer genes are occupied by the Polycomb (PcG) repressor complex in embryonic stem cells (ESCs). Their prevalence in the full spectrum of cancers, the exact context of chromatin involved, and their status in adult cell renewal systems are unknown. Using a genome-wide analysis, we demonstrate that ∼75% of hypermethylated genes are marked by PcG in the context of bivalent chromatin in both ESCs and adult stem/progenitor cells. A large number of these genes are key developmental regulators, and a subset, which we call the “DNA hypermethylation module,” comprises a portion of the PcG target genes that are down-regulated in cancer. Genes with bivalent chromatin have a low, poised gene transcription state that has been shown to maintain stemness and self-renewal in normal stem cells. However, when DNA-hypermethylated in tumors, we find that these genes are further repressed. We also show that the methylation status of these genes can cluster important subtypes of colon and breast cancers. By evaluating the subsets of genes that are methylated in different cancers with consideration of their chromatin status in ESCs, we provide evidence that DNA hypermethylation preferentially targets the subset of PcG genes that are developmental regulators, and this may contribute to the stem-like state of cancer. Additionally, the capacity for global methylation profiling to cluster tumors by phenotype may have important implications for further refining tumor behavior patterns that may ultimately aid therapeutic interventions

SIRT1 Inhibition Affects Key Phenotypic Aspects of Cancer Cells

<div><p>(A) MDA-MB-231 cells were infected for two rounds with RNAi-2 and −3 retrovirus, and puromycin-resistant colonies were counted after 3 d of selection. Error bars indicate standard deviation from the average of three experiments.</p><p>(B) RKO cells were transfected with 500 ng of pGL3-OT, a TCF-LEF−responsive reporter, or pGL3-OF, a negative control with a mutated TCF-LEF binding site in combination with 10 ng of pRL-CMV vector. Twenty-four hours post-transfection, cells were treated with either vehicle (DMSO) control or with 700 μM SPT for 24 h. <i>Firefly</i> luciferase activity was measured and normalized to the <i>Renilla</i> luciferase activities.</p><p>(C) As described in (A), pooled populations of MDA-MB-231 cells stably expressing RNAi-2 or RNAi-3 were harvested, protein concentrations were determined, and Western blot analysis was performed. An antibody that specifically recognizes the unphosphorylated (active) form of β-catenin was used, and on the same blot, β-actin was probed to ensure equal loading.</p><p>(D) Western blot analysis was performed on RKO cells expressing control or SIRT1 RNAi. Antibodies against SIRT1, phospho-GSK3β (inactive), cyclin D1, p27, and β-actin were used for Western blotting. On the same blot, β-actin was probed to ensure equal loading.</p></div