scholarly journals Bending and looping of long DNA by Polycomb repressive complex 2 revealed by AFM imaging in liquid

2020 ◽  
Vol 48 (6) ◽  
pp. 2969-2981 ◽  
Author(s):  
Patrick R Heenan ◽  
Xueyin Wang ◽  
Anne R Gooding ◽  
Thomas R Cech ◽  
Thomas T Perkins
Keyword(s):  
Gene Silencing ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Cpg Island ◽  
Specific Binding ◽  
Gc Content ◽  
Polycomb Repressive Complex ◽  
Apparent Dissociation Constant ◽  
Polycomb Repressive Complex 2 ◽  
Epigenetic Gene Silencing

Abstract Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that methylates histone H3 at Lysine 27. PRC2 is critical for epigenetic gene silencing, cellular differentiation and the formation of facultative heterochromatin. It can also promote or inhibit oncogenesis. Despite this importance, the molecular mechanisms by which PRC2 compacts chromatin are relatively understudied. Here, we visualized the binding of PRC2 to naked DNA in liquid at the single-molecule level using atomic force microscopy. Analysis of the resulting images showed PRC2, consisting of five subunits (EZH2, EED, SUZ12, AEBP2 and RBBP4), bound to a 2.5-kb DNA with an apparent dissociation constant ($K_{\rm{D}}^{{\rm{app}}}$) of 150 ± 12 nM. PRC2 did not show sequence-specific binding to a region of high GC content (76%) derived from a CpG island embedded in such a long DNA substrate. At higher concentrations, PRC2 compacted DNA by forming DNA loops typically anchored by two or more PRC2 molecules. Additionally, PRC2 binding led to a 3-fold increase in the local bending of DNA’s helical backbone without evidence of DNA wrapping around the protein. We suggest that the bending and looping of DNA by PRC2, independent of PRC2’s methylation activity, may contribute to heterochromatin formation and therefore epigenetic gene silencing.

scholarly journals Gene Silencing Triggers Polycomb Repressive Complex 2 Recruitment to CpG Islands Genome Wide

2014 ◽  
Vol 55 (3) ◽  
pp. 347-360 ◽  
Author(s):  
Eva Madi Riising ◽  
Itys Comet ◽  
Benjamin Leblanc ◽  
Xudong Wu ◽  
Jens Vilstrup Johansen ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Cpg Islands ◽  
Polycomb Repressive Complex ◽  
Polycomb Repressive Complex 2 ◽  
Genome Wide

scholarly journals The histone variant H2A.Z is required to establish normal patterns of H3K27 methylation in Neurospora crassa

2020 ◽  
Author(s):  
Abigail J. Courtney ◽  
Masayuki Kamei ◽  
Aileen R. Ferraro ◽  
Kexin Gai ◽  
Qun He ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Molecular Mechanisms ◽  
Histone Variant ◽  
Polycomb Group ◽  
Stable Gene ◽  
Polycomb Group Proteins ◽  
Polycomb Repressive Complex ◽  
Facultative Heterochromatin ◽  
Polycomb Repressive Complex 2 ◽  
H3k27 Methylation

ABSTRACTNeurospora crassa contains a minimal Polycomb repression system, which provides rich opportunities to explore Polycomb-mediated repression across eukaryotes and enables genetic studies that can be difficult in plant and animal systems. Polycomb Repressive Complex 2 is a multi-subunit complex that deposits mono-, di-, and tri-methyl groups on lysine 27 of histone H3, and tri-methyl H3K27 is a molecular marker of transcriptionally repressed facultative heterochromatin. In mouse embryonic stem cells and multiple plant species, H2A.Z has been found to be co-localized with H3K27 methylation. H2A.Z is required for normal H3K27 methylation in these experimental systems, though the regulatory mechanisms are not well understood. We report here that Neurospora crassa mutants lacking H2A.Z or SWR-1, the ATP-dependent histone variant exchanger, exhibit a striking reduction in levels of H3K27 methylation. RNA-sequencing revealed downregulation of eed, encoding a subunit of PRC2, in an hH2Az mutant compared to wild type and overexpression of EED in a ΔhH2Az;Δeed background restored most H3K27 methylation. Reduced eed expression leads to region-specific losses of H3K27 methylation suggesting that EED-dependent mechanisms are critical for normal H3K27 methylation at certain regions in the genome.AUTHOR SUMMARYEukaryotic DNA is packaged with histone proteins to form a DNA-protein complex called chromatin. Inside the nucleus, chromatin can be assembled into a variety of higher-order structures that profoundly impact gene expression. Polycomb Group proteins are important chromatin regulators that control assembly of a highly condensed form of chromatin. The functions of Polycomb Group proteins are critical for maintaining stable gene repression during development of multicellular organisms, and defects in Polycomb proteins are linked to disease. There is significant interest in elucidating the molecular mechanisms that regulate the activities of Polycomb Group proteins and the assembly of transcriptionally repressed chromatin domains. In this study, we used a model fungus to investigate the regulatory relationship between a histone variant, H2A.Z, and a conserved histone modifying enzyme complex, Polycomb Repressive Complex 2 (PRC2). We found that H2A.Z is required for normal expression of a PRC2 component. Mutants that lack H2A.Z have defects in chromatin structure at some parts of the genome, but not others. Identification of PRC2-target domains that are differentially dependent on EED provides insights into the diverse mechanisms that regulate assembly and maintenance of facultative heterochromatin in a simple model system.Data Reference NumbersGSE146611

scholarly journals Ligand-specific binding forces of LFA-1 and Mac-1 in neutrophil adhesion and crawling

2018 ◽  
Vol 29 (4) ◽  
pp. 408-418 ◽  
Author(s):  
Ning Li ◽  
Hao Yang ◽  
Manliu Wang ◽  
Shouqin Lü ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Adhesion Molecules ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Specific Binding ◽  
Bond Rupture ◽  
Dynamic Force ◽  
Lymphocyte Function ◽  
Underlying Mechanisms ◽  
Distinct Features

Lymphocyte function–associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1) and their counterreceptors such as intercellular cell adhesion molecules (ICAM-1 and ICAM-2), junctional adhesion molecules (JAM-A, JAM-C), and receptors for advanced glycation end products (RAGE) are crucial for promoting polymorphonuclear leukocyte (neutrophil, PMN) recruitment. The underlying mechanisms of ligand-specific bindings in this cascade remain incompletely known. We compared the dynamic force spectra for various LFA-1/Mac-1–ligand bonds using single-molecule atomic force microscopy (AFM) and tested their functions in mediating PMN recruitment under in vitro shear flow. Distinct features of bond rupture forces and lifetimes were uncovered for these ligands, implying their diverse roles in regulating PMN adhesion on endothelium. LFA-1 dominates PMN adhesion on ICAM-1 and ICAM-2, while Mac-1 mediates PMN adhesion on RAGE, JAM-A, and JAM-C, which is consistent with their bond strength. All ligands can trigger PMN spreading and polarization, in which Mac-1 seems to induce outside-in signaling more effectively. LFA-1–ICAM-1 and LFA-1/Mac-1–JAM-C bonds can accelerate PMN crawling under high shear stress, presumably due to their high mechanical strength. This work provides new insight into basic molecular mechanisms of physiological ligands of β2 integrins in PMN recruitment.

scholarly journals Downregulation of the Polycomb-Associated Methyltransferase Ezh2 during Maturation of Hippocampal Neurons Is Mediated by MicroRNAs Let-7 and miR-124

2020 ◽  
Vol 21 (22) ◽  
pp. 8472
Author(s):  
Laura Guajardo ◽  
Rodrigo Aguilar ◽  
Fernando J. Bustos ◽  
Gino Nardocci ◽  
Rodrigo A. Gutiérrez ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Hippocampal Neurons ◽  
Gene Sequence ◽  
Histone H3 ◽  
Catalytic Subunit ◽  
Maturation Process ◽  
Polycomb Repressive Complex 2 ◽  
Ezh2 Expression ◽  
Epigenetic Gene Silencing ◽  
Mature Neurons

Ezh2 is a catalytic subunit of the polycomb repressive complex 2 (PRC2) which mediates epigenetic gene silencing through depositing the mark histone H3 lysine 27 trimethylation (H3K27me3) at target genomic sequences. Previous studies have demonstrated that Enhancer of Zeste Homolog 2 (Ezh2) was differentially expressed during maturation of hippocampal neurons; in immature neurons, Ezh2 was abundantly expressed, whereas in mature neurons the expression Ezh2 was significantly reduced. Here, we report that Ezh2 is downregulated by microRNAs (miRs) that are expressed during the hippocampal maturation process. We show that, in mature hippocampal neurons, lethal-7 (let-7) and microRNA-124 (miR-124) are robustly expressed and can target cognate motifs at the 3′-UTR of the Ezh2 gene sequence to downregulate Ezh2 expression. Together, these data demonstrate that the PRC2 repressive activity during hippocampal maturation is controlled through a post-transcriptional mechanism that mediates Ezh2 downregulation in mature neurons.

scholarly journals Ikaros mediates gene silencing in T cells through Polycomb repressive complex 2

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Attila Oravecz ◽  
Apostol Apostolov ◽  
Katarzyna Polak ◽  
Bernard Jost ◽  
Stéphanie Le Gras ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Silencing ◽  
Polycomb Repressive Complex ◽  
Polycomb Repressive Complex 2

scholarly journals TheArabidopsisepigenetic regulator ICU11 as an accessory protein of Polycomb Repressive Complex 2

2020 ◽  
Vol 117 (28) ◽  
pp. 16660-16666 ◽  
Author(s):  
Rebecca H. Bloomer ◽  
Claire E. Hutchison ◽  
Isabel Bäurle ◽  
James Walker ◽  
Xiaofeng Fang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nucleation Site ◽  
The Body ◽  
Accessory Protein ◽  
Polycomb Repressive Complex ◽  
Chromatin States ◽  
Polycomb Repressive Complex 2 ◽  
Core Components ◽  
Direct Target

Molecular mechanisms enabling the switching and maintenance of epigenetic states are not fully understood. Distinct histone modifications are often associated with ON/OFF epigenetic states, but how these states are stably maintained through DNA replication, yet in certain situations switch from one to another remains unclear. Here, we address this problem through identification ofArabidopsisINCURVATA11 (ICU11) as a Polycomb Repressive Complex 2 accessory protein. ICU11 robustly immunoprecipitated in vivo with PRC2 core components and the accessory proteins, EMBRYONIC FLOWER 1 (EMF1), LIKE HETEROCHROMATIN PROTEIN1 (LHP1), and TELOMERE_REPEAT_BINDING FACTORS (TRBs).ICU11encodes a 2-oxoglutarate–dependent dioxygenase, an activity associated with histone demethylation in other organisms, and mutant plants show defects in multiple aspects of theArabidopsisepigenome. To investigate its primary molecular function we identified theArabidopsis FLOWERING LOCUS C(FLC) as a direct target and foundicu11disrupted the cold-induced, Polycomb-mediated silencing underlying vernalization.icu11prevented reduction in H3K36me3 levels normally seen during the early cold phase, supporting a role for ICU11 in H3K36me3 demethylation. This was coincident with an attenuation of H3K27me3 at the internal nucleation site inFLC, and reduction in H3K27me3 levels across the body of the gene after plants were returned to the warm. Thus, ICU11 is required for the cold-induced epigenetic switching between the mutually exclusive chromatin states atFLC, from the active H3K36me3 state to the silenced H3K27me3 state. These data support the importance of physical coupling of histone modification activities to promote epigenetic switching between opposing chromatin states.

scholarly journals The Histone H3 Lysine 9 Methyltransferases G9a and GLP Regulate Polycomb Repressive Complex 2-Mediated Gene Silencing

2014 ◽  
Vol 53 (2) ◽  
pp. 277-289 ◽  
Author(s):  
Chiara Mozzetta ◽  
Julien Pontis ◽  
Lauriane Fritsch ◽  
Philippe Robin ◽  
Manuela Portoso ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Histone H3 ◽  
Polycomb Repressive Complex ◽  
Polycomb Repressive Complex 2

scholarly journals Phosphorylation of the oncogenic transcription factor ERG in prostate cells dissociates polycomb repressive complex 2, allowing target gene activation

2017 ◽  
Vol 292 (42) ◽  
pp. 17225-17235 ◽  
Author(s):  
Vivekananda Kedage ◽  
Brady G. Strittmatter ◽  
Paige B. Dausinas ◽  
Peter C. Hollenhorst
Keyword(s):  
Transcription Factor ◽  
Cell Migration ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Cellular Migration ◽  
Serine Residue ◽  
Polycomb Repressive Complex ◽  
Polycomb Repressive Complex 2 ◽  
Prostate Cells ◽  
Prostate Cancers

In ∼50% of prostate cancers, chromosomal rearrangements cause the fusion of the promoter and 5′-UTR of the androgen-regulated TMPRSS2 (transmembrane protease, serine 2) gene to the open reading frame of ERG, encoding an ETS family transcription factor. This fusion results in expression of full-length or N-terminally truncated ERG protein in prostate epithelia. ERG is not expressed in normal prostate epithelia, but when expressed, it promotes tumorigenesis via altered gene expression, stimulating epithelial-mesenchymal transition, cellular migration/invasion, and transformation. However, limited knowledge about the molecular mechanisms of ERG function in prostate cells has hampered efforts to therapeutically target ERG. ERK-mediated phosphorylation of ERG is required for ERG functions in prostate cells, but the reason for this requirement is unknown. Here, we report a mechanism whereby ERK-mediated phosphorylation of ERG at one serine residue causes a conformational change that allows ERK phosphorylation at a second serine residue, Ser-96. We found that the Ser-96 phosphorylation resulted in dissociation of EZH2 and SUZ12, components of polycomb repressive complex 2 (PRC2), transcriptional activation of ERG target genes, and increased cell migration. Conversely, loss of ERG phosphorylation at Ser-96 resulted in recruitment of EZH2 across the ERG-cistrome and a genome-wide loss of ERG-mediated transcriptional activation and cell migration. In conclusion, our findings have identified critical molecular mechanisms involving ERK-mediated ERG activation that could be exploited for therapeutic intervention in ERG-positive prostate cancers.

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