The Cullin-4 Complex DCDC Does Not Require E3 Ubiquitin Ligase Elements To Control Heterochromatin in Neurospora crassa

ABSTRACT The cullin-4 (CUL4) complex DCDC ( D IM-5/-7/-9/ C UL4/ D DB1 c omplex) is essential for DNA methylation and heterochromatin formation in Neurospora crassa . Cullins form the scaffold of cullin-RING E3 ubiquitin ligases (CRLs) and are modified by the covalent attachment of NEDD8, a ubiquitin-like protein that regulates the stability and activity of CRLs. We report that neddylation is not required for CUL4-dependent DNA methylation or heterochromatin formation but is required for the DNA repair functions. Moreover, the RING domain protein RBX1 and a segment of the CUL4 C terminus that normally interacts with RBX1, the E2 ligase, CAND1, and CSN are dispensable for DNA methylation and heterochromatin formation by DCDC. Our study provides evidence for the noncanonical functions of core CRL components.

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The Cullin3 Ubiquitin Ligase Functions as a Nedd8-bound Heterodimer

Molecular Biology of the Cell ◽

10.1091/mbc.e06-06-0542 ◽

2007 ◽

Vol 18 (3) ◽

pp. 899-909 ◽

Cited By ~ 47

Author(s):

Wananit Wimuttisuk ◽

Jeffrey D. Singer

Keyword(s):

Ubiquitin Ligase ◽

Covalent Attachment ◽

Winged Helix ◽

Btb Domain ◽

C Terminus ◽

Ubiquitin Conjugating Enzyme ◽

Ubiquitination Pathway ◽

Insight Into ◽

Ligase Function

Cullins are members of a family of scaffold proteins that assemble multisubunit ubiquitin ligase complexes to confer substrate specificity for the ubiquitination pathway. Cullin3 (Cul3) forms a catalytically inactive BTB-Cul3-Rbx1 (BCR) ubiquitin ligase, which becomes functional upon covalent attachment of the ubiquitin homologue neural-precursor-cell-expressed and developmentally down regulated 8 (Nedd8) near the C terminus of Cul3. Current models suggest that Nedd8 activates cullin complexes by providing a recognition site for a ubiquitin-conjugating enzyme. Based on the following evidence, we propose that Nedd8 activates the BCR ubiquitin ligase by mediating the dimerization of Cul3. First, Cul3 is found as a neddylated heterodimer bound to a BTB domain-containing protein in vivo. Second, the formation of a Cul3 heterodimer is mediated by a Nedd8 molecule, which covalently attaches itself to one Cul3 molecule and binds to the winged-helix B domain at the C terminus of the second Cul3 molecule. Third, complementation experiments revealed that coexpression of two distinct nonfunctional Cul3 mutants can rescue the ubiquitin ligase function of the BCR complex. Likewise, a substrate of the BCR complex binds heterodimeric Cul3, suggesting that the Cul3 complex is active as a dimer. These findings not only provide insight into the architecture of the active BCR complex but also suggest assembly as a regulatory mechanism for activation of all cullin-based ubiquitin ligases.

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LSD1 prevents aberrant heterochromatin formation in Neurospora crassa

Nucleic Acids Research ◽

10.1093/nar/gkaa724 ◽

2020 ◽

Vol 48 (18) ◽

pp. 10199-10210

Author(s):

William K Storck ◽

Vincent T Bicocca ◽

Michael R Rountree ◽

Shinji Honda ◽

Tereza Ormsby ◽

...

Keyword(s):

Dna Methylation ◽

Neurospora Crassa ◽

Constitutive Heterochromatin ◽

Histone H3 ◽

Feedback Mechanism ◽

Histone Demethylase ◽

Histone Deacetylation ◽

Heterochromatin Formation ◽

Lysine Specific Demethylase ◽

Specialized Form

Abstract Heterochromatin is a specialized form of chromatin that restricts access to DNA and inhibits genetic processes, including transcription and recombination. In Neurospora crassa, constitutive heterochromatin is characterized by trimethylation of lysine 9 on histone H3, hypoacetylation of histones, and DNA methylation. We explored whether the conserved histone demethylase, lysine-specific demethylase 1 (LSD1), regulates heterochromatin in Neurospora, and if so, how. Though LSD1 is implicated in heterochromatin regulation, its function is inconsistent across different systems; orthologs of LSD1 have been shown to either promote or antagonize heterochromatin expansion by removing H3K4me or H3K9me respectively. We identify three members of the Neurospora LSD complex (LSDC): LSD1, PHF1, and BDP-1. Strains deficient for any of these proteins exhibit variable spreading of heterochromatin and establishment of new heterochromatin domains throughout the genome. Although establishment of H3K9me3 is typically independent of DNA methylation in Neurospora, instances of DNA methylation-dependent H3K9me3 have been found outside regions of canonical heterochromatin. Consistent with this, the hyper-H3K9me3 phenotype of Δlsd1 strains is dependent on the presence of DNA methylation, as well as HCHC-mediated histone deacetylation, suggesting that spreading is dependent on some feedback mechanism. Altogether, our results suggest LSD1 works in opposition to HCHC to maintain proper heterochromatin boundaries.

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Induction of H3K9me3 and DNA methylation by tethered heterochromatin factors in Neurospora crassa

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715049114 ◽

2017 ◽

Vol 114 (45) ◽

pp. E9598-E9607 ◽

Cited By ~ 11

Author(s):

Jordan D. Gessaman ◽

Eric U. Selker

Keyword(s):

Dna Methylation ◽

Gene Silencing ◽

Neurospora Crassa ◽

Dna Methyltransferase ◽

Catalytic Domain ◽

De Novo ◽

Histone Deacetylation ◽

Heterochromatin Formation ◽

Histone Deacetylase 1

Functionally different chromatin domains display distinct chemical marks. Constitutive heterochromatin is commonly associated with trimethylation of lysine 9 on histone H3 (H3K9me3), hypoacetylated histones, and DNA methylation, but the contributions of and interplay among these features are not fully understood. To dissect the establishment of heterochromatin, we investigated the relationships among these features using an in vivo tethering system in Neurospora crassa. Artificial recruitment of the H3K9 methyltransferase DIM-5 (defective in methylation-5) induced H3K9me3 and DNA methylation at a normally active, euchromatic locus but did not bypass the requirement of DIM-7, previously implicated in the localization of DIM-5, indicating additional DIM-7 functionality. Tethered heterochromatin protein 1 (HP1) induced H3K9me3, DNA methylation, and gene silencing. The induced heterochromatin required histone deacetylase 1 (HDA-1), with an intact catalytic domain, but HDA-1 was not essential for de novo heterochromatin formation at native heterochromatic regions. Silencing did not require H3K9me3 or DNA methylation. However, DNA methylation contributed to establishment of H3K9me3 induced by tethered HP1. Our analyses also revealed evidence of regulatory mechanisms, dependent on HDA-1 and DIM-5, to control the localization and catalytic activity of the DNA methyltransferase DIM-2. Our study clarifies the interrelationships among canonical aspects of heterochromatin and supports a central role of HDA-1–mediated histone deacetylation in heterochromatin spreading and gene silencing.

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Regulation of FLIP(L) and TRAIL-R2 signalling by the SCFSkp2 Ubiquitin Ligase Complex

10.1101/723718 ◽

2019 ◽

Author(s):

JZ Roberts ◽

C Holohan ◽

T Sessler ◽

J Fox ◽

C. Higgins ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Large Subunit ◽

Caspase 8 ◽

Apoptosis Induction ◽

Splice Form ◽

Expression Levels ◽

Ubiquitin Ligase Complex ◽

Direct Binding ◽

The Stability ◽

Ring E3

AbstractDepending on its expression levels, the long splice form of the pseudo-caspase FLIP (FLIP(L)) can act as an inhibitor (high expression) or activator (low expression) of apoptosis induction by the TRAIL-R2 death-inducing signalling complex (DISC); its expression levels are therefore tightly regulated. Here, we demonstrate that the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2) regulates the stability of FLIP(L) (but not the short splice form FLIP(S)), and, unusually, this is mediated by direct binding of FLIP(L) to Cullin-1 rather than via Skp2. By fine mapping the interaction of FLIP(L) with Cullin-1 to the large subunit of its pseudo-caspase domain, we found that the interaction is significantly stronger with FLIP(L)’s DISC-processed p43-form. Importantly, this interaction disrupts the ability of p43-FLIP to interact with FADD, caspase-8 and another DISC component, TRAF2. Moreover, we find that SCFSkp2 associates with TRAIL-R2 constitutively and does so independently of FLIP(L) and other canonical DISC components. Inhibition of Cullin-1 expression (using siRNA) or activity (using a NEDDylation inhibitor, MLN4924) enhanced FLIP(L) and TRAF2 levels at the TRAIL-R2 DISC and enhanced caspase-8 processing. This suggests that processing of FLIP(L) to p43-FLIP at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP’s association with the DISC thereby altering caspase-8 processing. These findings provide important new insights into how FLIP(L) expression and TRAIL-R2 signaling is controlled.

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Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain

Biochemical Journal ◽

10.1042/bj20071708 ◽

2008 ◽

Vol 415 (1) ◽

pp. 155-163 ◽

Cited By ~ 69

Author(s):

M. Christine Bruce ◽

Voula Kanelis ◽

Fatemeh Fouladkou ◽

Anne Debonneville ◽

Olivier Staub ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Down Regulation ◽

Hect Domain ◽

Ww Domains ◽

Protein Protein Interaction ◽

C Terminus ◽

Subsequent Degradation ◽

Interaction Domains ◽

The Stability ◽

Py Motif

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein–protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse–chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain–HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.

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The Stability of Histone Acetyltransferase General Control Non-derepressible (Gcn) 5 Is Regulated by Cullin4-RING E3 Ubiquitin Ligase

Journal of Biological Chemistry ◽

10.1074/jbc.m111.290767 ◽

2011 ◽

Vol 286 (48) ◽

pp. 41344-41352 ◽

Cited By ~ 14

Author(s):

Yongming Li ◽

Aimee Jaramillo-Lambert ◽

Jing Hao ◽

Yi Yang ◽

Wenge Zhu

Keyword(s):

Ubiquitin Ligase ◽

Histone Acetyltransferase ◽

E3 Ubiquitin Ligase ◽

General Control ◽

The Stability ◽

Ring E3

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Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614279113 ◽

2016 ◽

Vol 113 (41) ◽

pp. E6135-E6144 ◽

Cited By ~ 13

Author(s):

Shinji Honda ◽

Vincent T. Bicocca ◽

Jordan D. Gessaman ◽

Michael R. Rountree ◽

Ayumi Yokoyama ◽

...

Keyword(s):

Dna Methylation ◽

Neurospora Crassa ◽

Histone Deacetylase ◽

Histone Deacetylation ◽

H3k9 Methylation ◽

Heterochromatin Protein 1 ◽

Heterochromatin Protein ◽

Heterochromatin Formation ◽

H3k9 Trimethylation ◽

Shed Light

DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1–associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation.

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The stability of HIV-2 Vpx and Vpr proteins is regulated by the presence or absence of zinc-binding sites and poly-proline motifs with distinct roles

Journal of General Virology ◽

10.1099/jgv.0.001456 ◽

2020 ◽

Vol 101 (9) ◽

pp. 997-1007

Author(s):

Kazunori Shimagaki ◽

Ryoko Koga ◽

Haruna Fujino ◽

Ami Ahagon ◽

Hiroshi Tateishi ◽

...

Keyword(s):

Binding Site ◽

Zinc Binding ◽

Hydrophobic Region ◽

Unique Region ◽

Content Type ◽

C Terminus ◽

Immunodeficiency Virus ◽

Zinc Binding Site ◽

The Stability

The Vpx and Vpr proteins of human immunodeficiency virus type 2 (HIV-2) are important for virus replication. Although these proteins are homologous, Vpx is expressed at much higher levels than Vpr. Previous studies demonstrated that this difference results from the presence of an HHCC zinc-binding site in Vpx that is absent in Vpr. Vpx has another unique region, a poly-proline motif (PPM) of seven consecutive prolines at the C-terminus. Using PPM point mutants of Vpx, this study demonstrated that these seven consecutive prolines are critical for suppressing proteasome degradation of Vpx in the absence of Gag. Both the PPM and the zinc-binding site stabilize Vpx but do so via different mechanisms. PPM and zinc-binding site mutants overexpressed in Escherichia coli aggregated readily, indicating that these motifs normally prevent exposure of the hydrophobic region outside the structure. Furthermore, introduction of the zinc-binding site and the PPM into Vpr increased the level of Vpr expression so that it was as high as that of Vpx. Intriguingly, HIV-2 has evolved to express Vpx at high levels and Vpr at low levels based on the presence and absence of these two motifs with distinct roles.

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Permuting the PGF Signature Motif Blocks both Archaeosortase-Dependent C-Terminal Cleavage and Prenyl Lipid Attachment for the Haloferax volcanii S-Layer Glycoprotein

Journal of Bacteriology ◽

10.1128/jb.00849-15 ◽

2015 ◽

Vol 198 (5) ◽

pp. 808-815 ◽

Cited By ~ 16

Author(s):

Mohd Farid Abdul Halim ◽

Kelly R. Karch ◽

Yitian Zhou ◽

Daniel H. Haft ◽

Benjamin A. Garcia ◽

...

Keyword(s):

Molecular Mechanisms ◽

Bacterial Species ◽

Industrial Applications ◽

Haloferax Volcanii ◽

Covalent Attachment ◽

Lipid Modification ◽

Cell Surfaces ◽

Content Type ◽

C Terminus

ABSTRACTFor years, the S-layer glycoprotein (SLG), the sole component of many archaeal cell walls, was thought to be anchored to the cell surface by a C-terminal transmembrane segment. Recently, however, we demonstrated that theHaloferax volcaniiSLG C terminus is removed by an archaeosortase (ArtA), a novel peptidase. SLG, which was previously shown to be lipid modified, contains a C-terminal tripartite structure, including a highly conserved proline-glycine-phenylalanine (PGF) motif. Here, we demonstrate that ArtA does not process an SLG variant where the PGF motif is replaced with a PFG motif (slgG796F,F797G). Furthermore, using radiolabeling, we show that SLG lipid modification requires the PGF motif and is ArtA dependent, lending confirmation to the use of a novel C-terminal lipid-mediated protein-anchoring mechanism by prokaryotes. Similar to the case for the ΔartAstrain, the growth, cellular morphology, and cell wall of theslgG796F,F797Gstrain, in which modifications of additionalH. volcaniiArtA substrates should not be altered, are adversely affected, demonstrating the importance of these posttranslational SLG modifications. Our data suggest that ArtA is either directly or indirectly involved in a novel proteolysis-coupled, covalent lipid-mediated anchoring mechanism. Given that archaeosortase homologs are encoded by a broad range of prokaryotes, it is likely that this anchoring mechanism is widely conserved.IMPORTANCEProkaryotic proteins bound to cell surfaces through intercalation, covalent attachment, or protein-protein interactions play critical roles in essential cellular processes. Unfortunately, the molecular mechanisms that anchor proteins to archaeal cell surfaces remain poorly characterized. Here, using the archaeonH. volcaniias a model system, we report the firstin vivostudies of a novel protein-anchoring pathway involving lipid modification of a peptidase-processed C terminus. Our findings not only yield important insights into poorly understood aspects of archaeal biology but also have important implications for key bacterial species, including those of the human microbiome. Additionally, insights may facilitate industrial applications, given that photosynthetic cyanobacteria encode uncharacterized homologs of this evolutionarily conserved enzyme, or may spur development of unique drug delivery systems.

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