DCAF26, an Adaptor Protein of Cul4-Based E3, Is Essential for DNA Methylation in Neurospora crassa

Xu, Hui; Wang, Jiyong; Hu, Qiwen; Quan, Yun; Chen, Huijie; Cao, Yingqiong; Li, Chunbo; Wang, Ying; He, Qun

doi:10.1371/journal.pgen.1001132

Cited by 38 publications

(36 citation statements)

References 34 publications

(63 reference statements)

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“…The control strain, bearing FLAG-tagged wild-type cul4 ϩ , exhibited a protein doublet (Fig. 1B), presumably reflecting the neddylated and unneddylated forms of CUL4 (6,21). Mutation of the predicted neddylation site eliminated the slower-migrating band (Fig.…”

Section: Resultsmentioning

confidence: 98%

“…DIM-9/DCAF26 is required for the establishment of H3K9me3 by the DCDC (6,21). To address the possibility that neddylation of N. crassa CUL4 might be required for another function, such as DNA repair, we tested the sensitivity of wild-type and K863 mutants to the alkylating agent methyl methanesulfonate (MMS) and the topoisomerase inhibitor camptothecin (CPT).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2015

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The cullin-4 (CUL4) complex DCDC (DIM-5/-7/-9/CUL4/DDB1 complex) 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. U biquitination, the addition of ubiquitin moieties to proteins, is a multistep process that regulates the intracellular stability, localization, and function of numerous proteins (1, 2). Ubiquitin is activated by an E1 enzyme and then transferred to a substrate by an E2 ubiquitin-conjugating enzyme under the direction of multisubunit cullin-RING E3 ubiquitin ligases (CRLs) (3, 4). Cullins form the scaffold of CRLs by bridging substrate adaptor proteins that interact with their N termini and catalytic proteins that interact with their C termini (4).Cullin-4 (CUL4) complexes control cell cycle progression, DNA repair, and signal transduction (5, 6). The Neurospora crassa DCDC (DIM-5/-7/-9/CUL4/DDB1 complex) is essential for methylation of lysine 9 of histone H3 (H3K9) and DNA methylation (6). The DCDC resembles established CRLs, in which the substrate specificity adaptor protein DDB1/DIM-8 (DNA-damage-binding protein 1) serves as a bridge between CUL4 and the DCAF (DDB1 and CUL4-associated factor) DIM-9. These members of the DCDC, in turn, associate with the histone methyltransferase DIM-5 and its partner, DIM-7 (6). In addition, as in validated CRLs, CUL4 is modified by attachment of the ubiquitin-like protein NEDD8 in DCDC (6). Similarly, the Schizosaccharomyces pombe fission yeast CUL4 complex CLRC directs H3K9 methylation, although the potentially ubiquitinated substrate for this complex remains elusive (7-9), as with DCDC.Fruitless efforts to find putative substrates for ubiquitination by DCDC led us to explore the possibility that this complex has an ubiquitination-independent function. The C terminus of cullins, which is essential for the catalytic activity of CRLs, directly interacts with the E2 ligase and RBX1/ROC1, a small RING domain catalytic protein that facilitates the attachment of ubiquitin moieties onto substrates (10). The flexible backbone of cullins undergoes conformational changes to bring RBX1 and the E2 ligase in close proximity to the substrates, which are recruited by the adaptor protein bound to the N terminus (11). Covalent attachment of NEDD8 to an invariant lysine in the C terminus regulates the stability and activity of cullins (12)(13)(14)(15). To explore the contribution of neddylation in the reg...

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

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

et al. 2015

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“…Generation of Antiserum against SET-2-GST-SET-2 (containing SET-2 amino acids 308 -568) fusion protein was expressed in BL21 cells and the recombinant protein was purified and used as the antigen to generate rabbit polyclonal antiserum as described previously (25,36,37).…”

Section: Methodsmentioning

confidence: 99%

Suppression of WHITE COLLAR-independent frequency Transcription by Histone H3 Lysine 36 Methyltransferase SET-2 Is Necessary for Clock Function in Neurospora

Sun

Zhou

Xiao

et al. 2016

Journal of Biological Chemistry

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The circadian system in Neurospora is based on the transcriptional/translational feedback loops and rhythmic frequency (frq) transcription requires the WHITE COLLAR (WC) complex. Our previous paper has shown that frq could be transcribed in a WC-independent pathway in a strain lacking the histone H3K36 methyltransferase, SET-2 (su(var)3-9-enhancer-of-zestetrithorax-2) (1), but the mechanism was unclear. Here we disclose that loss of histone H3K36 methylation, due to either deletion of SET-2 or H3K36R mutation, results in arrhythmic frq transcription and loss of overt rhythmicity. Histone acetylation at frq locus increases in set-2 KO mutant. Consistent with these results, loss of H3K36 methylation readers, histone deacetylase RPD-3 (reduced potassium dependence 3) or EAF-3 (essential SAS-related acetyltransferase-associated factor 3), also leads to hyperacetylation of histone at frq locus and WC-independent frq expression, suggesting that proper chromatin modification at frq locus is required for circadian clock operation. Furthermore, a mutant strain with three amino acid substitutions (histone H3 lysine 9, 14, and 18 to glutamine) was generated to mimic the strain with hyperacetylation state of histone H3. H3K9QK14QK18Q mutant exhibits the same defective clock phenotype as rpd-3 KO mutant. Our results support a scenario in which H3K36 methylation is required to establish a permissive chromatin state for circadian frq transcription by maintaining proper acetylation status at frq locus.Despite evolutionary distance, circadian clock oscillators are conserved among the organisms to perform their cellular and behavioral activities. The eukaryotic circadian clock oscillator contains positive and negative elements that form auto-regulatory negative feedback loops (2-8).In the Neurospora circadian negative feedback loop, the GATA-family transcription factors WHITE COLLAR-1 (WC-1) 3 and WHITE COLLAR-2 (WC-2) serve as positive elements. Typically, WC-1 and WC-2 form a heterodimer through their Per-Arnt-Sim (PAS) domain and rhythmically bind the promoter of the clock gene frequency (frq) to activate its transcription. FREQUENCY (FRQ) and its partner FRQ-interacting RNA helicase (FRH) act as negative elements. FRQ is the core factor in Neurospora oscillator, which determines the normal clock rhythm (9 -16). The translated FRQ interacts with FRH and forms the FFC (FRQ-FRH complex) to inhibit its own transcription by suppressing the activity of the WC complex. FRQ is progressively phosphorylated and degraded through the ubiquitin-proteasome pathway. Degradation of FRQ derepresses the WC complex, initiating a new circle of frq transcription (14,(17)(18)(19). The circadian oscillator creates a robust rhythmic system with a period of ϳ22 h in Neurospora crassa (17,20). Rhythmic activation and repression of frq transcription result in cyclic changes of frq mRNA abundance. In Neurospora, histone modifiers and chromatin remodelers are required for rhythmic transcription of frq gene and clock-controlled genes (1, 9, 22-24). Howeve...

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“…At least 20 bona fide DCAFs likely exist in mammalian cells (Angers et al 2006;He et al 2006;Higa et al 2006b;Jin et al 2006;Higa and Zhang 2007;O'Connell and Harper 2007;Hu et al 2008;Lee et al 2008;McCall et al 2008;Scrima et al 2008;Choe et al 2009;Jackson and Xiong 2009;Xu et al 2010).…”

Section: Cdt2mentioning

confidence: 99%

Mechanism of CRL4^Cdt2, a PCNA-dependent E3 ubiquitin ligase

Havens¹,

Walter²

2011

Genes Dev.

197

254

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Eukaryotic cell cycle transitions are driven by E3 ubiquitin ligases that catalyze the ubiquitylation and destruction of specific protein targets. For example, the anaphasepromoting complex/cyclosome (APC/C) promotes the exit from mitosis via destruction of securin and mitotic cyclins, whereas CRL1 Skp2 allows entry into S phase by targeting the destruction of the cyclin-dependent kinase (CDK) inhibitor p27. Recently, an E3 ubiquitin ligase called CRL4 Cdt2 has been characterized, which couples proteolysis to DNA synthesis via an unusual mechanism that involves display of substrate degrons on the DNA polymerase processivity factor PCNA. Through its destruction of Cdt1, p21, and Set8, CRL4Cdt2 has emerged as a master regulator that prevents rereplication in S phase. In addition, it also targets other factors such as E2F and DNA polymerase h. In this review, we discuss our current understanding of the molecular mechanism of substrate recognition by CRL4 Cdt2 and how this E3 ligase helps to maintain genome integrity.

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DCAF26, an Adaptor Protein of Cul4-Based E3, Is Essential for DNA Methylation in Neurospora crassa

Cited by 38 publications

References 34 publications

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

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

Suppression of WHITE COLLAR-independent frequency Transcription by Histone H3 Lysine 36 Methyltransferase SET-2 Is Necessary for Clock Function in Neurospora

Mechanism of CRL4^Cdt2, a PCNA-dependent E3 ubiquitin ligase

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DCAF26, an Adaptor Protein of Cul4-Based E3, Is Essential for DNA Methylation in Neurospora crassa

Cited by 38 publications

References 34 publications

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

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

Suppression of WHITE COLLAR-independent frequency Transcription by Histone H3 Lysine 36 Methyltransferase SET-2 Is Necessary for Clock Function in Neurospora

Mechanism of CRL4Cdt2, a PCNA-dependent E3 ubiquitin ligase

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Mechanism of CRL4^Cdt2, a PCNA-dependent E3 ubiquitin ligase