Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

Zhao, Yuanbiao; Shen, Yating; Yang, Shu‐Hua; Wang, Jiyong; Hu, Qiwen; Wang, Ying; He, Qun

doi:10.1074/jbc.m109.034710

Cited by 59 publications

(77 citation statements)

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“…Chromatin Immunoprecipitation-ChIP assay was performed as described previously (1,25,36,38). Briefly, Neurospora tissues under the experimental conditions were fixed with 1% formaldehyde for 15 min at 25°C with shaking, the fixation reaction were stopped by adding 125 mM glycine with shaking for another 5 min.…”

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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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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“…In Neurospora, heterochromatin formation is initiated at A·T-rich repetitive DNA sequences by the H3K9 methyltransferase DIM-5 (defective in methylation 5), which exists in a multiprotein complex called DCDC (DIM-5/7/9 -CUL4/ DDB1 complex) (16)(17)(18)(19). According to proposed nomenclature conventions (20), DIM-5 is sometimes also referred to as KMT1 (lysine [K] methyltransferase 1), based on its structural and functional homologies with the mammalian Suv39H1 (KMT1A) and Suv39H2 (KMT1B) enzymes.…”

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Heterochromatin Controls γH2A Localization in Neurospora crassa

et al. 2014

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bIn response to genotoxic stress, ATR and ATM kinases phosphorylate H2A in fungi and H2AX in animals on a C-terminal serine. The resulting modified histone, called ␥H2A, recruits chromatin-binding proteins that stabilize stalled replication forks or promote DNA double-strand-break repair. To identify genomic loci that might be prone to replication fork stalling or DNA breakage in Neurospora crassa, we performed chromatin immunoprecipitation (ChIP) of ␥H2A followed by next-generation sequencing (ChIP-seq). ␥H2A-containing nucleosomes are enriched in Neurospora heterochromatin domains. These domains are comprised of A·T-rich repetitive DNA sequences associated with histone H3 methylated at lysine-9 (H3K9me), the H3K9me-binding protein heterochromatin protein 1 (HP1), and DNA cytosine methylation. H3K9 methylation, catalyzed by DIM-5, is required for normal ␥H2A localization. In contrast, ␥H2A is not required for H3K9 methylation or DNA methylation. Normal ␥H2A localization also depends on HP1 and a histone deacetylase, HDA-1, but is independent of the DNA methyltransferase DIM-2. ␥H2A is globally induced in dim-5 mutants under normal growth conditions, suggesting that the DNA damage response is activated in these mutants in the absence of exogenous DNA damage. Together, these data suggest that heterochromatin formation is essential for normal DNA replication or repair.

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“…Generation of antiserum against PKAC-1 or RCM-1. The glutathione S-transferase (GST)/PKAC-1 fusion protein (containing PKAC-1 amino acids 1 to 318) or GST/RCM-1 (containing RCM-1 amino acids 480 to 883) was expressed in BL21 cells, and the recombinant proteins were purified and used as the antigens to generate rabbit polyclonal antisera as described previously (36,37). RCO-1 antiserum was generated previously (29).…”

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Role for Protein Kinase A in the Neurospora Circadian Clock by Regulating White Collar-Independent frequency Transcription through Phosphorylation of RCM-1

Liu

et al. 2015

Molecular and Cellular Biology

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Rhythmic activation and repression of clock gene expression is essential for the eukaryotic circadian clock functions. In the Neurospora circadian oscillator, the transcription of the frequency (frq) gene is periodically activated by the White Collar (WC) complex and suppressed by the FRQ-FRH complex. We previously showed that there is WC-independent frq transcription and its repression is required for circadian gene expression. How WC-independent frq transcription is regulated is not known. We show here that elevated protein kinase A (PKA) activity results in WC-independent frq transcription and the loss of clock function. We identified RCM-1 as the protein partner of RCO-1 and an essential component of the clock through its role in suppressing WCindependent frq transcription. RCM-1 is a phosphoprotein and is a substrate of PKA in vivo and in vitro. Mutation of the PKAdependent phosphorylation sites on RCM-1 results in WC-independent transcription of frq and impaired clock function. Furthermore, we showed that RCM-1 is associated with the chromatin at the frq locus, a process that is inhibited by PKA. Together, our results demonstrate that PKA regulates frq transcription by inhibiting RCM-1 activity through RCM-1 phosphorylation. Circadian clocks allow prokaryotic and eukaryotic organisms to regulate their daily molecular, cellular, behavioral, and physiological activities. The eukaryotic circadian clocks are composed of autoregulatory negative-feedback loops, including positive and negative elements that form the core circadian oscillators (1-6). Rhythmic transcriptional activation of the negative elements by the positive elements and rhythmic repression of positive elements by the negative elements are thought to be the main molecular basis for the generation of endogenous rhythmicity in eukaryotic clock systems. In addition to regulation at the transcriptional level, posttranslational modification of clock proteins by phosphorylation plays essential roles in clock functions (7-10).In the filamentous fungus Neurospora crassa, the core circadian negative feedback loop is composed of the positive elements, White Collar 1 (WC-1) and White Collar 2 (WC-2), and the negative elements, FREQUENCY (FRQ) and its partner FRQ-interacting RNA helicase (FRH) (2,3,11,12). WC-1 and WC-2, two Per-Arnt-Sim (PAS) domain-containing transcription factors, form the WC heterodimeric complex that binds to the Clock (C)-box of frq promoter to activate frq transcription (13-18). On the other hand, FRQ and FRH form the FFC complex to inhibit frq transcription by suppressing the activity of the WC complex by promoting WC phosphorylation (19)(20)(21)(22)(23)(24). FRQ is progressively phosphorylated over time before its degradation through the ubiquitin-proteasome pathway mediated by FWD-1 (21, 25-28). Genetic analyses show that the FRQ-dependent phosphorylation of WC-1 and WC-2 that suppresses White Collar complex (WCC) activity is mostly mediated by CKI and CKII (21).WC complex was long thought to be the only transcriptional activator o...

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Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

Cited by 59 publications

References 50 publications

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

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

Heterochromatin Controls γH2A Localization in Neurospora crassa

Role for Protein Kinase A in the Neurospora Circadian Clock by Regulating White Collar-Independent frequency Transcription through Phosphorylation of RCM-1

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