JMJD5 links CRY1 function and proteasomal degradation.
ABSTRACT: The circadian oscillator is a molecular feedback circuit whose orchestration involves posttranslational control of the activity and protein levels of its components. Although controlled proteolysis of circadian proteins is critical for oscillator function, our understanding of the underlying mechanisms remains incomplete. Here, we report that JmjC domain-containing protein 5 (JMJD5) interacts with CRYPTOCHROME 1 (CRY1) in an F-box/leucine-rich repeat protein 3 (FBXL3)-dependent manner and facilitates targeting of CRY1 to the proteasome. Genetic deletion of JMJD5 results in greater CRY1 stability, reduced CRY1 association with the proteasome, and disruption of circadian gene expression. We also report that in the absence of JMJD5, AMP-regulated protein kinase (AMPK)-induced CRY1 degradation is impaired, establishing JMJD5 as a key player in this mechanism. JMJD5 cooperates with CRY1 to repress circadian locomotor output cycles protein kaput (CLOCK)-brain and muscle ARNT-like protein 1 (BMAL1), thus linking CRY1 destabilization to repressive function. Finally, we find that ablation of JMJD5 impacts FBXL3- and CRY1-related functions beyond the oscillator.
Project description:The mammalian circadian clock coordinates various physiological activities with environmental cues to achieve optimal adaptation. The clock manifests oscillations of key clock proteins, which are under dynamic control at multiple post-translational levels. As a major post-translational regulator, the ubiquitination-dependent proteasome degradation system is counterbalanced by a large group of deubiquitin proteases with distinct substrate preference. Until now, whether deubiquitination by ubiquitin-specific proteases can regulate the clock protein stability and circadian pathways remains largely unclear. The mammalian clock protein, cryptochrome 1 (CRY1), is degraded via the FBXL3-mediated ubiquitination pathway, suggesting that it is also likely to be targeted by the deubiquitination pathway. Here, we identified that USP2a, a circadian-controlled deubiquitinating enzyme, interacts with CRY1 and enhances its protein stability via deubiquitination upon serum shock. Depletion of Usp2a by shRNA greatly enhances the ubiquitination of CRY1 and dampens the oscillation amplitude of the CRY1 protein during a circadian cycle. By stabilizing the CRY1 protein, USP2a represses the Per2 promoter activity as well as the endogenous Per2 gene expression. We also demonstrated that USP2a-dependent deubiquitination and stabilization of the CRY1 protein occur in the mouse liver. Interestingly, the pro-inflammatory cytokine, TNF-?, increases the CRY1 protein level and inhibits circadian gene expression in a USP2a-dependent fashion. Therefore, USP2a potentially mediates circadian disruption by suppressing the CRY1 degradation during inflammation.
Project description:The circadian system ensures that plants respond appropriately to environmental change by predicting regular transitions that occur during diel cycles. In order to be most useful, the circadian system needs to be compensated against daily and seasonal changes in temperature that would otherwise alter the pace of this biological oscillator. We demonstrate that an evening-phased protein, the putative histone demethylase JMJD5, contributes to temperature compensation. JMJD5 is co-expressed with components of the Evening Complex, an agglomeration of proteins including EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRHYTHYMO (LUX), which also integrates temperature changes into the molecular clockwork. One role of the Evening Complex is to regulate expression of PSEUDORESPONSE REGULATOR9 (PRR9) and PRR7, important components of the temperature compensation mechanism. Surprisingly we find that LUX, but not other Evening Complex components, is dispensable for clock function at low temperatures. Further genetic analysis suggests JMJD5 acts in a parallel pathway to LUX within the circadian system. Although an intact JMJD5 catalytic domain is required for its function within the clock, our findings suggest JMJD5 does not directly regulate H3K36 methylation at circadian loci. Such data refine our understanding of how JMDJ5 acts within the Arabidopsis circadian system.
Project description:The circadian transcriptional repressors cryptochrome 1 (Cry1) and 2 (Cry2) evolved from photolyases, bacterial light-activated DNA repair enzymes. In this study, we report that while they have lost DNA repair activity, Cry1/2 adapted to protect genomic integrity by responding to DNA damage through posttranslational modification and coordinating the downstream transcriptional response. We demonstrate that genotoxic stress stimulates Cry1 phosphorylation and its deubiquitination by Herpes virus associated ubiquitin-specific protease (Hausp, a.k.a Usp7), stabilizing Cry1 and shifting circadian clock time. DNA damage also increases Cry2 interaction with Fbxl3, destabilizing Cry2. Thus, genotoxic stress increases the Cry1/Cry2 ratio, suggesting distinct functions for Cry1 and Cry2 following DNA damage. Indeed, the transcriptional response to genotoxic stress is enhanced in Cry1-/- and blunted in Cry2-/- cells. Furthermore, Cry2-/- cells accumulate damaged DNA. These results suggest that Cry1 and Cry2, which evolved from DNA repair enzymes, protect genomic integrity via coordinated transcriptional regulation.
Project description:In mammals, the circadian clock relies on interlocked feedback loops involving clock genes and their protein products. Post-translational modifications control intracellular trafficking, functionality and degradation of clock proteins and are keys to the functioning of the clock as recently exemplified for the F-Box protein Fbxl3. The SCF(Fbxl3) complex directs degradation of CRY1/2 proteins and Fbxl3 murine mutants have a slower clock. To assess whether the role of Fbxl3 is phylogenetically conserved, we investigated its function in the sheep, a diurnal ungulate. Our data show that Fbxl3 function is conserved and further reveal that its closest homologue, the F-Box protein Fbxl21, also binds to CRY1 which impairs its repressive action towards the transcriptional activators CLOCK/BMAL1. However, while Fbxl3 appears to be ubiquitously expressed, Fbxl21 expression is tissue-specific. Furthermore, and in sharp contrast with Fbxl3, Fbxl21 is highly expressed within the suprachiasmatic nuclei, site of the master clock, where it displays marked circadian oscillations apparently driven by members of the PAR-bZIP family. Finally, for both Fbxl3 and Fbxl21 we identified and functionally characterized novel splice-variants, which might reduce CRY1 proteasomal degradation dependent on cell context. Altogether, these data establish Fbxl21 as a novel circadian clock-controlled gene that plays a specific role within the mammalian circadian pacemaker.
Project description:Mammalian Cryptochromes, CRY1 and CRY2, function as principal regulators of a transcription-translation-based negative feedback loop underlying the mammalian circadian clockwork. An F-box protein, FBXL3, promotes ubiquitination and degradation of CRYs, while FBXL21, the closest paralog of FBXL3, ubiquitinates CRYs but leads to stabilization of CRYs. Fbxl3 knockout extremely lengthened the circadian period, and deletion of Fbxl21 gene in Fbxl3-deficient mice partially rescued the period-lengthening phenotype, suggesting a key role of CRY protein stability for maintenance of the circadian periodicity. Here, we employed a proteomics strategy to explore regulators for the protein stability of CRYs. We found that ubiquitin-specific protease 7 (USP7 also known as HAUSP) associates with CRY1 and CRY2 and stabilizes CRYs through deubiquitination. Treatment with USP7-specific inhibitor or Usp7 knockdown shortened the circadian period of the cellular rhythm. We identified another CRYs-interacting protein, TAR DNA binding protein 43 (TDP-43), an RNA-binding protein. TDP-43 stabilized CRY1 and CRY2, and its knockdown also shortened the circadian period in cultured cells. The present study identified USP7 and TDP-43 as the regulators of CRY1 and CRY2, underscoring the significance of the stability control process of CRY proteins for period determination in the mammalian circadian clockwork.
Project description:Nuclear hormone receptors (NRs) regulate physiology by sensing lipophilic ligands and adapting cellular transcription appropriately. A growing understanding of the impact of circadian clocks on mammalian transcription has sparked interest in the interregulation of transcriptional programs. Mammalian clocks are based on a transcriptional feedback loop featuring the transcriptional activators circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), and transcriptional repressors cryptochrome (CRY) and period (PER). CRY1 and CRY2 bind independently of other core clock factors to many genomic sites, which are enriched for NR recognition motifs. Here we report that CRY1/2 serve as corepressors for many NRs, indicating a new facet of circadian control of NR-mediated regulation of metabolism and physiology, and specifically contribute to diurnal modulation of drug metabolism.
Project description:Circadian clocks are near-ubiquitous molecular oscillators that coordinate biochemical, physiological, and behavioral processes with environmental cues, such as dawn and dusk. Circadian timing mechanisms are thought to have arisen multiple times throughout the evolution of eukaryotes but share a similar overall structure consisting of interlocking transcriptional and posttranslational feedback loops. Recent work in both plants and animals has also linked modification of histones to circadian clock function. Now, using data from published microarray experiments, we have identified a histone demethylase, jumonji domain containing 5 (JMJD5), as a previously undescribed participant in both the human and Arabidopsis circadian systems. Arabidopsis JMJD5 is coregulated with evening-phased clock components and positively affects expression of clock genes expressed at dawn. We found that both Arabidopsis jmjd5 mutant seedlings and mammalian cell cultures deficient for the human ortholog of this gene have similar fast-running circadian oscillations compared with WT. Remarkably, both the Arabidopsis and human JMJD5 orthologs retain sufficient commonality to rescue the circadian phenotype of the reciprocal system. Thus, JMJD5 plays an interchangeable role in the timing mechanisms of plants and animals despite their highly divergent evolutionary paths.
Project description:Using a forward genetics ENU mutagenesis screen for recessive mutations that affect circadian rhythmicity in the mouse, we isolated a long period (approximately 26 hr) circadian mutant named Overtime (Ovtm). Positional cloning and genetic complementation reveal that Ovtm is encoded by the F-box protein FBXL3, a component of the SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligase complex. The Ovtm mutation causes an isoleucine to threonine (I364T) substitution leading to a loss of function in FBXL3, which interacts specifically with the CRYPTOCHROME (CRY) proteins. In Ovtm mice, expression of the PERIOD proteins PER1 and PER2 is reduced; however, the CRY proteins CRY1 and CRY2 are unchanged. The loss of FBXL3 function leads to a stabilization of the CRY proteins, which in turn leads to a global transcriptional repression of the Per and Cry genes. Thus, Fbxl3(Ovtm) defines a molecular link between CRY turnover and CLOCK/BMAL1-dependent circadian transcription to modulate circadian period.
Project description:FBXL3 (F-Box and Leucine Rich Repeat Protein 3) encodes a protein that contains an F-box and several tandem leucine-rich repeats (LRR) domains. FBXL3 is part of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase complex that binds and leads to phosphorylation-dependent degradation of the central clock protein cryptochromes (CRY1 and CRY2) by the proteasome and its absence causes circadian phenotypes in mice and behavioral problems. No FBXL3-related phenotypes have been described in humans. By a combination of exome sequencing and homozygosity mapping, we analyzed two consanguineous families with intellectual disability and identified homozygous loss-of-function (LoF) variants in FBXL3. In the first family, from Pakistan, an FBXL3 frameshift variant [NM_012158.2:c.885delT:p.(Leu295Phefs*25)] was the onlysegregating variant in five affected individuals in two family loops (LOD score: 3.12). In the second family, from Lebanon, we identified a nonsense variant [NM_012158.2:c.445C>T:p.(Arg149*)]. In a third patient from Italy, a likely deleterious non-synonymous variant [NM_012158.2:c.1072T>C:p.(Cys358Arg)] was identified in homozygosity. Protein 3D modeling predicted that the Cys358Arg change influences the binding with CRY2 by destabilizing the structure of the FBXL3, suggesting that this variant is also likely to be LoF. The eight affected individuals from the three families presented with a similar phenotype that included intellectual disability, developmental delay, short stature and mild facial dysmorphism, mainly large nose with a bulbous tip. The phenotypic similarity and the segregation analysis suggest that FBXL3 biallelic, LoF variants link this gene with syndromic autosomal recessive developmental delay/intellectual disability.
Project description:JMJD5 is a Jumonji C (JmjC) protein that has been implicated in breast cancer tumorigenesis, circadian rhythm regulation, embryological development, and osteoclastogenesis. Recently, JMJD5 (also called KDM8) has been reported to demethylate dimethylated Lys-36 in histone H3 (H3K36me2), regulating genes that control cell cycle progression. Here, we report high-resolution crystal structures of the human JMJD5 catalytic domain in complex with the substrate 2-oxoglutarate (2-OG) and the inhibitor N-oxalylglycine (NOG). The structures reveal a ?-barrel fold that is conserved in the JmjC family and a long shallow cleft that opens into the enzyme's active site. A comparison with other JmjC enzymes illustrates that JMJD5 shares sequence and structural homology with the asparaginyl and histidinyl hydroxylase FIH-1 (factor inhibiting hypoxia-inducible factor 1 [HIF-1]), the lysyl hydroxylase JMJD6, and the RNA hydroxylase TYW5 but displays limited homology to JmjC lysine demethylases (KDMs). Contrary to previous findings, biochemical assays indicate that JMJD5 does not display demethylase activity toward methylated H3K36 nor toward the other methyllysines in the N-terminal tails of histones H3 and H4. Together, these results imply that JMJD5 participates in roles independent of histone demethylation and may function as a protein hydroxylase given its structural homology with FIH-1 and JMJD6.