The COP9 signalosome (CSN) is a highly conserved multifunctional complex that has two major biochemical roles: cleaving NEDD8 from cullin proteins and maintaining the stability of CRL components. We used mutation analysis to confirm that the JAMM domain of the CSN-5 subunit is responsible for NEDD8 cleavage from cullin proteins in Neurospora crassa. Point mutations of key residues in the metal-binding motif (EX_nHXHX₁₀D) of the CSN-5 JAMM domain disrupted CSN deneddylation activity without interfering with assembly of the CSN complex or interactions between CSN and cullin proteins. Surprisingly, CSN-5 with a mutated JAMM domain partially rescued the phenotypic defects observed in a csn-5 mutant. We found that, even without its deneddylation activity, the CSN can partially maintain the stability of the SCF^FWD-1 complex and partially restore the degradation of the circadian clock protein FREQUENCY (FRQ) in vivo. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, we found that deletion of the CAND1 ortholog in N. crassa had little effect on the conidiation circadian rhythm. Our results suggest that CSN integrity plays major roles in hyphal growth, conidial development, and circadian function in N. crassa. Cullin-RING E3 ubiquitin ligases (CRLs) play important roles in regulating a wide range of processes, such as signal transduction, transcription, cell cycle progression, circadian rhythm, and development, via the ubiquitin-proteasome pathway. The activity and stability of CRLs is precisely controlled by the COP9 signalosome (CSN), an evolutionarily conserved multisubunit protein complex. Under the control of the CSN, CRL activity can be either downregulated via cleavage of NEDD8 (an ubiquitin-like protein) from cullin proteins (deneddylation) or preserved by maintaining the stability of CRL components. We generated point mutations of key residues in the JAMM domain of the CSN-5 subunit to disrupt CSN deneddylation activity, thereby creating a series of mutants containing the intact CSN complex but lacking deneddylation activity. Surprisingly, hyphal growth, conidial development, circadian rhythm, and stability of the SCF^FWD-1 complex in these CSN-5 point mutants were comparable to that observed in wild-type N. crassa. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, deletion of the N. crassa ortholog of CAND1 (cullin-associated NEDD8-dissociated protein 1) had little effect on conidial development and the circadian clock. Our results suggest that the integrity of the CSN is important for growth and development in N. crassa.

Significance: Rhythmic clock gene transcription is essential for the functions of eukaryotic circadian clocks. In the Neurospora circadian oscillator, the WHITE COLLAR (WC) complex is responsible for rhythmic frequency ( frq ) transcription and was thought to be the only transcriptional activator for frq . Here, we show that frq can be constitutively transcribed in a WC-independent manner when the transcriptional corepressor rco-1 is deleted. In rco-1 mutants, high-level constitutive WC-independent frq transcription results in impaired WC activity and loss of circadian rhythmicity. Our results further indicate that RCO-1 acts together with the histone modifier SET-2 and the chromatin remodeling factor CHD-1 to regulate normal chromatin structure at the frq locus, which permits rhythmic frq transcription.

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.

The Cop9 signalosome (CSN) is an evolutionarily conserved multifunctional complex that controls ubiquitin-dependent protein degradation in eukaryotes. We found seven CSN subunits in Neurospora crassa in a previous study, but only one subunit, CSN-2, was functionally characterized. In this study, we created knockout mutants for the remaining individual CSN subunits in N. crassa. By phenotypic observation, we found that loss of CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, or CSN-7 resulted in severe defects in growth, conidiation, and circadian rhythm; the defect severity was gene-dependent. Unexpectedly, CSN-3 knockout mutants displayed the same phenotype as wild-type N. crassa. Consistent with these phenotypic observations, deneddylation of cullin proteins in csn-1, csn-2, csn-4, csn-5, csn-6, or csn-7 mutants was dramatically impaired, while deletion of csn-3 did not cause any alteration in the neddylation/deneddylation state of cullins. We further demonstrated that CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, and CSN-7, but not CSN-3, were essential for maintaining the stability of Cul1 in SCF complexes and Cul3 and BTB proteins in Cul3-BTB E3s, while five of the CSN subunits, but not CSN-3 and CSN-5, were also required for maintaining the stability of SKP-1 in SCF complexes. All seven CSN subunits were necessary for maintaining the stability of Cul4-DDB1 complexes. In addition, CSN-3 was also required for maintaining the stability of the CSN-2 subunit and FWD-1 in the SCF^FWD-1 complex. Together, these results not only provide functional insights into the different roles of individual subunits in the CSN complex, but also establish a functional framework for understanding the multiple functions of the CSN complex in biological processes. Protein degradation is precisely controlled in cells. The ubiquitin-mediated protein degradation pathway is highly conserved in eukaryotes, and the activity of ubiquitin ligases is regulated by the Cop9 signalosome (CSN), a multisubunit complex that is evolutionarily conserved from yeast to humans. Determining how the CSN complex functions biologically is crucial for understanding regulation of the ubiquitin-mediated protein degradation pathway. The filamentous fungus N. crassa is commonly used to study protein degradation. Its CSN complex contains seven subunits (CSN-1 to CSN-7). In this study, we generated knockout mutants of individual CSN subunits and observed the phenotypes of each mutant. We demonstrated that six of the seven CSN subunits were essential for cleaving the ubiquitin-like protein Nedd8 from cullin proteins (which act as scaffolds for ubiquitin ligases). In contrast, loss of the CSN-3 subunit had no effect on cullin neddylation. We also found that each CSN subunit had distinct roles in maintaining the stability of key components of cullin-based ubiquitin ligases. In summary, we systematically investigated the unequal contributions of CSN subunits to deneddylation and the maintenance of cullin-based ubiquitin ligases in N. crassa. Our work establishes a framework for understanding the function of CSN subunits in other eukaryotes.

MTOR complex-2 stimulates acetyl-CoA and de novo lipogenesis through ATP citrate lyase in HER2/PIK3CA-hyperactive breast cancer

DNA methylation is involved in gene silencing and genome stability in organisms from fungi to mammals. Genetic studies in Neurospora crassa previously showed that the CUL4-DDB1 E3 ubiquitin ligase regulates DNA methylation via histone H3K9 trimethylation. However, the substrate-specific adaptors of this ligase that are involved in the process were not known. Here, we show that, among the 16 DDB1- and Cul4-associated factors (DCAFs) encoded in the N. crassa genome, three interacted strongly with CUL4-DDB1 complexes. DNA methylation analyses of dcaf knockout mutants revealed that dcaf26 was required for all of the DNA methylation that we observed. In addition, histone H3K9 trimethylation was also eliminated in dcaf26^KO mutants. Based on the finding that DCAF26 associates with DDB1 and the histone methyltransferase DIM-5, we propose that DCAF26 protein is the major adaptor subunit of the Cul4-DDB1-DCAF26 complex, which recruits DIM-5 to DNA regions to initiate H3K9 trimethylation and DNA methylation in N. crassa. DNA associates with histones to form chromatin in eukaryotes. Epigenetics refers to DNA and histone modifications in chromatin that persist from one cell generation to the next, controlling gene expression and genome stability. These epigenetic changes are crucial for the development and differentiation of the various cell types in eukaryotes. In this study, we identified DCAF26 as a crucial regulator of DNA methylation. Inactivation of this gene in N. crassa resulted in loss of both DNA methylation and histone H3K9 trimethylation. We found that the resulting severe defects in the development and growth of dcaf26 mutants were similar to those found in cul4, ddb1, and dim-5 mutants, suggesting that these four genes function in the same pathway. Furthermore, we showed that DCAF26 functioned as an adaptor protein for the Cullin4-DDB1 complex to recruit the histone methyltransferase DIM-5 and regulate trimethylation of histone H3K9, which marks DNA for methylation. Our results reveal important roles for DCAF26 in H3K9 trimethylation and DNA methylation in N. crassa and suggest a conserved mechanism for DNA methylation in eukaryotic organisms.