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. 2015 Jan;14(1):25-8.
doi: 10.1128/EC.00212-14. Epub 2014 Oct 31.

The cullin-4 complex DCDC does not require E3 ubiquitin ligase elements to control heterochromatin in Neurospora crassa

Keyur K Adhvaryu  1 , Jordan D Gessaman  1 , Shinji Honda  1 , Zachary A Lewis  1 , Paula L Grisafi  1 , Eric U Selker  2
Affiliations

The cullin-4 complex DCDC does not require E3 ubiquitin ligase elements to control heterochromatin in Neurospora crassa

Keyur K Adhvaryu et al. Eukaryot Cell. 2015 Jan.

Abstract

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.

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Figures

FIG 1
FIG 1
Neddylation of CUL4 is dispensable for DNA methylation but not for DNA repair. (A) Schematic of CUL4 showing the NEDD8 attachment site (K863), cullin and cullin homology (CH) domains, and C-terminal region thought to interact with NEDD8 attachment machinery, E2 ligase, RBX1, CAND1, and CSN. (B) Western blots of extracts from an untransformed cul4RIP1 strain and cul4RIP1 strains expressing the indicated FLAG-CUL4 constructs. The strains are listed in Table S2 in the supplemental material. (C) DNA methylation analysis of wild-type (WT) and cul4RIP1 strains and cul4RIP1 strains expressing the indicated FLAG-tagged CUL4 constructs at the his-3 locus. DNA was digested with 5-methylcytosine-sensitive BfuCI (lanes B) or its 5-methylcytidine-insensitive isoschizomer, DpnII (lanes D), and the Southern blot was probed for methylated region 8:G3 (36); size standards (in kb) are indicated on the (left). Equivalent results were obtained for methylated regions 8:A6 and 5:B8 (36) (see Fig. S2 in the supplemental material). (D) Sensitivity of neddylation site mutants to DNA-damaging agents. Serial dilutions of conidia (indicated at the bottom) were tested on medium with or without MMS (0.015%), CPT (0.3 μg/ml), or TBZ (0.5 μg/ml).
FIG 2
FIG 2
The CUL4 C terminus is not required for DCDC function. (A) Normal DNA methylation in a CUL4 C terminus deletion mutant. DNA methylation was tested for the wild-type (WT) strain, a cul4RIP1 strain, and cul4RIP1 strains bearing either the wild-type cul4+ allele or the C terminus truncation allele (the strain contained cul4 residues 1 to 823 [cul41-823] and a deletion of residues 824 to 923; Fig. 1A), as described in the legend to Fig. 1C. (B) H3K9me3 is unaffected by deletion of the C terminus of CUL4. Nuclear extracts from the indicated strains were probed by Western blotting to detect global trimethylated H3K9 or histone H3 levels. (C) The CUL4 C-terminal deletion abrogates DNA repair. Strains of the indicated genotypes were tested as described in the legend to Fig. 1D.
FIG 3
FIG 3
RBX1, the putative RING protein for CUL4 complexes, is not required for control of DNA methylation by DCDC. (A) Normal DNA methylation in an rbx1 deletion strain. DNA methylation was tested for the wild type (WT), a cul4RIP1 strain, and the rbx1 deletion mutant, as described in the legend to Fig. 1C. (B) Compromised DNA repair in the rbx1 mutant. Serial dilutions of conidia from strains of the indicated genotype were tested on medium with or without MMS (0.015%). The strains are listed in Table S2 in the supplemental material.
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