Abstract

The CUL4–DDB1–ROC1 ubiquitin E3 ligase regulates cell-cycle progression, replication and DNA damage response^{1, 2, 3, 4}. However, the substrate-specific adaptors of this ligase remain uncharacterized. Here, we show that CUL4–DDB1 complexes interact with multiple WD40-repeat proteins (WDRs) including TLE1-3, WDR5, L2DTL (also known as CDT2) and the Polycomb-group protein EED (also known as ESC). WDR5 and EED are core components of histone methylation complexes that are essential for histone H3 methylation and epigenetic control at K4 or K9 and K27, respectively^{5, 6, 7}, whereas L2DTL regulates CDT1 proteolysis after DNA damage through CUL4–DDB1 (ref. 8). We found that CUL4A–DDB1 interacts with H3 methylated mononucleosomes and peptides. Inactivation of either CUL4 or DDB1 impairs these histone modifications. However, loss of WDR5 specifically affects histone H3 methylation at K4 but not CDT1 degradation, whereas inactivation of L2DTL prevents CDT1 degradation but not histone methylation. Our studies suggest that CUL4–DDB1 ligases use WDR proteins as molecular adaptors for substrate recognition, and modulate multiple biological processes through ubiquitin-dependent proteolysis.

Introduction

The CUL4–DDB1–ROC1 complex is a new class of cullin-containing ubiquitin E3 ligases¹. Previous studies have indicated that the CUL4–DDB1 E3 ligase regulates the proteolysis of the replication licensing protein CDT1 or fission yeast ribonucleotide reductase inhibitor, Spd1, in response to DNA damage or replication^{2, 3, 9}. The CUL4 protein shares significant homology with other cullin family members including CUL1–3 and CUL-5 (ref. 1). Cullin E3 ligases use specific substrate-targeting adaptors, such as F-box proteins in SCF (SKP1–CUL1–F-box) complexes or BTB–POZ proteins in CUL3–ubiquitin E3 complexes, for substrate recognition¹. CUL4 may use a similar substrate-specific adaptor mechanism to target various proteins for ubiquitin-dependent proteolysis. The CUL4–ROC1 complex binds to DDB1 (UV-damaged DNA-binding protein 1), which has been proposed to be adaptor protein for substrate-targeting^{1, 10}. However, it remains unclear whether the CUL4–DDB1 E3 ligase requires additional proteins to define substrate specificity.

We recently isolated a novel human WDR protein, L2DTL, which binds to the CUL4–DDB1 complex and CDT1 (ref. 8). We showed that L2DTL is required for CDT1 proteolysis in response to DNA damage through the CUL4–DDB1 ubiquitin E3 ligase⁸. L2DTL requires the WD40-repeat domain for DDB1 binding⁸ and DDB1 also directly interacts with WDR proteins DDB2 and the Cockayne syndrome protein, CSA¹¹. The CSA-containing CUL4A–DDB1 E3 ligase has recently been shown to target CSB for proteolysis, whereas DDB2 regulates the UV radiation-induced XPC translocation within chromatin^{12, 13}. Importantly, the binding of DDB2 and CSA to DDB1 seems to be mutually exclusive¹¹. These observations raise the possibility that WDR proteins constitute the substrate-specific adaptors that recruit substrates to the CUL4–DDB1–ROC1 core complexes. Here, we show that the isolated CUL4–DDB1 complex contains several additional WDR proteins. Our studies suggest that WDR proteins act as substrate-specific adaptors for the CUL4–DDB1 ubiquitin E3 ligases.

We previously isolated the CUL4B complex from human HeLa cells by affinity chromatography using the anti-CUL4B antibodies as the affinity resin⁸. The proteins associated with the CUL4B complexes were identified by mass spectrometry. This led to the identification of a novel WD40-repeat protein, human L2DTL, in the CUL4B–DDB1 complex⁸, which regulates CDT1 proteolysis in response to DNA damage⁸. Using similar purifications from293 cells, several other WD40-repeat proteins were identified, including WDR26, WD40 repeat–transducin-like enhancer proteins 1-3 (TLE1-3), WDR82, glutamate-rich WD40-repeat protein 1 (GRWD1) and Suppressor of mec-8 and unc-52 (SMU1), in addition to L2DTL, DDB1 and components of the COP9–signalosome complex (CSN; Fig. 1)⁸. To confirm these interactions, these WDR proteins were tagged with a Flag-epitope tag and expressed in human cells by transfection (Fig. 2). Our studies confirmed that these WDR proteins interact with the CUL4–DDB1 complexes in vivo (Fig. 2 and data not shown). Endogenous WDR proteins such as TLE2 and L2DTL were observed in complexes with the CUL4–DDB1 E3 ligases (Fig. 3a–d).

Figure 1: Isolation of various WDR proteins that interact with CUL4B.

Human CUL4B complexes were immuno-affinity purified from 293 cells using the affinity purified anti-CUL4B antibody and the associated WDR proteins were identified by mass spectrometry as indicated. IgG, control antibodies.

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Figure 2: WDR proteins interact with the CUL4–DDB1 complexes.

(a) Human L2DTL and other WDR proteins were Flag-epitope tagged and expressed in 293 or H1299 cells. The protein complexes were isolated from transfected cell lysates by immunoprecipitation with the anti-Flag antibody, pre-immune serum, and anti-CUL4CT antibodies that recognize both CUL4A and CUL4B and blotted for Flag-tagged and CUL4 proteins as indicated. (b) Flag-tagged WDR5 was transfected as in a and the complexes were immunoprecipitated by anti-Flag, CUL4, RBBP5 and L2DTL antibodies as indicated, then blotted with anti-Flag antibodies. (c, d) Flag-tagged WDR proteins were transfected as in a, their complexes immunoprecipitated by anti-Flag and the proteins separated by SDS–PAGE. The top half of the gel was blotted with anti-DDB1 antibodies and the bottom half with anti-Flag antibodies. Because the top halves also contain Flag–COP1, L2DTL and WDR26 in c, and RBBP5 and WDR59 in d, the anti-DDB1 blots were stripped and reblotted with anti-Flag antibodies for these proteins. The Flag–WDR26 blot in c was from a different exposure.

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Figure 3: Endogenous WDR proteins interact with the CUL4–DDB1 complexes.

(a–h) Protein complexes containing human WDR proteins L2DTL, TLE2, DDB2, COP1, EED and RBBP5, as well as CUL4A and CUL4B were isolated by specific antibodies as indicated from human H1299 or HeLa cells. Their association with CUL4A, CUL4B, DDB1 and WDR5 was analysed by western blotting as indicated. NRS, normal rabbit serum. In h, the proteins in the lysates are from a different exposure.

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As WDR proteins share substantial sequence homology within the WD40 repeat domain^{14, 15}, we tested the association of additional WDR proteins with the CUL4–DDB1 complexes. More than 20 WDR proteins were analysed, including WDR5, RBBP5, EED–ESC1–HEED, COP1 and Notchless (NLE1; see Supplementary Information, Fig. S1), and found that they can interact with the CUL4–DDB1 complexes when they are expressed in vivo (Figs 2, 3). The interaction of the endogenous CUL4–DDB1 with COP1, WDR5, RBBP5 and EED proteins was verified using their specific antibodies (Fig. 3d–h). These studies indicate that CUL4–DDB1 complexes can interact with a large number of WDR proteins and raise the possibility that these WDR proteins may serve as substrate-recognition subunits or adaptors of the CUL4–DDB1 ubiquitin E3 ligases. As the function of many WDR proteins was not previously defined, we named these proteins CDW proteins for CUL4- and DDB1-associated WDR proteins (see Supplementary Information, Fig. S1).

To determine the structural basis of the interaction between WDR proteins and CUL4–DDB1, the protein sequences of L2DTL proteins from fission yeast to human, as well as the WDR proteins originally isolated from CUL4B association, was compared (Fig. 1). This alignment revealed the presence of a centrally positioned and conserved tandem repeat of a DXXXR/KXWDXR/K motif (R/K; either Arg or Lys), between L2DTL, WDR82, GRWD1 and TLE1-3 proteins (see Supplementary Information, Fig. S2). This subdomain of WD40 repeats is also conserved in CSA and DDB2. The importance of the arginine (Arg 273) in DDB2 within the WDXR submotif is underscored by its mutation to histidine (R273H) in a xeroderma pigmentosum group E patient and the subsequent inability to bind DDB1 (ref. 16). This motif is present in most of the WDR proteins we found (see Supplementary Information, Fig. S3), suggesting that it is a key determinant for the interaction with DDB1. The crystal structures of WDR5 and TLE1 WD40-repeat domains reveal that the Asp and Arg/Lys in the WDXR/K submotif are exposed on the bottom surface of the barrel-shaped WD40 propeller folds^{17, 18}, and are thus available for interaction with CUL4–DDB1. The Arg/Lys in the DXXXR/K submotif is on the side to the bottom of the barrel, whereas the first Asp is on the opposite side of the barrel that binds to histone in WDR5. Thus the R/KXWDXR/K motif is more likely to be involved in DDB1 binding. However, we have noticed that although the last Arg/Lys in WDXR/K is required for DDB2 binding to DDB1, it is not strictly observed among the WDR proteins we identified. A few WDR proteins lack the first or second Arg/Lys in the WDXR/K submotif but still bind to CUL4–DDB1 (see Supplementary Information, Fig. S3). Interestingly, although EED strongly interacts with CUL4A–DDB1 (Fig. 3e), it does not have a recognizable WDXR/K submotif (it has WNIQ in the N repeat of the WD40-repeat domain and WRIN in the N+1 repeat instead; see Supplementary Information, Fig. S3 and data not shown)¹⁹. Thus, although WDR proteins containing the R/KXWDXR/K motif are enriched in CUL4–DDB1 complexes, other structural motifs may also mediate the interaction between WDR proteins and CUL4–DDB1.

If CUL4–DDB1 can associate with different WDR proteins to target distinct substrates, loss of L2DTL, but not other WDR proteins, should prevent CDT1 degradation. To test this hypothesis, we silenced the expression of various WDR proteins, such as Drosophila WDS (the orthologue of WDR5), RBBP5, ESC, GRWD1, and WDR26 in Drosophila S2 cells and analysed CDT1 degradation in response to DNA damage. As a positive control, we used Drosophila L2DTL, CUL4 and CSN5 RNA interference (RNAi) to suppress CDT1 degradation after DNA damage^{3, 8}.

As shown in Fig. 4a and b, although the treatment of Drosophila S2 cells with either CUL4, CSN5 or L2DTL double-stranded RNAs prevented the degradation of CDT1 in response to -irradiation, the inactivation of other WDR proteins by RNAi did not alter CDT1 proteolysis after DNA damage.

Figure 4: L2DTL, but not other WDR proteins, specifically targets CDT1 for proteolysis in response to DNA damage.

(a, b) Drosophila S2 cells were treated with 15 g each of specific double-stranded RNAs for Neo (control) and Drosophila orthologues of CUL4, L2DTL, CSN5, WDR5 (WDS), EED, RBBP5, GRWD1 and WDR26 for 60 h. The cells were -irradiated at 100 Gy and cells were harvested in SDS containing buffer 1 h later. The total CDT1 and geminin proteins were analysed by specific Drosophila anti-CDT1 and geminin antibodies. (c, d) Human L2DTL is specific for CDT1 proteolysis after DNA damage. Human H1299 (c) and HeLa (d) cells were transfected with 50 nM siRNAs for Luciferase (Luc; control), L2DTL, WDR5, RBBP5, CUL4A and CUL4B, and WDR26 for 72 h as indicated. The cells were -irradiated (10 Gy) and harvested after 1 h. The protein levels of CDT1, L2DTL, WDR5, RBBP5, WDR26, geminin, CUL2, and CUL4A and B were analysed by specific antibodies as indicated. CTD1*, a possible modified CDT1 protein.

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We also examined the requirement for WDR5, RBBP5 and WDR26 for proteolysis of CDT1 in human cells. Again, only the inactivation of human L2DTL prevented CDT1 proteolysis in response to -irradiation, whereas loss of other WDR proteins did not block CDT1 degradation after irradiation (Fig. 4c, d). These studies suggest that L2DTL is the only WDR protein tested so far that is required for CDT1 proteolysis.

If distinct WDR proteins define specific biological events for CUL4–DDB1 regulation, the function of CUL4–DDB1 complex should be required for biological processes specifically regulated by a particular WDR protein. We focused on testing whether CUL4–DDB1 regulates histone H3 methylation at K4, K9 and K27, as WDR proteins WDR5, RBBP5 and EED interact with the CUL4–DDB1 complexes (Figs 2, 3). WDR5 is an essential component of the MLL (mixed-lineage leukaemia) histone methylation complexes⁷, which are homologous to the Saccharomyces cerevisiae Set1 and the Drosophila Trithorax complexes that catalyze the critical trimethylation at K4 on histone H3 (ref. 5). RBBP5 is another WDR protein invariably associated with the MLL–WDR5 complexes^{20, 21}. The trimethylation at K4 marks transcription start sites of almost all active promoters, including the homeobox genes (Hox)^{7, 22}. It has been shown that WDR5 directly interacts with the methylated K4 of histone H3 in cell lysates and this interaction is required for the K4 trimethylation by the Set-domain containing MLL methylation complexes⁷. Loss of WDR5 by siRNA-mediated gene silencing has been shown to severely and specifically block the tri- and mono-methylation of histone H3 at K4, whereas dimethylation at this lysine is only modestly affected⁷.

Inactivation of WDR5 by small interfering RNA (siRNA) severely inhibited the tri- and mono-methylation of histone H3 at K4 (Fig. 5d), whereas K4 dimethylation was only slightly affected (Fig. 5d), consistent with previous reports⁷. Treatment with WDR5 siRNA did not significantly affect total histone H3 levels or trimethylation at K9 (Fig. 5d)⁷. To determine whether the regulation of histone H3 methylation at K4 is unique to WDR5–RBBP5, we examined whether loss of L2DTL or other WDR proteins affects this methylation. We found that the loss of L2DTL or DDB2 did not significantly affect K4 methylation status (Fig. 5c). These observations indicate that WDR5 and RBBP5 are specific WDR proteins for these K4 modifications in histone H3, as other WDR proteins, including L2DTL, cannot replace their function.

Figure 5: CUL4–DDB1 associates with histone H3 methylated at K4, K9 and K27 and regulates H3 methylation in vivo.

(a) Association of DDB1 and WDR5 with histone H3 methylated at K4. HeLa cells were harvested and lysed as described in the Methods. The mononucleosomes containing the methylated histone H3 were released from the insoluble chromatin preparation by micrococcal nuclease. The soluble nucleosomes were clarified by centrifugation and used as the source for immunoprecipitation with control normal rabbit serum (NRS), and antibodies against histone H3 that is tri- and dimethylated at K4 as indicated H3K4 (Me)2/3. The association of DDB1 and WDR5 with nucleosomes was analysed by western blotting using the indicated antibodies. The asterisk indicates another protein detected by the WDR5 antibody. (b) DDB1 is associated with histone H3 methylated at K4, K9 and K27 whereas CUL4A is enriched in histone H3 methylated at K4. The experiment was performed as in a, except mononucleosome fractions were immunoprecipitated with antibodies against tri-, di- and monomethylated histone H3K4; and trimethylated H3K9 and H3K27. These complexes were blotted with anti-CUL4A and CUL4B antibodies. CUL4A^Nedd8 is a ubiquitin-like modification of CUL4A by Nedd8. (c) The biotinylated N-terminal peptides of histone H3, with or without K4 methylation, was linked to streptavidin–Sepharose beads and incubated with the nuclear extract from HeLa cells. The binding of CUL4A, DDB1 and WDR5 to the peptides were analysed. (d) WDR5, but not other WDR proteins, regulates histone H3K4 methylatiom. Human HeLa cells were treated with 50 nM siRNAs for luciferase, WDR5, RBBP5, L2DTL and DDB2 for 72 h. Total cell lysates were prepared by lysing cells in SDS sample buffer and the levels of tri-, di- and monomethylated histone H3K4 and trimethylated H3K9 were analysed. Total histone H3 was used as a control. (e) CUL4 and DDB1 regulate histone methylation at H3K4, H3K9 and H3K27. HeLa cells were treated with 50 nM siRNAs for luciferase, CUL4A and CUL4B, and DDB1 for 72 h. The total cell lysates were prepared and the levels of tri-, di- and monomethylated histone H3K4 and trimethylated H3K9 and H3K27 were analysed. Total histone H3 was used as a control.

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Our previous studies showed that CUL4–DDB1 and L2DTL can interact with CDT1 to regulate its stability after DNA damage^{3, 8}. If WDR5 or RBBP5 are adaptors for CUL4–DDB1, CUL4–DDB1 should interact with the methylated histone H3 at K4. Furthermore, loss of either CUL4 or DDB1 should mimic the effects of WDR5 or RBBP1 inactivation on histone H3 methylation at K4 if the CUL4–DDB1 complexes are functionally involved. We tested this possibility by examining whether CUL4–DDB1 can interact with methylated mononucleosomes. Previous studies indicate that WDR5 directly binds to histone H3 containing di- and tri-methylated H3 at K4 (ref. 7) and this binding is considered as an essential and critical step that allows the MLL methylation complexes to methylate histone H3 at K4 (ref. 7). To monitor CUL4–DDB1 binding, the soluble mononucleosomes were released from the nuclear fraction after digestion with micrococcal nuclease²³. Mononucleosomes containing tri- and di-methylated histone H3 at K4 were then immunoprecipitated with ChIP grade anti-tri- and di-methylated K4 antibodies and their association with WDR5 or DDB1–CUL4 was examined⁷. Our studies revealed that WDR5 and DDB1 are indeed present in the nucleosomes immunoprecipitated by the di- and trimethylated K4 antibodies (Fig. 5a). In addition, DDB1 is associated with nucleosomes containing monomethylated histone H3 at K4, and trimethylated histone H3 at K9 and K27 (Fig. 5b). As the WDR protein EED regulates the methylation of histone H3 at K9 and K27 (Refs 5, 6), CUL4–DDB1 in complex with EED may participate in regulating the methylation of H3 at K9 and K27 (Fig. 3e).

We found that CUL4 was also present in the H3 methylated nucleosome fractions. Interestingly, CUL4A, but not its paralogue CUL4B, is enriched in methylated histone H3 nucleosomes (Fig. 5b), although only trace amounts of CUL4 can be detected with H3 methylated at K9 and K27. It has been shown that WDR5 can bind to the amino terminal peptide of histone H3 only when K4 is methylated⁷. We found that WDR5, DDB1 and CUL4A can interact with the H3 N-terminal peptide containing methylated K4, but not its unmodified form in cell lysates, suggesting that these interactions are specific for methylated histone H3 (Fig. 5c). In these experiments, although we could relatively easily find CUL4–DDB1 in the methylated nucleosomes, we could not detect L2DTL in these fractions, consistent with its inability to alter histone H3 methylation at K4 (Fig. 5a–c, and data not shown).

As CUL4–DDB1 interact with WDR5, RBBP5 and EED, and both DDB1 and CUL4 associated with methylated histone H3, we examined whether the CUL4–DDB1 complex is involved in histone H3 methylation. As predicted, inactivation of either CUL4 (CUL4A and CUL4B) or DDB1 by siRNA severely reduced the tri- and monomethylations of histone H3 at K4, with little or no significant effect on the H3K4 dimethylation (Fig. 5e). This selective inhibition of tri- and monomethylations of histone H3 at K4 after CUL4 or DDB1 inactivation mirrors the effect of WDR5 and RBBP5 inactivation, and supports the notion that WDR5 and RBBP5 function as adaptor proteins for CUL4–DDB1 to regulate H3 K4 methylation (Fig. 5d)⁷. In these experiments, inactivation of CUL4 or DDB1 caused a significant inhibition of histone H3 trimethylation at K9 and K27 (Fig. 5e). This effect is relatively modest compared to the impact of CUL4–DDB1 inactivation on histone H3 tri- and monomethylation at K4 (Fig. 5e). As this effect is not observed after WDR5 and RBBP5 inactivation (Fig. 5d), CUL4–DDB1 may use other WDR proteins for the regulation of histone H3 methylation at K9 and K27. The CUL4–DDB1 complex also associates with EED (Fig. 3e), suggesting that EED is a strong candidate for the CUL4–DDB1 effect on trimethylation of histone H3 at K9 and K27. The human Polycomb-group protein EED is the homologue of Drosophila PcG protein Esc, which forms a core complex with the SET-domain containing protein enhancer of zeste (E(Z), or its human homologue EZh2) and suppressor of zeste-12 (Su(z)12) to methylate K9 and K27 in histone H3 (Refs 24, 25) and repress transcription^{5, 6, 26}. The trimethylation at K27 in histone H3 is also associated with mammalian X chromosome inactivation and genomic imprinting, whereas K9 trimethylation is responsible for the transcriptionally repressive state in heterochromatin^{5, 6, 27}. However, because our EED siRNAs did not efficiently silence the expression of EED, we were not able to determine whether EED siRNA severely affects K9 and K27 methylation, as reported in mouse or Drosophila^{5, 6, 27}. However, the loss of L2DTL, WDR5, RBBP5 or DDB2, do not affect the trimethylation of histone H3 at K9 and K27. These studies indicate that the CUL4–DDB1 E3 ligase complexes interact with distinct WDR proteins (such as WDR5 and EED proteins) to differentially regulate histone methylation at K4, K9 and K27, and that they interact with L2DTL to target CDT1 for proteolysis in response to DNA damage.

We have shown that the CUL4–DDB1 ubiquitin E3 ligase interacts with multiple WDR proteins. WDR proteins constitute one of the largest gene families in human genome with more than 150 members^{14, 28}. As WDR proteins have been shown to be involved in regulating an array of important biological processes (such as transcription, cell-cycle regulation, signalling and chromatin modification^{14, 29}), it is possible that these processes are also controlled by the CUL4–DDB1 ubiquitin E3 ligase complex. Our data suggest that WDR proteins function as the substrate-specific adaptors for the core CUL4–DDB1 ubiquitin E3 ligase complex in specific biological events, such as histone methylation or CDT1 degradation after DNA damage. These data are consistent with the observation that CUL4, DDB1 or its paralogues are involved in regulating histone methylation in fission yeast³⁰. As L2DTL, DDB2 and CSA directly interact with DDB1, it is likely that DDB1 provides an interaction interface between WDR proteins and the CUL4–ROC E3 ligase complex to promote substrate recognition and polyubiquitination. Further work is required to determine how CUL4–DDB1 ubiquitin E3 ligases regulate a wide range of biological processes by targeting specific proteins for ubiquitin-dependent proteolysis.

Note added in proof: while this manuscript was in press, two papers were published that demonstrated that WD40-repeat domain proteins can act as substrate-specific adaptors for CUL4–DDB1 ubiquitin E3 ligases^{31, 32}.

Acknowledgements

This work was supported by the grants from National Institutes of Health to H.S. (CA77695) and H.Z. (CA72878 and CA98955). H.Z. was also supported in part by an US Army grant (W81XWH-04-1-0230). H.S. and H.Z. would like to thank the members of the Sun and Zhang laboratories for discussions, R.K. for mass spectrometry, and T.Y and J. Guan for sequence alignments. The 293 suspension cells were from the Cell Culture Centre (Cellex Sciences, Minneapolis, MN).

Competing interests statement

The authors declare no competing financial interests.

Received 23 August 2006; Accepted 28 September 2006; Published online 15 October 2006.

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