Codanin-1, mutated in the anaemic disease CDAI, regulates Asf1 function in S-phase histone supply
2012; Springer Nature; Volume: 31; Issue: 8 Linguagem: Inglês
10.1038/emboj.2012.55
ISSN1460-2075
AutoresKatrine Ask, Zuzana Jasencakova, P Ménard, Yunpeng Feng, Geneviève Almouzni, Anja Groth,
Tópico(s)Extracellular vesicles in disease
ResumoArticle9 March 2012free access Codanin-1, mutated in the anaemic disease CDAI, regulates Asf1 function in S-phase histone supply Katrine Ask Katrine Ask Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Zuzana Jasencakova Zuzana Jasencakova Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Patrice Menard Patrice Menard Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Yunpeng Feng Yunpeng Feng Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Geneviève Almouzni Geneviève Almouzni Institut Curie UMR218-CNRS, Paris Cedex 05, France Search for more papers by this author Anja Groth Corresponding Author Anja Groth Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Katrine Ask Katrine Ask Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Zuzana Jasencakova Zuzana Jasencakova Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Patrice Menard Patrice Menard Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Yunpeng Feng Yunpeng Feng Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Geneviève Almouzni Geneviève Almouzni Institut Curie UMR218-CNRS, Paris Cedex 05, France Search for more papers by this author Anja Groth Corresponding Author Anja Groth Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Author Information Katrine Ask1, Zuzana Jasencakova1, Patrice Menard1, Yunpeng Feng1, Geneviève Almouzni2 and Anja Groth 1 1Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark 2Institut Curie UMR218-CNRS, Paris Cedex 05, France *Corresponding author. Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark. Tel.: +45 353 25538; Fax: +45 353 25669; E-mail: [email protected] The EMBO Journal (2012)31:2013-2023https://doi.org/10.1038/emboj.2012.55 Correction(s) for this article Codanin-1, mutated in the anaemic disease CDAI, regulates Asf1 function in S-phase histone supply18 July 2012 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Efficient supply of new histones during DNA replication is critical to restore chromatin organization and maintain genome function. The histone chaperone anti-silencing function 1 (Asf1) serves a key function in providing H3.1-H4 to CAF-1 for replication-coupled nucleosome assembly. We identify Codanin-1 as a novel interaction partner of Asf1 regulating S-phase histone supply. Mutations in Codanin-1 can cause congenital dyserythropoietic anaemia type I (CDAI), characterized by chromatin abnormalities in bone marrow erythroblasts. Codanin-1 is part of a cytosolic Asf1–H3.1-H4–Importin-4 complex and binds directly to Asf1 via a conserved B-domain, implying a mutually exclusive interaction with the chaperones CAF-1 and HIRA. Codanin-1 depletion accelerates the rate of DNA replication and increases the level of chromatin-bound Asf1, suggesting that Codanin-1 guards a limiting step in chromatin replication. Consistently, ectopic Codanin-1 expression arrests S-phase progression by sequestering Asf1 in the cytoplasm, blocking histone delivery. We propose that Codanin-1 acts as a negative regulator of Asf1 function in chromatin assembly. This function is compromised by two CDAI mutations that impair complex formation with Asf1, providing insight into the molecular basis for CDAI disease. Introduction During DNA replication, parental histones segregate onto leading and lagging strands in a random fashion (Groth et al, 2007b). In parallel, new histones are deposited to maintain nucleosomal density. Nucleosome assembly is the first step towards restoration of chromatin on new DNA. Given the role of histones and higher order chromatin structures in epigenetic gene regulation and genome stability, histone supply pathways must be fine-tuned to meet the demands at replication forks (De Koning et al, 2007; Jasencakova and Groth, 2010; Annunziato, 2011). The histone H3-H4 chaperone anti-silencing function 1 (Asf1) is a key player in chromatin replication, donating new histones to CAF-1 (Tyler et al, 1999; Mello et al, 2002) and handling histones together with the MCM2-7 replicative helicase potentially for recycling (Groth et al, 2007a; Jasencakova et al, 2010). The two mammalian homologues, Asf1a and Asf1b, have largely redundant functions in S-phase histone dynamics, with Asf1b being more specialized to proliferating cells (Corpet et al, 2011). The current view is that Asf1 binds histones H3-H4 in the cytoplasm and in complex with Importin-4 accompanies histone dimers into the nucleus where they are transferred to downstream chromatin assembly factors (De Koning et al, 2007; Campos et al, 2010; Jasencakova et al, 2010; Alvarez et al, 2011). Asf1 binds canonical S-phase histones H3.1-H4 as well as replacement histones H3.3-H4, which are delivered to CAF-1 and HIRA, respectively (Tagami et al, 2004). It is not entirely clear how the specificity of Asf1 in these distinct assembly pathways is regulated, but CAF-1 p60 and HIRA bind in a mutually exclusive manner to the same binding pocket in Asf1 (Tang et al, 2006; Malay et al, 2008). Purification of soluble histone H3 complexes recently revealed that the HSC70, HSP90 and NASP chaperones act early in the histone supply pathway upstream of Asf1 and Importin-4 (Campos et al, 2010; Alvarez et al, 2011). Whereas HSC70 and HSP90 probably are important for folding, NASP is required to maintain a soluble pool of histones available for deposition (Campos et al, 2010; Cook et al, 2011). However, it remains unclear how histones H3-H4 are transferred to Asf1 and whether additional factors regulate Asf1 histone shuttling. As an entry point to understand Asf1 function, we have characterized human Asf1 complexes and recently reported a comprehensive profiling of modifications on Asf1-bound histones (Groth et al, 2007a; Jasencakova et al, 2010). In addition to cytosolic binding partners with predicted roles in histone metabolism (such as sNASP, RbAp46-HAT1, and Importin-4), a protein of unknown function, Codanin-1, caught our attention due to its abundance and link to disease. Mutations in CDAN1, the gene encoding Codanin-1, cause congenital dyserythropoietic anaemia type I (CDAI), a rare recessive anaemic disorder (Dgany et al, 2002; Iolascon et al, 2011). Codanin-1 is a 134-kDa ubiquitously expressed protein conserved in flies, frogs and fish, but with no apparent homologue in worms and yeast (Dgany et al, 2002). The Drosophila homologue, Discs lost (Dlt), is required for cell survival and cell-cycle progression (Pielage et al, 2003) and mice homozygous for a gene-trap in the CDAN1 locus die during early embryogenesis (Renella et al, 2011). This argues that Codanin-1 is an essential protein and consistently the majority of CDAI cases show missense mutations leading to single amino-acid substitutions in Codanin-1 (Dgany et al, 2002; Tamary et al, 2005; Heimpel et al, 2006; Ru et al, 2008). A principal cytological feature of bone marrow erythroblasts from CDAI patients is abnormal chromatin structure, known as ‘spongy heterochromatin’ having a Swiss cheese-like appearance (Wickramasinghe and Wood, 2005). Furthermore, cell-cycle analyses of patient samples show accumulation of erythroblasts in S phase (Wickramasinghe and Pippard, 1986; Tamary et al, 1996) suggesting replication defects. Given that Asf1 function is essential for DNA replication and chromatin assembly in human cells (Groth et al, 2005, 2007a), an appealing idea was that CDAI disease could be linked to defects in histone metabolism. Here, we characterize a molecular link between Codanin-1 and Asf1 function in histone supply and address the impact of CDAI mutations on this interplay. Results Codanin-1 is part of a cytosolic Asf1–H3-H4–Importin-4 complex We identified Codanin-1 by mass spectrometry of e-Asf1a and e-Asf1b complexes isolated from asynchronous HeLa S3 cells (Figure 1A; Supplementary Figure S1A). This finding is consistent with a high-throughput proteomic screen identifying Codanin-1 as an Asf1b-associated protein (Ewing et al, 2007) and we further confirmed the interaction by reciprocal immunoprecipitation of endogenous Asf1 and Codanin-1 (Figure 1B and D). We previously found that Importin-4 is specific to cytosolic Asf1 complexes, while MCM4, 6, 7 are part of a nuclear Asf1 complex (Groth et al, 2007a; Jasencakova et al, 2010). Western blot analysis showed that Codanin-1 is a cytosolic protein, mainly found in cytosolic Asf1 complexes similarly to Importin-4 (Supplementary Figure S1A and B). Cytosolic Asf1 separates into two major forms on a sizing column; a histone-bound complex and a histone-free form eluting at lower molecular weight (Groth et al, 2005). To address whether Codanin-1 could be part of an Asf1 complex containing histones, we analysed purified cytosolic e-Asf1b complexes by gel filtration. Codanin-1 co-eluted with histone-bound Asf1, showing an elution profile highly similar to Importin-4 (Figure 1C). We confirmed that Codanin-1 co-purified with soluble non-nucleosomal histone H3.1 (Figure 1E), using a cell line expressing low levels of epitope-tagged histone H3.1 (Tagami et al, 2004). Moreover, Importin-4 co-immunoprecipitated with Codanin-1 (Figure 1D) supporting that these factors are present together in a complex with Asf1 and histone H3-H4. However, the chaperone sNASP and RbAp46 thought to act upstream of Asf1 and the downstream chaperones HIRA and CAF-1 did not co-purify with Codanin-1 (Supplementary Figure S1C and D). These biochemical data identify Codanin-1 as a new member of a cytosolic Asf1–H3.1-H4–Importin-4 complex. We asked whether the interaction between Codanin-1 and Asf1 is histone dependent, taking advantage of Asf1 carrying a mutation in the histone-binding site, V94R (Mousson et al, 2005; Groth et al, 2007a). While the interaction with Importin-4 is lost in the Asf1a V94R mutant (Figure 1F; Jasencakova et al, 2010), Codanin-1 bound wild-type Asf1 and the V94R mutant equally well (Figure 1F). Thus, demonstrating that the interaction between Codanin-1 and Asf1 is histone independent. Figure 1.Codanin-1, a new partner of the cytosolic Asf1–H3-H4–Importin-4 complex. (A) Coomassie staining of Asf1a complexes isolated from asynchronous HeLa S3 cells stably expressing Onestrep-tagged (e−) Asf1a. Codanin-1 was identified in both cytosolic and nuclear e-Asf1a complexes by mass spectrometry analysis. Proteins annotated in black were previously reported (Groth et al, 2007a; Jasencakova et al, 2010). (B) Co-immunoprecipitation of Codanin-1 with endogenous Asf1 (a and b) from cytosolic HeLa S3 extracts. (C) Size-exclusion chromatography of cytosolic e-Asf1b complexes isolated as in (A). Codanin-1 co-elutes together with histone H3 and Importin-4. (D) Co-immunoprecipitation of Importin-4 and Asf1 (a and b) with endogenous Codanin-1 from cytosolic HeLa S3 extracts. (E) FLAG–HA-tagged (e−) H3.1 was immunoprecipitated from cytosolic extracts of HeLa S3 cells expressing e-H3.1 (Tagami et al, 2004) and analysed by western blotting. Pull down with sepharose beads was used as negative control. (F) Western blot analysis of cytosolic complexes containing wild-type e-Asf1a or the histone binding mutant, e-Asf1a V94R. Download figure Download PowerPoint Codanin-1 binds Asf1 via a B-domain similar to HIRA and CAF-1 p60 To dissect the nature of the Codanin-1–Asf1 interaction, we performed a series of pull-down experiments using recombinant GST–Asf1 and in-vitro translated 35S-labelled Codanin-1. Indeed, full-length Codanin-1 bound to both recombinant Asf1a and Asf1b (Figure 2A). Detailed mapping revealed that the N-terminal part of Codanin-1 interacts with the globular domain of Asf1a (Figure 2A; Supplementary Figure S2A). Additionally, Codanin-1 bound recombinant wild-type and the Asf1aV94R mutant equally well (Supplementary Figure S2A). Together, these data identify Codanin-1 as a direct Asf1 binding partner. Figure 2.Codanin-1 binds directly to Asf1 via the same pocket as HIRA and CAF-1. (A) (Top left) Summary of the in vitro analysis of Codanin-1–Asf1 binding. ‘+’, binding; ‘−’, no binding; n.d., not done. (Top right) Coomassie staining of GST–Asf1 used for the pull-down assay. (Lower panel) In vitro binding analysis using GST–Asf1 (a and b) to pull down in vitro translated 35S-labelled Codanin-1. Bound proteins were visualized by autoradiography. (B) (Left) Sequence alignment of the B-domains in Codanin-1, HIRA and CAF-1 p60. Note that CAF-1 p60 has two B-domain-like motifs designated (1) and (2). Conservation is indicated by red colour intensity. (Right) Sequence alignment showing conservation of the Codanin-1 B-domain in human (Homo sapiens, H.s.), mice (Mus musculus, M.m.), opossum (Monodelphis domestica, M.d.), frogs (Xenopus tropicalis, X.t.), fish (Danio rerio, D.r.) and flies (Drosophila melanogaster, D.m.). The degree of conservation is illustrated by red colour intensity. (C) (Left) Coomassie staining of GST–Asf1 used for the pull-down assay. (Right) In vitro binding analysis using GST fusions of Asf1a, Asf1b and Asf1b mutated in the B-domain binding pocket (Asf1b D36AD37A; Tang et al, 2006) and in vitro translated 35S-labelled wild-type Codanin-1 or B-domain mutant (SRR/AAA). Bound proteins were visualized by autoradiography. Download figure Download PowerPoint Of the many Asf1-associated proteins only a few are direct binding partners, including downstream histone chaperones HIRA and CAF-1 p60 that play key roles in chromatin assembly (Mello et al, 2002; Daganzo et al, 2003). These downstream histone chaperones interact with Asf1 in a mutually exclusive manner via a so-called B-domain motif (Kirov et al, 1998; Daganzo et al, 2003; Tang et al, 2006). Interestingly, closer examination of the N-terminal part of Codanin-1 revealed a putative B-domain with a high similarity to the domains present in CAF-1 and HIRA (Figure 2B, left). Importantly, the B-domain residues RRI, involved in direct contacts with Asf1 side chains (Tang et al, 2006), are also present in Codanin-1. Additionally, we noticed that the B-domain in Codanin-1 is evolutionary conserved (Figure 2B, right). Disruption of the B-domain in Codanin-1 reduced binding to Asf1 (a and b) in vitro (Figure 2C). Conversely, mutation of the Asf1b residues (D36A and D37A) critical for binding the B-domain in HIRA and CAF-1 p60 (Daganzo et al, 2003; Tang et al, 2006) also abolished Codanin-1 interaction (Figure 2C). Taken together, these results demonstrate that Asf1 binds Codanin-1 in a manner that compares with its interaction with HIRA and CAF-1, implying that these interactions are mutually exclusive. Codanin-1 is a negative regulator of chromatin replication Given the interaction with Asf1, it was important to analyse the effect of Codanin-1 downregulation on chromatin replication. We targeted Codanin-1 by an siRNA smart pool as well as an independent siRNA and both strategies efficiently downregulated Codanin-1 mRNA and protein levels (Figure 3A; Supplementary Figure S3A). Whereas depletion of Asf1 (a and b) leads to accumulation of cells in S phase due to inhibition of DNA replication (Groth et al, 2005, 2007a), cell cycle progression was not dramatically altered upon Codanin-1 knockdown (Supplementary Figure S3B). However, we consistently found higher cell counts in cultures depleted for Codanin-1 as compared with controls, suggesting a moderate proliferation advantage (Supplementary Figure S3C). To directly probe DNA synthesis, we labelled newly synthesized DNA with a short pulse of EdU (5-ethynyl-2-deoxyuridine), which can be visualized with Click-iT technology. Surprisingly, cells treated with Codanin-1 siRNAs for 56 h displayed stronger EdU signals than controls (Figure 3B) and quantitative analysis at several time points confirmed this observation (Figure 3C; Supplementary Figure S4A). The increase in EdU incorporation was not associated with DNA damage as indicated by the lack of γH2AX (Supplementary Figure S4B). This argues that the rate of DNA replication is increased in the absence of Codanin-1. Consistent with this observation, we noted that Codanin-1-depleted cells also had more PCNA (proliferating cell nuclear antigen) loaded onto chromatin as compared with control cells (Figure 3B and D; Supplementary Figure S4C). PCNA acts as a processivity clamp for DNA polymerases and marks sites of ongoing replication (reviewed in Moldovan et al, 2007). The higher levels of PCNA on replicating chromatin thus probably reflect that more replication forks are active in Codanin-1-depleted cells. Figure 3.Codanin-1 depletion enhances DNA replication and Asf1 binding to chromatin. (A) U-2-OS cells were treated with an independent siRNA (siCdan1 #1) or an siRNA smart pool (siCdan1 #2) targeting Codanin-1 for 56 or 70 h. Knockdown efficiency was assessed by western blot analysis and qPCR (Supplementary Figure S3A). (B) Immunofluorescence analysis of U-2-OS cells treated with siRNA for 56 h followed by EdU pulse labelling. PCNA staining served as a marker for S-phase cells. Scale bar, 20 μm. (C) Quantification of EdU incorporation. (Left) Dot plot illustrating the distribution of EdU intensities within one experiment. Cells were treated as in (B) and EdU intensities were measured in PCNA-positive cells. n>87 and ***P 97 and ***P 86; ***P<0.0001; NS, not significant P=0.66 calculated by Wilcoxon paired test. Download figure Download PowerPoint Discussion Here, we characterize Codanin-1 as a novel interactor of the histone chaperone Asf1 and conclude that it acts as a negative regulator of Asf1 (Figure 7). The salient pieces of evidence supporting this view are (i) Codanin-1 is found in a cytosolic complex together with Asf1, histones H3.1-H4 and Importin-4; (ii) Asf1 binds via its HIRA/CAF-1 interaction pocket directly to a highly conserved B-domain motif in Codanin-1, maki
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