RESEARCH ARTICLE

CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functionsin sister chromatid cohesion by regulating SMC1 expressionVarun Jayeshkumar Shah1,2 and Subbareddy Maddika1,*

ABSTRACTCullin–RING-type E3 ligases (CRLs) control a broad range ofbiological processes by ubiquitylating numerous cellular substrates.However, the role of CRL E3 ligases in chromatid cohesion isunknown. In this study, we identified a new CRL-type E3 ligase(designated as CRL7SMU1 complex) that has an essential role in themaintenance of chromatid cohesion. We demonstrate that SMU1,DDB1, CUL7 and RNF40 are integral components of thiscomplex. SMU1, by acting as a substrate recognition module, bindstoH2Bandmediatesmonoubiquitylation at the lysine (K) residueK120through CRL7SMU1 E3 ligase complex. Depletion of CRL7SMU1 leadsto loss of H2B ubiquitylation at the SMC1a locus and, thus,subsequently compromised SMC1a expression in cells. Knockdownof CRL7SMU1 components or loss of H2B ubiquitylation leads todefective sister chromatid cohesion, which is rescued by restoration ofSMC1a expression. Together, our results unveil an important role ofCRL7SMU1 E3 ligase in promoting H2B ubiquitylation for maintenanceof sister chromatid cohesion during mitosis.

This article has an associated First Person interview with the firstauthor of the paper.

KEY WORDS: E3 ligase, SMU1, H2B ubiquitylation, RNF40, CUL7,DDB1, Mitosis, Chromatid cohesion

INTRODUCTIONUbiquitylation is an essential post-translational modification thatregulates a wide array of cellular processes in eukaryotes (Hershkoand Ciechanover, 1998). Ubiquitin is covalently attached through itsC-terminal glycine (G) residue to the ε-amino group of lysine (K) or,occasionally, to the N-terminus of the protein substrate. Proteins canbe tagged with either single ubiquitin or with a polyubiquitin chain.Linkage of ubiquitin with substrates occurs in a three-enzyme cascadecatalyzed by ubiquitin like modifier activating enzymes (UBAs,hereafter referred to as E1), ubiquitin-conjugating enzymes (UBE2s,hereafter referred to as E2) and ubiquitin ligases (E3 ligases). E3ligases are the most heterogeneous class of enzymes, which bringtogether the correct E2 with the right substrate and, thus, are criticalfor defining substrate specificity during ubiquitylation process(Berndsen and Wolberger, 2014; Hershko and Ciechanover, 1998).Depending on the domain architecture and on the mechanism of

ubiquitin transfer to the substrate, E3 ligases have been divided

into three types: homologous to the E6-AP carboxyl-terminus(HECT) E3s, really interesting new gene (RING) E3s and ringbetween ring fingers (RBR) E3s. The HECT-type E3 ligases,characterized by the presence of a HECT domain, possess intrinsiccatalytic activity. HECT-type E3s load ubiquitin on themselvesthrough formation of an ubiquitin-thioester intermediate with thecatalytic cysteine residue within the HECT domain and thentransfers the ubiquitin to the target protein (Berndsen andWolberger, 2014; Rotin and Kumar, 2009). HECT-type E3spredominantly function as monomeric enzymes, but the existenceof functional multimeric HECT-type E3s in the cell has recentlyemerged (Maddika and Chen, 2009). For example, the HECT-typeE3 UBR5 (also known as and hereafter referred to as EDD) formsa multi-component E3 ligase with proteins DDB1, DYRK2 andVPRBP (officially known as DCAF1) as core components, toregulate G2/M transition. RING-type E3s, characterized by thepresence of a RING domain or an U-box domain, are the mostabundant type of ubiquitin ligases in the cell (Berndsen andWolberger, 2014). RING-type E3s do not have intrinsic catalyticactivity however, they function as scaffold between E2 andthe substrate and thereby mediate direct transfer of ubiquitin tothe substrate. RING-type E3s can function as monomers,homodimers, heterodimers and multi-protein complexes, such as inthose comprising cullin proteins and RING-type E3s ligases(CRLs) (Francis et al., 2013; Lydeard et al., 2013; Petroski andDeshaies, 2005; Zimmerman et al., 2010). However, RBR-type E3ligases share common features with both RING-type and HECT-typeE3 ligase families. They recruit ubiquitin-linked E2 through theirRING domain but, like HECT-type E3s, catalyze the ubiquitintransfer in a two-stepmechanism, with the first transfer to the E3 itselfand the second transfer to the substrate (Spratt et al., 2014).

CRLs are a superfamily of RING-type E3s responsible for asmuch as 20% of ubiquitin-dependent protein modification in cells(Bennett et al., 2010; Petroski and Deshaies, 2005). CRLs assembleinto multimeric complexes to enhance substrate diversity duringubiquitylation. CRLs consist of two distinct modules: (1) asubstrate-targeting unit composed of a substrate-recognitionprotein and an adaptor protein that links the module to the cullin,and (2) the RING component that is active in recruiting an ubiquitin-linked E2 enzyme (Zimmerman et al., 2010). CRLs ubiquitylateseveral cellular substrates and thus control broad range of biologicalprocesses, including cell growth, development, signal transduction,transcriptional control and tumor suppression (Harper and Tan,2012; Petroski and Deshaies, 2005). However, no CRL E3 ligase isimplicated in sister chromatid cohesion so far. In this study, weidentified a new CRL (i.e. the CRL7SMU1 complex, comprisingSMU1, DDB1, CUL7 and RNF40 as core components) that hasan essential role in maintenance of chromatid cohesion. Wedemonstrated that CRL7SMU1 E3 ligase supports sister chromatidcohesion through H2B ubiquitylation at SMC1a locus and therebycontrolling its expression.Received 7 December 2017; Accepted 26 February 2018

1Laboratory of Cell Death & Cell Survival, Centre for DNA Fingerprinting andDiagnostics, Hyderabad, India-500 039. 2Graduate studies, Manipal Academy ofHigher Education, Manipal, India-576 104.

*Author for correspondence (msreddy@cdfd.org.in)

S.M., 0000-0002-5880-391X

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RESULTSIdentification of CRL7SMU1 E3 ligase complexPrevious studies have demonstrated that VPRBP, a protein with aWD repeat region and a LIS1 homology (LisH) domain, acts as asubstrate recognition component of HECT-type as well as RING-type E3 ubiquitin ligase complexes (Maddika and Chen, 2009;Nakagawa et al., 2013). To identify proteins with a similarcombination of LisH and WD repeat organization that canassemble E3 ligase complexes, we performed a global search forLisH domain (ID: PS50896) using UniProt database. We retrieved28 human proteins that contain LisH domain, nine of which exhibita combination of LisH domain with WD repeats in their architecture(Fig. 1A). To test if these proteins assemble E3 ligase complexes,we isolated protein complexes associated with SMU1, one of thelisted proteins with such a domain organization. SMU1 (suppressorof mec-8 and unc-52) is a spliceosome accessory protein known forits role in DNA replication, mitotic spindle assembly andmaintenance of genomic stability (Ren et al., 2013; Sugaya et al.,2006, 2005). Tandem affinity purification of S-protein tag, FLAGtag, streptavidin-binding protein tag (SFB)-triple-tagged SMU1followed by mass spectrometry (MS) analysis revealed severalknown and unknown interacting partners (Fig. 1B). Interestingly,we found DDB1, CUL7 and RNF40 among SMU1-interactingproteins. CUL7 is a member of the cullin family of proteins thatfunction as scaffold for E3 ubiquitin ligases. Like other cullins,CUL7 also assembles into a CRL by associating with Skp1, Fbx29and ROC1 (Dias et al., 2002). However, DNA damage-bindingprotein 1 (DDB1) together with DNA damage-binding protein 2(DDB2) is known to function in nucleotide excision repair. At themolecular level, DDB1 functions as an adaptor protein for the Cul4ubiquitin E3 ligase complex, and DYRK2 in complex with theEDD, DDB1, VPRBP (EDVP)-comprising E3 ligase complex(DYRK2–EDVP) to regulate ubiquitin-dependent degradation ofvarious substrates, such as Cdt1, c-Jun, p21 and katanin p60 (Iovineet al., 2011). RNF40 is a RING-type E3 ligase that heterodimerizeswith RNF20 to monoubiquitylate histone H2B to H2Bub (Kimet al., 2009). As it is well known that CUL7 acts as a scaffoldingprotein (Dias et al., 2002) and DDB1 as adaptor (Iovine et al.,2011) for various E3 ligase complexes and, further, RNF40being an E3 ligase, we thus propose the existence of a new CRL7E3 ligase complex with SMU1 as the substrate recognitioncomponent.By performing immunoprecipitation using antibody against

SMU1, we validated the endogenous association of RNF40,DDB1 and CUL7 with SMU1 (Fig. 1C). Importantly, DYRK2 – ascaffolding subunit of the EDVP E3 ligase complex – does notinteract with SMU1, suggesting that CRL7SMU1 is a distinctcomplex. Further, exogenously expressed SMU1 efficientlyinteracted with CUL7, RNF40 and DDB1, but not with DYRK2(Fig. S1A). Likewise, we found no interaction of SMU1 withVPRBP (Fig. 1D), a known functional subunit of the DYRK2–EDVP complex as well as the CUL4–DDB1 complex, againsupporting our conclusion that CRL7SMU1 is a discrete E3 ligasecomplex in cells. In fact, SMU1 is not part of any known CRLsbecause SMU1 neither interacts with SKP1 (Fig. 1E) nor withROC1 (Fig. 1F), while ROC1 and SKP1 interacted positively.Whereas DDB1 can associate with CUL4A, CUL7, SMU1 andRNF40 (Fig. S1B), we found no interaction of CUL7 and RNF40with CUL4A (Fig. S1C,D), again supporting CRL7SMU1 as anindependent complex. Previously, RNF40 was known to act as afunctional E3 ligase in its RNF40/RNF20 heterodimeric form (Kimet al., 2009). Surprisingly, however, we found that SMU1 does not

interact with RNF20 (Fig. 1G), implying that the SMU1–RNF40 E3ligase complex is formed independently of RNF20.

Furthermore, to investigate whether SMU1 forms a stable E3ligase complex in vivo, we analyzed these proteins in HEK-293Tcell extracts by using size-exclusion chromatography. SMU1,DDB1, CUL7 and RNF40 were co-eluted in similar cell lysatefractions corresponding to a molecular mass of∼440 kDa (Fig. 1H),suggesting that CRL7SMU1 complex proteins physically interactwith each other and assemble a large complex in cells. It is wellestablished that WD repeats assist in the assembly of E3 ligasecomplexes, such as DDB1-CRLs. To delineate the regions of SMU1required for assembly of the E3 ligase complex, we made deletionconstructs of SMU1 that either lack the WD repeat region or theN-terminal LisH domain-containing region (Fig. 1I). Interestingly,we found that, while the WD repeat region is dispensable forassociation with components of the E3 ligase, the LisH domainregion is required for assembly of the E3 ligase complex (Fig. 1J).We demonstrate that SMU1, via its LisH domain region, assemblesa so-far-unknown E3 ligase complex by associating with DDB1,CUL7 and RNF40 in cells.

CRL7SMU1 complex regulates monoubiquitylation of H2B atK120In search of substrate(s) of the newly identified E3 ligase complex,we next examined theMS data derived from SMU1 purification. Wefound histone 2B (H2B) to be one of the interacting partners ofSMU1. H2B, together with H2A, H3 and H4, is a part of the corenucleosome and a well-known substrate of the RNF20–RNF40heterodimer that monoubiquitylates H2B at the K residue at position120 (K120) in humans (K123 in yeast) (Kim et al., 2009; Woodet al., 2003). However, no substrate recognition subunit for RNF20–RNF40 E3 ligase has yet been reported. As we identified RNF40 asa core component in the CRL7SMU1 complex, we proposed H2B tobe a probable substrate of this complex. By performing the co-immunoprecipitation experiments using streptavidin pull-downfollowed by immunoblotting with anti-H2B antibody, weconfirmed the specific interaction of H2B with CUL7, DDB1,RNF40 and SMU1 (Fig. 2A). To establish the architecture of thecomplex and further test if SMU1 acts as substrate recognitioncomponent in the E3 ligase complex, we performed knockdownexperiments. On one hand, interaction between RNF40 and H2Bwas severely hampered upon depletion of SMU1, whereas DDB1interaction with RNF40 was intact in these cells (Fig. 2B). On theother hand, interaction between SMU1-H2B and SMU1-DDB1 wasunaffected upon knockdown of RNF40 (Fig. 2C). However,depletion of DDB1 resulted in reduced binding of SMU1 toCUL7 (Fig. 2D). Also, depletion of CUL7 led to loss of interactionbetween RNF40 and DDB1 without having any effect on theinteraction between SMU1 and DDB1 (Fig. 2E). These data suggestthat H2B binds to SMU1, which associates to CUL7 and, further,RNF40 through DDB1 (Fig. 2F). To substantiate the role of SMU1as the substrate recognition protein of the complex, we next checkedthe direct interaction of recombinant bacterially expressedglutathione S-transferase (GST)-tagged SMU1 (GST-SMU1) withmaltose-binding protein (MBP)-tagged H2B (MBP-H2B). Indeed,we found that SMU1 interacts with H2B directly under in vitroconditions (Fig. 2G). Together, these experiments suggest thatSMU1 functions as a substrate-recognition component that linksH2B with DDB1–CUL7–RNF40. Next, to examine the role of theCRL7SMU1 complex in histone H2B ubiquitylation, we usedspecific small interfering (si)RNAs or short hairpin (sh)RNAs todeplete complex proteins in HeLa cells. It has been shown

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Fig. 1. SMU1 assembles CRL type E3 ligase complex by interacting with DDB1, CUL7 and RNF40. (A) Proteins that contain a LisH domain andWD repeats.(B) Partial list of SMU1-associated proteins identified by biochemical purification followed byMS analysis were listed together with the number of peptides for eachprotein. (C) Immunoprecipitation (IP) with control IgG or anti-SMU1 antibodywas performed with extracts prepared fromHEK-293T cells. The presence of RNF40,DDB1, CUL7 and DYRK2 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (D) SFB-tagged VPRBP, together witheither Myc-tagged SMU1 or Myc-tagged DYRK2, was expressed in cells and the interaction of the respective proteins was detected by immunoblotting with theindicated antibodies after pulling down the complexes with streptavidin Sepharose. (E,F) HA tagged-SKP1 together with either SFB-tagged SMU1 or SFB-taggedROC1 (E), and SFB-tagged ROC1 together with Myc-tagged SMU1 or HA-tagged SKP1 (F) were expressed in cells and their interaction was detected asdescribed in D. (G) HeLa cells expressing Myc-tagged RNF20 were lysed and immunoprecipitation was carried out using either IgG or anti-Myc antibody. Thepresence of SMU1 andRNF40 was detected in these immunoprecipitates by immunoblotting using specific antibodies. (H) HEK-293T cell extracts were analysedby size-exclusion chromatography using a Sephacryl 300 column. Proteins eluted from the different fractions were immunoblotted with antibodies against theindicated proteins. (I) Domain architecture of full-length SMU1 (SMU1 FL) and its deletion mutants. (J) SFB-tagged SMU1 FL and SMU1 deletion mutants weretransfected in HeLa cells. 24 h post transfection, cells were lysed and pull-down was carried out using Streptavidin-binding peptide (SBP) beads. The presence ofDDB1, CUL7 and RNF40 in these precipitates was evaluated by immunoblotting with their respective antibodies.

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that knockdown of RNF20/40 abolishes H2B ubiquitylation(Kim et al., 2009). In agreement with previous studies we foundthat knockdown of RNF40 led to downregulation of H2Bmonoubiquitylation at K120 (Fig. 2H). Interestingly, similar to

knockdown of RNF40, depletion of SMU1 (Fig. 2I), DDB1(Fig. 2J) and CUL7 (Fig. 2K) also led to significant downregulationof H2B ubiquitylation, suggesting that these proteins functiontogether to ubiquitylate H2B K120 in vivo. Further, RNF40-

Fig. 2. See next page for legend.

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mediated ubiquitylation of H2B is known to stimulate H3K4trimethylation via the complex of proteins associated with Set1(COMPASS) (Kim et al., 2009). Similar to RNF40 (Fig. 2H),depletion of SMU1, DDB1 and CUL7 resulted in reduction ofH3K4 trimethylation (Fig. 2I-K). DDB1 depletion, as well asRNF40 depletion, seems to affect H3K4 trimethylation levels morethan H2B ubiquitylation. Given that DDB1 is part of multipleindependent E3 ligase complexes, it might be possible that DDB1regulates H3K4me3 through additional mechanisms that areindependent of H2B ubiquitylation. In fact, it has been reportedthat CUL4-DDB1 ubiquitin E3 ligase interacts with multiple WDrepeat-containing proteins and regulates H3 methylations, includingthat of K4Me1, K4Me3, K9Me3 and K27Me3 (Higa et al., 2006).Likewise, RNF40 might also be forming other protein complexes todirectly regulate H3K4me3 independently of H2B ubiquitylation.Nonetheless, our data suggest that SMU1, DDB1, CUL7 andRNF40 are integral components of the functional E3 ligase complexthat regulate histone modifications.

Intact CRL7SMU1 complex is required for mitotic progressionNext, we sought to understand the functional role of the CRL7SMU1

complex in cells. We observed that cells depleted of SMU1 inresponse to SMU1 siRNA transfection (Fig. S2A) proliferated at asignificantly slower rate than control cells transfected with controlsiRNA (Fig. S2B). Furthermore, we found that depletion of SMU1led to the accumulation of 4N cells as well as polyploid (>4N ) cells(Fig. 3A). This increase in the 4N population is due to arrest in themitotic phase, as time-lapse imaging analysis confirmed that cellslacking SMU1 spent several hours in mitosis while control siRNAcells took 60 min on average to complete mitosis (Fig. 3B,C).Likewise, we found that knockdown of all individual components ofthe CRL7SMU1 complex led to accumulation of phosphorylated H3-positive cells (Fig. 3D,E) and significant accumulation of cells withround morphology in culture (Fig. S2C), typical of mitoticallyarrested cells. Since we found that CRL7SMU1 complex proteins are

required for normal progression of mitosis, we next tested if loss ofSMU1 leads to any mitotic defects. We observed variouschromosomal and spindle defects upon SMU1 knockdown(Fig. 4A). Loss of SMU1 resulted in significant increase in cellsdisplaying lagging chromosomes, anaphase/nuclear bridges andmultipolar spindles (Fig. 4B). Similarly, we also found thatdepletion of CUL7, DDB1 or RNF40 individually in cells led tosevere mitotic defects (Fig. 4C,D). Since loss of CRL7SMU1

complex proteins resulted in numerous mitotic defects, we nexttested if loss of H2B monoubiquitylation at K120 phenocopies theloss of E3 ligase complex from cells. Expression of the H2B K120Rmutant, but not wild type H2B, resulted in accumulation of cellswith multiple mitotic defects (Fig. 4E–G), thus suggesting that H2Bubiquitylation at K120 is critical for normal mitotic progression andfurther prevention of genomic instability.

The CRL7SMU1 complex drives SMC1a gene expressionIt is well known that H2B ubiquitylation is enriched at sites of activegene transcription and modulates transcription elongation. Since wefound that the CRL7SMU1 complex promotes H2B ubiquitylationand H3K4 trimethylation (sites of active gene transcription), wehypothesized that this complex controls transcription of a specificset of genes required for mitosis. Previously, a microarray analysisupon SMU1 depletion had revealed alterations in the expression ofvarious mitotic genes (Papasaikas et al., 2015). We tested if SMU1is enriched on the DNA of this set of mitotic genes by usingchromatin immunoprecipitation assay (ChIP). SMU1 wassignificantly enriched on different mitotic genes, such as thoseencoding SMC1a, ANAPC12, ANAPC5, Aurora A and CDCA2(Fig. 5A). Consequently, we tested if depletion of CRL7SMU1

components affects H2B ubiquitylation at these loci. Although,depletion (Fig. S3) of individual components of the E3 ligasecomplex reduced H2Bub levels at distinct loci, we found SMC1a tobe common locus where H2B ubiquitylation is hampered followingthe loss of the CRL7SMU1 complex (Fig. 5B). As H2Bubiquitylation is directly associated with gene transcription, wefurther tested whether this E3 ligase complex regulates transcriptlevels of mitotic genes. Again, we observed that expression levelsof only SMC1a were reduced upon depletion of CUL7, DDB1,RNF40 or SMU1 (Fig. 5C). Consequently, we also found asignificant reduction in SMC1a protein levels upon depletion ofindividual components of CRL7SMU1 complex (Fig. 5D). Notably,expression of the H2B K120R mutant also reduced the geneexpression of SMC1a (Fig. 5E) consistent with our hypothesis thatH2B ubiquitylation is required for expression of this crucialmitotic gene.

CRL7SMU1 complex is essential for sister chromatid cohesionStructural maintenance of chromosomes protein 1A (SMC1a) is acentral component of the cohesin complex, which is required for thecohesion of sister chromatids and essential for accurate chromosomesegregation at the onset of mitosis (Brooker and Berkowitz, 2014;Sumara et al., 2000). To determine if the CRL7SMU1 complex isrequired for sister chromatid cohesion we prepared metaphasespreads on HeLa cells that had been depleted of individualcomponents of the E3 ligase complex. Control cells displaynormal ‘X-shaped’ mitotic chromosomes, with sister chromatidstightly linked at the centromere and chromosome arms separated.However, strikingly, knockdown of CUL7, DDB1, RNF40 orSMU1 led to parallel chromatids and single chromatids in isolationwithin a large fraction of mitotic cells (Fig. 6A,B) suggesting apremature loss of sister chromatid cohesion in these cells. We next

Fig. 2. CRL7SMU1 complex regulates the monoubiquitylation of H2B atposition K120. (A) SFB-tagged CUL7, DDB1, RNF40, SMU1, Rab7 or emptyvector (EV) were transfected and interaction of H2B was detected byimmunoblotting with specific antibody after streptavidin Sepharose pull-down.(B) HeLa cells were transduced with either control or SMU1-specific shRNAfollowed by overexpression of SFB-tagged RNF40. 72 h post transduction,pull-down was performed with streptavidin Sepharose beads, and interactionof DDB1 and H2B with RNF40 was evaluated by immunoblotting with theirrespective antibodies. (C) SFB-tagged SMU1 was overexpressed in cellstransduced with either control or RNF40-specific shRNA. The interaction ofSMU1 with H2B and DDB1 was detected through immunoblotting usingspecific antibodies after immunoprecipitation. (D,E) Cells were transduced witheither control or DDB1 shRNA (E), and control or CUL7 shRNA containing viralparticles. Pull-down followed by detection of different indicated proteins inprecipitates were done as described in B. (F) Model shows the assembly ofCRL7SMU1 complex in association with its substrate H2B. (G) GST pull-downassay was performed with immobilized control GST or GST–SMU1 fusionproteins on glutathione beads, followed by incubation with bacterially purifiedMBP-H2B. The interaction of SMU1 with H2B was assessed byimmunoblotting with anti-MBP antibody. Expression of GST, recombinantGST-SMU1 and MBP-H2B was shown by Coomassie Blue staining. (H) HeLacells were transduced using either control or RNF40 shRNA. Post 72 h, cellswere collected and lysed to isolate soluble and histone fractions. Lysates weresubjected to SDS-PAGE followed by immunoblotting using the indicatedantibodies. (I) Cells were transfected/transduced with either control or SMU1siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid-extractedhistone fractions were subjected to SDS-PAGE followed by immunoblottingusing indicated antibodies. The data presented here represent threeindependent experiments.

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measured the distance between metaphase sister kinetochores inCRL7SMU1 complex-depleted cells by immunostaining CENP-A.Interkinetochore distances between sister chromatids weresignificantly increased in aligned metaphases of cells depleted ofSMU1, CUL7, DDB1 and RNF40 compared with controlmetaphases (Fig. 6C,D), further supporting a critical role ofCRL7SMU1 complex in establishing cohesion between sisterchromatids. Interestingly, expression of the H2B K120R mutantbut not of wild type H2B also resulted in defective chromatidcohesion (Fig. 6E,F) and enhanced interkinetochore distance(Fig. 6G,H), thus suggesting that H2B ubiquitylation byCRL7SMU1 complex is critical for maintenance of sister chromatidcohesion.Based on these observations, we hypothesized that expression of

H2B-Ub fusion protein, which mimics H2B K120 ubiquitylation(Zhang et al., 2013), rescues the cohesion defects caused bydepletion of CRL7SMU1 E3 ligase from cells. To our surprise,overexpression of H2B-Ub fusion protein could not rescue cellsfrom cohesion defects (Fig. S4A,B) caused by loss of E3 ligase.These data prompted us to reason that dynamic H2B ubiquitylationand deubiquitylation is necessary for maintenance and laterdissolution of sister chromatid cohesion during appropriate phasesof mitosis. Therefore, constitutive loss or presence of H2Bmonoubiquitylation might be deleterious for normal chromatidcohesion. This is in agreement with previous data, which showedthat loss of USP44, a deubiquitinase for H2B that mimicks

constitutive H2B ubiquitylation, also leads to various mitoticabnormalities (Zhang et al., 2012). In support of the dynamic natureof H2B ubiquitylation during chromatid cohesion, we found thattransient expression of H2B-Ub fusion protein alone is sufficient toinduce sister chromatid cohesion defects (Fig. S4C,D), increasedinterkinetochore distance (Fig. S4E) as well as mitotic defects incells (Fig. S4F,G). Nonetheless, we next tested if restoration ofSMC1a in cells through plasmid-based expression would rescue thechromatid cohesion defects caused by loss of E3 ligase. Indeed,exogenous expression of SMC1a (Fig. 7A) significantly preventeddefective chromatid cohesion in cells due to depletion of E3 ligasecomponents (Fig. 7B). Also, SMC1a expression in CUL7-, DDB1-,RNF40- or SMU1-depleted cells rescued the interkinetochoredistance that was enhanced due to loss of E3 ligase complex(Fig. 7C). In conclusion, we identified that SMU1, DDB1, CUL7and RNF40 assemble a CRL type E3 ligase complex and promotesmonoubiquitylation of H2B to drive the expression of SMC1a,which is essential for maintenance of sister chromatid cohesionduring mitosis.

DISCUSSIONH2B ubiquitylation is one of the critical histone modificationsassociated with gene expression (Cole et al., 2015; Xie et al., 2017).In humans, RNF20/40 is considered as a major E3 ligase for H2Bmonoubiquitylation (Kim et al., 2009). Although, H2Bub is knownto be globally associated with transcribed genes and its levels

Fig. 3. Intact CRL7SMU1 complex is required for mitotic progression. (A) HeLa cells transfected with either control siRNA or SMU1-specific siRNAs werestained with propidium iodide and cell cycle analysis (whether the cells were 2N, 4N or >4N) was performed by flow cytometry. (B) HeLa cells were transfectedwith indicated siRNAs. The transition of cells through mitosis was analyzed by live cell time-lapse microscopy after synchronizing cells by using doublethymidine block. (C) Time taken by each cell from mitotic entry to division was calculated and the data were plotted for control and SMU1-depleted cells (n=15).(D) HeLa cells were transduced with control shRNA, Cul7 shRNA, DDB1 shRNA, RNF40 shRNA and SMU1 shRNA separately. 72 h post transduction,cells were probedwith antibody against phosphorylated H3 (pH3) tomeasure themitotic index. Nuclei were counterstainedwith DAPI. (E) Quantification of mitoticindex results. Error bars indicate the mean+s.d.; *P<0.05, **P<0.01

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correlate positively with gene expression (Minsky et al., 2008),knockdown of RNF20 affects transcription of only a subset of genes(Shema et al., 2008). This suggests the presence of multiple E3ligase complexes that, independently of RNF20, regulate H2Bmonoubiquitylation in the cell. In this study, we provided multiplelines of evidence that support an essential role of the CRL7SMU1 E3

ligase complex in mediating H2B monoubiquitylation. ThoughH2B ubiquitylation has been implicated in a wide range of cellularprocesses such as transcription initiation and elongation, DNAdamage response, replication, stem cell differentiation, RNAprocessing and export, its role in chromatid cohesion and mitosisis unexplored. We clearly have shown that our newly identified E3

Fig. 4. Depletion of CRL7SMU1 complex proteins induce mitotic defects. (A) HeLa cells were transfected with either control or SMU1 siRNAs. Cells werestained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomaldefects (lagging chromosomes and anaphase bridges). Scale bar: 10 μm. (B) Quantification of results shown in A (n=50 cells each). (C) HeLa cells weretransduced with control shRNA, CUL7 shRNA, DDB1 shRNA or RNF40 shRNA. Cells were stained with antibody against α-tubulin to check the spindledefects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges).Scale bar: 5 μm. (D)Quantification of results shown in C (n=40 cells each). (E) Cells were transfected with H2Bwild type (WT) andH2BK120Rmutant. (F) Variousmitotic abnormalities in cells expressing H2BWT and H2B K120Rmutant were checked using immunofluorescence after staining with antibody against α-tubulinand DAPI. Scale bar: 5 μm. (G) Quantification of results shown in F (n=50 cells each). Error bars indicate the mean+s.d.; ***P<0.001, **P<0.01, *P<0.05;Student’s t-test.

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ligase complex promotes H2B ubiquitylation at SMC1a to maintainsister chromatid cohesion during mitosis. Moreover, ectopicexpression of the H2Bub1 mutant (H2B K120R) caused analteration of the transcription of SMC1a and a significant increasein mitotic defects, including cohesion loss, which is perhaps directevidence that H2B ubiquitylation is critical for mitotic progression.E3 ligases play an essential role in the final step of the

ubiquitylation process to catalyze the transfer of ubiquitin toappropriate substrates. E3 ligases, in particular the CRLs, oftenassemble into multimeric complexes to enhance substrate diversityand specificity during the process of ubiquitylation (Lydeard et al.,2013; Zimmerman et al., 2010). Several interaction domains, suchas F-Box, SOCS-box, β-domains, WD40 repeats, ankyrin, Kelch,WW and RLD motifs are known to assist in the assembly of multi-

component E3 ligases (Petroski and Deshaies, 2005). In this study,using SMU1 as an example, we demonstrated that LisH domainproteins participate in the organization of a functional E3 ligasecomplex. The LisH domain is a highly conserved domain ineukaryotic proteins and proposed to mediate protein–proteininteractions. Although proteins harboring LisH domain are knownto participate in processes, such as microtubule dynamics andchromosome segregation, and are implicated in pathogenesis (Emesand Ponting, 2001), studies on the molecular function of the LisHdomain are limited. There are certain examples to suggest that theLisH domain participates in the organization of multimericcomplexes to regulate protein stability. For instance, in S.cerevisiae, LisH domain proteins, such as GID1, GID7, GID8together with GID2, GID4, GID5 and GID9, assemble a multimeric

Fig. 5. CRL7SMU1 complex is necessary for driving SMC1 gene expression. (A) Exponentially growing HeLa cells were subjected to ChIP analysis usingeither anti-SMU1 or anti-IgG antibody. SMU1 enrichment at various loci is shown. The data shown is derived from three independent experiments. (B) Cellsexpressing control shRNA, CUL7 shRNA, DDB1 shRNA, RNF40 shRNA and SMU1 shRNA were subjected to ChIP analysis using H2Bub antibody. Foldchange of H2Bub enrichment at indicated loci with respect to control shRNA is shown. The data shown is derived from three independent experiments. (C) TotalRNA was extracted from HeLa cells transfected with control or SMU1 siRNA or CUL7 shRNA or DDB1 shRNA and RNF40 shRNA, and expression levelsof various genes measured by qRT-PCR from three independent experiments is shown. (D) HeLa cells transduced with control, CUL7 shRNA, DDB1 shRNA,RNF40 shRNA or SMU1 shRNA and levels of SMC1a protein was measured by immunoblotting with specific antibody. (E) HeLa cells were transfected withH2B wild type (WT) and H2B K120R mutant. Relative expression of indicated genes measured by using qRT-PCR from three independent experiments wasplotted. Error bars indicate the mean+s.d; ***P<0.001; **P<0.01; *P<0.05, Student’s t-test.

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glucose induced degradation deficient (GID) complex. The GIDcomplex mediates polyubiquitylation of fructose-1,6-bisphosphatase (FBPase) via the E3 ubiquitin ligase activity ofGID2, which contains a RING domain (Menssen et al., 2012).However, the role of LisH domain-containing proteins in theassembly of an E3 ligase in humans is unknown. Although we andothers have previously shown that VPRBP, which contains a LisHdomain, acts as a substrate recognition protein in RING-type E3s aswell as in HECT-type E3s (Maddika and Chen, 2009; Nakagawaet al., 2013), whether the LisH domain is required for the assemblyof E3 ligases is unexplored. In another example, WDR26 – incomplex with Axin1 – controls β-catenin levels and negatively

regulates Wnt signaling (Goto et al., 2016). Although, the LisHdomain of WDR26 is critical for β-catenin degradation, whether itparticipates in the organization of E3 ligase in this case is notstudied. Thus, our current study provides clues in corroborating thecritical role of LisH domain proteins in organizing E3 ligasecomplexes.

SMC1 is an essential component of cohesin – a multi-proteincomplex made up of four subunits [Smc1; Smc3; an α-kleisinsubunit, i.e. Mcd1/Scc1 (mitosis) or Rec8 (meiosis); and Irr1/Scc3]– that is conserved from yeast to humans. Cohesin has a well-documented role in chromatid cohesion, where it provides stablebut reversible connections between sister chromatids during both

Fig. 6. CRL7SMU1 complex regulates chromatid cohesion. (A) HeLa cells were transduced with Control, CUL7, DDB1, RNF40 and SMU1 shRNA separatelyand chromosome spreads were prepared after 96 h of transduction. Representative images of chromosome spreads are shown. Insets show intact chromosomeor single chromatids. (B) Quantification of data shown in A. For defective chromatid cohesion analysis, cells were scored as normal when two or fewerchromosomes showed defects, or defective when three or more chromosomes showed defects. Total 115 mitotic spreads were analysed per condition andP values were calculated (P<0.001) using two-tailed Fisher’s exact test (knockdown of complex proteins compared to control). (C) HeLa cells were transducedseparately with Control, CUL7, DDB1, RNF40 or SMU1 shRNAs and stained with anti-CENP-A antibody to stain kinetochores. (D) Distance betweenpaired kinetochores was measured at individual z-planes and plotted (n=150). ***P<0.001. (E) Chromosome spreads were prepared from HeLa cells transfectedwith either H2B wild type or H2B K120R mutant. Representative images of chromosome spreads were shown. Insets show intact chromosome or singlechromatids. (F) Quantification of data shown in E (n=100), P<0.001. (G,H) Cells expressing either H2B WT or K120R mutant were stained with anti-CENPAantibody (G) and the distance between paired kinetochores was measured (H); ***P<0.001.

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mitosis and meiosis (Nasmyth and Haering, 2009). However, recentfindings indicate that, in higher eukaryotes, sister chromatidcohesion is not the only major function of cohesin, but alsoregulates other processes including transcriptional regulation, DNArepair, chromosome condensation and morphogenesis (Dorsett,2011). Indeed, elegant studies in yeast indicate that severe reductionin the level of chromatin-bound cohesin does not entirely affect itsfunction of holding the sister chromatids together but drasticallyaffects the non-canonical functions (Dorsett, 2011; Heidinger-Pauliet al., 2010; Mehta et al., 2013). Thus, although our studies haveclearly demonstrated that defective sister chromatid cohesion due toloss of CRL7SMU1 complex is dependent on SMC1, it still remainsto be determined whether these effects are mediated directly throughSMC1 localization at centromeres or indirectly through regulationof the expression of unknown genes.In addition to H2B ubiquitylation, the CRL7SMU1 complex might

also directly regulate ubiquitylation of mitotic proteins. In supportof this speculation, we found proteins, such as Adenomatouspolyposis coli (APC) in the list of SMU1-associated proteins.Further studies are required to test if APC acts as a substrate of thisE3 ligase complex. However, our work revealed that the LisHdomain protein SMU1 associates with RNF40 to form a RING-typeCRL. Interestingly, the list of SMU1-associated proteins alsocontains HECT type E3s, EDD and HUWE1. In fact, proteins, suchas VPRBP and DDB1, were previously shown to associate withRING-type E3s as well as HECT-type E3s (Maddika and Chen,2009; Nakagawa et al., 2013). Thus, in future studies it would beinteresting to test if SMU1 participates in the assembly of bothRING- and HECT-type E3 ligases to regulate different cellularprocesses by controlling distinct set of substrates.

MATERIALS AND METHODSPlasmidsFull-length SMU1, DDB1, CUL7, RNF40, RNF20, SMC1a, Roc1,VPRBP, Rab7, H2B wild type or K120R mutant were cloned into S-protein tag, FLAG tag, streptavidin-binding protein tag (SFB)-triple-taggeddestination vectors using the Gateway cloning system (Invitrogen). H2B andSMU1 were cloned into GST and MBP destination vectors using the same

system. SMU1 and DYRK2 were also cloned into Myc destination vectors,and SKP1 was cloned into a HA destination vector by using the Gatewaycloning system. The point mutations for H2B were generated by PCR-basedsite-directed mutagenesis and cloned into SFB- and GST-tagged destinationvectors. RNF40 plasmid was a kind gift fromDr Steven Johnsen (UniversityMedical Center Göttingen, Germany). Myc-tagged RNF20 was kindlyprovided by Dr Jae Bum Kim (Seoul National University, South Korea).The FLAGH2B-Ub fusion construct was a kind gift fromDr HengbinWang(University of Alabama at Birmingham, AL). The cMyc-SMC1a plasmidwas a gift from Michael Kastan (Addgene plasmid #32363) (Kim et al.,2002), Myc3-CUL7 was a gift from Yue Xiong (Addgene plasmid #20695)(Andrews et al., 2006) and GFP-H2B was a gift from Geoff Wahl (Addgeneplasmid #11680) (Kanda et al., 1998).

AntibodiesAntibodies against SMU1 (Abgent #AT3965a; 1:1000); RNF40 (Sigma#R9029; 1:2000); CUL7 (Sigma #C1743; 1:2000); DDB1 (Bethyl #A300-462A; 1:5000); SMC1a (Abcam #ab133643; 1:1000); H2B (Millipore #07-371; 1:5000); histone H2B ubiquitylated at Lys120 (H2Bub; Cell SignalingTechnology #5546; 1:1000); cyclin A (BD #611269; 1:1000); CDT1(Bethyl #A300-786A; 1:1000); histone H3 phosphorylated at Ser10 (pH3;Cell Signaling Technology #9701L; for western blotting 1:1000, forimmunofluorescence 1:200); Cul4a (Bethyl #A300-739A; 1:5000); RNF20(Abcam #ab32629; 1:1000); Myc-tag (Santa Cruz #9E10; 1:1000); FLAG-tag (Sigma, #F3165; 1:10,000); HA (Bethyl, #A190-108A; 1:1000), actin(Sigma, #A5441; 1:10,000) and α-tubulin (Sigma, #T6074; for westernblotting 1:5000, for immunofluorescence 1:200) were used in this study.HRP-/FITC-conjugated anti-mouse and anti-rabbit secondary antibodieswere obtained from Jackson ImmunoResearch.

Cell lines and transfectionHEK-293T, HeLa and BOSC23 cell lines were used in this work. All celllines were purchased from American Type Culture Collection, and weretested and authenticated by the cell bank using their standard short tandemrepeats (STR)-based techniques. Cells were also continuously monitored bymicroscopy to maintain their original morphology and tested formycoplasma contamination by using DAPI staining. HEK-293T or HeLawere transfected with various plasmids using PEI (Polysciences)according to the manufacturer’s protocol. Briefly, the plasmid wasmixed with PEI (1 mg/ml) at a ratio of 1:3 in serum-free RPMImedium. Then, the DNA-PEI mixture was incubated for 20 min at room

Fig. 7. Exogenous SMC1a expression partially rescues cohesion defects caused by loss of CRL7SMU1 complex. (A) HeLa cells were transduced withcontrol shRNA, CUL7 shRNA, DDB1 shRNA, RNF40 shRNA and SMU1 shRNA alone or together with SMC1a plasmid and SMC1a levels were analysed byimmunoblotting. (B) Cells expressing control shRNA, Cul7 shRNA, DDB1 shRNA, RNF40 shRNA and SMU1 shRNA alone or together with SMC1a wereanalysed for chromatid cohesion (n=100), ***P<0.001 for knockdown of complex proteins compared to control and **P<0.01 between shRNA alone and alongwith SMC1a (rescue) (C) and inter kinetochore distance (n=190). Errors bars indicate the mean±s.d.; Student’s t-test.

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temperature (RT) and the complexes were added to cells to allow thetransfection of plasmid.

Tandem affinity purificationSMU1-associated proteins were isolated using tandem affinity purificationas described before (Maddika and Chen, 2009). Briefly, HEK-293T cellsexpressing SFB-triple-tagged SMU1 were lysed with NETN lysis buffer(20 mM Tris-HCl at pH 8.0, 100 mMNaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing protease inhibitors [phenylmethylsulfonyl fluoride (PMSF),Pepstatin A and aprotinin] on ice for 20 min. The cell lysates were added tostreptavidin–Sepharose beads (Amersham Biosciences) and incubated for1.5 h at 4°C. Then beads were washed thrice with lysis buffer and theassociated proteins were eluted using 2 mg/ml biotin (Sigma) for 1 h at 4°C.The eluates from the first step of purification were then incubated with S-protein–agarose beads (Novagen) for 1 h at 4°C. After clearing the unboundproteins by washing, the proteins associated with S-protein–agarose beadswere eluted by boiling in SDS-loading buffer for 10 min at 95°C. Proteins ofthe eluted lysate were separated by SDS-PAGE. The associated proteinswere identified by in-gel trypsin digestion followed by liquidchromatography–mass spectrometry (LC–MS)/MS analysis at the TaplinMass Spectrometry Facility (Harvard University).

Immunoprecipitation and western blottingFor immunoprecipitation assays, cells were lysed with NETN buffer.The whole-cell lysates obtained by centrifugation were incubatedwith 2 µg of specified antibody bound to protein G–Sepharose beads,or with streptavidin–sepharose beads (GE) for 1.5 h at 4°C. Theimmunocomplexes were then washed with NETN buffer three times andloaded onto an SDS polyacrylamide gel. Western blotting was carried outby following standard protocols. Proteins were separated by denaturingSDS–PAGE and then transferred onto polyvinylidene difluoride (PVDF)membrane. The membranes were blocked in 5% non-fat dried milk in Tris-buffered saline (TBS) and then incubated with the primary antibodiesovernight at 4°C. Then, the blots were incubated with the correspondingsecondary antibodies conjugated with HRP for 1 h at room temperature.Visualization was carried out by enhanced chemiluminescence detection(Thermo Fisher Scientific).

Histone extractionHistones were isolated according to the acid extraction protocol. Briefly,cells were lysed under hypotonic conditions and the intact nuclei werecollected. Isolated nuclei were acid extracted using 0.2M H2SO4. Histoneswere precipitated using 33% TCA and residual TCA was removed by ice-cold acetone. The pellets were air dried and Milli-Q water was added todissolve histones. Histone fractions were subjected to SDS-PAGE followedby western blotting with antibodies of interest.

GST pull-down assaysBacterially expressed GST and GST-SMU1 bound to glutathione-Sepharosebeads were incubated with eluted MBP-H2B for 1 h at 4°C. Beads werewashed and proteins eluted by boiling in 2×SDS Laemmli buffer and thenseparated by SDS-PAGE; the interactions were analysed by westernblotting.

RNA interference and lentiviral infectionControl siRNA and pre validated SMU1 siRNA were purchased fromDharmacon (Catalogue no: J-021129-10). TRC lentiviral SMU1 shRNA(RHS4533-EG55234), DDB1 shRNA (RHS4533-EG1642), CUL7 shRNA(RHS4533-EG9820) and RNF40 shRNA (RHS4533-EG9810) werepurchased from Dharmacon. Transfection was performed twice, 24 hapart, with 200 nM siRNA using Oligofectamine reagent in accordancewiththe manufacturer’s protocol (Invitrogen). shRNAs were transfectedtransiently using PEI (Invitrogen) in BOSC23 packaging cells along withpackaging vectors. 48 h post transfection, the viral medium was collectedand added to the target cells along with polybrene (8 mg/ml). 48 or 72 h postinfection, cells were collected and processed for various assays andimmunoblotting was performed with the specific antibodies to check theefficiency of knockdown.

Immunofluorescence stainingCells grown on coverslips were fixed with 3% paraformaldehyde solution inphosphate-buffered saline (PBS) containing 50 mM sucrose for 15 min atroom temperature. After permeabilization with 0.5% Triton X-100 buffercontaining 20 mM HEPES at pH 7.4, 50 mM NaCl, 3 mM MgCl2 and300 mM sucrose for 5 min at room temperature, cells were incubated with a1% BSA for blocking for 30 min at room temperature. After washing withPBS, cells were incubated with primary antibodies for 2–3 h at roomtemperature followed by three washes with 1×PBS for 5 min each. Then,cells were incubated with FITC- or Rhodamine-conjugated secondaryantibodies for 60 min at room temperature followed by three washes with1×PBS for 5 min each. Nuclei were counterstained with DAPI. After a finalwash with PBS, coverslips were mounted with glycerine-containingparaphenylenediamine. Images were taken using a Zeiss confocalmicroscope (LSM Meta 510 or LSM 700).

Cell cycle analysisHeLa cells transfected with the desired expression vectors and siRNA orshRNA were harvested, washed with phosphate-buffered saline and fixedwith ice-cold 70% ethanol for at least 1 h. Cells were washed thrice in PBSand treated for 30 min at 37°C with RNase A (5 μg/ml) and propidiumiodide (25 μg/ml), then analysed using a BD Accuri C6 flow cytometer.

Cell proliferation assayHeLa cells were transfected with Control and SMU1 siRNA. 48 h posttransfection 1×105 cells were seeded in five different plates and cellscounted based on the Trypan Blue dye exclusion test.

Gel filtrationA Sephacryl 300 column (GE Healthcare) was used to separate the proteincomplexes in the range of 10–1500 kDa. The column was calibrated usingfour gel filtration markers thyroglobulin (669 kDa), ferritin (440 kDa),conalbumin (75 kDa) and ovalbumin (44 kDa). After calibration, lysates(0.8–1 ml, total protein concentration 1–2 mg/ml) were injected andallowed to pass through the column. 1 ml fractions were collected using aBio-Rad 2110 fraction collector within the molecular weight range ofinterest and all 1 ml fractions were concentrated separately by usingprotein concentration columns to the final volume of 80 µl. All thefractions were subjected to SDS-PAGE followed by western blot stainingof proteins of interest.

Quantitative real-time PCRTotal RNA was isolated using Trizol reagent (Invitrogen) as permanufacturer’s instructions. 2 μg of total RNA was transcribed in thepresence of anchored oligo dT using Superscript-III (Invitrogen) as permanufacturer’s protocol. Quantitative PCR (qPCR) was then initiated usingthe SYBR Pre mix Ex Taq (Tli RNaseH plus) kit (Clontech Laboratories)in 7500 real-time (RT) PCR systems (Applied Biosystems) as permanufacturer’s protocol. The threshold cycle (Ct) values for particulargenes were normalized to GAPDH for each sample. Sequences for primersused for qRT-PCR analysis are included in Table S2.

ChIP assay4 μg mouse IgG (Bethyl Laboratories), 4 μg SMU1 (Abgent #AT3965a)or 4 µl of H2Bub (Cell Signaling Technology #5546; dilution 1:250)antibodies were used for chromatin immunoprecipitation (ChIP) assay.Briefly, HeLa cells were crosslinked by using 1% formaldehyde solution.Crosslinking was allowed to proceed for 10 min at room temperature andwas then stopped by addition of glycine to a final concentration of 0.125 M.Cells were washed twice with ice-cold 1×PBS and lysed using cell lysisbuffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40 and proteaseinhibitors) followed by nuclei lysis buffer (50 mM Tris pH 8.1, 10 mMEDTA, 1% SDS and protease inhibitors). Isolated chromatin was sonicatedusing a Diagenode Bioruptor at medium power and checked for fragmentsize. After microcentrifugation, the supernatant was diluted with 1×IPdilution buffer and preclearing was performed by using Protein G beads.Then equal amount of antibody was added and incubated on a rotatingplatform for 12 to 16 h at 4°C. Next day, protein G beads were added to

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chromatin. Beads were washed two times with IP Dialysis buffer (2 mMEDTA and 50 mM Tris pH 8) and three times with IP wash buffer (100 mMTris pH 8, 250 mM LiCl, 1% NP-40 and 1% Na deoxycholate) for 5 min atroom temperature. Then beads were quickly washed with 1×TE and boundDNA fragments were eluted using elution buffer (50 mMNaHCO3 and 1%SDS) at 65°C. Input and elution products were kept for decrosslinking at 65°C overnight. Then, samples were treated with RNase A and proteinase K for3 h at 37°C and DNAwas extracted using PCI treatment. qRT-PCRwas theninitiated using the SYBR Pre mix Ex Taq (Tli RNaseH plus) kit (ClontechLaboratories) in 7500 real-time PCR systems (Applied Biosystems) as permanufacturer’s protocol. Sequences for primers used for ChIP analysis wereincluded in Table S1.

Metaphase spreadsMetaphase chromosome spreads were performed as described before(Maddika et al., 2009). Briefly, cells treated with colcemid for 4 h werecollected, washed with PBS and treated with 75 mMKCl for 30 min at roomtemperature. The treated cells were then fixed in fresh solution of methanol:acetic acid (3:1) and dropped onto glass slides. Cells were allowed to air dry,stained with Giemsa’s solution (5%) and visualized under the microscope.

AcknowledgementsWe thank the Centre for DNA Fingerprinting and Diagnostics (CDFD) core facility aswell as Dr Rohit Joshi’s lab for their assistance in confocal imaging. We thank NanciRani for technical assistance, T. S. Shaffiqu for providing assistance in gel filtrationexperiment, Sawant Suresh, Zaffer Ullah Zargar and Amit M. Karole for their criticalinputs during gene expression and ChIP experiments, Dr Ashwin Dalal andDr Usha Dutta for their inputs during mitotic spread experiments. We also thank allmembers of LCDCS for their suggestions and critical inputs at various stages of theproject.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: S.M.; Methodology: V.J.S., S.M.; Validation: V.J.S.; Formalanalysis: S.M.; Investigation: V.J.S.; Resources: S.M.; Data curation: V.J.S.; Writing -original draft: V.J.S., S.M.;Writing - review & editing: S.M.; Supervision: S.M.; Projectadministration: S.M.; Funding acquisition: S.M.

FundingThis work was supported in part by a grant from the Council of Scientific andIndustrial Research, India [grant number: 37(6371)/17] to S.M.; and Centre for DNAFingerprinting and Diagnostics (CDFD) core funds. S.M. is a senior fellow of theWellcome Trust/Department of Biotechnology (DBT) India Alliance and a recipientof the Senior Innovative Young Biotechnologist Award (IYBA) from the Departmentof Biotechnology (DBT), Ministry of Science and Technology V.J.S. receivedfellowship support from the DBT, India Alliance. Deposited in PMC for release after 6months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.213868.supplemental

ReferencesAndrews, P., He, Y. J. and Xiong, Y. (2006). Cytoplasmic localized ubiquitin ligasecullin 7 binds to p53 and promotes cell growth by antagonizing p53 function.Oncogene 25, 4534-4548.

Bennett, E. J., Rush, J., Gygi, S. P. and Harper, J. W. (2010). Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics.Cell 143, 951-965.

Berndsen, C. E. and Wolberger, C. (2014). New insights into ubiquitin E3 ligasemechanism. Nat. Struct. Mol. Biol. 21, 301-307.

Brooker, A. S. and Berkowitz, K. M. (2014). The roles of cohesins in mitosis,meiosis, and human health and disease. Methods Mol. Biol. 1170, 229-266.

Cole, A. J., Clifton-Bligh, R. and Marsh, D. J. (2015). Histone H2Bmonoubiquitination: roles to play in human malignancy. Endocr Relat. Cancer22, T19-T33.

Dias, D. C., Dolios, G., Wang, R. and Pan, Z.-Q. (2002). CUL7: A DOC domain-containing cullin selectively binds Skp1*Fbx29 to form an SCF-like complex.Proc.Natl. Acad. Sci. USA 99, 16601-16606.

Dorsett, D. (2011). Cohesin: genomic insights into controlling gene transcriptionand development. Curr. Opin. Genet. Dev. 21, 199-206.

Emes, R. D. andPonting, C. P. (2001). A new sequencemotif linking lissencephaly,Treacher Collins and oral-facial-digital type 1 syndromes, microtubule dynamicsand cell migration. Hum. Mol. Genet. 10, 2813-2820.

Francis, O., Han, F. and Adams, J. C. (2013). Molecular phylogeny of a RINGE3 ubiquitin ligase, conserved in eukaryotic cells and dominated byhomologous components, the muskelin/RanBPM/CTLH complex. PLoS ONE 8,e75217.

Goto, T., Matsuzawa, J., Iemura, S.-I., Natsume, T. and Shibuya, H. (2016).WDR26 is a new partner of Axin1 in the canonical Wnt signaling pathway. FEBSLett. 590, 1291-1303.

Harper, J. W. and Tan, M.-K. M. (2012). Understanding cullin-RING E3 biologythrough proteomics-based substrate identification. Mol. Cell. Proteomics 11,1541-1550.

Heidinger-Pauli, J. M., Mert, O., Davenport, C., Guacci, V. and Koshland, D.(2010). Systematic reduction of cohesin differentially affects chromosomesegregation, condensation, and DNA repair. Curr. Biol. 20, 957-963.

Hershko, A. and Ciechanover, A. (1998). The ubiquitin system. Annu. Rev.Biochem. 67, 425-479.

Higa, L. A., Wu, M., Ye, T., Kobayashi, R., Sun, H. and Zhang, H. (2006). CUL4-DDB1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulateshistone methylation. Nat. Cell Biol. 8, 1277-1283.

Iovine, B., Iannella, M. L. and Bevilacqua, M. A. (2011). Damage-specific DNAbinding protein 1 (DDB1): a protein with a wide range of functions. Int. J. Biochem.Cell Biol. 43, 1664-1667.

Kanda, T., Sullivan, K. F. and Wahl, G. M. (1998). Histone-GFP fusion proteinenables sensitive analysis of chromosome dynamics in living mammalian cells.Curr. Biol. 8, 377-385.

Kim, S.-T., Xu, B. and Kastan, M. B. (2002). Involvement of the cohesin protein,Smc1, in Atm-dependent and independent responses to DNA damage. GenesDev. 16, 560-570.

Kim, J., Guermah, M., McGinty, R. K., Lee, J.-S., Tang, Z., Milne, T. A.,Shilatifard, A., Muir, T. W. and Roeder, R. G. (2009). RAD6-Mediatedtranscription-coupled H2B ubiquitylation directly stimulates H3K4 methylation inhuman cells. Cell 137, 459-471.

Lydeard, J. R., Schulman, B. A. and Harper, J. W. (2013). Building andremodelling Cullin-RING E3 ubiquitin ligases. EMBO Rep. 14, 1050-1061.

Maddika, S. and Chen, J. (2009). Protein kinase DYRK2 is a scaffold that facilitatesassembly of an E3 ligase. Nat. Cell Biol. 11, 409-419.

Maddika, S., Sy, S. M.-H. and Chen, J. (2009). Functional interaction between Chfrand Kif22 controls genomic stability. J. Biol. Chem. 284, 12998-13003.

Mehta, G. D., Kumar, R., Srivastava, S. and Ghosh, S. K. (2013). Cohesin:functions beyond sister chromatid cohesion. FEBS Lett. 587, 2299-2312.

Menssen, R., Schweiggert, J., Schreiner, J., Kusevic, D., Reuther, J., Braun, B.and Wolf, D. H. (2012). Exploring the topology of the Gid complex, the E3ubiquitin ligase involved in catabolite-induced degradation of gluconeogenicenzymes. J. Biol. Chem. 287, 25602-25614.

Minsky, N., Shema, E., Field, Y., Schuster, M., Segal, E. and Oren, M. (2008).Monoubiquitinated H2B is associated with the transcribed region of highlyexpressed genes in human cells. Nat. Cell Biol. 10, 483-488.

Nakagawa, T., Mondal, K. and Swanson, P. C. (2013). VprBP (DCAF1): apromiscuous substrate recognition subunit that incorporates into both RING-family CRL4 and HECT-family EDD/UBR5 E3 ubiquitin ligases. BMC Mol. Biol.14, 22.

Nasmyth, K. and Haering, C. H. (2009). Cohesin: its roles and mechanisms. Annu.Rev. Genet. 43, 525-558.

Papasaikas, P., Tejedor, J. R., Vigevani, L. and Valcarcel, J. (2015). Functionalsplicing network reveals extensive regulatory potential of the core spliceosomalmachinery. Mol. Cell 57, 7-22.

Petroski, M. D. and Deshaies, R. J. (2005). Function and regulation of cullin-RINGubiquitin ligases. Nat. Rev. Mol. Cell Biol. 6, 9-20.

Ren, L., Liu, Y., Guo, L., Wang, H., Ma, L., Zeng, M., Shao, X., Yang, C., Tang, Y.,Wang, L. et al. (2013). Loss of Smu1 function de-represses DNA replication andover-activates ATR-dependent replication checkpoint. Biochem. Biophys. Res.Commun. 436, 192-198.

Rotin, D. and Kumar, S. (2009). Physiological functions of the HECT family ofubiquitin ligases. Nat. Rev. Mol. Cell Biol. 10, 398-409.

Shema, E., Tirosh, I., Aylon, Y., Huang, J., Ye, C., Moskovits, N., Raver-Shapira,N., Minsky, N., Pirngruber, J., Tarcic, G. et al. (2008). The histone H2B-specificubiquitin ligase RNF20/hBRE1 acts as a putative tumor suppressor throughselective regulation of gene expression. Genes Dev. 22, 2664-2676.

Spratt, D. E., Walden, H. and Shaw, G. S. (2014). RBR E3 ubiquitin ligases: newstructures, new insights, new questions. Biochem. J. 458, 421-437.

Sugaya, K., Hongo, E. and Tsuji, H. (2005). A temperature-sensitive mutation intheWD repeat-containing protein Smu1 is related to maintenance of chromosomeintegrity. Exp. Cell Res. 306, 242-251.

Sugaya, K., Hongo, E., Ishihara, Y. and Tsuji, H. (2006). The conserved role ofSmu1 in splicing is characterized in its mammalian temperature-sensitive mutant.J. Cell Sci. 119, 4944-4951.

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llScience

Sumara, I., Vorlaufer, E., Gieffers, C., Peters, B. H. and Peters, J.-M. (2000).Characterization of vertebrate cohesin complexes and their regulation inprophase. J. Cell Biol. 151, 749-762.

Wood, A., Krogan, N. J., Dover, J., Schneider, J., Heidt, J., Boateng, M. A.,Dean, K., Golshani, A., Zhang, Y., Greenblatt, J. F. et al. (2003). Bre1, an E3ubiquitin ligase required for recruitment and substrate selection of Rad6 at apromoter. Mol. Cell 11, 267-274.

Xie, W., Nagarajan, S., Baumgart, S. J., Kosinsky, R. L., Najafova, Z., Kari, V.,Hennion, M., Indenbirken, D., Bonn, S., Grundhoff, A. et al. (2017). RNF40regulates gene expression in an epigenetic context-dependent manner. GenomeBiol. 18, 32.

Zhang, Y., Foreman, O., Wigle, D. A., Kosari, F., Vasmatzis, G., Salisbury, J. L.,van Deursen, J. and Galardy, P. J. (2012). USP44 regulates centrosomepositioning to prevent aneuploidy and suppress tumorigenesis. J. Clin. Invest.122, 4362-4374.

Zhang, Z., Jones, A., Joo, H.-Y., Zhou, D., Cao, Y., Chen, S., Erdjument-Bromage, H., Renfrow, M., He, H., Tempst, P. et al. (2013). USP49deubiquitinates histone H2B and regulates cotranscriptional pre-mRNA splicing.Genes Dev. 27, 1581-1595.

Zimmerman, E. S., Schulman, B. A. and Zheng, N. (2010). Structuralassembly of cullin-RING ubiquitin ligase complexes. Curr. Opin. Struct. Biol. 20,714-721.

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RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs213868. doi:10.1242/jcs.213868

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