© 2018. Published by The Company of Biologists Ltd.

CRL7SMU1 E3 ligase complex driven H2B ubiquitination functions in sister

chromatid cohesion by regulating SMC1 expression

Varun Jayeshkumar Shah1, 2, & Subbareddy Maddika1*

1Laboratory of Cell Death & Cell Survival, Centre for DNA Fingerprinting and Diagnostics,

Hyderabad, INDIA

2Graduate studies, Manipal Academy of Higher Education, Manipal, INDIA

*To whom correspondence should be addressed

Dr. Subbareddy Maddika

Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500 001, INDIA

Tel: +91-40-24749353

Fax: +91-40-24749448

E-mail: msreddy@cdfd.org.in

Key words: E3 ligase, SMU1, H2B ubiquitination, RNF40, CUL7, DDB1, mitosis, chromatid

cohesion

Summary statement

We identified that SMU1, DDB1, CUL7 and RNF40 assemble an E3 ligase complex and

promotes H2B monoubiquitination to drive SMC1 expression, which is essential for sister

chromatid cohesion.

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JCS Advance Online Article. Posted on 5 March 2018

Abstract

Cullin–RING E3 ligases (CRLs) control broad range of biological processes by ubiquitinating

numerous cellular substrates. However, role of CRL E3 ligases in chromatid cohesion is

unknown. In this study, we identified a new CRL type E3 ligase (designated as CRL7SMU1

complex) that has an essential role in maintenance of chromatid cohesion. We demonstrate that

SMU1, DDB1, CUL7 and RNF40 as integral components of this complex. SMU1 by acting as a

substrate recognition module, binds to H2B and mediates monoubiquitination at K120 site

through CRL7SMU1 E3 ligase complex. Depletion of CRL7SMU1 leads to loss of H2B

ubiquitination at SMC1a locus and thus subsequently compromised SMC1a expression in cells.

Knock down of CRL7SMU1 components or loss of H2B ubiquitination leads to defective sister

chromatid cohesion, which is rescued by restoration of SMC1a expression. Together, our results

unveil an important role of CRL7SMU1 E3 ligase in promoting H2B ubiquitination for

maintenance of sister chromatid cohesion during mitosis.

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Introduction

Ubiquitination is an essential posttranslational modification that regulates wide array of cellular

processes in eukaryotes (Hershko and Ciechanover, 1998). Ubiquitin is covalently attached

through its C-terminal glycine to the ε-amino group of lysine or occasionally to the N-terminus

of the protein substrates. Proteins can be tagged with either single ubiquitin or with a

polyubiquitin chain. Linkage of ubiquitin with substrates occurs in a three-enzyme cascade

catalyzed by E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3

(ubiquitin ligase) enzymes. E3 ligases are the most heterogeneous class of enzymes, which bring

together the correct E2 with the right substrate and thus are critical for defining substrate

specificity during ubiquitination 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 classified into three types: HECT E3s, RING E3s and RBR type

E3s. The HECT-type E3 ligases characterized by the presence of HECT (homologous to E6-AP

carboxyl terminus) domain possess intrinsic catalytic activity. HECT-E3s load ubiquitin on

themselves through formation of ubiquitin-thioester intermediate with the catalytic cysteine

residue within the HECT domain and then transfers the ubiquitin to the target protein (Berndsen

and Wolberger, 2014; Rotin and Kumar, 2009). HECT-E3s predominantly function as

monomeric enzymes, but existence of functional multimeric HECT E3s in the cell are recently

emerging (Maddika and Chen, 2009). For example, HECT-E3 EDD forms a multi-component E3

ligase with DDB1, DYRK2 and VPRBP as core components to regulate G2/M transition. RING-

class of E3s characterized by the presence of RING (Really Interesting New Gene) or U-box

domain are the most abundant type of ubiquitin ligases in the cell (Berndsen and Wolberger,

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2014). RING-type E3s do not have intrinsic catalytic activity however, they function as scaffold

between E2 and the substrate and thereby mediate direct transfer of ubiquitin to the substrate.

RING E3s can function as monomers, homodimers, heterodimers and multi-protein complexes,

such as the cullin-RING ligases (CRLs) (Francis et al., 2013; Lydeard et al., 2013; Petroski and

Deshaies, 2005; Zimmerman et al., 2010). On the other hand, the RBR-type E3 ligases share

common features with both RING and HECT E3 ligase families. They recruit ubiquitin charged

E2 through the RING domain but, like HECT-E3s, catalyze the ubiquitin transfer through two-

step mechanism where first transfer occur on to E3 itself and later to the substrate (Spratt et al.,

2014).

CRLs [Cullin–RING E3 ubiquitin ligases] are a superfamily of RING E3s responsible for

as much as 20% of ubiquitin-dependent protein modification in cells (Bennett et al., 2010;

Petroski and Deshaies, 2005). Cullin based E3 ligases assemble into multimeric complexes to

enhance substrate diversity during ubiquitination. CRLs consist of two distinct modules: 1) a

substrate-targeting unit composed of a substrate-recognition protein and an adaptor protein that

links the module to the cullin, and 2) the RING component that is active in recruiting an

ubiquitin charged E2 enzyme(Zimmerman et al., 2010). CRLs ubiquitinate several cellular

substrates and thus control broad range of biological processes, including cell growth,

development, signal transduction, transcriptional control and tumor suppression (Harper and

Tan, 2012; Petroski and Deshaies, 2005). However, no CRL E3 ligase is implicated in sister

chromatid cohesion so far. In this study, we identified a new CRL type E3 ligase (CRL7SMU1

complex with SMU1-DDB1-CUL7 and RNF40 as core components) that has an essential role in

maintenance of chromatid cohesion. We demonstrated that CRL7SMU1 E3 ligase supports sister

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chromatid cohesion through H2B ubiquitination at SMC1a locus and thereby controlling its

expression.

Results

Identification of CRL7SMU1 E3 ligase complex

Previous studies have demonstrated that DCAF1/VprBP, a protein with WD repeat region and

LisH domain acts as a substrate recognition component of HECT-type as well as RING-based E3

ubiquitin ligase complexes (Maddika and Chen, 2009; Nakagawa et al., 2013). To identify

proteins with a similar combination of LisH and WD repeat organization that may assemble E3

ligase complexes, we performed a global search for LisH domain (ID: PS50896) using UniProt

database. We retrieved 28 human proteins that contain LisH domain of which nine of them

exhibit a combination of LisH domain with WD repeats in their architecture (Figure 1A). To test

the possibility if these proteins assemble E3 ligase complexes, we isolated protein complexes

associated with SMU1, one of the listed proteins with such a domain organization. SMU1

(suppressor of mec-8 and unc-52) is a spliceosome accessory protein known for its role in DNA

replication, mitotic spindle assembly and maintenance of genomic stability (Ren et al., 2013;

Sugaya et al., 2006; Sugaya et al., 2005). Tandem affinity purification of SBP-Flag-S peptide

tagged (SFB)- SMU1 followed by mass spectrometric analysis revealed several known and

unknown interacting partners (Figure 1B). Interestingly, we found DDB1, CUL7 and RNF40 in

the list of SMU1 interacting proteins. CUL7 is a member of the Cullin family of proteins that

function as scaffold for E3 ubiquitin ligases. Like other cullins, CUL7 also assembles CRL by

associating with Skp1, Fbx29, and ROC1 (Dias et al., 2002). On the other hand DDB1 (Damaged

DNA-binding protein 1) along with DDB2 (damaged DNA binding protein 2) is known to

function in nucleotide excision repair. At the molecular level, DDB1 functions as an adaptor

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protein for Cul4 ubiquitin E3 ligase complex and DYRK2-EDVP complex to regulate ubiquitin-

dependent degradation of various substrates like Cdt1, c-Jun, p21 and katanin p60 (Iovine et al.,

2011). RNF40 is a RING type of E3 ligase which heterodimerize with RNF20 to

monoubiquitinate histone H2B (Kim et al., 2009). As it is well known that CUL7 acts as a

scaffolding protein and DDB1 as adaptor for various E3 ligase complexes and further RNF40

being an E3 ligase, we thus propose a new CRL7 E3 ligase complex with SMU1 as substrate

recognition component.

By performing immunoprecipation using SMU1 antibody, we validated the endogenous

association of RNF40, DDB1 and CUL7 with SMU1 (Figure 1C). Importantly, DYRK2, a

scaffolding subunit of EDVP E3 ligase complex, do not interact with SMU1 suggesting that

CRL7SMU1 is a distinct complex. Further, exogenously expressed SMU1 efficiently interacted

with CUL7, RNF40 and DDB1 but not with DYRK2 (Figure S1A). Likewise, we found no

interaction of SMU1 with VPRBP (Figure 1D), which was known functional subunit of DYRK2-

EDVP complex as well as CUL4-DDB1 complex, again supporting our conclusion that

CRL7SMU1 is a discrete E3 ligase complex in cells. In fact, SMU1 is not part of any known

Cullin-RING based E3 ligase complexes since SMU1 neither interacts with SKP1 (Figure 1E)

nor ROC1 (Figure 1F), while ROC1 and SKP1 interacted positively. While DDB1 can associate

with CUL4A as well as CUL7 along with SMU1 and RNF40 (Figure S1B), we found no

interaction of CUL7 and RNF40 with CUL4A (Figure S1C & S1D), again supporting CRL7SMU1

as an independent complex. Previously, RNF40 was known to act as functional E3 ligase in

RNF40/RNF20 heterodimeric form. Surprisingly, however, we found that SMU1 does not

interact with RNF20 (Figure 1G), implying that SMU1-RNF40 E3 ligase complex is formed

independent of RNF20.

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Furthermore, to investigate whether SMU1 forms a stable E3 ligase complex in vivo, we

analyzed these proteins in 293T cell extracts by using size-exclusion chromatography. SMU1,

DDB1, CUL7 and RNF40 were co-eluted in similar cell lysate fractions corresponding to a

molecular mass of approximately 440 kDa (Figure 1H), suggesting that CRL7SMU1 complex

proteins physically interact with each other and assemble a large complex in cells. WD repeats

are well established to assist in the assembly of E3 ligase complexes such as DDB1-CRLs. To

delineate the regions of SMU1 required for assembly of the E3 ligase complex, we made deletion

constructs of SMU1 that either lack WD repeat region or the N-terminal LisH domain-containing

region (Figure 1I). Interestingly, we found that that while WD repeat region is dispensable for

association with components of E3 ligase, LisH-domain region is required for assembly of E3

ligase complex (Figure 1J). Collectively, we demonstrate that SMU1 via its LisH domain region

assembles a novel E3 ligase complex by associating with DDB1, CUL7 and RNF40 in cells.

CRL7SMU1 complex regulates mono-ubiquitination of H2B at K120 site.

In search of substrate(s) of newly identified E3 ligase complex, we next examined the mass spec

data derived from SMU1 purification. We found Histone 2B (H2B) as one of the interacting

partners of SMU1. H2B along with H2A, H3 and H4 is a part of core nucleosome and is a well-

known substrate of RNF20/40 complex, which monoubiquitinates H2B at Lysine 120 site in

humans (K123 in yeast) (Kim et al., 2009; Wood et al., 2003). However, no substrate recognition

subunit for RNF40/RNF20 E3 ligase is reported yet. As we identified RNF40 as a core

component in the CRL7SMU1 complex, we proposed H2B as a probable substrate of this complex.

By performing the co-immunoprecipitation experiments using streptavidin pull down followed

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by immunoblotting with H2B antibody, we confirmed the specific interaction of H2B with

CUL7, DDB1, RNF40 and SMU1 (Figure 2A). To establish the architecture of the complex and

further test if SMU1 acts as substrate recognition component in the E3 ligase complex, we

performed knockdown experiments. Interaction between RNF40 and H2B is severely hampered

upon depletion of SMU1 where as DDB1 interaction with RNF40 is intact in these cells (Figure

2B). On the other hand, interaction between SMU1-H2B and SMU1-DDB1 was unaffected upon

knockdown of RNF40 (Figure 2C). However, depletion of DDB1 resulted in reduced binding of

SMU1 with CUL7 (Figure 2D). Also, depletion of CUL7 led to loss of RNF40-DDB1 interaction

with no effect on SMU1-DDB1 interaction (Figure 2E). This data suggest that H2B binds to

SMU1, which associates to CUL7 and further RNF40 through DDB1 (Figure 2F). To

substantiate the role of SMU1 as substrate recognition protein of the complex, we next checked

the direct interaction of recombinant bacterially expressed glutathione S-transferase (GST)-

tagged SMU1 (GST-SMU1) with Maltose binding protein (MBP) - tagged H2B (MBP-H2B).

Indeed, we found that SMU1 interacts with H2B directly under in vitro conditions (Figure 2G).

Together, these experiments suggest that SMU1 functions as a substrate recognition component

that links H2B with DDB1-CUL7-RNF40. Next, to examine the role of CRL7SMU1 complex in

histone H2B ubiquitination, we used specific si/shRNA to deplete complex proteins in HeLa

cells. It has been shown that knockdown of RNF20/40 abolishes H2B ubiquitination (Kim et al.,

2009). In agreement with previous studies we found knock down of RNF40 led to

downregulation of H2B monoubiquitination at K120 (Figure 2H). Interestingly, similar to

knockdown of RNF40, depletion of SMU1 (Figure 2I), DDB1 (Figure 2J) and CUL7 (Figure 2K)

also led to significant downregulation of H2B ubiquitination suggesting that these proteins

function together to ubiquitinate H2B K120 in vivo. Further, RNF40 mediated H2B

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ubiquitination is known to stimulate H3K4 trimethylation via COMPASS complex (Kim et al.,

2009). Similar to RNF40 (Figure 2H), depletion of SMU1, DDB1 and CUL7 resulted in

reduction of H3K4 trimethylation (Figure 2I-2K). DDB1 depletion as well as RNF40 depletion

seems to affect H3K4 trimethylation levels more than H2B ubiquitination. Given that DDB1 is

part of multiple independent E3 ligase complexes, it might be possible that DDB1 regulates

H3K4me3 through additional mechanisms independent of H2B ubiquitination. Infact, it has been

reported that CUL4-DDB1 ubiquitin E3 ligase interacts with multiple WD repeat containing

proteins and regulate H3 methylations including H3 K4Me1, K4Me3, K9Me3 and K27Me3

(Higa et al., 2006). Likewise, RNF40 could also be forming other protein complexes to directly

regulate H3K4me3 independent of H2B ubiquitination. Nonetheless, our data suggest that

SMU1, DDB1, CUL7 and RNF40 are integral components of functional E3 ligase complex that

regulate histone modifications.

Intact CRL7SMU1 complex is required for mitotic progression

Next, we sought to understand the functional role of CRL7SMU1 complex in cells. We observed

that cells depleted with SMU1 (Figure S2A) proliferated at a significantly slower rate than

control cells (Figure S2B). Furthermore, we found that depletion of SMU1 led to the

accumulation of 4N cells as well as polyploid (>4N) cells (Figure 3A). This increase in the 4N

population is due to arrest in the mitotic phase, as time-lapse imaging analysis confirmed that

cells lacking SMU1 spent several hours in mitosis while control siRNA cells took 60 minutes on

an average to complete mitosis (Figure 3B & 3C). Likewise, we found that knock down of all the

individual components of CRL7SMU1 complex led to accumulation of phospho-H3 positive cells

(Figure 3D & 3E), and significant accumulation of morphologically round cells in culture

(Figure S2C), typical of possibly mitotically arrested cells. Since we found that CRL7SMU1

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complex proteins are required for normal progression of mitosis, we next tested if loss of SMU1

leads to any mitotic defects. We observed various chromosomal and spindle defects upon SMU1

knockdown (Figure 4A). Loss of SMU1 resulted in significant increase in cells displaying

lagging chromosomes, anaphase/nuclear bridges and multipolar spindles (Figure 4B). Similarly,

we also found that depletion of CUL7, DDB1 or RNF40 individually in cells led to severe

mitotic defects (Figure 4C & 4D). Since loss of CRL7SMU1 complex proteins resulted in

numerous mitotic defects, we next tested if loss of H2B monoubiquitination at K120

phenocopies loss of E3 ligase complex from cells. Expression of H2B K120R, but not wild type

H2B, resulted in accumulation of cells with multiple mitotic defects (Figure 4E, 4F & 4G), thus

suggesting that H2B ubiquitination at K120 position is critical for normal mitotic progression

and further prevention of genomic instability.

CRL7SMU1 complex drives SMC1a gene expression

It is well known that H2B ubiquitination is enriched at sites of active gene transcription and

modulates transcription elongation. Since we found that CRL7SMU1 complex promotes H2B

ubiquitination and H3K4 trimethylation (sites of active gene transcription), we hypothesized that

this complex may control transcription of specific set of genes required for mitosis. Previously, a

microarray analysis upon SMU1 depletion had revealed alterations in the expression of various

mitotic genes (Papasaikas et al., 2015). We tested if SMU1 is enriched on these set of mitotic

genes by using chromatin immunoprecipitation assay (ChIP). SMU1 was significantly enriched

on different mitotic genes such as SMC1a, ANAPC12, ANAPC5, Aurora A and CDCA2 (Figure

5A). Consequently, we tested if depletion of CRL7SMU1 components affects H2B ubiquitination

at these loci. Although, depletion (Figure S3) of individual components of the E3 ligase complex

reduced H2Bub levels at distinct loci, we found SMC1a to be common locus where H2B

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ubiquitination is hampered with loss of CRL7SMU1 complex (Figure 5B). As H2B ubiquitination

is directly associated with gene transcription, we further tested whether this E3 ligase complex

regulate transcript levels of mitotic genes. Interestingly, again we observed that expression levels

of only SMC1a were reduced upon depletion of CUL7, DDB1, RNF40 and SMU1 (Figure 5C).

Consequently, we also found significant reduction in SMC1a protein levels upon depletion of

individual components of CRL7SMU1 complex (Figure 5D). Notably, expression of H2B K120R

mutant also reduced the gene expression of SMC1a (Figure 5E) consistent with our hypothesis

that H2B ubiquitination is required for expression of this critical mitotic gene.

CRL7SMU1 complex is essential for sister chromatid cohesion

SMC1a (Structural maintenance of chromosomes protein 1A) is a central component of cohesin

complex, which is required for the cohesion of sister chromatids and is essential for accurate

chromosome segregation at the onset of mitosis (Brooker and Berkowitz, 2014; Sumara et al.,

2000). To determine if CRL7SMU1 complex is required for sister chromatid cohesion we prepared

metaphase spreads on HeLa cells depleted with individual components of E3 ligase complex.

Control cells display normal “X-shaped” mitotic chromosomes with sister chromatids tightly

linked at the centromere and chromosome arms separated. However, strikingly, knockdown of

CUL7, DDB1, RNF40 or SMU1 led to parallel chromatids and single chromatids in isolation in a

large fraction of mitotic cells (Figure 6A & 6B) suggesting a premature loss of sister chromatid

cohesion in these cells. We next measured the distance between metaphase sister kinetochores in

CRL7SMU1 complex -depleted cells using CENP-A immunostaining. Sister chromatid

interkinetochore distances were significantly increased in aligned metaphases of SMU1, CUL7,

DDB1 and RNF40 depleted cells compared with control metaphases (Figure 6C & 6D), further

supporting a critical role of CRL7SMU1 complex in establishing cohesion between sister

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chromatids. Interestingly, expression of H2B K120R mutant, but not wild type H2B, also

resulted in defective chromatid cohesion (Figure 6E & 6F) and enhanced interkinetochore

distance (Figure 6G & 6H), thus suggesting that H2B ubiquitination by CRL7SMU1 complex is

critical for maintenance of sister chromatid cohesion.

Based on these observations, we hypothesized that expression of H2B-Ub fusion protein,

which mimics H2B K120 ubiquitination (Zhang et al., 2013), could rescue the cohesion defects

caused due to depletion of CRL7SMU1 E3 ligase from cells. To our surprise, over-expression of

H2B-Ub fusion protein could not rescue cells from cohesion defects (Figure S4A & S4B) caused

due to E3 ligase loss. This data prompted us to reason that dynamic H2B ubiquitination and

deubiquitination might be necessary for maintenance and later dissolution of sister chromatid

cohesion during appropriate phases of mitosis. Therefore, constitutive loss or presence of H2B

monoubiquitination might be deleterious for normal chromatid cohesion. This is in agreement

with previous data that loss of USP44, a deubiquitinase for H2B, (mimicking constitutive H2B

ubiquitination) also led to various mitotic abnormalities (Zhang et al., 2012). In support of the

dynamic nature of H2B ubiquitination during chromatid cohesion, we found that transient

expression of H2B-Ub fusion protein alone is sufficient to induce sister chromatid cohesion

defects (Figure S4C & S4D), increased interkinetochore distance (Figure S4E) as well as mitotic

defects in cells (Figure S4F & S4G). Nonetheless, we next tested if restoration of SMC1a in cells

through plasmid-based expression would rescue the chromatid cohesion defects caused due to E3

ligase loss. Indeed, exogenous expression of SMC1a (Figure 6A) significantly prevented

defective chromatid cohesion in cells resulted due to depletion of E3 ligase components (Figure

6B). Also, SMC1a expression in CUL7, DDB1, RNF40 or SMU1 depleted cells rescued the

interkinetochore distance that was enhanced due to loss of E3 ligase complex (Figure 6C). In

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conclusion, we identified that SMU1, DDB1, CUL7 and RNF40 assemble a CRL type E3 ligase

complex and promotes monoubiquitination of H2B to drive the expression of SMC1a, which is

essential for maintenance of sister chromatid cohesion during mitosis.

Discussion

H2B ubiquitination is one of the critical histone modifications associated with gene expression

(Cole et al., 2015; Xie et al., 2017). In humans, RNF20/40 is considered as a major E3 ligase for

H2B monoubiquitination (Kim et al., 2009). Although, H2Bub1 is known to be globally

associated with transcribed genes and its levels correlate positively with the gene expression

(Minsky et al., 2008), knockdown of RNF20 affects transcription of only a subset of genes

(Shema et al., 2008). This possibly suggests the presence of multiple E3 ligase complexes,

independent of RNF20, to regulate H2B monoubiquitination in the cell. In this study, we

provided multiple lines of evidence supporting an essential role of CRL7SMU1 E3 ligase complex

in mediating H2B monoubiquitination. Though H2B ubiquitination has been implicated in wide

range of cellular processes such as transcription initiation and elongation, DNA damage

response, replication, stem cell differentiation, RNA processing and export, its role in chromatid

cohesion and mitosis is unexplored. We clearly have shown that our newly identified E3 ligase

complex promotes H2B ubiquitination at SMC1a to maintain sister chromatid cohesion during

mitosis. Moreover, ectopic expression of the H2Bub1 mutant (H2BK120R) caused an alteration

of the transcription of SMC1a and a significant increase in mitotic defects including cohesion

loss, which is perhaps direct evidence that H2B ubiquitination is critical for mitotic progression.

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E3 ligases play an essential role in the final step of ubiquitination process to catalyze the

transfer of ubiquitin to appropriate substrates. E3 ligases, in particular cullin based class, often

assemble into multimeric complexes to enhance substrate diversity and specificity during the

process of ubiquitination (Lydeard et al., 2013; Zimmerman et al., 2010). Several interaction

domains such as F-Box, SOCS-box, β-domains, WD40 repeats, ankyrin, Kelch, WW and RLD

motifs were known to assist in the assembly of multi-component E3 ligases. In this study, by

using SMU1 as an example, we demonstrated that LisH domain proteins participate in the

organization of a functional E3 ligase complex. LisH (LIS1 homology) is a highly conserved

domain found in eukaryotic proteins that is proposed to mediate protein-protein interactions.

Although proteins harboring LisH domain are known to participate in processes such as

microtubule dynamics, chromosome segregation and are implicated in pathogenesis (Emes and

Ponting, 2001), studies on molecular function of LisH domain are limited. There are certain

examples to suggest that LisH domain participates in organization of multimeric complexes to

regulate protein stability. For instance, in S. cerevisiae, LisH domain proteins such as GID1,

GID7 and GID8 along with GID2, GID4, GID5, and GID9 assemble a multimeric glucose

induced degradation deficient (GID) complex. The GID complex mediates polyubiquitination of

fructose-1,6-bisphosphatase (FBPase) via E3 ubiquitin ligase activity of GID2, that contains a

RING domain (Menssen et al., 2012). However, the role of LisH domain containing proteins in

the assembly of an E3 ligase in humans is unknown. Although earlier, we and others have shown

that DCAF1/VprBP that contains LisH domain acts as a substrate recognition protein in RING

E3s as well as HECT type of E3 ligase complexes (Maddika and Chen, 2009; Nakagawa et al.,

2013), whether LisH domain is required for the assembly of E3 ligases is unexplored. In another

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example, WDR26 in complex with Axin1 controls β-catenin levels and negatively regulate Wnt

signaling (Goto et al., 2016). Although, LisH domain of WDR26 is critical for β-catenin

degradation, whether it participates in the organization of E3 ligase in this case is not studied.

Thus our current study provides clues in corroborating the critical role of LisH domain proteins

in organizing E3 ligase complexes.

SMC1 is an essential component of Cohesin, a multi-protein complex (made up of four

subunits Smc1, Smc3, an α-kleisin subunit – Mcd1/Scc1 (mitosis)/Rec8 (meiosis), and

Irr1/Scc3), which is conserved from yeast to humans. Cohesin has a well-documented role in

chromatid cohesion where it provides stable but reversible connections between sister

chromatids during both mitosis and meiosis (Nasmyth and Haering, 2009). However, recent

findings indicate that in higher eukaryotes sister chromatid cohesion may not be the only major

function of cohesin, but may also regulate other processes including transcriptional regulation,

DNA repair, chromosome condensation and morphogenesis (Dorsett, 2011). Indeed, elegant

studies in yeast indicate that severe reduction in the level of chromatin bound cohesin does not

entirely affect its function of holding the sister chromatids together, but drastically affects the

non-canonical functions (Dorsett, 2011; Heidinger-Pauli et al., 2010; Mehta et al., 2013). Thus,

although our studies have clearly demonstrated defective sister chromatid cohesion due to

CRL7SMU1 complex loss is dependent on SMC1, it still remains to be determined whether these

effects are mediated directly through SMC1 localization at centromeres or indirectly through

regulation of unknown gene expression.

In addition to H2B ubiquitination, CRL7SMU1 complex may also directly regulate

ubiquitination of mitotic proteins. In support of this speculation, we found proteins such as

Adenomatous polyposis coli (APC) in the list of SMU1 associated proteins. Further studies are

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required to test if APC acts as a substrate of this E3 ligase complex. Lastly, our work revealed

that a LisH domain protein SMU1 associate with RNF40 to form a RING type CRL E3 ligase.

Interestingly, the list of SMU1 associated proteins also contains HECT type E3 ligases, EDD and

HUWE1. In fact, proteins such as VPRBP and DDB1 were shown previously to associate with

RING as well as HECT E3s (Maddika and Chen, 2009; Nakagawa et al., 2013). Thus, in future

studies it would be interesting to test if SMU1 may participate in the assembly of both RING and

HECT-type of E3 ligases to regulate different cellular processes by controlling distinct set of

substrates.

Materials and methods

Plasmids

Full length SMU1, DDB1, CUL7, RNF40, RNF20, SMC1a, Roc1, VprBP, Rab7, H2B wild type

and K120R mutant were cloned into SFB triple tagged (S protein/Flag/Streptavidin binding

protein) destination vectors using Gateway cloning system (Invitrogen). H2B and SMU1 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 HA destination vector using

gateway cloning method. The point mutations for H2B was generated by PCR- based site –

directed mutagenesis and cloned into SFB and GST-tagged destination vectors. RNF40 plasmid

was a kind gift from Dr. Steven Johnsen (University Medical Center Göttingen, Göttingen).

Myc-tagged RNF20 was kindly provided by Dr. Jae Bum Kim (Seoul National University, South

Korea). Flag H2B-Ub fusion construct was a kind gift from Dr. Hengbin Wang (UBA,

Alabama). cMyc-SMC1a plasmid was a gift from Michael Kastan (Addgene plasmid #

32363)(Kim et al., 2002), myc3-CUL7 was a gift from Yue Xiong (Addgene plasmid #

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20695)(Andrews et al., 2006) and GFP-H2B was a gift from Geoff Wahl (Addgene plasmid #

11680)(Kanda et al., 1998).

Antibodies

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), Ubiquityl-Histone H2B (Lys120) (CST # 5546) (1:1000),

Cyclin A (BD # 611269) (1:1000), CDT1 (Bethyl # A300-786A) (1:1000), pH3 (S10) (Cell

signaling technologies #9701L) (WB- 1:1000, IF- 1:200), Cul4a (Bethyl # A300-739A) (1:5000),

RNF20 (abcam # ab32629) (1:1000), Myc (Santa Cruz # 9E10) (1:1000), Flag (Sigma, F3165)

(1:10000), HA (Bethyl, A190-108A) (1:1000), Actin (Sigma, A5441) (1:10000), and Alpha

tubulin (Sigma, T6074) (WB, 1:5000, IF- 1:200) antibodies were used in this study. HRP/ FITC

conjugated anti-Mouse and anti-Rabbit secondary antibodies were obtained from Jackson

Immunologicals.

Cell lines and transfection

HEK 293T, HeLa and BOSC23 cell lines were used in this work. All cell lines were purchased

from American Type Culture Collection, which were tested and authenticated by the cell bank

using their standard STR (short tandem repeats) based techniques. Cells were also continuously

monitored by microscopy to maintain their original morphology and also tested for mycoplasma

contamination by using DAPI staining. HEK 293T or HeLa were transfected with various

plasmids using PEI (Polysciences) according to the manufacturer’s protocol. Briefly, the plasmid

was mixed with PEI (1 mg/ml) in 1:3 ratio in serum free RPMI medium. Then, DNA-PEI

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mixture was incubated at room temperature (RT) for 20 min and the complexes were added to

cells to allow the transfection of plasmid.

Tandem affinity purification

SMU1-associated proteins were isolated by using tandem affinity purification as described

before (Maddika and Chen, 2009). Briefly, 293T cells expressing SFB-triple tagged SMU1 were

lysed with NETN lysis buffer (20mM Tris-HCl at pH 8.0, 100mM NaCl, 1mM 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 on to streptavidin–sepharose

beads (Amersham Biosciences) and incubated for 1.5 h at 4°C. Then beads were washed thrice

with lysis buffer and the associated 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 unbound proteins by washing, the proteins

associated with S-protein–agarose beads were eluted by boiling in SDS-loading buffer for 10

min at 95 °C. Eluted protein lysate was loaded on SDS-PAGE. The associated proteins were

identified by in gel trypsin digestion followed by LC-MS/MS analysis at Taplin biological mass

spectrometry facility (Harvard University).

Immunoprecipitation and Western blotting

For immunoprecipitation assays, cells were lysed with NETN buffer. The whole-cell lysates

obtained by centrifugation were incubated with 2 µg of specified antibody bound to protein G–

sepharose beads or with streptavidin–sepharose beads (GE) for 1.5 h at 4 °C. The

immunocomplexes were then washed with NETN buffer three times and applied to SDS–PAGE.

Western blotting was carried out by following standard protocols. Proteins were separated by

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denaturing SDS–PAGE and then transferred on to polyvinylidene difluoride (PVDF) membrane.

The membranes were blocked in 5% non-fat dried milk in Tris-buffered saline (TBS) and then

incubated overnight with the primary antibodies at 4 °C. Then, the blots were incubated with the

corresponding secondary antibodies conjugated with HRP at room temperature for 1 h.

Visualization was carried out by enhanced chemiluminescence detection (Thermo Fisher

Scientific).

Histone extraction

Histones were isolated using acid extraction protocol. Briefly, cells were lysed under hypotonic

conditions and the intact nuclei were collected. Isolated nuclei were acid extracted using 0.4N

H2SO4. Histones were precipitated using 33% TCA and residual TCA was removed by ice-cold

acetone. The pellets were air-dried and milli Q water was added to dissolve histones. Histone

fractions were subjected to SDS-gel followed by western blotting with antibodies of interest.

GST pulldown assays

Bacterially expressed GST and GST-SMU1 bound to glutathione-Sepharose beads were

incubated with eluted MBP-H2B for 1 hr, at 4°C. Beads were washed and proteins were eluted

by boiling in 2X SDS Laemmli buffer and then separated by SDS-PAGE; the interactions were

analysed by western blotting.

RNA interference and lentiviral infection

Control siRNA and pre validated SMU1 siRNA were purchased from Dharmacon (Catalogue no:

J-021129-10). TRC Lentiviral SMU1 shRNA (RHS4533-EG55234), DDB1 shRNA (RHS4533-

EG1642), CUL7 shRNA (RHS4533-EG9820) and RNF40 shRNA (RHS4533-EG9810) were

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purchased from Dharmacon. Transfection was performed twice, 24 h apart, with 200 nM siRNA

using Oligofectamine reagent in accordance with the manufacturer’s protocol (Invitrogen).

shRNAs were transfected transiently using PEI (Invitrogen) in BOSC23 packaging cells along

with packaging vectors. Forty-eight hours post transfection, the viral medium was collected and

added to the target cells along with polybrene (8 mg/ml). 48 or 72 hours post infection, cells

were collected and processed for various assays and immunoblotting was performed with the

specific antibodies to check the efficiency of knockdown.

Immunofluorescence staining

Cells grown on coverslips were fixed with 3% paraformaldehyde solution in PBS containing

50mM sucrose at room temperature (RT) for 15 min. After permeabilization with 0.5% Triton X-

100 buffer containing 20 mM HEPES at pH 7.4, 50 mM NaCl, 3 mM MgCl2 and 300 mM

sucrose at RT for 5 min, cells were incubated with a 1% BSA for blocking at RT for 30 min.

After washing with PBS, cells were incubated with primary antibodies for 2–3 hrs at RT

followed by three times washes with 1X PBS 5min each. Then, cells were incubated with FITC

or Rhodamine-conjugated secondary antibodies at RT for 60 min followed by three times wash

with 1X PBS 5min each. Nuclei were counterstained with DAPI. After a final wash with PBS,

coverslips were mounted with glycerine containing paraphenylenediamine. Images were taken

using Zeiss confocal microscope (LSM Meta 510 or 700).

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Cell cycle analysis

HeLa cells transfected with the desired expression vectors and si/sh RNA were harvested,

washed with phosphate-buffered saline and fixed with ice-cold 70% ethanol for at least 1 h. Cells

were washed thrice in PBS and treated for 30 min at 37 °C with RNase A (5 μg/ml) and

propidium iodide (25 μg/ml), then analysed using BD accuri C6 flow cytometer.

Cell proliferation assay

HeLa cells were transfected with Control and SMU1 siRNA. 48 hr post transfection 1x10^5 cells

were seeded in five different plates and cell counting was done using trypan blue dye exclusion

method.

Gel filtration

Sephacryl 300 column from GE healthcare was used to separate the protein complexes in the

range of 10 kDa –1500kDa. Column was calibrated using four gel filtration markers

Thioglobulin (669 kDa), Ferritin (440 kDa), Conalbumin (75 kDa) and Ovalbumin (44 kDa).

After calibration, lysates (0.8-1 ml, Concentration 1-2 mg/ml) were injected and allowed to pass

through column. 1 ml fractions were collected using a Bio-Rad 2110 fraction collector within the

molecular weight range of interest and all 1 ml fractions were concentrated separately using

protein concentration columns to final volume of 80 µl. All the fractions were subjected to SDS-

gel followed by western blotting with antibodies of interest.

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Quantitative RT-PCR

Total RNA was isolated using Trizol reagent (Invitrogen) as per manufacturer's instructions.

Two microgram of total RNA was reverse transcribed in the presence of anchored oligo dT using

Superscript-III (Invitrogen) as per manufacturer's protocol. Quantitative PCR (qPCR) was then

initiated using the SYBR Pre mix Ex Taq (Tli RNaseH plus) kit (Clontech Laboratories) in 7500

real time PCR systems (Applied Biosystems) as per manufacturer's protocol. The threshold cycle

(Ct) values for particular genes were normalized to GAPDH for each sample. Sequences for

Primers used for qRT-PCR analysis were included in supplementary information.

Chromatin Immunoprecipitation (ChIP) assay

4ug Mouse IgG (bethyl), 4ug SMU1 (Abgent # AT3965a) or 4 µl of H2Bub1 (CST # 5546)

antibodies were used for ChIP assay. Briefly, HeLa cells were crosslinked using 1%

formaldehyde solution. Cross-linking was allowed to proceed for 10 min at room temperature

and was then stopped by the addition of glycine to a final concentration of 0.125 M. Cells were

washed twice with ice-cold 1X PBS and cells were lysed using cell lysis buffer (5mM PIPES

pH-8, 85mM KCL, 0.5% NP-40 and protease inhibitors) followed by nuclei lysis buffer (50mM

Tris pH-8.1, 10mM EDTA, 1% SDS and protease inhibitors). Isolated chromatin was sonicated

using Diagenode Bioruptor at medium power and checked for fragment size. After

microcentrifugation, the supernatant was diluted with 1X IP dilution buffer and preclearing was

performed using Protein G beads. Then equal amount of antibody was added and incubated on a

rotating platform for 12 to 16 h at 4°C. Next day, protein G beads were added to chromatin.

Beads were washed two times with IP Dialysis buffer (2mM EDTA and 50mM Tris pH-8) and

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three times with IP wash buffer (100mM Tris pH-8, 250mM LiCl, 1% NP-40 and 1% Na

deoxycholate) for five minutes at RT. Then beads were quickly washed with 1X TE and bound

DNA fragments were eluted using elution buffer (50mM NaHCO3 and 1% SDS) at 65°C. Input

and elution products were kept for decrosslinking at 65°C O/N. Then, samples were treated with

RNase A and proteinase K for 3 hours at 37°C and DNA was extracted using PCI treatment.

Quantitative PCR (qPCR) was then initiated using the SYBR Pre mix Ex Taq (Tli RNaseH plus)

kit (Clontech Laboratories) in 7500 real time PCR systems (Applied Biosystems) as per

manufacturer's protocol. Sequences for primers used for ChIP analysis were included in

supplementary information.

Metaphase spreads

Metaphase chromosome spreads were performed as described before(Maddika et al., 2009).

Briefly, cells treated with colcemid for 4 hours were collected, washed with phosphate buffered

saline (PBS) and treated with 75 mM KCl at room temperature for 30 min. The treated cells were

then fixed in fresh methanol:acetic acid (3:1) solution and dropped onto glass slides. Cells were

allowed to air dry and were stained with giemsa (5%) and visualized under the microscope.

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Author contributions

SM conceptualized and managed the project. SM and VJS designed the experiments, analysed

the data and wrote the manuscript. VJS performed all the experiments.

Acknowledgements

This work was supported in part by a grant from Council of Scientific & Industrial Research,

India (to S.M; 37(6371)/17) and CDFD core funds. S.M is a senior fellow of Wellcome

Trust/DBT India Alliance and a recipient of Department of Biotechnology’s Sr. IYBA award.

V.J.S acknowledge the fellowship support from Department of Biotechnology (DBT), India. We

thank CDFD core facility as well as Dr. Rohit Joshi’s lab for their assistance in confocal

imaging. We thank Nanci Rani for technical assistance, Shaffiqu T S for providing assistance in

gel filtration experiment, Sawant Suresh, Zaffer Ullah Zargar and Amit M Karole for their

critical inputs during gene expression and ChIP experiments, Dr, Ashwin Dalal and Dr. Usha

Dutta for their inputs during mitotic spread experiments. We also thank all members of LCDCS

for their suggestions and critical inputs at various stages of the project.

Competing financial interests

The authors declare no competing financial interests.

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Figures

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Figure 1: SMU1 assembles CRL type E3 ligase complex by interacting with DDB1, CUL7

and RNF40. (A) Graphical representation of proteins containing LisH domain and WD repeats.

(B) Partial list of SMU1-associated proteins identified by biochemical purification followed by

mass spectrometric analysis were listed together with the number of peptides for each

protein. (C) Immunoprecipitation (IP) with control IgG or anti-SMU1 antibody was performed

with extracts prepared from 293T cells. The presence of RNF40, DDB1, CUL7 and DYRK2 in

these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (D)

SFB-VPRBP along with either Myc-SMU1 or Myc-DYRK2 was expressed in cells and the

interaction of these proteins was detected by immunoblotting with the indicated antibodies after

pulling down the complexes with streptavidin sepharose. (E) HA tagged-SKP1 along with either

SFB-SMU1 or SFB-ROC1 and (F) SFB-ROC1 along with Myc-SMU1 or HA-SKP1 were

expressed in cells and the their interaction was detected as described in figure 1D. (G) HeLa

cells expressing Myc-RNF20 were lysed and immunoprecipitation was carried out using either

IgG or Myc antibody. The presence of SMU1 and RNF40 was detected in these

immunoprecipitates by immunoblotting using specific antibodies. (H) 293T cell extracts was

analysed by size-exclusion chromatography using Sephacryl 300 column. Proteins eluted from

the different fractions were immunoblotted with the indicated antibodies. (I) Graphical

representation of domain architecture of SMU1 and its deletion mutants. (J) SFB tagged SMU1

FL and deletion mutants were transfected in HeLa cells. 24hr Post transfection, cells were lysed

and pulldown was carried out using SBP beads. The presence of DDB1, CUL7 and RNF40 in

these precipitates were evaluated by immunoblotting with their respective antibodies.

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Figure 2: CRL7SMU1 complex regulates the mono-ubiquitination of H2B at K120 site. (A)

SFB tagged CUL7, DDB1, RNF40, SMU1, Rab7 or empty vector (EV) were transfected and the

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interaction of H2B was detected by immunoblotting with specific antibody after streptavidin

sepharose pull down. (B) Hela cells were transduced with either control or SMU1 specific

shRNA followed by over expression of SFB-RNF40. 72 hours post transduction, pulldown was

performed with streptavidin sepharose beads and the interaction of DDB1 and H2B with RNF40

was evaluated by immunoblotting with their respective antibodies. (C) SFB tagged SMU1 was

overexpressed in cells transduced with either control or RNF40 shRNA. The interaction of

SMU1 with H2B and DDB1 was detected through immunoblotting using specific antibodies

after immunoprecipitation. (D) Cells were transduced with either control or DDB1 shRNA and

(E) Control or CUL7 shRNA containing viral particles. Pulldown followed by detection of

different indicated proteins in precipitates were done as described in figure 2B. (F) Model shows

the assembly of CRL7SMU1 complex in association with its substrate H2B. (G) GST pulldown

assay was performed with immobilized control GST or GST–SMU1 fusion proteins on

glutathione beads, followed by incubation with bacterially purified MBP-H2B. The interaction of

SMU1 with H2B was assessed by immunoblotting with MBP antibody. Expression of the GST,

recombinant GST-SMU1 and MBP-H2B was shown by coomassie staining. (H) HeLa cells were

transduced using either control or RNF40 shRNA. Post 72 hours, cells were collected and lysed

to isolate soluble and histone fractions. Lysates were subjected to SDS-PAGE followed by

immunoblotting using the indicated antibodies. (I) Cells were transfected/transduced with either

control or SMU1 siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid extracted

histone fractions were subjected to SDS-PAGE followed by immunoblotting using indicated

antibodies. The data presented here is representation of three independent experiments.

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Figure 3: Intact CRL7SMU1 complex is required for mitotic progression. (A) HeLa cells

transfected with either control siRNA or SMU1 specific siRNAs were stained with Propidium

Iodide and cell cycle analysis was performed by flow cytometry. (B) HeLa cells were transfected

with indicated siRNAs. The transition of cells through mitosis was analyzed by live cell time-

lapse microscopy after synchronizing cells using double thymidine block. (C) Time taken by

each cell from mitotic entry to division was calculated and the data was 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 hrs post transduction, cells

were probed with pH3 antibody to measure mitotic index. Nuclei were counterstained with

DAPI. (E) Quantification of mitotic index results is shown.

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Figure 4: Depletion of CRL7SMU1 complex proteins induce mitotic defects. (A) HeLa cells

were transfected with either control or SMU1 siRNAs. Cells were stained with alpha- tubulin

antibody to check the spindle defects (multipolar spindles) and nuclei were counterstained with

DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges) (scale

bar, 10μm). (B) Quantification of results from figure 4A is shown (n=50 cells each). (C) HeLa

cells were transduced with control shRNA, CUL7 shRNA, DDB1 shRNA or RNF40 shRNA.

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Cells were stained with alpha- tubulin antibody to check the spindle defects (multipolar spindles)

and nuclei were counterstained with DAPI to check for chromosomal defects (lagging

chromosomes and anaphase bridges) (Scale bar, 5μm). (D) Quantification of results from figure

4C is shown, (n=40 cells each). (E) Cells were transfected with H2B Wild type (WT) and K120R

mutant. (F) Various mitotic abnormalities in cells expressing H2B Wild type (WT) and K120R

mutant were checked using immunofluorescence after staining with alpha- tubulin antibody and

DAPI (Scale bar, 5μm). (G) Quantification of results from figure 4F is shown (n=50 cells each).

Statistical analysis: error bars- standard deviation, ***P<0.001 **P<0.01; *P<0.05, Student’s t-

test.

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Figure 5: CRL7SMU1 complex is necessary for driving SMC1 gene expression. (A)

Exponentially growing HeLa cells were subjected to ChIP analysis using either SMU1 or IgG

antibody. SMU1 enrichment at various loci is shown. The data shown is derived from three

independent experiments. (B) Cells expressing control shRNA, CUL7 shRNA, DDB1 shRNA,

RNF40 shRNA and SMU1 shRNA were subjected to ChIP analysis using H2Bub antibody. Fold

change of H2Bub enrichment at indicated loci with respect to control shRNA was shown. The

data shown is derived from three independent experiments. (C) Total RNA was extracted from

HeLa cells transfected with control or SMU1 siRNA or CUL7 shRNA or DDB1 shRNA and

RNF40 shRNA, and expression levels of various genes measured by qRT-PCR from three

independent experiments was shown. (D) HeLa cells transduced with control, CUL7 shRNA,

DDB1 shRNA, RNF40 shRNA or SMU1 shRNA and levels of SMC1a protein was measured by

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immunoblotting with specific antibody. (E) HeLa cells were transfected with H2B wild type and

K120R mutant. Relative expression of indicated genes measured by using qRT-PCR from three

independent experiments was plotted. Statistical analysis: error bars- standard deviation,

***P<0.001; **P<0.01; *P<0.05, Student’s t-test.

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Figure 6: CRL7SMU1 complex regulates chromatid cohesion. (A) HeLa cells were transduced

with Control, CUL7, DDB1, RNF40 and SMU1 shRNA separately and chromosome spreads

were prepared after 96 hours of transduction. Representative images of chromosome spreads are

shown. (B) Quantification of data from figure 6A is shown. For defective chromatid cohesion

analysis, cells were scored as normal when two or fewer chromosomes showed defects, or

defective when three or more chromosomes showed defects. Total 115 mitotic spreads were

analysed per condition and p value calculation was done using two-tailed Fisher’s exact test. P

value is less than 0.001 for knockdown of complex proteins compared to control. (C) HeLa cells

were transduced with Control, CUL7, DDB1, RNF40 and SMU1 shRNA separately and stained

with anti-CENP-A antibody to stain kinetochores. (D) Distance between paired kinetochores was

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measured at individual Z planes and plotted (n= 150). (E) Chromosome spreads were prepared

from HeLa cells transfected with either H2B wild type or H2B K120R mutant. Representative

images of chromosome spreads were shown. (F) Quantification of data from figure 6E was

shown (n= 100) p < 0.001. (G) Cells expressing either H2B WT or K120R mutant were stained

with anti-CENPA antibody and (H) the distance between paired kinetochores was measured.

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Figure 7: Exogenous SMC1a expression partially rescues cohesion defects caused due to

loss of CRL7SMU1 complex. (A) HeLa cells were transduced with control shRNA, CUL7

shRNA, DDB1 shRNA, RNF40 shRNA and SMU1 shRNA alone or together with SMC1a

plasmid and SMC1a levels were analysed by immunoblotting. (B) Cells expressing control

shRNA, Cul7 shRNA, DDB1 shRNA, RNF40 shRNA and SMU1 shRNA alone or together with

SMC1a were analysed for chromatid cohesion (n= 100, P value is less than 0.001 for knockdown

of complex proteins compared to control and less than 0.01 between shRNA alone and along

with SMC1a (rescue) (C) and inter kinetochore distance (n= 190). Statistical analysis: error bars-

standard deviation, ***P<0.001; **P<0.01, Student’s t-test.

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Supplementary Data

Supplementary table 1: List of primers used in this study for ChIP experiments were shown.

Gene name Primer sequence Location chr12:121756213-121756365, strand= reverse

ANAPC5 For: GGAGATGTGGTGGTGGATCT ANAPC5 Rev: ACCGGTACTCCTTGGAGAGG SMC1A For: CGGTGAACCTCTGAAATGGT chrX: 53449441-53449547,

strand= forward SMC1A Rev: TGGGGTTCCTGAAACTGATT MUS81 For: TCTCCAAACCCTCTCTGCTC chr11:65632546-65632715,

strand= forward MUS81 Rev: AAACACTTCTCGCACCGACT CDCA2 For: CCTGAAACCAAGGAGTCTGC chr8:25317784-25317933,

strand= forward CDCA2 Rev: TCTTCCCAGTTCCCAAAATG ANAPC12 For: GACCCGTAAGAAACAGAAGGAA chr9:116029543-116029694, ANAPC12 Rev: TGAACGATTATTGGGCTTGG strand= reverse AURKA For: CAGGCCAATCGGCTTTCTA chr20:54967248-54967345,

strand= reverse AURKA Rev: TTCCAAGAGCTCAGCCGTTA

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Gene name Primer sequence

DDB1 For: TGCCTACAAGCCCATGGAAG DDB1 Rev: TGGCAGCGCTATCCTTTTGA RNF40 For: AAGAGGGCTCAGGAGGACAT RNF40 Rev: CATCTGCGTAGACCTCCACC DDB1 For: ATCATTGACCCTGAGTGCCG DDB1 Rev: CCTGAGGGTCCTGGTAGACA CUL7 For: TACCAGGAGGGGTCCTCAAG CUL7 Rev: TTCTCCAAGTTCTGGCCGTC SMU1 For: TGGCACTGTAAAGATCTGGAA SMU1 Rev: CTGTTGCACACCACAAAGTG SMC1a_For: TACCTCAGTTCTCGGGCGTA SMC1a_Rev: TGACTTACCAGAGCCATTGGG MUS81_For: GGCCCAGGAGACCAATCCT MUS81_Rev: TTCAGCCGGAACTTCTGCTC AURKA_For: GTCAAGTCCCCTGTCGGTTC AURKA_Rev: GCAATGGAGTGAGACCCTCT ANAPC5_For: CGGTCACTATCAACAGGCAGA ANAPC5_Rev: GCGAGGTACGGTAACCCAAA ANAPC12_For: AGGCCGGCTTGAAAAGAGATTA ANAPC12_Rev: GGGCCAAACCCAGTGAACTA CDCA2_For: CCGAAGACTGGGTTCAGGTTA CDCA2_Rev: TGCCAAGCGATGAGGAACTT

Supplementary table 2: List of primers used in this study for qRT-PCR analysis were

shown.

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C

Inpu

t

SFB

- EV

SFB

- CU

L7

Flag (CUL7)

DDB1

RNF40

SMU1

CUL4a

IP :

SBP

(CUL

7)

SFB

- EV

SFB

- CU

L7

DDB1

RNF40

SMU1

CUL4a

CUL7

DDB1

SMU1

CUL4a

SFB

- EV

SFB

- RN

F40

IP :

SBP

(RNF

40)

Flag (RNF40)

DDB1

SMU1

CUL4a

CUL7

SFB

- EV

SFB

- RN

F40

Inpu

t

SMU1

CUL4a

CUL7

RNF40

IP :

SBP

(DDB

1)

SFB

- EV

SFB

- DD

B1

Flag (DDB1)

SMU1

CUL4a

CUL7

RNF40

SFB

- EV

SFB

- DD

B1

Inpu

t

A BS

BP

IgG

Flag (SMU1)

Myc (CUL7)

SFB

-SM

U1

Myc

-DY

RK

2

Flag (SMU1)

SFB

-SM

U1

Myc

-CU

L7

Myc (DYRK2)

IP

Inpu

t

Flag (SMU1)

SFB

-SM

U1

Fla

g-D

DB

1 S

FB-S

MU

1 M

yc-R

NF4

0

Myc (RNF40)

Flag (SMU1)

Flag (DDB1)

D

Figure S1

Supplementary Figure S1: (A) 293T cells were transfected with SFB tagged SMU1 along

with Myc- CUL7, MYC-RNF40, Flag-DDB1 and Myc-DYRK2. The presence of RNF40,

DDB1, CUL7 and DYRK2 in SMU1 complex proteins was evaluated by immunoblotting

with the indicated antibodies. (B) Cells were transfected with either SFB-Empty vector (EV)

or SFB-DDB1 (C) SFB-CUL7 (D) and SFB-RNF40. Interaction of SMU1-DDB1-CUL7-

RNF40 was assessed by immunoblotting with respective antibodies after pulling down with

streptavidin sepharose.

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Figure S2A B

Day 1 2 3 4 5

20

15

10

5

0

Control siRNA SMU1 siRNA

Prol

ifera

tion

(105

)

Control shRNA SMU1 shRNA

DDB1 shRNA CUL7 shRNA

RNF40 shRNA AN

Ris lortnoC

AN

Ris 1U

MS

SMU1

Actin

C

Supplementary Figure S2: (A) HeLa cells transfected with control or SMU1 siRNA. Cell

lysates were checked for knockdown of SMU1 using SMU1 specific antibody and actin was

used as loading control. (B) HeLa cells were transfected with control and SMU1 siRNA

separately. 48 hr post transduction 1*10^5 cells were seeded in five different plates and rate

of cell proliferation was measured by cell counting after staining with trypan blue dye. (C)

Cells were transduced with control shRNA, RNF40 shRNA, SMU1 shRNA, DDB1 shRNA

or CUL7 shRNA. 72 hours post transduction, mitotic cells (rounded off cells) were imaged in

phase contrast microscope and representative images were shown.

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Figure S3

0

0.2

0.4

0.6

0.8

1

Control shRNA

CUL7 shRNA

CUL7

0

0.2

0.4

0.6

0.8

1

DDB1 shRNA

DDB1

eneg evitaleR

noisserpxe

eneg evitaleR

noisserpxe

eneg evitaleR

noisserpxe

eneg evitaleR

noisserpxe

**

**

0

0.2

0.4

0.6

0.8

1

RNF40 shRNA

RNF40

0

0.2

0.4

0.6

0.8

1

SMU1 shRNA

SMU1

* *

Control shRNA

Control shRNA

Control shRNA

Supplementary Figure S3: Total RNA was extracted from HeLa cells transfected with

control or SMU1 siRNA or CUL7 shRNA or DDB1 shRNA and RNF40 shRNA, and

expression levels of various genes measured by qRT-PCR was shown. Statistical analysis:

error bars- standard deviation, ***P<0.001 **P<0.01; *P<0.05, Student’s t-test.

J. Cell Sci. 131: doi:10.1242/jcs.213868: Supplementary information

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Figure S4

Cohesion DefectsNormal

H2B - Ub fusion

H2B WT

% of chromosome spreads

0 20 40 60 80 100

H2B - Ub fusionH2B WT

NormalDefective

C

A B

Cohesion Defects

Normal

Control shRNA

+ H2B ub fusion

D

0

20

40

60

80

100

shRNA : Control CUL7 DDB1 RNF40 SMU1 H2B Ub – – + – + – + – +Fusion:

Defective Normal

% o

f Ch

rom

osom

e s

prea

ds

*** *** **

Multipolar spindles

Anaphase Bridges

Lagging chromosomes

0

10

20

30

% o

f cel

ls

H2B WT H2B-Ub fusion

F

H2B WT

H2B ub fusion

Lagging chromosomes

Anaphase bridges

Multipolar spindles

DAPI

E

Inte

rkin

etoc

hore

dist

ance

(µm

)

H2B WT Ub fusion

2.0

1.0

0

3.0 ***

G

CUL7 shRNA

DDB1 shRNA

RNF40shRNA

SMU1 shRNA

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Supplementary Figure S4: (A) Cells expressing control shRNA, CUL7 shRNA, DDB1

shRNA, RNF40 shRNA and SMU1 shRNA alone or together with H2B-Ub fusion were

analysed for chromatid cohesion (n= 75). (B) Percentage of chromosome spreads showing

normal and defective cohesion was plotted. P value is less than 0.001 for knockdown of

complex proteins compared to control and non-significant between shRNA alone and along

with H2B-Ub fusion (C) Chromosome spreads were prepared form HeLa cells transfected

with either H2B wild type or H2B-Ub fusion. Representative images of chromosome spreads are shown. (D) Quantification of data from figure S3C is shown. (n= 90), p < 0.001. (E) The

distance between paired kinetochores was measured for cells transfected with either H2B WT

or H2B-Ub fusion. (F) Cells were transfected with H2B Wild type (WT) or H2B-Ub fusion

protein. Mitotic defects were detected using immunofluorescence after staining with DAPI

(Scale bar, 10µm). (G) Quantification of the data from figure S5F is shown (n=25 cells each).

Statistical analysis: error bars- standard deviation, ***P<0.001 **P<0.01; *P<0.05, Student’s

t-test. (Scale bar, 5µm).

J. Cell Sci. 131: doi:10.1242/jcs.213868: Supplementary information

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