The Tudor protein survival motor neuron (SMN) is a ... · relocation to damaged centromeres requires its functional Tudor domain and activity of the H3K79 methyltransferase DOT1L.

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The Tudor protein survival motor neuron (SMN) is achromatin-binding protein that interacts withmethylated lysine 79 of histone H3

Mirna Sabra1,2,3,*, Pascale Texier1,2,3,*, Jhony El Maalouf1,2,3 and Patrick Lomonte1,2,3,`

1Centre de Genetique et Physiologie Moleculaire et Cellulaire CNRS, UMR5534, Universite Claude Bernard Lyon 1, 69622 Villeurbanne, France2Universite de Lyon 1, F-69000 Lyon, France3Laboratoire d’excellence, Labex DEVweCAN, F-69000 Lyon, France

*These authors contributed equally to this work`Author for correspondence ([email protected])

Accepted 16 May 2013Journal of Cell Science 126, 3664–3677� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.126003

SummarySpinal muscular atrophy (SMA) is a muscular disease characterized by the death of motoneurons, and is a major genetic cause of infantmortality. Mutations in the SMN1 gene, which encodes the protein survival motor neuron (SMN), are responsible for the disease. SMN

belongs to the Tudor domain protein family, whose members are known to interact with methylated arginine (R) or lysine (K) residues.SMN has well-defined roles in the metabolism of small non-coding ribonucleoproteins (snRNPs) and spliceosome activity. Wepreviously showed that SMN relocated to damaged interphase centromeres, together with the Cajal-body-associated proteins coilin and

fibrillarin, during the so-called interphase centromere damage response (iCDR). Here we reveal that SMN is a chromatin-binding proteinthat specifically interacts with methylated histone H3K79, a gene expression- and splicing-associated histone modification. SMNrelocation to damaged centromeres requires its functional Tudor domain and activity of the H3K79 methyltransferase DOT1L. In vitro

pulldown assays showed that SMN interacts with H3K79me1,2 at its functional Tudor domain. Chromatin immunoprecipitation

confirmed that SMN binds to H3K79me1,2-containing chromatin in iCDR-induced cells. These data reveal a novel SMN property in thedetection of specific chromatin modifications, and shed new light on the involvement of a putative epigenetic dimension to theoccurrence of SMA.

Key words: Survival motor neuron protein (SMN), Methylated H3K79, Centromeres, Cajal bodies, HSV-1 ICP0 protein

IntroductionThe survival motor neuron (SMN) gene is present at an inverted

duplicated locus on chromosome 5 in human cells (Lefebvre

et al., 1995). The telomeric copy, called SMN1, differs from the

centromeric copy, called SMN2, by five nucleotides. Both genes

are transcribed, and one of the nucleotide changes in SMN2

favors the deletion of one exon (the exon 7), resulting in the

synthesis of a truncated unstable protein called SMND7 and very

little full-length functional SMN (Kashima and Manley, 2003;

Lorson and Androphy, 2000; Lorson et al., 1999; Monani et al.,

1999; Singh et al., 2006; Singh and Singh, 2011; Singh et al.,

2009)}. Homozygous deletions or mutations in SMN1 are

responsible for the development of the autosomal recessive

disorder proximal spinal muscular atrophy (SMA), the most

common genetic cause of infant mortality (Burghes and Beattie,

2009; Coovert et al., 1997; Melki, 1997). The severity of the

disease depends on the ability of SMN2 to compensate for the

absence of SMN protein (Lefebvre et al., 1997). The absence of

functional SMN protein primarily affects motoneurons, leading

to muscle paralysis and atrophy (Monani, 2005).

SMN is expressed ubiquitously and forms a large multiprotein

complex with Gemin proteins (2 to 8) and Unrip proteins

(Baccon et al., 2002; Carissimi et al., 2005; Carissimi et al., 2006;

Charroux et al., 1999; Charroux et al., 2000; Gubitz et al., 2002;

Pellizzoni et al., 2002). This complex is implicated in the nucleo-

cytoplasmic transport of small nuclear RNAs (snRNAs) and in

their maturation to small nuclear ribonucleoproteins (snRNPs) as

part of the spliceosomal complex (Akten et al., 2011; Deryusheva

et al., 2012; Fischer et al., 1997; Leung et al., 2011; Liu et al.,

1997; Makarov et al., 2012; Massenet et al., 2002; Matera and

Shpargel, 2006; Sebbag-Sznajder et al., 2012). Studies using

different models of SMA have suggested that the severity of the

disease could correlate with the inefficiency of snRNP

maturation, and as a consequence, to defective splicing of

target pre-mRNAs (Chari et al., 2009; Gabanella et al., 2007;

Imlach et al., 2012; Lotti et al., 2012; Pellizzoni, 2007; Winkler

et al., 2005; Workman et al., 2009; Zhang et al., 2008). The

implication of SMN in such a fundamental molecular pathway in

cells of all origins is difficult to reconcile with the selective

involvement of the neuromuscular system in SMA. To that

extent, recent studies suggested that loss of other non-snRNP

biogenesis-related functions of SMN is important in the

development of SMA pathology (Baumer et al., 2009; Praveen

et al., 2012). Recently, SMN2 exon 7 splicing was shown to be

dependent on functional SMN protein, with an increased

requirement in motoneurons (Jodelka et al., 2010; Ruggiu et al.,

2012). Other recent studies suggested that SMN loss-of-function

preferentially affected the U12-dependent minor spliceosome,

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which could account for the misregulation of U12-dependent

specifically processed genes (Boulisfane et al., 2011; Lotti et al.,2012), possibly in a non-cell-autonomous-dependent manner(Imlach et al., 2012; Lotti et al., 2012). This implies that SMN

may contribute, directly or indirectly, to a specific machineryinvolved in the coupled transcription/splicing of RNAs that areessential for the survival of motoneurons.

SMN is a Tudor domain-containing protein. Tudor domains are

part of the ‘royal’ domain superfamily and recognize methylatedarginine (R) or lysine (K) residues (Botuyan et al., 2006; Chenet al., 2011; Huang et al., 2006; Huyen et al., 2004). The SMN

Tudor domain binds preferentially to symmetric dimethyl-arginine(sDMA) residues in Arg–Gly (RG)-rich sequences of spliceosome-associated Sm family proteins and in coilin, the major structuralcomponent of the nuclear domains known as Cajal bodies (CBs)

(Boisvert et al., 2002; Cote and Richard, 2005; Friesen et al., 2001;Liu et al., 2012; Raska et al., 1991; Selenko et al., 2001; Tripsianeset al., 2011). The localization of SMN to CBs depends on its

interaction with methylated and phosphorylated coilin (Hearstet al., 2009; Hebert et al., 2002; Hebert et al., 2001; Toyota et al.,2010) and phosphorylation of SMN itself (Petri et al., 2007;

Renvoise et al., 2012). Following hypomethylation of coilin, SMNcomplexes form individual nuclear bodies called Gemini bodies orgems juxtaposed to CBs (Liu and Dreyfuss, 1996). Apart from

these two nuclear domains, SMN does not accumulate elsewherein the nucleus under normal conditions, although diffuse nuclearstaining can be observed by immunofluorescence (IF) (Renvoiseet al., 2006). However, following destabilization of interphase

centromeres by the viral E3 ubiquitin ligase protein ICP0 (fromherpes simplex virus type 1), we observed centromericaccumulation of three CB proteins: coilin, fibrillarin and SMN

(Morency et al., 2007). We named this response the interphasecentromere damage response (iCDR). We showed that coilininteracted with the damaged centromeric chromatin, suggesting

that epigenetic changes to centromeric chromatin induced by ICP0might be responsible for triggering the iCDR. In this study weanalyzed the interdependency between coilin and SMN for their

accumulation at damaged centromeres, and found that coilin wasnecessary but not sufficient for the targeting of SMN tocentromeres. The Tudor domain of SMN was required for itscentromeric accumulation. We investigated the requirement for

both R- and K-methyltransferase activities, and found thatdepletion of K-methyltransferase disruptor of telomeric silencing1-like (DOT1L) protein significantly affected the targeting of

SMN to damaged centromeres. DOT1L induces the mono- and di-methylation histone H3 at K79. We therefore analyzed the capacityof SMN to interact with methylated H3K79. We found that SMN

interacted with mono- and di-methylated H3K79 through its Tudordomain. We then performed chromatin immunoprecipitationanalysis of centromeric SMN in iCDR-induced cells, and foundthat SMN interacted with mono- and di-methylated H3K79 within

nucleosomes in centromeric chromatin. Overall, these datademonstrate that SMN is a chromatin-binding protein thatrecognizes specific histone markers through its Tudor domain.

ResultsCoilin is necessary but not sufficient for SMN relocation todamaged centromeres

The iCDR implicates three proteins that are normallyconcentrated in CBs. To determine whether all three proteinsare dependent on each other for their relocation to damaged

centromeres, the iCDR was induced by expression of ICP0 in

cells depleted of one of these proteins. The efficiency of siRNAs

targeting coilin, fibrillarin and SMN was tested by WB and IF

(supplementary material Fig. S1A,C; Fig. S2A). The absence of

centromeric colocalization of all proteins within each depletion

background was also verified in the absence of ICP0 expression

(supplementary material Fig. S1B,D; Fig. S2B). The nuclear

distribution of coilin could appear ‘spotty’ in a subset of SMN-

depleted cells, as reported previously (Girard et al., 2006;

Shpargel and Matera, 2005); however, coilin did not colocalize

with centromeres (supplementary material Fig. S1Bv). Overall,

our data showed that depletion of SMN did not prevent the

relocation of coilin (Fig. 1Aiv) or fibrillarin (supplementary

material Fig. S2Civ) to damaged centromeres. Similar results

were obtained for the centromeric relocation of coilin and SMN in

fibrillarin-depleted cells (Fig. 1Aiii,Biii, respectively). Depletion

of coilin did not affect the behavior of fibrillarin at damaged

Fig. 1. Coilin is necessary for relocation of SMN to damaged

centromeres. HeLa cells were first transfected with control (siCtrl; i), coilin

(siCo_1; ii), fibrillarin (siFib_1; iii) or SMN (siSMN_1; iv) siRNA. After

depletion of the target protein (see supplementary material Figs S1, S2), cells

were transfected with an ICP0-expressing plasmid. IF was performed to co-

detect coilin (A, green) or SMN (B, green), centromeres (red) and ICP0 (gray/

blue in merged images). Enlarged views of the boxed areas in the merged

images are shown in the ‘close-up’ column. Scale bars: 10 mm.

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centromeres (supplementary material Fig. S2Cii), whereas itabrogated SMN relocation (Fig. 1Bii). All these data were

reproduced with a second siRNA targeting coilin, fibrillarin or

SMN (data not shown). These data suggest that SMN, but notfibrillarin, is dependent on coilin for its relocation to damaged

centromeres. To confirm these behaviors in another biologicalcontext, ICP0 was co-expressed with a coilin mutant depleted of

its RG box (HA–CoDRG; Fig. 2). HA–CoDRG was dominant-negative towards endogenous coilin because it induced the

disappearance of coilin from CBs (Fig. 2ii). Similarly, fibrillarinand SMN did not show characteristic CB labeling in cells

expressing HA–CoDRG (Fig. 2iv,vi). None of these observationswere made in cells expressing HA-tagged unmodified coilin

(HA–Co; Fig. 2i,iii,v). Co-expression of HA–Co and ICP0 didnot prevent relocation of coilin, fibrillarin or SMN to damaged

centromeres (Fig. 2vii,ix,xi). In contrast, co-expression of HA–CoDRG and ICP0 prevented relocation of coilin and SMN, but

not fibrillarin, to damaged centromeres (Fig. 2viii,x,xii). It is nottechnically possible to check for the presence of ICP0 and the

HA-tagged protein in the same cell together with labeling theendogenous CB protein of interest and centromeres; however our

experimental procedure was designed so that all ICP0-positivecells were also positive for expression of the HA-tagged protein

(data not shown). These data, together with those obtained by the

knockdown approach, confirm that coilin is necessary for therelocation of SMN to damaged centromeres.

HeLaPV cells are known to express hypomethylated coilin

(Hebert et al., 2002), and to display a subset of gems that areseparate from CBs, suggesting a weak interaction between coilin

and SMN in these cells (Liu and Dreyfuss, 1996) (Fig. 3Ai).Expression of ICP0 in HeLaPV cells induced the relocation of

coilin and fibrillarin, but not SMN, to damaged centromeres,

(Fig. 3Aii–iv). It is known that the RG box of coilin is modified

by methylation of R residues, and that this modification favorsthe binding of SMN to coilin (Hebert et al., 2001; Hebert et al.,

2002). Therefore, the latter results suggest that the presence ofSMN at damaged centromeres might simply be a consequence ofits interaction with fully methylated coilin. To test the validityof this assumption, we used the SYM10 antibody, which directly

recognizes coilin methylated in its RG box (Boisvert et al., 2002),to detect the presence of methylated coilin at centromeres.Although SYM10 clearly labeled CBs in cells not expressing

ICP0 (Fig. 3Bi,iii), no colocalization between coilin and SYM10or centromeres and SYM10 was observed in ICP0-expressingcells (Fig. 3Bii,iv, respectively). Of note, the expression of ICP0

in cells is easily identified by the specific multiple spot patternadopted by coilin (Fig. 3Bii) (see Morency et al., 2007) (Figs 1,2; supplementary material Figs S1, S2). Although we cannotexclude the possible lack of sensitivity of the SYM10 antibody

towards low amounts of fully methylated centromeric coilin,these data, together with those obtained in HeLaPV cells, suggestthat coilin does not need to be methylated in its RG box to

accumulate at damaged centromeres. Altogether, these datasuggest that: (i) relocation of SMN to damaged centromeres isunlikely to be only a consequence of its interaction with

methylated coilin (i.e., that other activities are required), and(ii) although centromeric coilin is necessary to induce thecentromeric targeting of SMN, it is not sufficient.

The Tudor domain is necessary but insufficient for theaccumulation of SMN at damaged centromeres

We then asked whether specific domains in SMN were required

for its centromeric targeting. In SMN, several domains that aremore or less important for its targeting to CBs have beendescribed (Renvoise et al., 2006). We focused our analysis

mainly on two domains: (i) the Tudor domain, because it

Fig. 2. Dominant-negative CoDRG

prevents relocation of SMN to damaged

centromeres. HeLa cells were transfected

with a plasmid expressing HA-tagged coilin

(HA-Co; i, iii, v, vii, ix and xi) or a coilin

mutant missing its RG box (HA-CoDRG, ii,

iv, vi, viii, x and xii), with (+ICP0) or

without (2ICP0) co-transfection of an

ICP0-expressing plasmid. IF was then

performed to detected coilin (green, i, ii, vii

and viii), fibrillarin (green, iii, iv, ix and x)

or SMN (green, v, vi, xi and xii), together

with HA (red, i–vi) or centromeres (red, vii–

xii) and ICP0 (gray, vii–xii). Enlarged

views of the boxed areas in the merged

images are shown in the ‘close-up’ column.

Scale bars: 10 mm.

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mediates the interaction of SMN with the sDMA isoforms of RG

motif-containing proteins such as coilin and Sm core proteins

(Boisvert et al., 2002; Hebert et al., 2002); and (ii) the ex7

domain, because its loss leads to the synthesis of an unstable

SMN_Dex7 protein with a reduced capacity to self-oligomerize,

which could partly explain the inability of SMN_Dex7 to

compensate for the absence of full-length SMN in SMA

patients (Lorson and Androphy, 2000; Lorson et al., 1998).

Five EGFP–SMN fusion proteins expressing full-length or

truncated SMN were evaluated by IF in terms of their ability to

accumulate at centromeres in ICP0-co-expressing cells (Fig. 4).

The proper expression and biological properties of all these

proteins have been characterized in the presence and absence of

endogenous SMN and in various cell types (Renvoise et al.,

2006). EGFP–SMN_FL expresses full-length SMN; EGFP–

SMN_Dex7 (aa 1–278 plus the first four aa encoded by ex8)

expresses SMN_Dex7 protein; EGFP–SMN_472D5 (aa 1–146)

lacks the C-terminal half of SMN, but contains the Tudor

domain; EGFP–SMN_N86 (aa 1–86) lacks the C-terminal half of

SMN, including the Tudor domain; and EGFP–SMN_E143K is a

mutant harboring a glutamic acid (E) to K substitution at position

134 in the Tudor domain, which has been identified in type I

SMA patients (Lefebvre et al., 1998) (Fig. 4A). EGFP–SMN_FL

was found in CBs in the absence of ICP0, and colocalized with

centromeres in ICP0-expressing cells (70.6617.9%; Fig. 4Bi,ii).

EGFP–SMN_Dex7 and EGFP–SMN_472D5 showed ‘spotty’

nuclear patterns and colocalized with coilin in CBs in ICP0-

negative cells, as reported previously (Renvoise et al., 2006).

Their re-location at centromeres in cells expressing ICP0 was

significantly reduced compared to EGFP–SMN_FL [12.564.6%

(P50.029) and 4.161.3% (P50.026) for EGFP–SMN_Dex7 and

EGFP–SMN_472D5, respectively; Fig. 4Biii–vi]. Additional

deletion of the Tudor domain induced the nucleoplasmic

distribution of EGFP–SMN_N86, but no colocalization with

centromeres in ICP0-expressing cells (Fig. 4Bvii,viii). Finally,

EGFP–SMN_E143K, which could colocalize with coilin in a

subset of CBs (Renvoise et al., 2006), was also significantly

affected for its accumulation at centromeres in ICP0-expressing

cells (10.961.2%, P50.025) (Fig. 3Bix,x). In order to rule out

any discrepancies in the EGFP–SMN proteins behavior due to

variations in the levels of ICP0 rather than to the absence of

specific SMN protein domains, we performed WB on co-

transfected cells. No variations of ICP0 levels could be detected

in the different samples (supplementary material Fig. S3). These

data suggest that: (i) although a functional Tudor domain is

required, its presence is insufficient for significant accumulation

of SMN at damaged centromeres; (ii) the SMN_Dex7 protein,

although able to colocalize with coilin in CBs, is significantly

reduced in its capacity to relocate to centromeres, which confers

to ex7, and therefore to the capacity of SMN to self-oligomerize,

a particular importance for the interaction of SMN with

destabilized centromeres.

Activity of type I and II PRMTs is not required to target SMN

to damaged centromeres

The data concerning the probable hypomethylation of

centromeric coilin and the requirement for the Tudor domain of

SMN led us to hypothesize that, although coilin is required, other

biological events are likely responsible for the relocation of SMN

to damaged centromeres. Because Tudor domains are implicated

in the interaction with methylated R and K, we decided to

investigate the requirement for the activity of the following for

the induction of centromeric SMN during the iCDR: type I

PRMTs (PRMT-1, -3, -4 and -6), which induce asymmetric R di-

methylation (aDMA); type II PRMT-5, which induces sDMA;

and HKMT PR-Set7, which induces di-methylation of histone H4

at K20. We inactivated PRMTs and PR-Set7 using specific

siRNAs and then induced the iCDR by expressing ICP0. siRNAs

Fig. 3. Hypomethylated coilin is targeted to

damaged centromeres. (A) HeLaPV cells

were left untransfected (i) or transfected with

an ICP0-expressing plasmid (ii–iv).

Untransfected cells were stained for SMN

(green) and coilin (red). ICP0-expressing cells

were stained for coilin (green; ii), fibrillarin

(green; iii) or SMN (green; iv) plus

centromeres (centro; red) and ICP0 (gray).

Enlarged views of the boxed areas in the

merged images are shown in the ‘close-up’

column. Scale bars: 10 mm. (B) Centromeric

coilin lacks a methylated RG box. HeLa cells

were left untransfected (i and iii) or transfected

with an ICP0-expressing plasmid (ii and iv). IF

was performed to detect coilin (green) using a

coilin-specific mouse monoclonal antibody

and/or methylated coilin in its RG box using

the SYM10 antibody (Boisvert et al., 2002), as

well as centromeres (Centro, red) and ICP0

(gray/blue in merged images). Scale bars: 10

mm.

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were first verified for their effect on their target protein (Fig. 5A).

Decreases in PRMT5 and PR-Set7 substrates [histone H4

symmetrically di-methylated at R3 (H4R3me2s) and K20

(H4K20me2), respectively] were also assessed. Depletion of

PRMT-1, -3, -4, -5, -6 or PR-Set7, and subsequent expression of

ICP0, did not modify the behavior of SMN towards destabilized

centromeres (Fig. 5Bi,ii; Table 1). Accordingly, addition of the

methyltransferase inhibitor 59-deoxy-59-(methylthio)adenosine

(MTA) prior to ICP0 expression did not prevent the iCDR,

including centromeric SMN accumulation (data not shown).

Overall, these data suggest that the absence of R methylation

activity did not directly affect SMN redistribution to damaged

centromeres, reinforcing the conclusion that centromeric

accumulation of SMN is not dependent on fully methylated coilin.

DOT1L H3K79 methyltransferase activity is required for

SMN accumulation at damaged centromeres

53BP1 is a double Tudor-domain-containing protein that was shown

to interact with H4K20me2, and to a lesser extent H3K79me2

(Botuyan et al., 2006; Huyen et al., 2004). Single, double and triple

methylation of H3K79 is dependent on DOT1L methyltransferase

activity (Feng et al., 2002; Jones et al., 2008; Steger et al., 2008). We

next performed experiments similar to those described above with

inactivation of DOT1L. We were unable to validate an antibody that

efficiently recognizes DOT1L by WB or IF; therefore, the efficiency

of the DOT1L siRNA was confirmed by detection of a decrease in

the amount of the DOT1L mRNA (Fig. 6A), and its substrate

H3K79me2 (Fig. 6B). We initially confirmed that depletion of

DOT1L did not affect the activity of ICP0 in centromeric protein

Fig. 4. The Tudor domain of SMN is required for its targeting to damaged centromeres. (A) Left, upper: schematic representation of the SMN protein sub-

domains and their exons positions. SMN is encoded by eight exons, generating a multi-domain polypeptide of 294 aa. Left, lower: EGFP-tagged SMN proteins

used in the experiments. Right: WB analysis of EGFP-tagged SMN proteins expressed in transfected HeLa cells. For details of the different EGFP–SMN proteins,

see Results and Renvoise et al. (Renvoise et al., 2006). (B) Left (i, iii, v, vii, ix): distribution of the EGFP–SMN proteins in control transfected cells (control).

Middle (ii, iv, viii, x): detection of EGFP–SMN proteins (green), centromeres (red), and ICP0 (gray/blue in merged images) in cells co-transfected with an

ICP0-expressing plasmid (+ICP0). In the merged images arrows indicate colocalization of EGFP–SMN proteins with centromeres. Enlarged views of the boxed

areas in the merged images are shown in the ‘close-up’ column. Scale bars: 10 mm. Right: the percentage of cells (means 6 s.d.) showing colocalization of SMN

with at least one centromere signal was determined for each protein. Significance of the results was calculated using a Student’s paired t-test. Results were

considered as significant if P,0.05.

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degradation (data not shown). Depletion of DOT1L did not affect

the location of SMN in CBs (Fig. 6Ci, ICP02 cell). Further

expression of ICP0 in DOT1L-knockdown cells showed the

classical ‘multi-dotted’ pattern typical of coilin accumulation at

centromeres (Fig. 6Ci, ICP0+ cells). This confirmed that ICP0 was

active when DOT1L was depleted and that the targeting of coilin

Fig. 5. PRMT and HKMT activity is not required for the targeting of SMN to damaged centromeres. (A) HeLa cells were transfected with siRNAs targeting

type I and type II PRMTs, which induce aDMA and sDMA, respectively, and HKMT PR-Set7, which induces di-methylation of H4K20. Detection of the

corresponding PRMTs and HKMT was performed by WB using appropriate antibodies (see Materials and Methods). For PRMT5 and PR-Set7, decreases in their

specific substrates, H4R3me2s and H4K20me2, respectively, were also evaluated. Actin and/or H4 were detected as loading controls. (B) Images of SMN

accumulation at damaged centromeres in ICP0-expressing HeLa cells previously transfected with siRNAs targeting type I PRMT1 (i and ii). After depletion of the

target protein, cells were transfected with an ICP0-expressing plasmid. Then, SMN (green) was detected together with coilin (red; i) or centromeres (red; ii) and

ICP0 (gray/blue in the merged image, ii). In PRMT1-depleted cells negative for ICP0 expression (arrowhead, ICP02, i), coilin and SMN colocalize within CBs. In

ICP0-positive cells (arrowhead, ICP0+, i), the ‘multispots’ adopted by coilin and SMN are representative of the iCDR occurring at centromeres (see Morency et al.,

2007) (Figs 1–3; supplementary material Figs S1, S2). Boxed areas show the colocalization of SMN and coilin (i) or SMN and centromeres (ii) in ICP0-expressing

cells. The images in B are representative of SMN behavior in all the other PRMTs or PR-Set7-depleted cells. Scale bars: 10 mm.

Table 1. SMN behavior in cells depleted of lysine or arginine methyltransferases

SamplesFrequency of nuclei with gems in

non-ICP0-expressing cellsFrequency of ICP0-expressing

cells showing centromeric SMNIntensity of the

centromeric SMN signal Remarks

Control ++++ ++++ ++++siCtrl +++ ++++ ++++siDOT1L +++ + + a,bsiPer-Set7 +++ +++ +++siPRMT1 +++ ++++ ++++siPRMT3 ++ +++ ++siPRMT4 +++ +++ +++siPRMT5 +c –d N/AsiPRMT6 +++ +++ +++

+, ,50%; ++, 50–70%; +++, 70–90%; ++++, .90%.aBased on the centromere signal obtained with the human anti-centromere antibody (CREST) by immunofluorescence the ICP0 activity on centromeres in the

absence of DOT1L seems much greater than in control cells.bWhen present, the number of centromeric SMN spots in ICP0-expressing cells is often fewer than in control cells.cThe depletion of PRMT5 provokes the accumulation of cells without gems.dAll ICP0-expressing cells show diffuse nucleoplasmic SMN.N/A, not applicable.

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to damaged centromeres was not affected by the absence of DOT1L.

The same ICP0+ cells also showed that SMN did not colocalize with

centromeric coilin. Co-staining of SMN and centromeres confirmed

that SMN did not accumulate at ICP0-damaged centromeres in most

DOT1L-depleted cells (Fig. 6Cii, arrow; Table 1), and that when it

did so it was with a dramatically reduced signal (Fig. 6Cii,

arrowhead; Table 1). To quantify this event precisely, DOT1L

KD ICP0-transfected cells were counted (about 500 ICP02 or

ICP0+ cells, in triplicate) and divided into three groups: (i) cells

showing centromeric SMN; (ii) cells with SMN in gems/CBs; and

(iii) cells with no visible gems/CBs (Fig. 6D). For control no-siRNA

and siRNA control (siCtrl)-transfected cells, more than 80% of cells

positive for ICP0 showed SMN at the centromeres. Very few ICP0+

cells (,5%) showed SMN in gems/CBs and very few ICP02 cells

(,10%) did not contain gems/CBs. Cells depleted of DOT1L with

siDOT1L showed a clear decrease in centromeric SMN (35613%)

and a concomitant increase in the proportion of ICP0+ cells with

SMN in gems/CBs (50614%). In addition, absence of DOT1L did

not affect gem/CB formation as only 1364% of cells did not contain

gems/CBs, similar to the results of control and siCtrl-transfected

cells. Overall, these data show that DOT1L activity is required for

the targeting of SMN to damaged centromeres and suggest that

SMN may relocate to damaged centromeres in a methylated

H3K79-dependent manner.

SMN binds to mono- and di-methylated H3K79

Both a functional SMN Tudor domain and DOT1L are required

for the targeting of SMN to damaged centromeres. This

Fig. 6. Depletion of DOT1L prevents the targeting of SMN to damaged centromeres. (A) RT-qPCR data from three independent experiments to assess the

decrease of the DOT1L mRNA in siDOT1L-treated cells. Ctrl, non-transfected control HeLa; siCtrl, HeLa cells transfected with control siRNA; siDOT1L, HeLa

cells transfected with DOT1L siRNA. (B) WB to assess the efficiency of siRNA treatment on DOT1L depletion. HeLa cells were left untransfected (Ctrl) or

transfected with a control siRNA (siCtrl) or an siRNA targeting DOT1L methyltransferase (siDOT1L). No commercial antibody that specifically recognizes

DOT1L in WB is available. Therefore, the efficiency of the siRNA against DOT1L was assessed by detection of its substrate H3K79me2. Detection of H4K20me2

was performed as a control for specificity. Actin and H3 were detected as loading controls. (C) After depletion of DOT1L, cells were transfected with an ICP0-

expressing plasmid. IF was performed to detect SMN (green) and coilin (red; i), or centromeres (Centro, red; ii) and ICP0 (gray; ii). SMN colocalized with coilin

in CBs in ICP0-negative cells (Ci; ICP02, two upper cells in the merged image). In ICP0-positive cells (Ci; ICP0+, two lower cells in the merged image) coilin

shows the typical ‘multispot’ pattern of the iCDR, whereas SMN does not predominantly colocalize with centromeres (Cii). Arrows and arrowheads in Cii indicate

nuclei positive for ICP0 with no SMN or a weak SMN signal at centromeres, respectively. Scale bars: 10 mm. (D) Data of three individual experiments (means 6

s.d.), showing the percentage of nuclei with specific SMN behaviors (SMN at centromeres, SMN in gems/CBs, and no gems/CBs detectable) in control, siCtrl, and

siDOT1L cells not expressing or expressing ICP0. In B and D *P,0.05 and **P,0.01 (Student’s paired t-test).

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suggested that SMN binds to methylated H3K79 through its

Tudor domain. GST pulldown experiments confirmed that SMN

interacted with mono- and di-methylated, but not tri-methylated,

H3K79. This interaction implied a functional Tudor domain

because the SMN_E134K protein was unable to bind to

methylated H3K79 (Fig. 7A). This interaction was specific for

methylated H3K79 because di-methylated H4K20 did not interact

with SMN. One should be cautious about the specificity of

the antibodies for histone modifications. Therefore, we next

performed pulldown assays using biotinylated peptides spanning

the H3K79 region (aa 61–90) with unmodified, mono- or di-

methylated K79 or a control peptide from histone H4 (aa 2–24).

SMN from cellular extracts was captured by the mono- and di-

methylated K79 peptides, although unmethylated H3K79 peptide

also pulled down SMN to some extent, unlike H4 peptide

(Fig. 7B). This confirmed that SMN could interact with

methylated H3K79 and ruled out any artifact due to antibodies

interacting non-specifically with modified H3K79. Together,

these results demonstrate that SMN interacts with histone H3

molecules that are mono- or di-methylated at K79 and that the

interaction occurs through its functional Tudor domain.

SMN is a chromatin-binding protein

To confirm that SMN could interact with methylated H3K79 in a

chromatin context, we performed chromatin immunoprecipitation

(ChIP) analysis of SMN in cells expressing ICP0 or its non-

functional mutant FXE (ICP0mut) under tetracycline induction

(Fig. 7C) (Gross et al., 2012). Expression of ICP0 induces the

iCDR with centromeric SMN, whereas ICP0mut does not

(Morency et al., 2007); therefore, binding of SMN to

methylated H3K79 should increase in ICP0-expressing cells

compared to controls. Cells were treated with tetracycline or not

for 24 hours and then analyzed by ChIP (see Materials and

Methods). We first verified that the amount of chromatin used for

ChIP was similar for all samples, and that the sonicated

chromatin in all samples was homogenous (supplementary

material Figs S4, S5). ChIP analysis showed mono- and di-

methylated H3K79 bound to SMN in ICP0-expressing cells (i.e.

cells with centromeric SMN), confirming the binding of SMN to

methylated H3K79 in the chromatin context (Fig. 7C). Overall,

these data demonstrate that SMN is a chromatin-binding protein

that specifically interacts with mono- and di-methylated H3K79

in damaged centromeric chromatin.

DiscussionOur data demonstrate that the Tudor protein SMN, whose lack

of function is responsible for the occurrence of the infant

motoneuron decay-associated disease SMA, is a chromatin-

binding protein that interacts with histone H3 that is mono- or di-

methylated at K79 (H3K79me1,2). The chromatin-binding ability

of SMN was elucidated by analysis of the iCDR, an enigmatic

response to damage occurring at centromeres. Although its

physiological significance remains elusive, study of the iCDR

enables us to uncover unknown molecular features of the proteins

involved. Centromeric accumulation of the iCDR-associated

protein SMN is dependent on upstream events requiring the

targeting of coilin to damaged centromeres. Although coilin is

necessary, it is clearly insufficient to induce centromeric

localization of SMN; activity of the H3K79 methyltransferase

DOT1L is also required. The iCDR was discovered recently as a

cellular response occurring after the destabilization of interphase

centromeres by the viral protein ICP0 of HSV-1 (Morency et al.,

2007). This response implies the relocation to damaged

centromeres of three CB proteins: coilin, fibrillarin and SMN

Fig. 7. SMN is a chromatin-binding protein that interacts with mono- and di-methylated H3K79. (A) WB was performed after GST pulldown experiments

using GST, GST-SMN or GST-SMN_E134K and histone extracts. A Coomassie-stained gel is shown below the WB images to show the amounts of GST

proteins and histone extracts (input) used. (B) Pulldown experiments performed using biotinylated peptides (5 mg, shown by Coomassie gel staining) and cell

extracts (input). After peptide pulldown, WB was performed to detect SMN retrieved under the different conditions. (C) SMN binds mono- and di-methylated

H3K79 within chromatin. TR (Control), TR-ICP0 (ICP0), and TR-FXE (ICP0mut) HeLa cells were exposed (+) or not (2) to tetracycline (Tet) for 24 hours

to induce ICP0 expression. ChIP experiments were performed using a specific anti-SMN antibody (see Materials and Methods for procedure) followed by WB to

detect immunoprecipitated SMN (upper gel) and H3K79me1 or H3K79me2 (lower gel) under the different conditions. The control samples on SMN and H3K79

gels are cell and histone extracts, respectively. HIg and LIg are for heavy and light immunoglobulin chains, respectively.

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(see model in Fig. 8). SMN partners such as Gemin or Sm

proteins, and U2 snRNAs, were not similarly detected at

centromeres (Morency et al., 2007). This suggested that the

molecular features that lead to the accumulation of the three

proteins at centromeres are likely to be different from those that

attract them to CBs. Coilin was shown to locate on metaphase

chromosome from different Drosophila cells, possibly

accumulating at centromeres (Liu et al., 2009). SMN was

recently involved in proliferation and differentiation of stem cells

in larval Drosophila CNS and male germline suggesting a role of

SMN in cell division (Grice and Liu, 2011). Whether this SMN

function could be related to its capacity to interact with

centromeres in order to protect them in fast growing cells

becomes a sensible question in light of the conclusion of the

Grice and Liu study (Grice and Liu, 2011). Coilin is the main

structural component of CBs and its absence provokes the

disappearance of CBs (Hebert et al., 2001; Tucker et al., 2001).

However, coilin deficiency in mouse and Drosophila models

does not affect their viability at least in normal, unstressed

conditions (Liu et al., 2009; Walker et al., 2009). CB formation

depends on the post-translational modification of coilin by

methylation and phosphorylation (Hebert et al., 2002; Toyota

et al., 2010). We show in this study that the presence of coilin

at damaged centromeres is necessary for the centromeric

recruitment of SMN, but not of fibrillarin. Data obtained both

in HeLaPV cells and by SYM10 antibody labeling suggest that

centromeric coilin is hypomethylated, unlike CB-associated

coilin; however, the data concerning the dependency of

centromeric SMN localization on coilin methylation seem

contradictory. On the one hand, hypomethylated centromeric

coilin (in HeLaPV cells) is not sufficient to induce centromeric

SMN, which suggests that centromeric coilin needs to be fully

methylated to induce centromeric SMN; and on the other hand,

data obtained by SYM10 labeling of ATCC HeLa cells, which

showed centromeric SMN, suggest that centromeric coilin is

indeed hypomethylated. Accordingly, the Y12 antibody, which

recognizes Sm proteins and more generally sDMA in RG-rich

repeats (Brahms et al., 2000), including those present in the coilin

RG box (Hebert et al., 2002), does not label centromeres during

the iCDR (Morency et al., 2007). To exclude any potential

artifact due to lack of ICP0 activity in HeLaPV cells, which

would prevent SMN from being targeted to damaged

centromeres, we confirmed that ICP0 induces the proteasomal-

dependent degradation of CENPs and their disappearance from

centromeres. We did not observe any differences compared to

ATCC HeLa cells (data not shown). A mutation of SMN in

HeLaPV cells that would explain its absence at damaged

centromeres can be ruled out because sequencing of SMN

cDNA obtained from a pool of HeLaPV mRNAs did not reveal

any amino acid modification compared to the original SMN

sequence (data not shown). These results suggest that HeLaPV

cells are likely to be defective in other activities that could

explain the lack of targeting of SMN to damaged centromeres in

spite of the presence of centromeric coilin. In contrast, the

Fig. 8. Working model for the iCDR. Step 0: the structure of the interphase centromere chromatin with interspersed stretches of H3-containing nucleosomes

(purple) and CENP-A-containing nucleosomes (red) according to the common model of centromere structure (Amor et al., 2004). CENP-B (green sticks)

maintains the chromatin structure and nucleosomes organization by its binding to a 17-nt DNA sequence (CENP-B box, green circle) present in the repetitive

alphoid sequence of the centromeric DNA (Tanaka et al., 2005). Two protein complex, namely NAC (CENP-A nucleosomes associated complex) and CAD

(CENP-A distal complex) are associated with the centromeric chromatin during interphase (Foltz et al., 2006; Okada et al., 2006). Step1: as soon as it enters the

nucleus the E3 ubiquitin ligase viral protein ICP0 temporarily localizes to centromeres (Everett et al., 1999). Step 2: CENP-A, -B, -C and proteins from the NAC

and the CAD are degraded in an ICP0- and proteasome-dependent manner (Everett et al., 1999; Gross et al., 2012; Lomonte and Morency, 2007; Lomonte et al.,

2001), which destabilizes the centromeric chromatin resulting in loss of nucleosomes (Gross et al., 2012). Simultaneously, ICP0 leaves the centromeres and coilin

and fibrillarin independently relocate to the damaged centromeres to initiate the iCDR (Gross et al., 2012; Morency et al., 2007; and this study). Step 3:

enlargement of the region of the centromeric chromatin (box region in the Step2 drawing). SMN relocates to the damaged centromeres in a coilin- and DOT1L-

dependent manner, and binds to histone H3 methylated on K79. We do not currently know if DOT1L accumulates at damaged centromeres because of the lack of

DOT1L antibodies suitable for immunofluorescence. Step 4: the iCDR leads to a hypothetical repair of the centromere structure or to the death of the cell.

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absence of certain post-translational modifications of coilin inHeLaPV cells could affect its activity at centromeres, which

would indirectly affect centromeric SMN accumulation. Forinstance, the phosphorylation status of coilin might be different in

HeLaPV cells, weakening its interaction with SMN. It would beinteresting to assess the requirement of coilin phosphorylation forthe targeting of both coilin and SMN at centromeres. SMN is a

Tudor protein that interacts preferentially with sDMA of non-histone proteins, including coilin (Boisvert et al., 2002; Cote and

Richard, 2005; Liu et al., 2012; Tripsianes et al., 2011). Inmammalian cells, Tudor domains are found in about 30 proteins,

which differ in terms of their capacity to bind either methyl-K ormethyl-R residues (Chen et al., 2011; Taverna et al., 2007; Yapand Zhou, 2010). So far, none of these proteins has been shown to

interact with both methylated K and R. Our study shows thatSMN, in addition to its previously described capacity to interact

with methylated R, also interacts with H3K79me1,2 through itsfunctional Tudor domain. A recent in vitro study described theinability of the SMN Tudor domain to bind to methyl-K residues

in synthetic peptides, as measured by isothermal titrationcalorimetry (ITC) binding assays (Tripsianes et al., 2011). In

that study, a truncated version of SMN spanning the Tudordomain (aa 84 to 147) was used to perform ITC assays with

peptides harboring methyl-R or -K residues. In accordance withthese results, our data show that the inactivation of the H4K20methyltransferase PR-Set7, unlike DOT1L inactivation, does not

prevent relocation of SMN to damaged centromeres, and in factno binding of SMN to H4K20me2 was detected. This suggests

that the amino acid environment of the recognized methyl-K isimportant. It is also likely that the SMN Tudor domain, when

contained within the functional full-length protein, possessesadditional binding properties towards methyl-K. We showedthat SMN_Dex7, although it contains an intact Tudor domain,

was significantly affected for its accumulation at damagedcentromeres. SMN_Dex7 has a reduced ability to self-

oligomerize, which confers different biological functions on theprotein (Lorson et al., 1998). Because basic Tudor domains, suchas that in SMN, are likely to function in a protein homodimer to

recognize ligands harboring methyl-R residues (Tripsianes et al.,2011), it is possible that SMN dimerization is required for its

Tudor domain to be fully compatible with methyl-K residues. Inaddition, we showed that an SMN protein mutated in its Tudor

domain (SMN_E134K), which matches an SMN mutant proteinfound in SMA patients (Lefebvre et al., 1998), did not bind toH3K79me1,2, which suggests that the SMN Tudor domain has

specificity towards methylated H3K79. We also showed that afunctional Tudor domain was required for SMN to efficiently

accumulate at damaged centromeres. This suggests that the SMNTudor domain is essential for centromeric SMN activity during

iCDR.

Other important data supporting the link between H3K79meand SMN show a significant decrease in the number of ICP0-

expressing cells with centromeric SMN upon DOT1Lknockdown. The yeast protein DOT1 and its human homolog,

DOT1L, are the only known methyltransferases with specificityfor H3K79 (Nguyen and Zhang, 2011; Steger et al., 2008). Itsabsence in knockout yeast, flies, and mice results in the complete

loss of all forms of methylated H3K79 (Jones et al., 2008;Shanower et al., 2005; van Leeuwen et al., 2002). The

methylation of H3K79 by DOT1/DOT1L results from trans-histone cross-talk that was initially found in combination with

mono-ubiquitylation of H2B at K123 and K120 in yeast andmammals, respectively (Briggs et al., 2002; Krogan et al., 2003;

Ng et al., 2003b; Ng et al., 2002; Sun and Allis, 2002; Wood

et al., 2003). However, recent reports showed that several othertypes of H2B K ubiquitylation could lead to H3K79 methylation

(Chatterjee et al., 2010; Whitcomb et al., 2012; Wu et al., 2011).Therefore, although exclusively catalyzed by DOT1/DOT1L, the

methylation of H3K79 is a complex event in which severalprotein complexes are involved. H3K79 methylation is a

hallmark of actively transcribed chromatin, but is also involvedin DNA damage repair (DDR) of double-strand breaks (DSB) and

ultraviolet radiation-associated DNA lesions (Giannattasio et al.,2005; Huyen et al., 2004; Ng et al., 2003a; Schubeler et al., 2004;

Steger et al., 2008). SMN is unlikely to be recruited to damagedcentromeres due to NER or a DSB-associated H3K79me-

dependent DDR mechanism, as occurs with 53BP1, anotherTudor domain protein (Giannattasio et al., 2005; Huyen et al.,

2004). Indeed, proteins implicated in various repair mechanisms

(non-homologous end joining, nucleotide excision repair,homologous recombination) and phosphorylation of H2Ax at

S139 (a hallmark of DDR) do not accumulate at centromeresduring the iCDR (Morency et al., 2007). One study reported that

H3K79me2 associates with centromeric minor satellite chromatinin mouse cells (Jones et al., 2008). Together with other

heterochromatin marks (H3K9me2 and H4K20me3), levels ofthis histone mark are significantly reduced in ES cells from

DOT1L-knockout mice (Jones et al., 2008). This results in theaccumulation of aneuploid cells during mouse embryogenesis

and in death of the embryos at around 10 days post-coitum (Joneset al., 2008). No such data are available for centromeric

chromatin in human cells. However, our ChIP experiments

using SMN obtained from ICP0-expressing cells as the targetconfirmed that SMN interacts with H3K79me1,2 in the

centromeric nucleosome, at least during the iCDR. At thisstage, we cannot tell whether methylated H3K79 is associated

with centromeric nucleosomes before ICP0 is expressed (itsaccessibility is increased after ICP0 chromatin destabilization

(Gross et al., 2012), or whether destabilization of centromeresby ICP0 activates DOT1L, which in turn induces H3K79

methylation in centromeric nucleosomes. ICP0 is a RINGfinger (RF) protein whose RF-associated E3 ubiquitin ligase

activity has been described in vivo and in vitro (Boutell andEverett, 2003; Boutell et al., 2002; Hagglund and Roizman,

2004). Many ICP0 substrates have been identified, but given themany interactions of ICP0 with cellular proteins, it is highly

unlikely that all its partners and substrates have been discovered.

One can hypothesize that ICP0 directly or indirectly induces H2BK ubiquitylation, which in turn activates DOT1L to induce

methylation of H3K79. During the iCDR, coilin acts upstream ofSMN and is required for the presence of SMN at centromeres.

This suggests that whatever the effect of ICP0 on histonemodifications, other epigenetic features, probably associated with

the presence of coilin, induce trans-histone cross-talk leading tothe activation of DOT1L, the methylation of centromeric H3K79,

and finally the targeting of SMN to damaged centromeres.Multiple attempts to detect a direct interaction between in vitro

synthesized SMN and biotinylated/methylated H3K79 peptidesfailed. Therefore, we cannot rule out an interaction between SMN

and DOT1L or one of its partners that would connect SMN tomethylated H3K79. However, (i) DOT1L does not contain an RG

box and is not known to be methylated at R residues, which

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would account for a possible interaction with the SMN Tudordomain; (ii) no studies conducted to detect DOT1/DOT1L

partners revealed the presence of SMN in DOT1/DOT1L

protein complexes (Bitoun et al., 2007; Mohan et al., 2010;Mueller et al., 2007; Mueller et al., 2009; Park et al., 2010); (iii)

SMN does not bind to H3K79me3 whose formation is alsocatalyzed by DOT1/DOT1L; and (iv) SMN functional Tudor

domain is required for its interaction with methylated H3K79. Allthese data highly suggest that SMN directly bind to methylated

H3K79. Although SMA is one of the most studied geneticdisorders, the mechanism that leads to motoneuron loss is poorly

understood. Because SMN is involved in the biogenesis ofspliceosomal snRNPs, many studies of molecular aspects of SMA

are focused on deciphering the impact of SMN loss on snRNPbiogenesis and activities (Chari et al., 2009; Gabanella et al.,

2007; Imlach et al., 2012; Lotti et al., 2012; Pellizzoni, 2007;Winkler et al., 2005; Workman et al., 2009; Zhang et al., 2008).

SMN makes a crucial contribution to the activity of the splicing

machinery by interacting with spliceosome-associated proteins,whereas methylation of H3K79 is positively correlated with the

transcription process. It is now widely accepted that maturationof nascent primary transcripts, including splicing, could occur co-

transcriptionally (Kornblihtt, 2007; Manley, 2002; Neugebauer,2002; Pandit et al., 2008; Perales and Bentley, 2009). Recently,

another level in the cross-talk between transcription and splicinghas been identified: histone modifications of chromatin

(Alexander and Beggs, 2010; Brown et al., 2012; Hnilicovaand Stanek, 2011; Luco et al., 2011). Notably, H3K79me1 was

found to be enriched, together with other histone modifications,in nucleosomes positioned over exons, suggesting that H3K79

methylation has a role in splicing (Andersson et al., 2009; Dhami

et al., 2010; Ernst and Kellis, 2010; Schwartz et al., 2009).Although the hypothesis that widespread splicing abnormalities

cause SMA is still a matter of debate (Baumer et al., 2009;Praveen et al., 2012), the requirement for functional SMN during

early postnatal development for the splicing of specifictranscripts involved in motoneuron survival has been suggested

(Baumer et al., 2009; Zhang et al., 2008). Accordingly, it hasrecently been shown that SMN loss-of-function affects the U12

splicing pathway in Drosophila, mammalian cells and humanlymphoblasts from SMA patients (Boulisfane et al., 2011; Lotti

et al., 2012). U12 splicing pathway is related to the so calledminor spliceosome involved in the processing of a small

proportion (about 1%) of introns (Patel and Steitz, 2003).Therefore, one of the ideas currently proposed is that the

absence of functional SMN may selectively target genes

processed through specific pathways; U12-dependent splicingbeing one of them (Imlach et al., 2012; Lotti et al., 2012). Even

so, not all U12 intron-containing genes are affected by theabsence of SMN (Lotti et al., 2012), and other studies in

Drosophila suggested that viability defects in Smn null mutantsare unlikely due to minor spliceosome level perturbations

(Praveen et al., 2012). By demonstrating for the first time thecapacity of SMN to bind to H3K79me1,2-containing chromatin,

our study suggests an additional molecular level concerning therole of SMN in SMA. It is indeed tempting to speculate that

beside regulating a specific splicing pathway, SMN plays anessential role in motoneuron and/or muscle cell homeostasis by

connecting H3K79 methylation-dependent gene expression andsplicing of particular transcripts, some of which potentially

involved in motoneuron and/or muscle cell survival.

Materials and MethodsCells and plasmids

HeLa cells were grown at 37 C in Glasgow’s modified Eagle’s medium. HeLaPV,HeLa TREX (TR, Invitrogen), TREX-ICP0 (TR-ICP0), and TREX-FXE cells weregrown at 37 C in DMEM. All media were supplemented with 10% fetal bovineserum, L-glutamine (1% v/v), 10 U/ml penicillin, and streptomycin. For T-REXcells, blasticidin (5 mg/ml) was added to the medium; for TR-ICP0 and TR-FXEcells, blasticidin and zeocin (100 mg/ml) were added. TR-ICP0 and TR-FXE celllines, which stably and inducibly express ICP0 and FXE, respectively, wereconstructed by transfecting T-REX cells with a pcDNA 4/TO plasmid (Invitrogen),which was designed to express ICP0 or FXE. Cell clones were then isolated underZeocin selection. Expression of ICP0 or FXE was induced by addition oftetracycline (1 mg/ml) to the medium and was confirmed by western blotting andimmunocytochemistry (Gross et al., 2012).

The pci110 plasmid expressing ICP0 protein, has been described previously(Everett et al., 1993). The plasmid pHA-coilin was constructed by in-frame cloningof the coilin cDNA into a pcDNA3.1 plasmid containing the HA tag sequence.pHA-coilinDRG expresses a coilin protein in which amino acids (aa) 388–419(including its RG box) are deleted. Plasmids expressing various EGFP-taggedSMN proteins (pEGFP-SMN, pEGFP-SMNDex7, pEGFP-SMN_472D5, pEGFP-SMN_N86) have been described previously (Renvoise et al., 2006) and were agenerous gift from S. Lefebvre (Institut Jacques Monod, Paris). The plasmidpGFP-SMN_E134K was constructed from pGFP-SMN by introduction of a singlemutation that replaces the glutamine residue at position 134 with a lysine residueusing the QuickChange Site-Directed Mutagenesis Kit (Stratagene). Plasmidsexpressing GST-SMN and GST-SMN_E134K were constructed by in-framecloning of SMN and SMN_E134K cDNA into pGEX vectors (Clontech). All newconstructs were checked for their sequences and in-frame cloning by sequencing.

Cellular and histone extracts, GST pulldown, peptide pulldown, andwestern blotting

To obtain cellular extracts, cells were washed once with phosphate-buffered saline(PBS), scraped off the flask and centrifuged at 1000 g for 5 minutes. The resultingcell pellet was resuspended in 200 ml of lysis buffer [15 mM Tris-HCl (pH 7.5),2 mM EDTA, 0.25 mM EGTA, 15 mM NaCl, 0.3 mM sucrose, 0.5% Triton X-100, 300 mM KCl, protease inhibitor cocktail; Roche]. Samples were incubated onice for 30 minutes, and centrifuged at 6000 g for 5 minutes at 4 C to remove alldebris. Histone extraction was performed by scraping cells off the plates,resuspending them in PBS-phenylmethylsulfonyl fluoride (PMSF) and sonicatingthem in an ultrasonic water bath twice for 15 seconds. Nuclei and debris werecentrifuged at 12,000 g for 10 minutes at 4 C. Pellets were resuspended in HCl(250 mM) and incubated on ice for 30 minutes before centrifugation at 12,000 g

for 20 minutes at 4 C. Trichloroacetate was added to the supernatant at a finalvolume of 20%, and incubation was carried out for a further 30 minutes on icebefore centrifugation at 12,000 g for 20 minutes at 4 C. Pellets were washed oncewith 500 ml of a mixed acetone/HCl (10 mM) solution and once with acetonealone, and were then dried in a SpeedVac concentrator (Savant) for 5 minutesbefore resuspension in water. For GST pulldown assays, 5 mg of GST fusionprotein preparation was mixed with 10 mg of histone extract in Bis-Tris-propane(BTP) buffer [25 mM BTP (pH 6.8), 0.5% Triton X-100, 1 M KCl, proteaseinhibitor cocktail]. All extracts were first incubated for 1 hour at 4 C withcontinuous mixing with beads linked to the GST protein alone in order to reducethe background signal. The pre-cleared extracts were then incubated overnight at4 C with continuous mixing with the appropriate GST fusion protein beads or anegative GST bead control. The beads were harvested by brief centrifugation, andwere then washed five times with BTP buffer [25 mM BTP (pH 6.8), 1 M KCl].Protein complexes were directly eluted from the beads by adding Laemmli bufferand were then boiled.

For peptide pulldown, 5 mg of biotinylated/methylated peptides correspondingto histone H3K79, H3K79me1 or H3K79me2 (aa 61–90; biot-lirklpfqrlvreiaqdfk79tdlrfqssavm; ProteoGenix) or histone H4 [aa 2–24; Millipore(12-372)] were first incubated with streptavidin–agarose beads (Pierce) for 2 hoursat 4 C in PBS. The beads were then extensively washed with PBS to removeunbound peptides. Cellular extracts were then added to the beads/peptides andincubated in BTP buffer [25 mM BTP (pH 6.8) 0.5 M KCl, protease inhibitorcocktail] for 18 hours at 4 C. Samples were washed six times with BTP buffer andthen Laemmli buffer was added. For western blotting (WB), samples were loadedon SDS-polyacrylamide gels before electrophoresis, transfer, and detection usingappropriate antibodies.

Chromatin immunoprecipitation assays

Cells were seeded on Petri dishes (56106 per dish). The following day, ICP0expression was induced by adding tetracycline (1 mg/ml) to the medium for24 hours. Formaldehyde was added to the medium at a final concentration of 0.5%for 5 minutes at room temperature (RT). Cells were washed twice with cold PBS,scraped off the dish, resuspended in 1 ml of buffer A [100 mM Tris-HCl (pH 9.4),10 mM DTT] at a concentration of 56106 cells per ml, and incubated at roomtemperature (RT) for 15 minutes. They were then incubated at 30 C for a further

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15 minutes and centrifuged at 1000 g. Cells were lysed in 1 ml of buffer B[10 mM HEPES (pH 6.5), 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100] at4 C for 5 minutes and centrifuged at 3000 g. They were then resuspended in 1 mlof buffer C [10 mM HEPES (pH 6.5), 10 mM EDTA, 0.5 mM EGTA, 200 mMEGTA] and incubated at 4 C for 5 minutes, before centrifugation at 3000 g. Nucleiwere then resuspended in 300 ml of buffer D [50 mM Tris-HCl (pH 8), 10 mMEDTA, 1% SDS, protease inhibitor cocktail; Roche] and incubated at RT for10 minutes, before sonication with a Bioruptor device (Diagenode). The samplevolume was adjusted to 3 ml with buffer D9 [50 mM Tris-HCl (pH 8), 10 mMEDTA; protease inhibitor cocktail; Roche] and an antibody specific for SMN(2 mg/ChIP) was added before incubation overnight at 4 C. Salmon sperm DNA/protein G agarose (200 ml; 50% slurry; Millipore) was added to each sample at 4 Cfor 2 hours. Then, the beads were washed five times with buffer D, and Laemmlibuffer was added. De-crosslinking was performed by heating the samples at 95 Cfor 90 minutes. Samples were then loaded on SDS-PAGE gels for WB.

siRNA transfection

Cells were seeded at a very low density before siRNAs transfection. Two rounds ofsiRNA transfection with Effectene Transfection Reagent (Qiagen) were performedat 48-hour intervals, according to the manufacturer’s protocol. The amount ofsiRNA was adjusted to the number of cells. At the end of the experiment, cellswere harvested and protein levels were quantified. The proteins were thenresuspended in Laemmli buffer and analyzed by WB.

The following siRNAs were used: control, 59-UACAGCUCUCUCUCGACCC-39; coilin_1, 59-CGACUGCCUCAGAGUUAAA-39; coilin_2, 59-CCAACUG-AGUUAUCAAAGG-39; fibrillarin_1, 59-UGAGGGUGUCUUCAUUUGU-39;fibrillarin_2, 59-GGACCAACAUCAUUCCUGU-39; SMN_1, 59-GCAUGCUC-UAAAGAAUGGU-39; SMN_2, 59-AGACGGUUGCAUUUACCCA-39; DOT1L,59-GCUCGCUAUGGAGAAUUAC-39; PR-Set7, 59-AAUCGCCUAGGAAGA-CUGAUC-39; and PRMT5, 59-GGCCAUCUAUAAAUGUCUG-39. The siRNAstargeting PRMT1 (L-010102), PRMT3 (L-026786), PRMT4 (L-004130) andPRMT6 (L-007773) we used were ON-TARGET plus SMARTpool siRNAs(Thermo Scientific).

RNA extraction and RT-qPCR

HeLa cells not-transfected or transfected with control siRNA (siCtrl) or siDOT1Lfollowing the above described procedure were harvested, and total RNA extractedusing Nucleosin RNA II columns (Macherey-Nagel). Reverse transcription (RT)was performed with 1 mg of RNA from each sample and using random primer[0.2 mm] and 200 units of Revert-Aid H M-MulV Reverse Transcriptase(Fermentas). Quantitative PCR was performed on cDNAs obtained from the RT,using light cycler 480 SYBR green I Master (Roche), and ran on a MX 3000 Papparatus (Stratagene). Data were normalized on human beta actin.

Primers used for the qPCR were as follow: DOT1L_fwd: 59-CATCC-GATGGGTCTGTGA-39, DOT1L_rev: 59-TGGTGTCATAGTCAATTAAA-ACGTAATTC-39, b-actin_fwd: 59-CGGGAAATCGTGCGTGACATTAAG-39,b-actin_rev: 59-GAACCGCTCATTGCCAATGGTGAT-39.

Immunofluorescence and confocal microscopy

IF was performed as described previously (Morency et al., 2007). Cells wereseeded at 1.56105 per well in 24-well plates containing a round coverslip prior toIF analysis. All samples were examined under a meta-confocal microscope (LSM510; Carl Zeiss, Wetzlar, Germany) at a resolution of 5126512 pixels using 0.8–1.0-mm-thick optical slices. A Zeiss Axiovert 200 M microscope was used at 636(NA 1.25) or 1006 (NA 1.3) magnification under oil-immersion objectives.Datasets were processed using LSM 510 software, and then ImageJ software(Rasband, W.S., ImageJ, US, National Institutes of Health, Bethesda, Maryland,USA. http://imageJ.nih.gov/ij/) and Adobe Photoshop.

Antibodies

The following antibodies were used: mouse monoclonal antibodies (mAbs) againstcoilin (Sigma), fibrillarin [38F3] (Abcam) and [72B9] (a kind gift from J. Cavaille,LBME, Toulouse), SMN (BD Biosciences), ICP0 [11060] (a kind gift from R.D.Everett, CVR-Glasgow), and H4 (clone 62-141-13; Millipore); rabbit polyclonalantibodies against actin (Sigma), coilin (coilin 20410; Dundee Cell Products), GFP(Roche), PRMT1 (Millipore), PRMT3 (ab3765; Abcam), PRMT4/CARM1 (CellSignaling, kindly provided by Muriel Leromancer, CRCL, Lyon), PRMT5(Millipore), PRMT6 (ab47244; Abcam); PR-Set7 (kindly provided by E. Julien,IGMM, Montpellier), H3 (Millipore), H3K79me1 (pAb-082; Diagenode), anti-H3K79me2 (ab3594, Abcam), anti-H3K79me3 (pAb-068, Diagenode),H4K20me2 (Millipore), H4R3me2s (ab5823; Abcam), and dimethyl-argininesymmetric (SYM10; Millipore); and a human autoimmune serum (huACA) againstcentromeric proteins (Antibodies Incorporated). For IF, the secondary antibodiesused were goat anti-rabbit, anti-mouse, and anti-human antibodies coupled toAlexa Fluor 488, 555, or 647 (Molecular probes). For WB, the secondaryantibodies used were rabbit anti-mouse and swine anti-rabbit antibodies coupled tohorseradish peroxidase (Dako).

AcknowledgementsWe thank Suzie Lefebvre of Institut Jacques Monod, Paris forproviding the EGFP-SMN-expressing plasmids; Gregory Matera ofthe University of North Carolina, Chapel Hill for providing theHeLaPV cells; and Jerome Cavaille of LBME, Toulouse, MurielLeromancer of CRCL, Lyon, and Eric Julien of IGMM, Montpellierfor providing antibodies. The Zeiss LSM 510 confocal microscopeimages were captured at the Centre Technologique desMicrostructures, Plateforme de l’Universite Claude Bernard Lyon1. P.L. is a CNRS researcher.

Author contributionsM.S., P.T. and P.L. conceived and designed experiments; M.S., P.T.,J.E.M. and P.L. performed the experiments; M.S., P.T., J.E.M. andP.L. analyzed the data; M.S., P.T., J.E.M. and P.L. contributedreagents/materials/analysis tool; and P.L. wrote the paper.

FundingThis work was supported by the Association Francaise contre lesMyopathies [Trampoline grant number 15033 to P.L.]; the FINOVIFoundation [grant number 004 to P.L.]; the Association pour laRecherche contre le Cancer [grant number ARC-7979 and ARC-4910 to P.L.]; the Ligue Nationale Contre le Cancer; the LabEXDEVweCAN of the Universite de Lyon, within the program‘Investissements d’Avenir’ operated by the French NationalResearch Agency [grants numbers: ANR-10-LABX-61 and ANR-11-IDEX-0007]; the Centre National de la Recherche Scientifique;and a studentship from the Ligue National Contre le Cancer (Comitedu Rhone) to M.S.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.126003/-/DC1

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SMN interaction with nucleosomal H3K79me1,2 3677