# Systematic yeast two-hybrid screening identifies novel functions for SET1C/COMPASS

Pierre Luciano, Kihyun Park, Stéphane Audebert, Luc Camoin, Carlos A Niño, da Kyeong Park, Isabella E Maudlin, Marion Dubarry, Lara Lee, Marlene Oeffinger, et al.

► To cite this version:

Pierre Luciano, Kihyun Park, Stéphane Audebert, Luc Camoin, Carlos A Niño, et al. Systematic yeast two-hybrid screening identifies novel functions for SET1C/COMPASS. 2025. hal-05398521

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# Systematic yeast two-hybrid screening identifies novel functions for SET1C/COMPASS

## Short title: SET1C methylates the ARTSTRGR motif of Snf2 AT-hook

Pierre Luciano¹, ¶, Kihyun Park², ¶, Stéphane Audebert¹, Luc Camoin¹, Carlos A. Niño¹, Da Kyeong Park³, Isabella E. Maudlin⁴, Marion Dubarry¹, Lara Lee¹, Marlene Oeffinger⁵,⁶,⁷, Jean D. Beggs⁴, Young Hye Kim³, Jaehoon Kim²,*, Bernhard Dichtl⁸,*, Vincent Géli¹,*.

¹Marseille Cancer Research Center (CRCM), U1068 Inserm, UMR7258 CNRS, Aix Marseille University, Institut Paoli-Calmettes, France. Equipe labellisée Ligue.

²Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, South Korea.

³Ochang Institute of Biological and Environmental Science, Korea Basic Science Institute, Cheongju 28119, South Korea

⁴Wellcome Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3BF, UK.

⁵ Institut de recherches cliniques de Montréal, Center for Genetic and Neurological Diseases, 110 avenue des Pins Ouest, Montréal, Québec, H2W 1R7, Canada

⁶Département de biochimie et médecine moléculaire, Faculté de Médicine, Université de Montréal, Québec, H3C 3J7, Canada

⁷Division of Experimental Medicine, Faculty of Medicine, McGill University, Montréal, Québec, H4A 3J1, Canada

⁸School of Life and Environmental Sciences, Deakin University, Geelong, Australia.

* To whom correspondence should be addressed

vincent.geli@inserm.fr

kimjaehoon@kaist.edu

bernhard.dichtl@deakin.edu.au

¶ Joint authors

Present Addresses:

Isabella E. Maudlin, Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK.

Marion Dubarry, Univ Lyon, Université Claude Bernard Lyon 1, INSA-Lyon, CNRS, UMR5240, Microbiologie, Adaptation et Pathogénie, 10 rue Raphaël Dubois, F-69622, Villeurbanne, France

Carlos A. Niño, IFOM ETS, The AIRC Institute of Molecular Oncology, Milan, Italy.

Vincent Géli, Institute for Research on Cancer and Aging, Nice (IRCAN), Nice, France
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ABSTRACT

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INTRODUCTION

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Set1 is the catalytic subunit of SET1C or COMPASS, which methylates histone H3K4 and serves as a scaffold for the association of seven tightly bound polypeptides. We have employed yeast two-hybrid screenings to determine the interactome of Set1 and each subunit, providing a unique resource for exploring known and novel roles of the complex. Our screenings identified a multitude of interactors involved in chromatin regulation, DNA replication, meiotic breaks, and Ty transposition, processes previously associated with SET1C. Consistent with Set1 being an RNA-binding protein, the screens link SET1C to multiple aspects of RNA biogenesis, including pre-mRNA splicing and polyadenylation. The results reveal that Set1 interacts with several importins and with RGG motif-containing proteins, providing insights into the mechanisms by which Set1 moves between cytoplasmic and nuclear compartments. We demonstrate that the transcriptional corepressor Nrm1 is methylated by SET1C *in vitro* suggesting that H3K4-like domains may represent a class of non-histone substrates for SET1C. We further reveal that reconstituted SET1C interacts with the AT hook domain of the chromatin remodeler Snf2 and methylates multiple arginines within this domain. *In vivo*, we report that the ARTSTRGR AT-hook motif is methylated in a Set1-dependent manner revealing new interplay between lysine and arginine methylation.

Post-translational modifications of histones shape the chromatin landscape and provide essential mechanisms of regulating DNA accessibility, thereby controlling gene expression, genome maintenance and transmission. Multiprotein complexes of the SET/MLL family methylate histone H3 on lysine 4 (H3K4), which modulates multiple aspects of genome biology (Ruthenburg et al, 2007). Each complex contains additional factors specifying their recruitment to explicit chromatin domains and their specific biological effects (Cenik & Shilatifard, 2021). The budding yeast Set1 complex called COMPASS (for Complex of Proteins Associated with Set1) or SET1C has proved to be an excellent model to study the SET1/MLL family complexes. In *Saccharomyces cerevisiae*, all H3K4 methylation is carried out by SET1C that is composed of Set1, the catalytic subunit, acting as a scaffold for seven other components (Swd1 [RbBP5], Swd2 [WDR82], Swd3 [WDR5], Bre2 [ASHL2], Sdc1 [DPY30], Spp1 [CFP1] and Shg1 [BOD1]) (Nagy et al, 2002; Briggs et al, 2001; Roguev et al, 2001; Miller et al, 2001). Swd1, Swd3, Bre2 and Sdc1 copurify with the SET domain of Set1, yet none of these proteins can interact alone with the SET domain, suggesting that interactions between subunits are required for catalytic domain formation (Kim et al, 2013). The crystal structure of the SET domain of Set1 associated with Swd1, Swd3, Bre2, Sdc1 shows that the catalytic module is organized by Swd1, whose C-terminal tail nucleates Swd3 and a sub-complex formed by Bre2-Sdc1 (Qu et al, 2018; Hsu et al, 2018). For its side, Spp1 associates with the N-SET domain of Set1, while Shg1 binds to the central region of Set1 and Swd2 contacts the N-terminal region of Set1 (Kim et al, 2013; Roguev et al, 2001; Dehe et al, 2006; Halbach et al, 2009). Swd1 contacts Spp1 and also interacts with the N-terminal region of Set1, suggesting that the C- and N-terminal regions of Set1 may interact with each other (Qu et al, 2018; Acquaviva et al, 2013b; Jeon et al, 2018). Such SET1C organization was
confirmed by cryo-electron microscopy and cross-linking experiments performed on the full-length complex (Wang *et al*, 2018). Interestingly, SET1C has a remarkable mode of assembly that is initiated in the cytoplasm while the nascent Set1 polypeptide emerges from the ribosome. Set1 is initially bound during its translation, by Shg1, Spp1 and Swd1, then Swd2, Swd3, Bre2 and Sdc1 associate with the initial pre-complex to form the full SET1C. This explains why Set1 is associated with its own mRNA (Luciano *et al*, 2017; Halbach *et al*, 2009).

Swd2, the only essential SET1C subunit, is also part of the APT complex (for Pta1-associated), a subcomplex of the Cleavage Polyadenylation Factor (CPF) involved in the transcriptional termination of mRNA and snoRNA suggesting a functional link between SET1C and 3' end formation/termination (Cheng *et al*, 2004; Nedea *et al*, 2003; Dichtl, 2004). Set1 has also been linked genetically to the Nrd1-Nab3-Sen1 (NNS) complex (Kim *et al*, 2006; Arigo *et al*, 2006), but intriguingly, it was reported to have two seemingly contradictory effects on ncRNA termination: on the one hand Set1 was suggested to promote efficient snoRNA termination mediated by Nrd1 (Terzi *et al*, 2011), and on the other hand it appeared to interfere with the early termination of a large number of ncRNAs (Castelnuovo *et al*, 2014; Margaritis *et al*, 2012). Interestingly, various lysines distributed between Nrd1, Nab3 and Sen1, are methylated, notably lysine 363 in the RNA Recognition Motif (RRM) of Nab3 whose mono methylation depends on Set1 and Set3 (Lee *et al*, 2020).

Set1 interacts co-transcriptionally with the RNA polymerase II Carboxy Terminal Domain (PolII CTD) phosphorylated on Ser5 of the heptad repeats, producing an H3K4 methylation gradient that starts at nucleosome +1 and fades away from the promoter (Soares *et al*, 2017; Ng *et al*, 2003). It was recently shown that the N-terminal region of Set1 and Swd2 interact cooperatively with the CTD (Carboxy Terminal Domain) of Rbp1 (RNA polymerase II large subunit) promoting SET1C recruitment to transcription elongation complexes at the 5' ends of genes (Bae *et al*, 2020). Deletion of residues 200-210 of Set1 abolishes interaction with the Rbp1-CTD and Swd2, while deletion of the first 200 amino acids of Set1 strongly reduces trimethylation of H3K4 (H3K4me3) at the 5' end of transcribed genes (Bae *et al*, 2020). Interestingly, Swd2 is ubiquitinated by Rad6/Bre1 and preventing Swd2 ubiquitination affects H3K4 trimethylation with a concomitant reduction in Spp1 recruitment to chromatin suggesting a cross-talk between Swd2 and Spp1 (Vitaliano-Prunier *et al*, 2008). In a similar vein, di- and tri-methylation of H3K4 by Set1, which depends on prior ubiquitination of histone H2B by Rad6/Bre1 (Dover *et al*, 2002; Sun & Allis, 2002), requires a contact between Spp1 and Swd1 (Hsu *et al*, 2019; Jeon *et al*, 2018). Other modes of SET1C recruitment to chromatin must exist in the absence of the Set1 N-terminal region, probably via direct interactions between SET1C's catalytic domain and the nucleosome (Jeon *et al*, 2018; Dehé & Géli, 2006; Thornton *et al*, 2014).

The central region of Set1 contains two RRMs positioned in tandem (Trésaugues *et al*, 2006) through which Set1 binds directly to RNA. This binding contributes to retain Set1 in the 5' region of genes thus facilitating their H3K4 trimethylation (Luciano *et al*, 2017; Battaglia *et al*, 2017; Sayou *et al*, 2017). Both the N-SET domain and Spp1 also contribute to Set1 binding to RNA. Set1 associates post-transcriptionally with transcripts produced by specific classes of genes including snRNA, Ty1 and a number of genes involved in adaptive responses (Luciano *et al*, 2017). The dRRM itself is flanked by an autoinhibitory region that
134 negatively regulates H3K4me3 (Schlichter & Cairns, 2005). It is possible that in the context
135 of full-length Set1, the alternative mode of SET1C recruitment may be inhibited by the
136 autoinhibitory domain.

137 In S. cerevisiae, H3K4me3 methylation is counteracted by the demethylase Jhd2, a
138 conserved JARID1 family protein (Huang et al, 2010; Liang et al, 2007). The erasure of
139 ancestral histone methylation states results from both active enzymatic demethylation by Jhd2
140 and passive dilution of parental histones during replication (Radman-Livaja et al, 2010).
141 SET1C is a dimer and its dimerization depends on the Sdc1 subunit (Choudhury et al, 2019).
142 It has been proposed that the symmetrical methylation of H3K4 on nucleosomes is a
143 consequence of the dimeric nature of SET1C and that Jhd2 preferentially demethylates
144 asymmetrical H3K4me3 (Choudhury et al, 2019). Interestingly, H3K4me3 at environmental
145 stress genes depends on H3-P16 isomerisation, a process that controls K4me3 by balancing
146 the actions of Jhd2 and Spp1 (Howe et al, 2014).

147 The complexity of the S. cerevisiae SET1C, the specific arrangement of subunits within
148 the complex, and the specific roles of certain subunits, notably Spp1 and Swd2 (Acquaviva et
149 al, 2013a), raise the question of the contribution of SET1C and of its subunits to multiple
150 processes. Overall, H3K4 methylation, SET1C and its individual subunits have been involved
151 in multiple processes in S. cerevisiae such as meiotic recombination (He et al, 2019; Sollier et
152 al, 2004; Borde et al, 2008; Acquaviva et al, 2013b; Sommermeyer et al, 2013; Karányi et al,
153 2018; Adam et al, 2018), stress response and epigenetic transcriptional memory (D'Urso et al,
154 2016; Kim & Buratowski, 2009; Weiner et al, 2012), DNA repair (Faucher & Wellinger,
155 2010), telomere and rDNA silencing (Jezek et al, 2023; Corda et al, 1999; Briggs et al, 2001;
156 Bryk et al, 2002; Santos-Rosa et al, 2004; Nislow et al, 1997), cell wall biogenesis (Nislow et
157 al, 1997), chromosome segregation (Beilharz et al, 2017; Zhang et al, 2000), antisense
158 transcription (Murray et al, 2015; van Dijk et al, 2011; Margaritis et al, 2012; Castelnuovo et
159 al, 2014), transcription termination (Kaczmarek Michaels et al, 2020; Nedea et al, 2003;
160 Dichtl, 2004; Cheng et al, 2004; Nedea et al, 2008; Terzi et al, 2011; Soares & Buratowski,
161 2012; Castelnuovo et al, 2013; Lee & Wang, 2018), Ty silencing (Luciano et al, 2017;
162 Berretta et al, 2008), chronological aging (Gong et al, 2023; Walter et al, 2014; Mei et al,
163 2019), ergosterol homeostasis (South et al, 2013), stress response (Deshpande et al, 2022;
164 Nadal-Ribelles et al, 2015), lipid homeostasis (Giaever et al, 2019), and DNA replication
165 (Ghaddar et al, 2023; Sollier et al, 2004; Rizzardi et al, 2012; Chong et al, 2020; Delamarre et
166 al, 2020; Santos-Rosa et al, 2021; de La Roche Saint-André & Géli, 2021; Serra-Cardona et
167 et al, 2022).

168 The multiple roles of Set1 and its subunits led us to perform global two-hybrid screening to
169 identify interactors either of Set1-full length, or of its N- and C-terminal regions and of each
170 of the individual subunits (Swd2, Shg1, Spp1, Swd1, Swd2, Sdc1, Bre2). The identification
171 of interactors is discussed not only for each bait, but also as a whole, revealing new potential
172 functions for the complex. In addition, we demonstrate that the transcriptional corepressor
173 Nrm1 and the Snf2 AT-hook are both methylated by SET1C in vitro. In vivo, deleting SET1
174 abrogates arginine methylation within the ARTSTRGR motif of Snf2 AT-hook. These results
175 suggest that H3K4-like sequences may represent a class of non-histone substrates for SET1C
176 and reveal new interplay between Lysine and arginine methylation. This work is an invaluable
177 resource for further exploring known and unsuspected roles of the SET1C complex and its
178 subunits.

179

180 RESULTS

181 Identification of Set1 and Set1 subunits interactors

182 We produced a total of ten yeast two-hybrid (Y2H) screens (Hybrigenics, See Methods). For
183 the Set1 protein, we performed a total of three screens using as bait the full-length Set1
184 protein (Set1 FL), the amino-terminal region including amino acids 1-754 or a C-terminal
185 fragment including amino acids 754-1081 (Fig. 1A). We have chosen to separate Set1 into
186 these 2 regions because they have been described as having well-defined properties. The Set1
187 1-754 fragment includes the domain involved in Set1 recruitment to chromatin, the double
188 RRM (Trésaugues et al, 2006), and the central self-inhibitory domain (Schlichter & Cairns,
189 2005) that all have regulatory roles (Dehé & Géli, 2006). The Set1 region 754-1081 contains
190 the N-SET domain, the SET domain and the post-SET domain: the latter is capable of
191 methylating H3K4 on its own (Thornton et al, 2014) (Fig. S1). For each of the subunits (SU)
192 of the complex whose organization is described in Fig. 1A we used the whole protein as bait.
193 Each gene encoding Set1 (and its fragments) or its subunits was cloned downstream the Gal4-
194 BD except for Swd2 that was placed upstream the Gal4-BD. These screens have proven their
195 power and effectiveness. In particular, they identified Mer2 as an interactor of Spp1
196 (Acquaviva et al, 2013b), and the CTD of Rpb1 as an interactor of the N-terminal region of
197 Set1 (Bae et al, 2020) (Fig. S1). The results of the ten Y2H screens are presented in Fig. 1 and
198 Table S2. Each interactor is characterised with a confidence score based on several criteria,
199 including the frequency with which a prey protein is found for a particular screen and the
200 presence of overlapping fragments, which allow the delineation of the interaction domain
201 involved (see Methods). Very high confidence interactors (indicated by a color code) are
202 likely to interact directly with their bait. For Set1 and its N- and C-terminal fragments, the
203 high confidence Y2H interactors are shown in Fig. 1B. All interactors are shown in Table S2.
204 The predicted protein-protein interactions within the Set1 interactors reveal functional
205 information that is detailed in the following paragraphs. The high confident interactors of the
206 seven SET1C subunits are shown in Fig. 1C-E. We found that Spp1, Shg1 and Swd2 interact
207 alone with Set1 (Fig. 1C). The minimum Set1 region for which an interaction is found for
208 each of these 3 subunits is shown in Fig. 1C. The high confidence interactors of the seven
209 SET1C subunits are shown in Fig. 1C-E. We found that Spp1, Shg1 and Swd2 display Y2H
210 interactions with Set1 (Fig. 1C). The high confidence interactors of Spp1, Shg1 and Swd2 are
211 indicated in Fig. 1D (see also Table S2). In contrast, we did not find any interaction between
212 Swd1, Swd3, Bre2 and Sdc1 (SET-c components) and Set1 in the various Y2H screens,
213 suggesting that these subunits must cooperate to bind to Set1. Only Bre2 was found to interact
214 with Sdc1 in both Sdc1 and Bre2 Y2H screens (Fig. 1E). These results are consistent with the
215 structure of the extended SET1C catalytic module and full-length SET1C (Wang et al, 2018;
216 Hsu et al, 2018; Qu et al, 2018).

217

218 Set1 1-754 interacts with the importin Kap104

219 We found that Set1 1-754 interacted with very high confidence with the importin Kap104,
220 suggesting a direct interaction (Fig. 2A). Kap104 has been reported to recognize specific
221 cargos containing a specific class of NLS termed PY-NLS (Soniat *et al*, 2013; Xu *et al*, 2010).
222 This PY-NLS includes a N-terminal or central hydrophobic or basic motif, which often
223 contains hydrophobic (R/H/K) residues and which can include RGG repeats (arginine-
224 glycine-glycine motifs) and a C-terminal Proline-Tyrosine (PY) dipeptide near the C-
225 terminus. Such cargos include the mRNA export factor Nab2, the subunit of the THO/TREX
226 complex Hrp1, and the transcription factor Tfg2 (Lee & Aitchison, 1999; Süel *et al*, 2008).
227 Interestingly, Nab2 is a confident interactor of Sdc1 (Fig. 2A). The Set1 interacting domain
228 (SID) of Kap104 extends from residue 359 to 621 and includes the HEAT-like repeat
229 (Yoshimura & Hirano, 2016) (Fig. 2B). Consistent with this result, we identified two PY-NLS
230 in the N-terminal region of Set1 that are likely recognized by Kap104 (Fig. 2C). Along the
231 same line, Shg1, which binds RRM2, interacted with very high confidence with Mog1
232 (Oliete-Calvo *et al*, 2018). Mog1 has been involved in the modulation of the nucleotide state
233 of Ran-GTP in the nucleus and of Ran-GDP in the cytoplasm, thereby conferring
234 directionality to the nuclear import pathway (Baker *et al*, 2001). Mog1 has been reported to
235 interact directly with the Ran homologue Gsp1 (Oki & Nishimoto, 1998) (Fig. 2A). We have
236 also identified, Kap123, Msn5 (Kap142), Nup82 and Mtr10 as interactors of Set1 1-754 that
237 are also involved in protein import/export (Fig. 2A). Kap123 is a major karyopherin that
238 recognizes NLS of cytoplasmic H3 and H4 (An *et al*, 2017) while Msn5/Kap142 was shown
239 to mediate the import into the nucleus of the subunits of RPA (Yoshida & Blobel, 2001).
240 Nup82 for its part belongs to a module at the cytoplasmic face of the NPC and interacts with
241 karyopherins (Beck & Hurt, 2017) (Fig. 2A). Finally, Mtr10 has been implicated in the
242 nuclear import of the mRNA-binding protein Npl3 (Senger *et al*, 1998; Pemberton *et al*,
243 1997). Collectively, these multiple interactions reveal insights for understanding the nuclear
244 import of Set1, which was reported to bind co-translationally with Shg1, Swd1, and Spp1
245 (Halbach *et al*, 2009).
246

246

247 Set1 1-754 interacts with Snf2 and RGG motif proteins

248 Set1 1-754 and Set1 FL interacted with very high confidence with Snf2, the catalytic subunit
249 of the SWI/SNF chromatin remodeling complex (Côté *et al*, 1994) (Fig. 1B). Interestingly,
250 two additional Snf2 complex components, Snf5 and Swi1, have been identified in the
251 Set1FL screen (Table S2). Together these observations strongly support the idea that the
252 suggested interaction of Set1 and SWI/SNF is of biological significance (Hirschhorn *et al*,
253 1992). The various Y2H screens revealed Set1 and its subunits interacted with a number of
254 proteins involved in chromatin structure regulation although the relevance of these
255 interactions remains to be demonstrated (Fig. 3). Interestingly, the SID region of Snf2 is
256 juxtaposed to an RGG motif composed of several RGG/RG repeats that is often found in
257 RNA-binding proteins (Thandapani *et al*, 2013) and may be the substrate for arginine
258 methyltranferases (McBride *et al*, 2005) (Fig. 2A, 2D). RGG repeats are multifunctional
259 motifs that mediate RNA and DNA binding and nuclear import. They have been involved in
260 RNA metabolism and chromatin dynamics possibly via arginine methylation, which
261 modulates RNA affinity and nuclear localization (108). Along the same line, Set1 1-754 also
262 interacted with the mRNA export factor Gbp2 (Poornima *et al*, 2021) and the nucleolar
263 protein Nop1, both of which contain an RGG motif (Fig. 2A, 2D). Other RGG proteins such
264 as polyadenylated RNA-binding protein Nab2 and the RNA-helicase Dbp1 interacted with
265 Set1 754-1081 (and Sdc1) and Spp1, respectively (Fig. 2A, 2D). For each of these proteins, 266 the putative SID includes the RGG motif. Of note, both Nop1 and Nab2 are methylated by the 267 Arginine methyltransferase Hmt1 (Smith *et al*, 2020; Green *et al*, 2002).

268 Our screens indicate that the minimal region of Snf2 interacting with Set1 1-754 was located 269 between Snf2 residues 1430-1499. We further refined this interaction by Y2H testing of five 270 Set1 fragments covering the entire Set1 length against the Snf2 1349-1650 region. The two- 271 hybrid analysis revealed that Snf2 (1349-1650) interacted with the N-terminal domain (Set1 272 1-230) and the N-SET region (Set1 762-938) (Fig. S2). To validate these interactions we 273 obtained recombinant protein where GST was fused to the C-terminal domain of Snf2 274 (Snf2C), the AT-hook (Snf2C-AT-hook) and the Bromo domain (Snf2C-Bromo) (Kim *et al*, 2010) (Fig. 4A). We performed pull-down assays to test the interaction of the GST-fusion 275 proteins with reconstituted SET1C or with the SET1C762 complex in which Set1 has a N- 276 terminal truncation of the first 761 residues, respectively (See METHODS) (Fig. S3 A-E). We 277 found that SET1C as well as the SET1C762 complex were both associated with Snf2C and 278 Snf2C-AT-hook but not to Snf2C-Bromo (Fig. 4 B, C), indicating that AT-hook is a key 279 region for interaction with SET1C. We then delineated the minimal AT-hook region required 280 for interaction with SET1C and found that residues 1461-1547 are essential for binding to 281 SET1C (Fig. 4 D-E). Collectively, these results indicate that SET1C interacts with Snf2C-AT- 282 hook region suggesting that the Set1 and Snf2 complexes cooperate to modify chromatin 283 structure. 284

285 286 SET1C is involved in multiple aspects of RNA biogenesis

287 Set1 was previously shown to bind RNA nascent transcripts through its dRRM contributing to 288 position Set1 and H3K4me3 predominantly to the 5' regions of genes. Of note, Set1 showed a 289 higher occupancy within introns, at transcripts from ribosomal DNA (rDNA), and tRNAs 290 (Luciano et al, 2017; Battaglia et al, 2017; Sayou et al, 2017). Moreover, Set1 also binds 291 post-transcriptionally to Ty1 retrotransposon transcripts and mRNA encoding specific 292 transcription factors genes (Luciano et al, 2017). The SET1C Y2H interactome revealed that 293 many proteins involved in RNA biogenesis were interacting directly or indirectly with Set1 or 294 its subunits (Fig. 5). We find a strong connection between SET1C and pre-mRNA processing, 295 as highlighted by the identification of multiple spliceosome subunits (Fig. 5). Some 296 spliceosome subunits were found in several screens (Fig. 1B, D; Table S2). For instance, the 297 Prp8 subunit of the U5 snRNP that function in critical molecular rearrangements during the 298 splicing process (Grainger & Beggs, 2005) interacted with Set1 FL, Spp1, and Swd1. 299 Similarly, Prp22 interacts with Set1FL and Set1 754-1080. Prp22 is a DEAH-box helicase 300 that associates with newly spliced mRNA and promotes its release from the spliceosome (Will 301 & Lührmann, 2011). We have refined the interaction region between Set1, Prp8 and Prp22, 302 showing that Prp8 and Prp22 interact strongly with Set1-F4. (n-SET). Prp22 interacts in 303 addition with Set1-F1 (Fig. S2). We have further strengthened the link between Set1 and 304 Prp22 by showing that Set1 is co-immunoprecipitated with Prp22 *in vivo* in an RNA- 305 independent manner. (Fig S4A, B). The preferential binding of Set1 to genes with introns 306 (Luciano et al, 2017) and its interaction with splicing factors, in particular Prp22, suggest that 307 Set1 may be involved in late splicing events. Alternatively, Prp22 and/or other splicing factors 308 could be involved in H3K4 methylation.
We also found a number of factors involved in rRNA processing that could be related to the binding of Set1 to ncRNA transcripts derived from the rDNA intergenic spacer regions (Sayou et al, 2017) (Fig. 5). Next, and consistent with the observation that SET1C regulates the choice of the polyadenylation site and the recruitment of the cleavage/polyadenylation complex (Kaczmarek Michaels et al, 2020), the Y2H screens revealed multiple potential interactions between SET1C and factors involved in RNA polyadenylation (Fig. 5). Some of these interactions are known, such as the interactions between Swd2 with Pta1 and Ref2, all three proteins belonging to the APT complex (Nedea et al, 2008). Interestingly, we found that Set1 1-754 interacts with Not1 and Not4 that belongs to the ubiquitin-protein ligase CCR4-NOT (Liu et al, 2001) (Fig. 5). This complex was shown to be involved in the regulation of H3K4me3 via a ubiquitin-dependent pathway (Mulder et al, 2007; Laribee et al, 2007) that was subsequently linked to Jhd2 degradation (Huang et al, 2010; Mersman et al, 2009). These results raise the question of which of the Y2H interactions described in this study are linked to the regulation of H3K4 methylation states.

Finally and somewhat surprisingly, we found that many Y2H interactors of Set1 1-754 were involved in tRNA nuclear transport, modification, and synthesis (Fig. 5). Swd2, which interacts with the Set1 N-terminus, exhibits a 2H interaction with a high degree of confidence with the cytoplasmic asparaginyl-tRNA synthetase Ded81 and the prolyl-tRNA synthetase Yhr020 while Sdc1 interacts with the glycyl-tRNA synthase Grs1 and Bre2 with the glutamine tRNA synthetase Gln4. Of note, Trm1 (Liu et al, 1998) and Trm732 (Guy et al, 2012) are both directly or indirectly involved in tRNA methylation and form part of what has been defined as the cell's global methyltransferome (Giaever et al, 2019).

## SET1C Y2H interactors are involved in several aspects of DNA transactions

As mentioned in the introduction, it has been shown that SET1C, and in particular Spp1, regulate the selection of meiotic breaks, the progression of the replication fork, DNA repair, chromosome segregation, and transposition of Ty elements (Deshpande & Bryk, 2023). We have classified all Set1 and subunit interactors according to these SET1C roles (Fig. S5). We found a number of interactors involved in meiosis. In particular, Spp1 not only interacts with Mer2, but also with the meiosis specific DNA helicase Mer3 (Nakagawa & Kolodner, 2002) that interacts also with Swd1 (Fig. S5). We further found Set1 interactions with the kinetochore proteins Spc25 and Cbf2 suggesting that Set1 could be transiently localized at the spindle pole body. Regarding the biology of retrotransposons, we previously reported that Set1 binds post-transcriptionally to Ty1 mRNA and represses Ty1 mobility (Luciano et al, 2017). Interestingly, the two-hybrid screens reveal that Set1 1-754 interacted with Gag capsid-like proteins of Ty1 (Fig. S5) raising the possibility that Set1 binding to Ty1 mRNA is linked to the interaction of Set1 1-754 with Gag. Concerning the role of Set1 and subunits in DNA replication and repair, we report multiple potential interactions (Fig. S5). In particular, Swd2, Swd1, and Spp1 interacted with high confidence with Orc6, Nrm1, and Mcm2, respectively (Fig. 1 and Fig. S5). Orc2 was previously described to interact physically with Spp1 (Kan et al, 2008). We recently reported that Spp1 is recruited at replication forks stalled at the Tus/Ter barrier independently of its interaction with Set1 (Ghaddar et al, 2023). Interestingly, in the Y2H screen Spp1 interacted with the extreme C-terminal region of Mcm2 (791-867) (Fig. S6A) that corresponds to a non-conserved accessible alpha-helix within the
353 MCM complex (Li *et al*, 2015b). We fused Spp1 and Mcm2 to GST and performed GST pull-down experiments. We confirmed *in vitro* in both directions a weak interaction between Spp1 and Mcm2 (Fig. S6B). Whether Spp1 is recruited by Mcm2 at stalled replication fork remains to be determined. On its side Nrm1 inactivates MBF, a major regulator of the G1/S transcription (de Bruin *et al*, 2006). During replication stress, Nrm1 phosphorylation by the checkpoint kinase prevents its binding to MBF target promoters leading to the activation of G1/S transcription (Travesa *et al*, 2012). An exciting idea is that Swd1 recruits Nrm1 to stalled forks by promoting its phosphorylation by Rad53. Swd1 would play a role in linking replication stress and transcriptional regulation via Nrm1. Of note, Nrm1 was identified as a gene required for the cell-cycle pattern of H3K79me2 during early S phase (Schulze *et al*, 2009). The interaction between Swd1 and Nrm1 is described in more detail below.

364 Finally, Y2H screening indicated that Set1 and its subunits interacted with a number of proteins involved in protein SUMOylation (Fig. S5). The proteins were either involved in SUMO conjugation or SUMO-dependent degradation. Remarkably, the C-terminus of Nis1 (360-407) that contains a potential SUMO-binding site (Hannich *et al*, 2005) was identified a high-confidence interactor of Spp1, Shg1, and Sdc1 (Fig. 1). Nis1 is localized at the bud neck (Iwase & Toh-e, 2001) at the vicinity of the septin collar containing several highly SUMOylated proteins (Shs1, Cdc11) (Wykoff & O'Shea, 2005) and has been implicated in preventing bud recovery at the site of division (Meitinger *et al*, 2014). We thus sought to confirm biochemically the interaction of Nis1 with the three SET1C subunits. We fused the GST to Spp1, Sdc1, and Sgh1 to perform pull-down experiments with *in vitro* translated Nis1. We confirmed the interaction of Nis1 with Spp1, and Sdc1, but not with Sgh1 (Fig. S7). However, mass spectrometry analyses on TAP-Nis1 did not reveal the presence of SET1C subunits (Table S3) suggesting that interaction between Nis1 and Spp1/Sdc1 might be transient. The relevance of the Nis1 and other putative interactors remains unclear, especially as many of those proteins are not known to be located in the nucleus. Interestingly, Nis1 has been reported to shuttle from the bud neck to the nucleus when overexpressed (Perez & Thorner, 2019). In Fig. S8, we have identified a number of interactors, including Sdc1 and Spp1, showing changes in localization following hypoxia (Henke *et al*, 2011). Of note, one of the two human SET1C homolog SET1B has a cytoplasmic location with functions unrelated to H3K4 methylation (Wang *et al*, 2017). Combined, these observations raise the question of the minor or transient localization of Set1 and its subunits outside the nucleus, or conversely of the transient localization of interactors within the nucleus.

386

387 Set1 is SUMOylated

388 Global analyses of SUMOylated proteins in fission yeast revealed that Set1 and Spf1 (Spp1) were SUMOylated (Shin *et al*, 2005; Nie *et al*, 2015). As Set1 had two-hybrid interactions with Slx5 and Wss1, both of which act on SUMOylated proteins as they undergo protein degradation (Mullen *et al*, 2010), we tested whether Set1 can be SUMOylated. Cells expressing Myc-Set1 (Dehe *et al*, 2006) or transformed with pPB66-SET1 expressing GBD-Set1-FL (GAL4 binding domain) were transformed with a plasmid encoding His₆-SUMO or a control plasmid. Proteins were purified on Ni-NTA agarose beads and detected by Western blot either using anti-MYC or anti-GAL4 antibodies. The results revealed that Set1 can be mono-SUMOylated (Fig. 6A) or di-SUMOylated (Fig. 6B). This difference may be due to the
397 fact that GBD-SET1-FL is under the control of the ADH promoter in plasmid pGB66 and thus
398 overexpressed.

399 We sought to refine the SUMOylated Set1 region. We transformed the plasmid encoding
400 His6-SUMO and the control plasmid into cells expressing Set1 fragments F1, F2, F3, F4,
401 F3+F4, and F5 (Fig. S9A). We expressed F3+F4 instead of the isolated F3 and F4 fragments
402 in order to preserve the K769-centered motif predicted to be highly SUMOylated (Fig. S9B).
403 F1, F2, F3, and F4 were not SUMOylated (not shown) while the F3+F4 fragment was clearly
404 SUMOylated (Fig. 6C). In order to identify whether K769 is important for F3+F4
405 SUMOylation, we mutated K769 to R. We show that introduction of K769R into F3+F4
406 abolishes its SUMOylation (Fig. 6C), indicating that K769 is likely to be the SUMOylated
407 lysine within the F3+F4 fragment. We then found that F5 was also SUMOylated (Fig. 6D). In
408 this case, the substitution K1055R, K1060R, or the double substitution, does not affect the
409 SUMOylation of F5 indicating that this motif also predicted to be SUMOylated with a high
410 score is not the SUMOylated motif (Fig. 6D). Taken together, these experiments indicate that
411 Set1 can be SUMOylated in the N-SET and SET domains, in the interaction region of Spp1
412 and Swd1-Swd2-Bre2-Sdc1, respectively. This raises the possibility that SUMOylation
413 regulates the interaction of Set1 with its subunits, in particular with Spp1, which interacts
414 dynamically with SET1C (Ghaddar et al, 2023; D'Urso et al, 2016; Karányi et al, 2018;
415 Serra-Cardona et al, 2022). Interestingly, in mammals, the SUMO peptidase SENP3 (the
416 ortholog of Ulp1 in budding yeast) interacts with MLL1 and MLL2, catalyzing the
417 deSUMOylation of RbBP5 (Swd1). This process regulates the association of specific subunits
418 of MLL1/MLL2, such as menin and Ash2L (Bre2), with the DLX3 gene, which plays a role in
419 osteogenic differentiation (Nayak et al, 2014).
420

421 The transcriptional corepressor Nrm1 interacts with SET1C

422 Screening with SWD1 yielded a total of forty clones, fourteen of which contained fragments
423 of the *NRM1/YNR009w* gene, which encodes a basic protein of 249 amino-acids (Fig. 7A). Its
424 amino-terminus carries a putative D-box motif that was found to destabilize the protein (de
425 Bruin *et al*, 2006). The Y2H results furthermore delineated a central domain (amino-acids 58
426 to 195) mediating the interaction with Swd1. A sequence motif within this domain displays
427 strong similarity to the amino-terminus of histone H3; lysine 118 within Nrm1 (Nrm1-K118)
428 aligns with lysine 4 of histone H3, the substrate of SET1C. We refer to this sequence as
429 H3K4-like domain. The similarity of the H3K4 like domain and the H3K4 modification site
430 suggested that Nrm1 may represent a non-histone substrate for SET1C. To test this idea, we
431 initially performed *in vitro* methylation experiments with partially purified SET1C, S-
432 adenosyl (methyl-³H) methionine and peptide substrates. We found that a H3K4 peptide was
433 efficiently methylated, but a peptide carrying a H3K4A substitution was not (Fig. 7B). Also,
434 no methylation was observed with a peptide encompassing K118 of Nrm1. The possibility
435 remained that this peptide lacked parts of Nrm1 that were required for methylation. For this
436 purpose, we obtained recombinant protein where the N-terminus of Nrm1 was fused to the 42
437 kDa maltose binding protein (MBP). Interestingly, we observed *in vitro* methylation of this
438 protein by SET1C but not with MBP only (Fig. 7C). Note, however, that histone methylation
439 occurred much more efficiently under these reaction conditions. Next, we asked whether
440 K116 and K118 of Nrm1 were required for the observed methylation activity. Fig. 7D shows
441 that wild-type and mutant MBP-fusion proteins were found to be methylated suggesting that
442 the H3K4-like domain is not required for the observed SET1C methylation activity.

443 The H3K4-like domain in Nrm1 raised our attention to other yeast proteins that carry such
444 sequences. We used the scansite search algorithm (http://scansite.mit.edu) to systematically
445 identify sequence motifs that are related to the SET1C modification site in histone H3. Search
446 parameters included four to six identical residues of the ARTKQT sequence that is found at
447 the H3K4 modification site. In addition, we did allow for biochemically equivalent amino
448 acid changes. Fig. 7E shows eight candidate proteins based on their function and the sequence
449 context of the H3K4-like domain. Importantly, some of these gene products are involved in
450 cellular pathways that functionally overlap with SET1C, e.g. transcription (Not5), rDNA
451 silencing (Irs4) and cell-cycle control (Dbf2 and Dbf20), increasing the possibility of
452 occurring physical and functional interactions.

453

454 SET1C Y2H interactors regulate metabolism and stress responses

455 Set1 was reported to regulate ergosterol levels (South *et al*, 2013). We identified Erg9, Ugt51,  
456 Vhr1, Vhr2 and Ste20, all involved in ergosterol metabolism (Daicho *et al*, 2020; Lees *et al*,  
457 1995; Warnecke & Heinz, 1994). Erg9 and Ste20 interacted with Set1 while Ugt51 and  
458 Vhr1/Vhr2 were high confidence interactors of Shg1 and Sdc1, respectively (Fig. S10).  
459 Along the same line, the various Y2H screens revealed numerous genes involved in  
460 phosphatidyl inositol metabolism interacting with Set1 and its subunits. In particular Vip1 is  
461 a high-confidence interactor of Set1 754-1081 (Fig. 1B). Vip1 is a bifunctional inositol  
462 pyrophosphate kinase and phosphatase that regulates IP7 levels in the inositol pyrophosphate  
463 (PP-IP) synthesis pathway (Lee *et al*, 2007; Mulugu *et al*, 2007). Interestingly, Vip1 was  
464 reported to regulate the environmental stress response (ESR) through IP7 that activates the  
465 HDAC Rdp3 (Worley *et al*, 2013) suggesting potential new avenues to explain ESR  
466 regulation by Set1 (Weiner *et al*, 2012). Along the same line, Swd1 and Swd3 were identified  
467 in a screen aimed to identify genes that negatively regulate the PHO pathway in a Vip1-  
468 dependent manner (Choi *et al*, 2017). How the putative interaction between Set1 754-1081,  
469 which also interacts with Pho23 subunit of the Rpd3L complex, and Vip1 fits into these  
470 processes remains to be discovered. Of note, Vip1 has been shown in *Arabidopsis thaliana* to  
471 change localization upon hypo-osmotic stress from the cytosol to the nucleus (Takeo & Ito,  
472 2017). On their side, Y2H Interactors identified as stress-responsive genes interact either with  
473 Set1 1-754 or Swd2 (Fig. S10). Swd2 interacted with high confidence with Nar1, an essential  
474 Fe/S protein required for the assembly of cytosolic Fe/S proteins (Balk *et al*, 2004) and the  
475 calcineurin phosphatase Cmp2 activated in response to ER stress (Mizuno *et al*, 2018). The  
476 Swd2/Nar1 interaction can be linked to the fact that Set1 754-1081 interacts with relatively  
477 good confidence with TyW1 according to hybrigenics criteria (see Methods), an iron-sulfur  
478 protein required for synthesis of wybutosine modified tRNA (Noma *et al*, 2006). In contrast  
479 to the stress genes mentioned above, many interactors involved in glucose metabolism  
480 (repression) interact only with the Set1 1-1081 or with members of the nSET module (Fig.  
481 S10).

482

483 Reconstituted Set1C methylates the Snf2 RG motif within the AT-hook in vitro
We have shown above that SET1C interacts with Snf2C-AT-hook region (Figure 4). We thus tested whether the Snf2C-AT-hook could be methylated by Set1C. Set1C and truncated Set1C were reconstituted and affinity purified from Sf9 cells coinfect ed with baculoviruses that express FLAG-Set1 (or truncated Set1) and the seven other untagged subunits (Kim et al, 2013)(Fig. S3). We incubated reconstituted Set1C with the Snf2C, AT-hook and Bromo fragments in the presence of radioactive S-adenosylmethionine (³H-SAM). The results indicate that Set1C methylates *in vitro* purified Snf2C and Snf2C-AT-hook, but not Bromo (Fig. 8A, B) and that Snf2C-AT-hook methylation requires Set1 FL (Fig. 8C). Collectively these results show that the Snf2C-AT-hook (1384-1547) interacts and is methylated by Set1 FL *in vitro*. Lys 1494 (K1494) and Lys 1498 (K1498) located between the AT-hook domains of Snf2 were previously shown to be acetylated by Gcn5 (Kim et al, 2010). We individually mutated all the Lys of the Snf2-AT-hook into Arg and tested the methylation of the mutated fragments. None of the substitutions abolished or decreased the methylation of the AT-hook indicating that he AT-hook could be methylated on multiple sites or on other type of residues (Fig. S11). We thus sought to identify the minimal region within the Snf2-AT-hook that is methylated by Set1C *in vitro*. The Snf2-AT-hook domain was divided in 6 regions that were fused to the GST (Fig. 8D). *In vitro* methylation assays indicated that the Snf2-B1, B2, B3 were methylated by the reconstituted Set1C (Fig. 8E). As the Snf2-B3 fragment that contains the last four lysines (K1488, K1494, K1498, K1526) of Snf2-AT-hook domain was still methylated, it suggested that one of several of these lysines are methylated by Set1C. We then examined which of these 4 lysine residues are methylated by Set1C *in vitro*. To our surprise, mutating all the lysine residues of the Snf3-B3 fragment did not abolish the methylation of Snf3-B3 (Fig. 8F). These results led us to think that Set1C could methylate arginine residues, in particular those contained in the RGG motif of the Snf2-B3 fragment. We thus deleted the RG repeats of Snf2-B3 (Fig. 8G) and purified the *Snf2-B3ΔRG*. We found that deletion of the Snf2-B3 RG repeats abolished the interaction between Snf2-B3 and Set1 and methylation of Snf2-B3 (Fig. 8H, I). The fact that both, interaction and methylation are lost upon deletion of the RG motif argues in favor that Set1C, and not a potential contaminant from the reconstituted Set1C, is responsible for the methylation of the Snf2-B3 RG repeats. Individual deletion of pairs of arginine residues in the RG motif did not suppress methylation of the Snf2-B3 fragment, suggesting flexibility in Set1C's ability to methylate Snf2-B3 (Fig. S12). To further confirm that Snf2-B3 is methylated on arginine, we incubated a mutant Snf2-B3 (in which lysines were mutated in alanine to limit protease digestion prior mass spectroscopy analysis) with reconstituted Set1C and SAM and further purified the Snf2-B3 mutant on a Ni-NTA resin (Fig. 9A-D). We then analyzed by mass spectroscopy arginine methylation in Snf2-B3. We found that R1490, R1501, R1505, R1507 and R1517 were mono- and dimethylated while R1509, R1513, and R1519 were monomethylated (Fig. 9E). We thus confirmed that reconstituted Set1C has the ability to methylate in vitro multiple arginines in the Snf2B3 region.

## Snf2 is methylated *in vivo* on its ARTSTRGR AT-hook motif in a Set1-dependent way

Our results show that an activity associated with reconstituted Set1C is capable of methylating arginines in the vicinity and in the RG repeats of Snf2C-AT-hook. The fact that only Set1FL is capable of methylating the AT-hook is in favor of this activity being associated with Set1C.
Nevertheless, one cannot formally rule out that methylation the AT-hook is due to a contaminant from insect cells from which Set1C has been reconstituted.

We thus investigated the interaction between Snf2 and Set1 in vivo and whether Snf2 is methylated by Set1C. We found that Set1 was coimmunoprecipitated with Snf2 tagged with a C-terminal Myc epitope (Fig. 10A). In contrast to what we observed in vitro, deletion of the RG motif of Snf2 did not affect the interaction between Snf2 and Set1, which is consistent with the fact that the SID of Snf2 is located upstream of the RG repeats (Fig. 10A).

To further investigate Snf2 methylation by Set1C in vivo, we purified Snf2-GFP and its subunits with GFP nanobodies in the presence or absence of Set1 (Fig. 10B). The protein composition of the complex was determined by mass spectrometry in the presence of absence of Set1 (Figure 10B, Table S5). We recovered in both cases all the known components of the Snf2 complex (Snf2, Swi1, Swi3, Snf5, Snf12, Swp82, Arp9, Arp7, Snf6, Rtt102, Snf11, Taf14) (Table S5, Fig. S13A). Interestingly, we found that the arginine methyltransferase Rmt2 was found specifically enriched with the Snf2-GFP complex in set1Δ cells (Fig. S13).

We then excised the Snf2-GFP gel band and subjected it to mass spectrometry. We examine the post-translational modifications (PTM) in the Snf2 (1485-1549) region in either WT or set1Δ strains (Table S6). We were unable to purify a peptide containing the RG repeats but we could identify the peptides flanking the RG motif (Fig. 10C). Fig. 10C represents the combined results of two independent experiments. In WT cells, we found that the region between K1494 and R1505 was reproducibly subjected to multiple PTM (Fig. 10C, upper panel). K1494 and K1498 that were previously found acetylated by Gcn5 (Kim et al, 2010) were found acetylated, however both lysines were also found to be methylated (Fig. 10C). Of note H3K4 that is methylated by Set1C has been reported to be also acetylated by Gcn5 (Guillemette et al, 2011). T1995, S1996, S1999, T1502, S1503, and T1504 were found phosphorylated. Interestingly, R1501 was found mono-methylated while R1505 and R1507 were found di-methylated in the two independent Snf2-GFP purifications. These 3 arginines belong to a ARTSTRGR motif that lies in the Snf2-B3 fragment. They were also found to be methylated in vitro by the reconstituted Set1C (Fig. 9E). We then examine the posttranslational modifications of the peptides flanking the RG motif in the set1Δ strain (Fig. 10C, lower panel). Phosphorylation of serines and threonines was not modified in the set1Δ strain except for T1502. In the set1Δ strain, K1494 and K1498 remained methylated indicating that methylation of both lysines does not depend on Set1C. Strikingly, deleting SET1 abolished the mono-methylation of R1501 and the di-methylation of R1505, R1507 (Fig. 10C, lower panel). The results thus show that SET1C is required for the methylation of the three arginines of the ARTSTRGR motif, in agreement with the in vitro results.

# DISCUSSION
We have exploited the power of Y2H screening technology to establish an extended interaction network for the histone H3K4 methylase complex SET1C. Comprehensive data integration unveiled many potential functions of either the whole complex or individual subunits. This study is partly validated by the fact that we found interactors in known functions of SET1C, or putative functions for which the mechanism is unknown. We have also provided a number of validations by confirming the interaction through biochemical
approaches. It seems likely that some proteins interact transiently with Set1 and its subunits, or under specific conditions of stress or nutrient limitations, explaining why these interactors have not previously been found associated with SET1C in biochemical approaches. The most illustrative case for this is the high-affinity interaction between Spp1 and Mer2 that takes place during the first meiotic division, which we had identified from the Spp1 screen and which has been extensively characterized (Acquaviva et al, 2013b). Nevertheless, considering the multitude of interactions identified, it seems unlikely that they all represent direct contacts, especially where multiple subunits of complexes or multiple components of a given metabolic pathways interact with Set1C. One caveat is that Y2H interactions can be mediated by endogenous proteins that bridge the interactions. But this does not make them less functionally significant. We anticipate that this systematic screen will be an invaluable resource for further investigation of the role of SET1C in known and novel processes revealed in this study. Processes discussed here include Set1's export/import mechanism through the nucleus, interaction with RGG proteins some of which are targeted by arginine methyltransferase, cooperation of SET1C with chromatin remodeling factors in particular Snf2C, interaction with splicing factors notably with Prp8 and Prp22, coupling replication with histone deposition (Mcm2/Spp1), SET1C plasticity regulated by SUMOylation, and Ty element transposition. A recurrent question related to the identification of potential SET1C interactors is whether they could be methylated by SET1C. Based on the identification of domains that closely resembles the histone H3 modification site, we have shown that the Swd1 interactor Nrm1 is methylated by SET1C in vitro suggesting that proteins with H3K4-like domains represent a class of non-histone substrates that may be regulated by SET1C.

We show that Set1C interacts both *in vitro* and *in vivo* with Snf2. Strikingly, reconstituted Set1C from insect cells is able to methylate *in vitro* the three arginines located within the **ARTSTRGR** motif of the AT-hook of Snf2. The *in vivo* results confirm that Set1 is required for the methylation of these arginines within the **ARTSTRGR** motif. It is interesting to note that this motif resembles the **ARTKQTAR** motif of the N-terminal H3. In yeast, H3R2 is mono- and di-methylated and its asymmetric di-methylation prevents H3K4me3 (Kirmizis et al, 2009, 2007). In contrast symmetric di-methylation of H3R2 correlates with H3K4me3 and requires Set1 *in vivo* but not the classical arginine methyltransferases: Hmt1, Hmt2, Rkm2, Rkm3, Rkm4, Hsl7, and Efm1 (Li et al, 2015a; Yuan et al, 2012). It has been proposed that Set1C could be responsible for both H3R2me2s and H3K4me3 or that H3R2me2s deposited by a yet unidentified arginine methyltransferase requires the prior deposition of H3K4me3 (Yuan et al, 2012).

Interestingly, previous tandem affinity protein purification of the mono- and asymmetric arginine methyltransferase Hmt1 (also termed Rmt1) identified 108 proteins associated with it (Jackson *et al*, 2012), a number of which were shown to be methylated. Snf2 was found to be copurified with Hmt1 *in vivo* and methylated *in vitro* by Hmt1 (Jackson *et al*, 2012) making Hmt1 a potential candidate for methylating Snf2. *In vitro*, purified Hmt1 catalyzes mono-methylation and asymmetric di-methylation of H3R2, however, deleting *HMT1* does not affect the asymmetric di-methylation of H3R2 *in vivo*, which suggests that several methyltransferases could act redundantly on H3R2 (Li *et al*, 2015a). In mammals, H3R2 and H3R8 are asymmetrically di-methylated by PRMT6 (Hamey *et al*, 2021; Guccione *et al*, 2007; Hyllus *et al*, 2007). It has been also reported that PRMT5 can be found
associated with hSWI/SNF and has the ability to methylate H3R8 (Pal *et al*, 2004). We did not identify Rmt1 in our purification but found that the arginine mono-methyl transferase Rmt2 enriched with Snf2-GFP in *set1Δ* cells. Rmt2 is a type IV methyl transferase that was reported to methylate the ribosomal protein L12 (Low & Wilkins, 2012; Chern *et al*, 2002). We could envision the possibility that Set1C could cooperate with distinct protein arginine methyl transferases to promote the mono-methylation of R1501 and the di-methylation R1505 and R1507 within the **ARTSTRGR** motif. However, it was previously reported that the combined inactivation of Rmt1, Rmt2 and Hsl7 did not affect the mono-methylation of H3R2, weakening this hypothesis (Kirmizis *et al*, 2009). It is possible that in yeast there is a redundancy of enzymes capable of methylating the Snf2 **ARTSTRGR** motif, but that for each of them their activity on this motif depends on the presence of Set1. Alternatively, Set1C could directly methylate the **ARTSTRGR** motif as discussed for the **ARTKQTAR** H3 motif (Yuan *et al*, 2012). The fact that Set1C interacts with Snf2, Gbp2, Nop1, Nab2, Dbb1 (Fig. 2), all of which have RG motifs and are mostly Hmt1 substrates, raises the possibility of a general interplay between methylarginine and methyllysine.

Interestingly, a very recent article shows that PRMT1 binds to the N-terminal region of MLL2 and methylates multiple arginine residues within its RGG/RG motifs (An *et al*, 2025).

634

## 635 MATERIALS and METHODS

### 636 Strain construction

All strains and plasmids used in this study are listed in Table S1. To obtain gene deletions and expression of tagged proteins, we amplified by PCR a disruption or a tagging cassette containing the appropriate marker as described (Janke *et al*, 2004).

640

### 641 Yeast two-hybrid analysis

Yeast two-hybrid screening was performed by Hybrigenics Services, S.A.S., Evry, France (73). The coding sequence for Set1-FL, Set1 1-754, Set1 754-1081 and Bre2 were cloned into pB66 (GAL4-bait) as C-terminal fusions to the Gal4 DNA-binding domain while those of Swd1, Swd3, Sdc1, Spp1, and Shg1 were cloned into pB27 as a C-terminal fusion to LexA (LEXA-bait). SWD2 was cloned into pB43 as a N-terminal fusion (SWD2-GAL4). Individual bait cloning was performed by using specific primers for PCR for every bait. Every PCR fragment subcloned as bait is entirely sequenced to avoid eventual mismatches in the coding sequence. The constructs were used as baits to screen a genomic *S. cerevisiae* library constructed into pP6 based on the pGADGH plasmid (Wilson *et al*, 1993). pB6, pB66 and pB43 are derived from the original pAS2Δ vector (Fromont-Racine *et al*, 1997). The library was submitted to very strict quality controls. Each protein is represented by several fragments (domains) and the library has been screened with a set of 6 reference baits before any other bait proteins are screened.

The Gal4 constructs were screened using a mating approach with YHGX13 (Y187 *ade2-101::loxP-kanMX-loxP*, *MATalpha*) and CG1945 (*MATa*) yeast strains. The LexA constructs were screened using a mating approach with YHGX13 (Y187 *ade2-101::loxP-kanMX-loxP*, *mat alpha*) and L40ΔGal4 (*MATa*) yeast strains as previously described (Fromont-Racine *et al*, 1997). Positive clones were selected on a medium lacking tryptophan, leucine and
histidine and supplemented with 3-aminotriazole (3AT) if necessary to handle bait auto-activation. The prey fragments of the positive clones were amplified by PCR and sequenced at their 5' and 3' junctions. The resulting sequences were used to identify the corresponding interacting proteins in the GenBank database (NCBI) using a fully automated procedure.

A confidence score (PBS, for Predicted Biological Score) was attributed to each interaction as previously described (Formstecher et al, 2005). The confidence score relies on two different levels of analysis. Firstly, a local score takes into account the redundancy and independency of prey fragments, as well as the distribution of reading frames and stop codons in overlapping fragments. Secondly, a global score takes into account the interactions found in all the screens performed at Hybrigenics using the same library. This global score represents the probability of an interaction being nonspecific. For practical use, the scores were divided into four categories, from A (highest confidence) to D (lowest confidence). A fifth category (E) specifically flags interactions involving highly connected prey domains previously found several times in screens performed on libraries derived from the same organism. Finally, several of these highly connected domains have been confirmed as false-positives of the technique. The PBS scores have been shown to positively correlate with the biological significance of interactions. When possible, the bait interacting domain of each prey is provided. The PBS scores have been shown to positively correlate with the biological significance of interactions (Rain et al, 2001). e-values for the interactions are available in the Hybrigenics database.

Protein expression and protein interaction assays

Protein expression was done in *Escherichia coli* BL21 cells and purification of GST-fusion proteins were as described (Dichtl *et al*, 2002). MBP-fusion proteins were purified according to manufacturer's instructions (New England BioLabs, Beverly, MA, USA). For His-tagged proteins expressed in bacteria, cDNAs were inserted into the pET28 vector (Novagen), expressed in *Escherichia coli*, and purified using Ni-NTA beads (Qiagen) following the previously described procedure (Kim & Roeder, 2011). GST pull-down assays were done as described (Dichtl *et al*, 2002).

A baculovirus expression system was used to express and reconstitute Set1 complexes containing either FLAG-Set1 or FLAG-Set1-C762. cDNAs were subcloned into pFASTBAC1, with or without an epitope tag, and baculoviruses were produced following the manufacturer's instructions (Gibco-Invitrogen). Sf9 cells were infected with various combinations of baculoviruses, and the complexes were purified by affinity chromatography using M2 agarose, as previously described (Kim & Roeder, 2011).

TAP-Nis1 affinity purification and mass spectrometry analysis

TAP-Nis1 and control cells (W303a) were grown in YPD, harvested in logarithmic phase (OD600 0.6-0.85) and cryo-lysed as previously described (Trahan & Oeffinger, 2022). Affinity purification was performed in RNP100 (20 mM HEPES-KOH pH 7.4, 100 mM NH4OAc, 0.5% Triton X-100, 0.1% Tween 20, 1:100 solution P, 1:5000 antifoam A, 100 mM NaCl) as described in (Trahan & Oeffinger, 2022). Following washes, the samples were on-bead trypsin digested in a volume of 50µl (f. c. 20 µg/mL trypsin (Sigma, proteomics grade)
704 in 20 mM Tris-HCl (pH 8.0), 37 °C, 900 rpm, 16-20 h; stopped with 2% formic acid) and
705 analyzed by tandem mass spectrometry as described in (Trahan & Oeffinger, 2022) using a
706 70-minute gradient on a LTQ Orbitrap Velos is a hybrid mass spectrometer (ThermoFisher
707 Scientific) in Data-dependent mode. Data were processed with Thermo Excalibur to generate
708 a raw file. Mascot search server (53 Version 2.3.02) was used with a parent tolerance of 10
709 ppm for precursor ions, 0.52Da for fragments, and only considering 1 possible missed
710 cleavage as well as a mass change of +16 for methionine oxidations in the mass calculation.
711 Data were searched against S. cerevisiae NCBI database and analyzed in Scaffold (version
712 3.6.4). The threshold and false discovery rates (FDR) were set to 80% and 0.37% respectively
713 for peptides, and to 95% (1 peptide minimum) and 1.9% for respectively for proteins.
714 Exclusive spectrum counts (ESC) were used for analysis and for each prey, the highest values
715 obtained in the controls were removed from those of the samples during analysis.
716

716

717 **In vitro** methylation

718 *In vitro* methylation reactions were done essentially as described (Roguev *et al*, 2001). 30 ml
719 reactions typically contained 2 to 4 ml of partially purified SET1C complex, 2 to 4 mg
720 substrate (either peptide, or core histone mixture, or recombinant H3) and 2 ml S-adenosyl
721 (methyl-³H) methionine in MTA buffer (50 mM Tris 8.5, 20 mM KCl, 10 mM MgCl₂, 250
722 mM sucrose). Reactions with core histones and recombinant H3 were resolved on 4-20%
723 NuPAGE gels and subjected to fluorography. For Snf2 methylation assays, reaction mixtures
724 containing purified SET1C (with 30 ng of the Bre2 subunit) and 200 ng of Snf2 fragments in
725 20 µl of reaction buffer (25 mM HEPES [pH 7.6], 50 mM KCl, 5 mM MgCl2, 0.1 mM
726 EDTA, and 10% glycerol) were supplemented with 1 µCi of S-adenosyl (methyl-³H)
727 methionine (PerkinElmer) and incubated at 30°C for 1 hour. Proteins were resolved by SDS-
728 PAGE and subjected to fluorography. For fluorography, gels were fixed for 30 min in 40%
729 methanol, 10% acetic acid, treated with EN³HANCE solution (Perkin Elmer) for 60 min,
730 washed in cold dH₂O for 30 min, dried and exposed for five to fourteen days to photographic
731 film.
732

732

733 **Mass spectrometry-based identification of Arg methylation in Snf2-B3**

734 Mutant Snf2-B3 underwent a methylation reaction with SET1C and SAM, followed by
735 purification via Ni-NTA affinity chromatography and separation by SDS-PAGE. The gel
736 bands corresponding to the mutant Snf2-B3 were excised and subjected to in-gel digestion
737 with AspN enzyme, followed by peptide extraction. The resulting peptide fractions were
738 analyzed using an Easy-nLC 1200 coupled to an Orbitrap Fusion Lumos mass spectrometer
739 (Thermo Fisher Scientific, MA, USA) at the Korea Basic Science Institute (Ochang). Peptides
740 were separated on a C18 column using a 150-minute gradient, and data were acquired in data-
741 dependent acquisition (DDA) mode. Full MS scans were acquired in the Orbitrap at a
742 resolution of 60,000 over an m/z range of 350-2,000. Precursors for MS/MS analysis were
743 selected for higher-energy collisional dissociation (HCD) fragmentation at a normalized
744 collision energy of 30%. Raw MS data were processed using Proteome Discoverer with the
745 SEQUEST search engine. The data were searched against the sequence of Snf2-B3 with a
746 precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.02 Da. AspN was set
747 as the digestion enzyme, allowing for up to two missed cleavages. Methylation,
748 dimethylation, and methionine oxidation were specified as variable modifications. The false
749 discovery rate (FDR) was set to 1% at the peptide level.

750

751 **Y2H interaction of Set1 fragments with selected preys**

752 The Set1 fragments were amplified from the pB66-Set1-FL plasmid and cloned into the SfiI
753 site of pB66. The selected preys (Snf2, Prp8, and Prp22) into p6 (Hybrigenics) were extracted
754 from the screen. Plasmid pB66 contains the Gal4 DNA binding domain and the TRP1 marker
755 while pP6 expresses the Gal4 activating domain and the LEU2 marker. TOTO cells were
756 transformed with the different pB66-Set1-Fragments and the pP6-interactors and incubated 3
757 days at 30°C on SD-LEU-TRP. The transformant colonies were then streaked on SD-LEU-
758 TRP-HIS containing either 5 mM or 20 mM of 3AT. To visualize interaction between Set1
759 fragments and the interactors, yeast cells were incubated 2 days at 30°C and cell growth was
760 examined.

761

762 **CO-IP of Prp22-FLAG with Myc-Set1.**

763 Co-immunoprecipitation experiment was performed in W303 expressing chromosomally
764 encoded Myc-Set1 (Dehe *et al*, 2006) and Prp22AID-FLAG (Mendoza-Ochoa *et al*, 2019).
765 250 ml of culture at an OD600 of 0.8 was harvested by centrifugation at 1000 x g and washed
766 twice in ice-cold 1 X PBS. The cell pellet was re-suspended in 900 µl lysis buffer (50 mM
767 Tris-HCl pH 7.5, 2 mM Mg2Cl2, 150 mM NaCl, 0.2% NP-40 and one complete EDTA-free
768 proteinase inhibitor tablet (Roche #11836145001) and 400 µl zirconia beads. Cells were lysed
769 using a Mini-Beadbeater-24 (BioSpec Products) twice at 2000 rpm for 2 min followed by 2
770 min on ice. The sample was centrifuged at 1000 x g for 2 min, the supernatant was collected
771 and additionally centrifuged at 20,000 x g for 30 min at 4°C and used for
772 immunoprecipitation. The concentration of protein was measured using the Bradford assay,
773 and 1 mg of protein used per IP. Prior to immunoprecipitation, extract was pre-cleared by
774 adding ½ volume unconjugated Protein A/G Dynabeads (Life Technologies
775 ##10001D/10003D). 50 µl Protein A/G Dynabeads conjugated to antibody were incubated
776 with the appropriate volume of extract on a rotating wheel overnight at 4°C. The next day,
777 beads were washed 8 times in lysis buffer (non-bound fraction kept for analysis). 20 µl of
778 loading buffer was added to the beads, input and non-bound samples, which were boiled for
779 10 min before loading on a NuPAGE 4-12% Bis-Tris gel Bis-Tris (Invitrogen) and western
780 blotting was performed.

781

782 **Set1 SUMOylation analysis**

783 Cells were transformed with a plasmid encoding 6His-SUMO under the CUP1 promoter
784 (YEp352-6His-SUMO) or the corresponding empty vector (Niño et al, 2016). The
785 transformed cells were grown on selective medium and stimulated overnight with 0.1 mM
786 CuSO4. 200 OD600 of cells were collected and lysed with glass beads in a 20% TCA solution,
787 the final TCA concentration was adjusted to 12%. Cell lysates were incubated at 4°C during
788 45 min, and precipitated proteins were collected by centrifugation. Proteins were resuspended
789 in loading buffer (6 M guanidinium-HCl, 100 mM KH2PO4, 20 mM Tris-HCl, pH 8.0, 100
790 mM NaCl, 0.1% Triton X-100, and 10 mM imidazole). 6His-SUMOylated proteins were
791 purified using Ni-NTA agarose beads (Qiagen), pre-equilibrated with loading buffer, and
792 incubated for 1 h at room temperature. After incubation the beads were collected by 793 centrifugation (3000 rpm, 2min) and washed twice with wash buffer (8 M urea, 100 mM 794 Na₂HPO₄/NaH₂PO₄ pH 6.4, 10 mM Tris-HCl, pH 6.4, 10 mM Imidazole, 10 mM β- 795 mercaptoethanol, and 0.1% Triton X-100). 6His-SUMOylated proteins were eluted with 50 796 ml 2X Laemmli buffer (95°C, 5min). The proteins in the eluted fraction were analyzed by 797 Western blot using anti-MYC (for endogenous Myc-Set1), anti-GAL4 (for GBD-Set1-full 798 length and fragments) and polyclonal rabbit anti-Smt3. 799

800 **Interaction Snf2-AT-hook with SET1C and SET1C-762**

801 GST-tagged Snf2 fragments (final concentration 250 nM) were combined with either the full- 802 length SET1C or C762SET1C (final concentration 25 nM), along with 15 µl of glutathione- 803 Sepharose 4B resin and BSA (final concentration 0.2 mg/ml) in a binding buffer containing 804 20 mM Tris-Cl (pH 7.9), 150 mM NaCl, 0.2 mM EDTA, 20% glycerol, 0.1% NP-40, and 1 805 mM PMSF, making a total volume of 300 µl. The mixtures were rotated at 4°C for 3 hours, 806 followed by four washes with the binding buffer. The proteins bound to the resin were then 807 eluted, separated by SDS-PAGE, and analyzed by immunoblotting. 808

809 **CO-IP of Snf2-Myc and Snf2ΔRG-Myc with Set1**

810 WT cells or expressing *Snf2-Myc* and *Snf2ΔRG-Myc* (200 ml) were grown at 30°C in YPD to 811 and O.D₆₀₀ = 0.8. Cells were collected by centrifugation, washed once with 10 mM Tris-HCL, 812 pH 8.0 and snap-frozen in liquid nitrogen. Cells were lysed using Retsch with the following 813 parameters :2 times 2 min at 30 m/S. The powder was resuspended in 3 ml of TMG 50 (10 814 mM Tris-HCL, pH 8.0, 0,1 mM MgCl₂, 10% (V/V) glycerol, 50 mM NaCl, 0.1 mM EDTA, 815 0.1 mM DTT) containing protease inhibitors, 10 mM MG-132 and 1 mM PMSF. Cell lysates 816 were clarified by centrifugation (13 300 rpm, 15', 4°C) and 1.4 ml of supernatant were 817 recovered. Protein concentration was determined using nanodrop and samples were adjusted 818 to the same concentration by adding lysis buffer. Immunoprecipitation was performed 819 overnight with 5 ml of 9E10 antibody (Santa Cruz Biotechnology) following by an incubation 820 for 3 hours with 25 ml of pre-equilibrated protein-G dynabeads (Invitrogen). 821 Immunoprecipitations were washed 3X with TMG 50 buffer and eluted using 25 ml of 1X 822 Laemmli loading Buffer. Samples were resolved on a 7.5% Acrylamide gel, transferred on a 823 nitrocellulose membrane and reveal with either anti-Myc 9E10 (Santa Cruz Biotechnology) 824 and anti-Set1 antibodies. 825

826 **Snf2-GFP Complex purification**

827 800 ml of *Snf2-GFP* and *Snf2-GFP set1Δ* cells were grown at 30°C in YPD to O.D₆₀₀ = 0.8. 828 Cell pellets were treated as described above except that supernatants were incubated with 25 829 ml of GFP nanobody coated Dynabeads (Gift from M. Modesti, CRCM Marseille) for 250 830 min, wash 3X with TMG 50 buffer and eluted in 30 ml of 2X Laemmli Loading buffer 831 containing 50 mM DTT. For Western blotting, 1 ml was resolved on a NuPAGE 4-12% Bis- 832 Tris Gel (Invitrogen), transferred on a nitrocellulose membrane and revealed using an anti- 833 GFP antibody (A-11122-Invitrogen). For the Silver Staining, 2,5 ml were resolved on a 834 NuPAGE 4-12% Bis-Tris Gel (Invitrogen) and stained using Pierce™ Silver Stain kit 835 (Thermo Scientific).
Mass spectrometry-based identification of post-translational modifications in Snf2-GFP

836
837 Purified Snf2-GFP (n=3, biological replicates) from WT and set1Δ strains were loaded on
838 NuPAGE™ 4–12% Bis–tris acrylamide gels according to the manufacturer’s instructions
839 (Life Technologies). Running of protein was stopped as soon as proteins stacked in a single
840 band. Protein containing bands were stained with Imperial Blue (Pierce), cut from the gel and
841 digested with high sequencing grade trypsin (Promega) before mass spectrometry analysis.
842 Briefly, gel pieces were washed and destained using few steps of 100 mM NH4HCO3.
843 Destained gel pieces were shrunk with 100 mM ammonium bicarbonate in 50% acetonitrile
844 and dried at RT. Protein spots were then rehydrated using 10mM DTT in 25 mM ammonium
845 bicarbonate pH 8.0 for 45 min at 56°C. This solution was replaced by 55 mM iodoacetamide
846 in 25 mM ammonium bicarbonate pH 8.0 and the gel pieces were incubated for 30 min at
847 room temperature in the dark. They were then washed twice in 25 mM ammonium
848 bicarbonate and finally shrunk by incubation for 5 min with 25 mM ammonium bicarbonate
849 in 50% acetonitrile. The resulting alkylated gel pieces were dried at room temperature. The
850 dried gel pieces were reswollen by incubation in 25 mM ammonium bicarbonate pH 8.0
851 supplemented with 12.5 ng/µl trypsin (Promega) for 1h at 4°C and then incubated overnight at
852 37°C. Peptides were harvested by collecting the initial digestion solution and carrying out two
853 extractions; first in 5% formic acid and then in 5% formic acid in 60% acetonitrile. Pooled
854 extracts were dried down in a centrifugal vacuum system. Samples were reconstituted in 0.1%
855 TFA 4% acetonitrile before mass spectrometry using an Orbitrap Fusion Lumos Tribrid Mass
856 Spectrometer (ThermoFisher Scientific, San Jose, CA) online with an Ultimate
857 3000RSLCnano chromatography system (ThermoFisher Scientific, Sunnyvale, CA). Peptides
858 were separated at 40°C using a two steps linear gradient (4–20% acetonitrile/H2O; 0.1%
859 formic acid for 110 min and 20-32% acetonitrile/H2O; 0.1% formic acid for 10 min). An
860 EASY-Spray nanosource was used for peptide ionization (2,200 V, 275°C). MS was
861 conducted using a data-dependent acquisition mode (DDA). The Orbitrap Lumos was used in
862 data dependent mode to switch consistently between MS and MS/MS. Time between Masters
863 Scans was set to 3 seconds. MS spectra were acquired with the Orbitrap in the range of m/z
864 400-1600 at a FWHM resolution of 120 000 measured at 400 m/z. AGC target was set at
865 4.0e5 with a 50 ms Maximum Injection Time. For internal mass calibration the 445.120025
866 ion was used as lock mass. The more abundant precursor ions were selected and collision-
867 induced dissociation fragmentation was performed in the ion trap to have maximum
868 sensitivity and yield a maximum amount of MS/MS data. Number of precursor ions was
869 automatically defined along run in 3s windows using the “Inject Ions for All Available
870 parallelizable time option” with a maximum injection time of 300 ms. The signal threshold for
871 an MS/MS event was set to 5000 counts. Charge state screening was enabled to exclude
872 precursors with 0 and 1 charge states. Dynamic exclusion was enabled with a repeat count of
873 1 and duration of 60 s.
874

For identification of PTMs the region corresponding to Snf2-GFP was excised from the gel
875 and subjected to enzymatic digestion under the same conditions as above. Raw mass
876 spectrometry files were analysed using MaxQuant software (version 1.6.3.4) as above with
877 the exception of the Max Missed cleavage set at 5, the FDR at peptide and proteins levels at
878 5% and with the following variable modifications activated; Lysine acetylation (+42.0106);
879
880 Serine, threonine and tyrosine phosphorylation (+79.966); Lysine and arginine methylation
881 (+14.0157), dimethylation (+28.0313) and trimethylation (+42.0470).

882

883 **Data Processing Protocol**

884 Relative intensity-based label-free quantification (LFQ) was processed using the MaxLFQ
885 algorithm from the freely available MaxQuant computational proteomics platform, version
886 1.6.2.1 (Cox et al. 2014, Cox et al. 2008). The acquired raw LC Orbitrap MS data were first
887 processed using the integrated Andromeda search engine (Cox et 2011). Spectra were
888 searched against the Saccharomyces cerevisiae (organism ID 4932) extracted from UniProt
889 containing 7904 entries. The following parameters were used for searches: (i) trypsin allowing
890 cleavage before proline; (ii) two missed cleavage was allowed; (iii) cysteine
891 carbamidomethylation (+57.02146) as a fixed modification and methionine oxidation
892 (+15.99491) and N-terminal acetylation (+42.0106) as variable modifications. The match
893 between runs option was enabled. The false discovery rate (FDR) at the peptide and protein
894 levels were set to 1% and determined by searching a reverse database. For protein grouping,
895 all proteins that cannot be distinguished based on their identified peptides were assembled
896 into a single entry according to the MaxQuant rules. The statistical analysis was done with
897 Perseus program (version 1.6.1.3) (Tyanova and Cox 2018) from the MaxQuant environment
898 (www.maxquant.org). Quantifiable proteins were defined as those detected in above 70% of
899 samples in one condition or more. Protein LFQ normalized intensities were base 2
900 logarithmized to obtain a normal distribution. Missing values were replaced using data
901 imputation by randomly selecting from a normal distribution centered on the lower edge of
902 the intensity values that simulates signals of low abundant proteins using default parameters
903 (a downshift of 1.8 standard deviation and a width of 0.3 of the original distribution). The
904 protein composition of the complex was determined using, a two-sample t-test using
905 permutation-based FDR-controlled at 0.01 and employing 250 permutations and a scaling
906 factor s0 with a value of 3. All proteins passing these criteria with a positive fold change are
907 considered to be the potential interactome of Snf2.
908

909

910 **Antibodies**

910 For IP: Mouse anti-FLAG (Sigma M2 #F1804); 10 µl/IP with Dynabeads and Protein G.
911 For westerns - primary antibodies: Mouse anti-c-MYC (Santa Cruz #SC-40x) 1:1000; Rat
912 anti-FLAG (Agilent #200474) 1:1000; Mouse anti-Gal4 Binding Domain (Euromedex)
913 1:5000; Rabbit anti-Smt3 is a gift from B. Palancade (Institut Jacques Monod, Paris). Anti-
914 Bre2 is a gift from Peter Nagy. The rabbit anti-Spp1 antibody was developed in V. Géli's lab.
915 The anti-Spp1 antibody is showing significant cross-reactivity with other cellular proteins but
916 did allow the identification of Spp1 in purified and partially purified fractions. Secondary
917 antibodies: Goat anti-mouse IRDye680RD (LI-COR #926-68070) 1:10,000, Goat anti-rat
918 IRDye800RD (LI-COR #926-32219) 1:10,000.

919

920

921 **DATA AVAILABILITY**

922 Additional data and reagents are available upon request to the corresponding authors. The
923 mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium
924 (www.proteomexchange.org) via the PRIDE partner repository
925 (https://www.ebi.ac.uk/pride/login): Snf2-B3 data (accession number PXD061448), Snf2-
926 GFP complex (accession number PXD061496), Snf2 PTM (accession number PXD061531).

927
928
929

930 ACKNOWLEDGEMENTS

931 We are grateful to Sandra Holbein and Andre Halbach for help with MBP-Nrm1 fusion
932 proteins. We thank Benoit Palancade for his help in the interpretation of the Y2H results and
933 reagents.
934

935
936
937
938

FUNDING

This work is supported by the "Ligue Nationale Contre le Cancer" (LNCC) (Equipe 938 labellisée, GELI/2019). Funding to B.D. was provided by grants from the Swiss National 939 Science Foundation (PP00A--102941/1 and 31003A_1327010). I.E.M. was funded by 940 Wellcome Trust PhD Studentship (105256) and work in the Wellcome Centre for Cell 941 Biology was supported by Wellcome core funding (092076). Funding to M. O. was provided 942 by the Natural Sciences and Engineering Research Council of Canada to M.O. (RGPIN-2020- 943 06924). Funding to J. K was provided by the National Research Foundation of Korea (NRF- 944 2022R1A2C3012803). Y. H. K is supported by the National Research Foundation of Korea 945 (NRF) grant funded by the Korea government (MSIT) (No. RS-2024-00454407). Proteomics 946 analyses using the mass spectrometry facility of Marseille Proteomics (marseille- 947 proteomique.univ-amu.fr) are supported by IBISA, the Cancéropôle PACA, the Provence- 948 Alpes-Côte d'Azur Region, the Institut Paoli-Calmettes, and Fonds Européens de 949 Développement Regional (FEDER). 950

951
952
953
954

CONFLICT OF INTEREST

The authors declare no conflict of interest.

955
956
AUTHOR CONTRIBUTIONS

B.D. J. K and V.G. designed the study and secured the funding. All the Y2H screens and prey 957 identification were performed by Hybrigenics in close interaction with B.D. and V. G. P.L. 958 performed molecular cloning, contributed to validation experiments, and purified Snf2-GFP 959 for subsequent analyses. SET1C and SET1C-C762 purification and interaction assays with 960 Snf2-AT-hook were performed by J. K. and K.P. Set1 SUMOylation experiments were 961 performed by C. N. M. D and B. D. organized the results shown in Table S2. L. L 962 constructed the F1-F5 fragments and contributed to validation experiments. Pull-down 963 experiments and Nrm1 methylation assay were performed by B. D. TAP-Nis1 data were 964 provided by M.O. I.M. and J. B. performed and discuss the Set1/Prp22 interaction. D.K. P 965 and H. H. K performed mass spectrometry analysis of the Snf2-B3. S.A. and L.C. performed 966
967 mass spectrometry analysis of the Snf2-GFP. V.G. and B. D. wrote the manuscript with inputs
968 of the authors.

969

970

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1383 Karyopherin β2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2. *J Struct*
1384 *Funct Genomics* 14: 31-35

1385 South PF, Harmeyer KM, Serratore ND & Briggs SD (2013) H3K4 methyltransferase Set1
1386 is involved in maintenance of ergosterol homeostasis and resistance to Brefeldin
1387 A. *Proc Natl Acad Sci U S A* 110: E1016-25

1388 Süel KE, Gu H & Chook YM (2008) Modular organization and combinatorial energetics of
1389 proline-tyrosine nuclear localization signals. *PLoS Biol* 6: e137

1390 Sun Z-W & Allis CD (2002) Ubiquitination of histone H2B regulates H3 methylation and
1391 gene silencing in yeast. *Nature* 418: 104-108

1392 Takeo K & Ito T (2017) Subcellular localization of VIP1 is regulated by phosphorylation
1393 and 14-3-3 proteins. *FEBS Lett* 591: 1972-1981

1394 Terzi N, Churchman LS, Vasiljeva L, Weissman J & Buratowski S (2011) H3K4
1395 Trimethylation by Set1 Promotes Efficient Termination by the Nrd1-Nab3-Sen1
1396 Pathway. *Mol Cell Biol* 31: 3569-3583

1397 Thandapani P, O'Connor TR, Bailey TL & Richard S (2013) Defining the RGG/RG motif.
1398 *Mol Cell* 50: 613-623

1399 Thornton JL, Westfield GH, Takahashi Y-H, Cook M, Gao X, Woodfin AR, Lee J-S, Morgan
1400 MA, Jackson J, Smith ER, *et al* (2014) Context dependency of Set1/COMPASS-
1401 mediated histone H3 Lys4 trimethylation. *Genes Dev* 28: 115-120

1402 Trahan C & Oeffinger M (2022) Single-Step Affinity Purification (ssAP) and Mass
1403 Spectrometry of Macromolecular Complexes in the Yeast S. cerevisiae. *Methods*
1404 *Mol Biol Clifton NJ* 2477: 195-223

1405 Travesa A, Kuo D, de Bruin RAM, Kalashnikova TI, Guaderrama M, Thai K, Aslanian A,
1406 Smolka MB, Yates JR, Ideker T, *et al* (2012) DNA replication stress differentially
1407 regulates G1/S genes via Rad53-dependent inactivation of Nrm1. *EMBO J* 31:
1408 1811-1822

1409 Trésaugues L, Dehé P-M, Guérois R, Rodriguez-Gil A, Varlet I, Salah P, Pamblanco M,
1410 LUCIANO pierre, Quevillon-Cheruel S, Sollier J, *et al* (2006) Structural
1411 Characterization of Set1 RNA Recognition Motifs and their Role in Histone H3
1412 Lysine 4 Methylation. *J Mol Biol* 359: 1170-1181

1413 Vitaliano-Prunier A, Menant A, Hobeika M, Géli V, Gwizdek C & Dargemont C (2008)
1414 Ubiquitylation of the COMPASS component Swd2 links H2B ubiquitylation to
1415 H3K4 trimethylation. *Nat Cell Biol* 10: 1365-1371

1416 Walter D, Matter A & Fahrenkrog B (2014) Loss of histone H3 methylation at lysine 4
1417 triggers apoptosis in Saccharomyces cerevisiae. *PLoS Genet* 10: e1004095

1418 Wang L, Collings CK, Zhao Z, Cozzolino KA, Ma Q, Liang K, Marshall SA, Sze CC,
1419 Hashizume R, Savas JN, *et al* (2017) A cytoplasmic COMPASS is necessary for cell
1420 survival and triple-negative breast cancer pathogenesis by regulating
1421 metabolism. *Genes Dev* 31: 2056-2066

1422 Wang Y, Ding Z, Liu X, Bao Y, Huang M, Wong CCL, Hong X & Cong Y (2018) Architecture
1423 and subunit arrangement of the complete Saccharomyces cerevisiae COMPASS
1424 complex. *Sci Rep* 8: 17405

1425 Warnecke DC & Heinz E (1994) Purification of a Membrane-Bound UDP-Glucose:Sterol
1426 [beta]-D-Glucosyltransferase Based on Its Solubility in Diethyl Ether. *Plant
1427 Physiol* 105: 1067-1073

1428 Weiner A, Chen HV, Liu CL, Rahat A, Klien A, Soares L, Gudipati M, Pfeffner J, Regev A,
1429 Buratowski S, et al (2012) Systematic Dissection of Roles for Chromatin
1430 Regulators in a Yeast Stress Response. *PLoS Biol* 10: e1001369

1431 Will CL & Lührmann R (2011) Spliceosome structure and function. *Cold Spring Harb
1432 Perspect Biol* 3: a003707

1433 Wilson TE, Padgett KA, Johnston M & Milbrandt J (1993) A genetic method for defining
1434 DNA-binding domains: application to the nuclear receptor NGFI-B. *Proc Natl Acad
1435 Sci USA* 90: 9186-9190

1436 Worley J, Luo X & Capaldi AP (2013) Inositol pyrophosphates regulate cell growth and
1437 the environmental stress response by activating the HDAC Rpd3L. *Cell Rep* 3:
1438 1476-1482

1439 Wykoff DD & O'Shea EK (2005) Identification of sumoylated proteins by systematic
1440 immunoprecipitation of the budding yeast proteome. *Mol Cell Proteomics MCP* 4:
1441 73-83

1442 Xu D, Farmer A & Chook YM (2010) Recognition of nuclear targeting signals by
1443 Karyopherin-β proteins. *Curr Opin Struct Biol* 20: 782-790

1444 Yoshida K & Blobel G (2001) The karyopherin Kap142p/Msn5p mediates nuclear import
1445 and nuclear export of different cargo proteins. *J Cell Biol* 152: 729-740

1446 Yoshimura SH & Hirano T (2016) HEAT repeats - versatile arrays of amphiphilic helices
1447 working in crowded environments? *J Cell Sci* 129: 3963-3970

1448 Yuan C-C, Matthews AGW, Jin Y, Chen CF, Chapman BA, Ohsumi TK, Glass KC, Kutateladze
1449 TG, Borowsky ML, Struhl K, et al (2012) Histone H3R2 Symmetric Dimethylation
1450 and Histone H3K4 Trimethylation Are Tightly Correlated in Eukaryotic Genomes.
1451 *Cell Rep* 1: 83-90

1452 Zhang Z, Shibahara K & Stillman B (2000) PCNA connects DNA replication to epigenetic
1453 inheritance in yeast. *Nature* 408: 221-225

1454 FIGURES AND LEGENDS

1455

1456 FIGURE 1
<figure><img src="image_2.png" /><figcaption>Figure 1. Schematic representation of the Set1 and subunit major interactors identified in the systematic yeast two-hybrid screens.</figcaption></figure>

A) Schematic representation of Set1 FL and Set1 fragments 1-754 and 754-1081. B) Set1 FL, 1-754 and 754-1081 major Y2H interactors. C) Interacting regions of Set1 with Swd2, Shg1 and Spp1. D) Swd2, Spp1, Shg1 major Y2H interactors. E) Swd1, Swd3, Bre2, Sdc1 major Y2H interactors. The term “interactor” is used to mean a high confidence two-hybrid interaction, with the limitations that this entails. The color reflects the Predicted Biological Score (see METHODS). Red, highest confidence; Blue, high confidence; Green, good confidence.

1457

1458

1459

1460

1461

1462

1463

1464

1465

1466

1467

1468

1469

1470

1471

1472
1473 FIGURE 2

1474

1475

<figure><img src="image_3.png" /></figure>

1476

1477

1478 **Figure 2. Set1 1-754 interacts with RGG proteins and the importin Kap104.**

1479 A) RGG proteins and import/export proteins interacting with Set1 1-754, Set1 754-1081 and

1480 Spp1, Shg1, and Sdc1. B) Set1 interacting domain (SID) (blue) within Kap104. Heat like

1481 repeat 9 and 10 are represented in purple C) PY-NLS in the N-terminal region of Set1. D) SID (blue) and RGG motif (green) within the RGG proteins. The interaction domains

1482 indicated represent the minimal overlapping DNA sequence present in multiple independent

1483 Y2H interacting clones of the same gene. Each genomic fragment of a Y2H clone was

1484 analyzed, and the shared overlapping region for given gene was determined to be the only

1485 common element among all interacting clones. As such, this region represents the minimal

1486 sequence required for interaction.

1487
1488

FIGURE 3

1489

1490

<figure><img src="image_4.png" /></figure>

1491

1492

1493

1494

1495

1496

1497

1498

1499

1500

1501

**Figure 3.** Chromatin regulators identified in all the two-hybrid screens. The lines indicate the individual proteins involved in the Y2H interaction. Red line refers to a very high confidence Y2H interaction. All Y2H interactors are described in Table S2. Interactors are grouped according to the complex to which they belong.
1502 FIGURE 4

1503
1504

<figure><img src="image_5.png" /><figcaption>Figure 4. SET1C interacts *in vitro* with Snf2C-AT-hook.</figcaption></figure>

1505
1506
1507
1508
1509
1510
1511
1512
1513
1514
1515
1516
1517
1518
1519
1520
1521

A) A schematic diagram depicting the domains of Snf2 and the Snf2 fragments used in this study, along with the SDS-PAGE/Coomassie staining of the purified GST-tagged Snf2 fragments. B and C) GST pull-down assay using purified GST-tagged Snf2 fragments. The purified SET1C (B) or SET1C-C762 complex (C) was mixed with GST-tagged Snf2 fragments, followed by GST pull-down, and the bound proteins were analyzed by immunoblotting. D) A schematic diagram illustrating Snf2 fragments with a more detailed breakdown of the AT-hook domain, along with the SDS-PAGE/Coomassie Blue staining of the purified Snf2 fragments. The lysines present in the AT-hook are represented by the letter K. E) GST pull-down assay using purified GST-tagged Snf2 fragments and SET1C.
1522 FIGURE 5

1523

<figure><img src="image_6.png" /></figure>

1524
1525
1526
1527 Figure 5. The SET1C Y2H interactome identifies proteins involved in RNA biogenesis.
1528 All Y2H interactors are described in Table S2. The processes linked to RNA metabolism in
1529 which the different interactors are involved are shown in the figure. The green lines linking
1530 Prp22 to Set1FL/Set1 754-1080 and Prp8 to Set1 FL and Spp1 indicate interactions with a
1531 high degree of confidence.
1532
1533
1534
1535
1536
1537
1538
1539
1540
1541
1542
1543
1544

<caption>Figure 5. The SET1C Y2H interactome identifies proteins involved in RNA biogenesis.</caption>

All Y2H interactors are described in Table S2. The processes linked to RNA metabolism in which the different interactors are involved are shown in the figure. The green lines linking Prp22 to Set1FL/Set1 754-1080 and Prp8 to Set1 FL and Spp1 indicate interactions with a high degree of confidence.
1545 FIGURE 6

1546

<figure><img src="image_7.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_10.png" /></figure>

1547

1548

1549 **Figure 6. Set1 is SUMOylated.**

1550 6His-SUMO-conjugated proteins were purified from cells transformed (+) or not transformed
1551 (-) with a plasmid encoding 6His-SUMO under control of the *CUP1* promoter. Cell lysates
1552 (Input) and Ni-purified material (Elutes) were analyzed by Western blotting with an anti-
1553 MYC antibody (A) or and anti-GAL4 antibody (B-D). Analysis of 6His-SUMO -conjugated
1554 forms of (A) genomically MYC-tagged Set1 or (B) GB-Set1 transformed cells was performed
1555 (left panels), in both the cases SUMO expression and efficiency of purification were
1556 controlled using an anti-SUMO antibody (right panels). C) SUMOylation analysis of Set1
1557 fragment F3+F4 (aa. 351-956) WT and the K769R mutant. D) SUMOylation analysis of Set1
1558 fragment F5 (aa. 956-1080) WT, single mutants K1055R and K1060R, and the double mutant
1559 K1055R/K1060R mutant.

1560

1561
<figure><img src="image_11.png" /><figcaption>1563
1564
1565
1566 Figure 7. Nrm1 is methylated *in vitro* by SET1C.</figcaption></figure>

1567 A) Schematic representation of the *S. cerevisiae* Nrm1 protein. Nrm1 carries a D-box sequence at its
1568 amino-terminus and a central domain that mediated interaction with the Swd1 subunit of SET1C in a
1569 yeast two-hybrid screen. A H3K4 like domain that closely resembles the modification site of SET1C
1570 in histone H3 is contained within the Swd1 interaction domain. Identical and similar amino-acid
1571 positions are shown in italic. Lysine 118 (K118) of Nrm1 aligns with lysine 4 of histone H3; also
1572 indicated are K116R and K118A mutant sequences. B) *In vitro* methylation reactions using partially
1573 purified SET1C, ³H-SAM and the indicated peptides. Radioactive reaction products retained on
1574 Whatman P81 filter paper following extensive washes were measured by scintillation counting. Each
1575 reaction was done in triplicate and error bars indicate the standard deviation. C) *In vitro* methylation
1576 reactions as in (B), however, core histone (2, 4 and 6 mg) and recombinant MBP and MBP-Nrm1
1577 fusion protein (0.5, 1 and 2 mg) were tested as substrates for methylation. Reaction products were
1578 separated on 4 to 12% NuPAGE gels and analyzed by fluorography. D) *In vitro* methylation reactions
1579 as in (C), however, core histone (2 mg) and recombinant MBP-Nrm1 wild-type and K118A and
1580 K116R fusion protein (0.5, 1 and 2 mg) were tested as substrates for methylation. (upper panel). Note
1581 that the molecular mass marker proteins (MM) have been resolved together with the core histone
1582 methylation reaction in the same lane. The asterisk indicates migration of a non-specific background
1583 signal of unclear origin. The substrate proteins included in the reactions were analyzed on a parallel
gel and stained with colloidal Coomassie G-250 (lower panel). E) H3K4-like proteins. Shown are
1584 selected proteins containing sequences similar to the modification site found in histone H3. An
1585 exhaustive list can be found in Table S4.
1586

1587
1588

1589

1590

1591
1592

FIGURE 8

1593

1594

<figure><img src="image_12.png" /></figure>

1595

1596

1597

<caption>Figure 8. Snf2 is methylated in tandem AT-hook domain by reconstituted Set1C.</caption>

1598

1599

1600

1601

1602

1603

1604

1605

1606

1607

1608

1609

1610

1611

1612

1613

1614

A) *In vitro* methyltransferase assay using purified SET1C and Snf2 fragments. H-SAM was used as a methyl-donor and methylated proteins were detected by autoradiography. The band marked with a red star is a degradation product of Snf2C. B) *In vitro* methyltransferase assay using SET1C and two Snf2-AT-hook fragments with two different tags. C) Schematic diagram of N-terminal truncated SET1 complexes (left) and *in vitro* methyltransferase assay with GST-Snf2-AT-hook and truncated SET1 complexes. D) Schematic diagram showing the positions of all lysines in Snf2-AT-hook and the further cleaved fragments of Snf2-AT-hook. The red box indicates the two lysines that are acetylated by Gcn5. E) Coomassie staining of purified Snf2 fragments (lower) and an *in vitro* methyltransferase assay using these fragments with SET1C (upper). F) *In vitro* methyltransferase assay by SET1C when each of the four lysines in the C-terminal region of the Snf2-AT-hook is substituted with arginine or when both lysines known to be acetylated by Gcn5 are substituted. G) A schematic diagram showing the position of the RG-repeat region and the design of Snf2-AT-hook with RG-repeat truncation. H and I) GST pull-down assay (H) and *in vitro* methyltransferase assay (I) using purified SET1C and GST-Snf2-AT-hook with or without RG-repeats.
<figure><img src="image_13.png" /><figcaption>1615 FIGURE 9</figcaption></figure>

<table><thead><tr><th>Peptide Sequence</th><th># Missed Cleavages</th><th>Modifications</th><th>Charge Rank</th><th>m/z [Da]</th><th>MH+ [Da]</th><th>DeltaM [ppm]</th><th>Confidence</th><th>X Corr</th></tr></thead><tbody><tr><td rowspan="4">DIPAPRTAGATSVASARTSTRGRGRGRGRGRGRGRGRPPAARNGLDYVRTPAAATSPI</td><td rowspan="4">0</td><td>R6(DM)</td><td>11</td><td>1</td><td>438.06381</td><td>4808.62917</td><td>-7.19</td><td>High</td><td>9.24</td></tr><tr><td>R6(M); R17(M)</td><td>10</td><td>1</td><td>481.77057</td><td>4808.6402</td><td>-4.9</td><td>High</td><td>8.2</td></tr><tr><td>R33(DM)</td><td>9</td><td>1</td><td>535.18964</td><td>4808.64851</td><td>-3.17</td><td>High</td><td>5.83</td></tr><tr><td>R33(M); R35(M)</td><td>9</td><td>1</td><td>535.18964</td><td>4808.64851</td><td>-3.17</td><td>High</td><td>5.83</td></tr><tr><td>DIPAPRTAGATSVASARTSTRGRGRGRGRGRGRGRGRPPAARNGLDYVRTPAAATSPI</td><td>1</td><td>R6(DM); R17(DM); R21(DM); R23(DM)</td><td>10</td><td>1</td><td>624.4541</td><td>6235.47553</td><td>4.77</td><td>High</td><td>5.34</td></tr><tr><td>DIPAPRTAGATSVASARTSTRGRGRGRGRGRGRGRGRPPAARNGLDYVRTPAAATSPI</td><td>2</td><td>R6(M); R17(DM); R21(DM); R23(DM)</td><td>11</td><td>1</td><td>566.50507</td><td>6221.48296</td><td>8.49</td><td>High</td><td>5.22</td></tr><tr><td rowspan="2">DNSPSEDFMDIPAPRTAGATSVASARTSTRGRGRGRGRGRGRGRGRPPAARNGL</td><td>2</td><td>M9(Oxydation); R36(M)</td><td>10</td><td>1</td><td>584.20435</td><td>5832.97797</td><td>-5.17</td><td>High</td><td>3.51</td></tr><tr><td>2</td><td>M9(Oxydation); R34(M)</td><td>10</td><td>1</td><td>584.20551</td><td>5832.98856</td><td>-3.18</td><td>High</td><td>2.93</td></tr><tr><td>DFMDIPAPRTAGATSVASARTSTRGRGRGRGRGRGRGRGRGRPPAARNGLDYVRTPAAATSPI</td><td>2</td><td>R24(M); R26(M); R54(DM)</td><td>10</td><td>1</td><td>658.15411</td><td>6572.47565</td><td>-6.6</td><td>High</td><td>3.4</td></tr></tbody></table>

1616
1617
1618 Figure 9. The arginines in the RG-repeat of Snf2 are methylated by reconstituted SET1C.

1620 A) A diagram showing the WT Snf2-B3 fragment and the Mut Snf2-B3 with all four lysines substituted with alanine. B) Coomassie staining of purified WT and Mut Snf2-B3. C) Mass-spectrometry experiment design to identify the methylation sites of Snf2-B3. D) Coomassie staining of Mut Snf2-B3 after methylation reaction and an additional purification step using Ni-NTA. The band corresponding to Mut Snf2-B3 (~18 KDa) was excised and used for mass spectrometry analysis. The ~37 KDa band observed in lanes 5–8 appears to be a SET1C subunit that binds non-specifically to Ni-NTA, likely SWD2 based on its size. E) Mass spectrometry analysis result of Snf2-B3 methylation sites revealed that multiple arginines in the RG-repeat were methylated. Amino acid sequence of Snf2 highlighting the arginines methylated by SET1. The sequence of the B3 fragment is shown in bold, and the arginines methylated by SET1 are marked in red. Methylation and demethylation are denoted as M and DM, respectively.

1632
1633 FIGURE 10

1634

<figure><img src="image_14.png" /></figure>

1635

1636

1637 Figure 10. The arginines of the motif ARTSTRGR within the AT hook are methylated in vivo in a Set1-dependent way.

1638
A) Set1 interacts *in vivo* with Snf2 and Snf2-ΔRG. Myc-tagged Snf2 and Snf2ΔRG were
1639
immunoprecipitated with 9E10 Myc antibodies (see Methods) and revealed with either
1640
9E10 (Upper panel) or Set1 antibodies (lower panel). B) Snf2C complex was purified
1641
from WT and *set1Δ* strains, separated on a 4-12% Bis-Tris Gel and Silver Stained (Left);
1642
the presence of Snf2-GFP is detected by Western-blotting with anti-GFP antibody
1643
(Right). The area corresponding to Snf2-GFP was excised from the gel and used for mass
1644
spectrometry analysis. Peptides flanking the RG repeats (1485-1549) with their PTM are
1645
shown in Fig. S14. C) Panel C show a focus on the amino sequence flanking the RG
1646
repeats. The positions of residues from D<sub>1485</sub> to V<sub>1549</sub> are indicated on the figure. Peptides
1647
identified after digestion of Snf2-GFP are indicated in color with their identified PTM
1648
indicated by the color code shown in the top of the panel. The small numbers above the
1649
amino acids indicate the probability of localisation according to the MS2 peaks. It should
1650
be noted that for wild-type K1488 and R1490, a peptide with dimethylation is detected,
1651
but no discriminating MS2 peak allows us to conclude whether K or R are dimethylated.
1652
This is also the case for dimethylation on R1528 and R1535. The results presented
1653
represent the observed PTMs from two independent experiments, each containing 3
1654
replicates (Table S6).
1655
1656

1657

1658

1659

1660

1661

1662

1663

1664

1665

1666

1667

1668