Phase Separation by the Sterile Alpha Motif of Polyhomeotic Compartmentalizes Polycomb Group 2 Proteins and Enhances Their Activity

1 Phase separation by the Sterile Alpha Motif of Polyhomeotic compartmentalizes Polycomb Group 2 proteins and enhances their activity

4 Elias Seif1, Jin Joo Kang1,2, Charles Sasseville1, Olga Senkovitch3, Alexander Kaltashov1, Elodie L. Boulier1, 5 Ibani Kapur1,2, Chongwoo A. Kim3, and Nicole J. Francis1,2,4* 6 7 1Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, QC, H2W 1R7, 8 Canada 9 2Division of Experimental Medicine, McGill University, 1001 Decarie Boulevard, Montreal, QC H4A 3J1, 10 Canada 11 3Department of Biochemistry and Molecular Genetics, Midwestern University, 19555 N. 59th St., 12 Glendale, AZ 85308 13 4Département de biochimie et médecine moléculaire Université de Montréal, 2900 Boulevard Edouard- 14 Montpetit, Montréal, QC H3T 1J4, Canada. 15 *Correspondence: [email protected] 16

17 18 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

19 Abstract: 20 Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A 21 conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit 22 Polyhomeotic (Ph) is important for chromatin compaction and large-scale chromatin organization. Like 23 many SAMs, Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between 24 chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To 25 understand mechanistically how this occurs, we analyzed the effects of Ph SAM on chromatin in vitro. 26 We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in 27 formation of concentrated, phase-separated condensates. Condensate formation depends on Ph SAM, 28 and is enhanced by but not strictly dependent on, its polymerization activity. Ph SAM-dependent 29 condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone 30 H2A. Overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, phase 31 separation is an activity of the SAM, which, in the context of Ph, can mediate large-scale compaction of 32 chromatin into biochemical compartments that facilitate histone modification.

33 34 35 36 Introduction: 37 Polycomb Group (PcG) proteins repress gene expression by modifying chromatin at multiple 38 scales, ranging from post-translational modification of histone proteins to organization of megabase 39 scale chromatin domains 1-6. Two main PcG complexes, PRC1 and Polycomb Repressive Complex 2 40 (PRC2), are central to PcG function and conserved across evolution 1-4. Both complexes can carry out 41 post-translational modification of histones (methylation of histone H3 on lysine 27 (H3K27me) for PRC2 42 and ubiquitylation of lysine 118/119 of histone H2A (H2A-Ub) for PRC1. PRC1, and to a lesser extent, 43 PRC2, are also implicated directly in long rage organization of chromatin and clustering of PcG proteins 44 into foci in cells. Two classes of PRC1 complexes have been defined, canonical (cPRC1), and non- 45 canonical (ncPRC1). Both types of complexes contain two ring finger proteins required for E3 ubiquitin 46 ligase activity towards H2A (Psc and dRING in Drosophila, Pcgf and Ring1A or B in mammals) 3,4. cPRC1 47 additionally contains a Cbx protein (Pc in Drosophila), and a PHC (Ph in Drosophila). In ncPRC1, RYBP 48 replaces the Cbx protein, PHCs are absent, other accessory proteins are variably present, depending on 49 the Pcgf subunit 4. At least in mouse embryonic stem cells, ncPRC1 is responsible for the bulk of 50 ubiquitylated H2A 7,8. This suggests histone modification and chromatin organization may be partitioned 51 between nc and cPRC1s, although both types of complexes share many genomic targets 7-10. All cPRC1 52 subunits can interact with DNA and/or chromatin, and both canonical and non-canonical PRC1s can 53 compact chromatin in vitro 9,11, but Polyhomeotic (Ph), and thus canonical PRC1, is the most implicated 54 in large-scale chromatin organization 3,12-17. 55 Ph is a core subunit of canonical PRC1, and its most notable feature is the presence of a 56 conserved Sterile Alpha Motif (SAM) in its C-terminus that can assemble into helical polymers 18. SAMs 57 are present in many different types of proteins and in many cases can mediate protein polymerization 58 19. The SAM of Ph is required for Ph function in Drosophila, and its full polymerization activity is 59 important for gene repression 20,21. PRC1 forms visible foci both in Drosophila and in mammalian 60 cells13,22, and, in Drosophila cells, a much larger number of diffraction-limited clusters 14. Disrupting the 61 Ph SAM impairs formation of PcG protein clusters and reduces long-range contacts among PcG bound 62 loci, suggesting the two processes are related 13,14. Despite the wealth of in vivo data supporting the bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

63 critical function of Ph SAM in large-scale organization of PcG proteins and chromatin, and in gene 64 regulation, the biochemical mechanisms by which Ph SAM links protein and chromatin organization are 65 not known. 66 In recent years, an important role for liquid-liquid phase separation (LLPS) in organizing 67 macromolecules in cells has been defined 23-26. This mechanism is increasingly accepted as being 68 important in formation of protein-RNA membraneless organelles26,27, and has more recently been 69 implicated in chromatin compartmentalization and genome organization 28-33, transcription activation 34- 70 37, DNA repair 38,39, and PcG protein organization 40-42. LLPS by nuclear/chromatin associated proteins 71 may concentrate proteins and RNAs, enhance or inhibit reactions, exclude other factors, and even 72 physically move genomic regions 23,25,43. Nucleated phase separation at super-enhancers mediated by 73 disordered regions in transcription factors and co-activators is believed to be important for driving 74 cycles of active transcription 34,35. Phase separation is also implicated in the formation of 75 heterochromatin and its function as a distinct chromatin environment 30,32, although the precise role of 76 LLPS is debated 44. The mammalian PcG protein Cbx2 (part of certain cPRC1s) has also been shown to 77 undergo LLPS in vitro with chromatin, and to form foci in mammalian cells, suggesting a link between 78 LLPS and PcG function 40,42. 79 Here, we consider the hypothesis that Ph SAM can organize chromatin though phase separation 80 by analyzing Ph-chromatin interactions in vitro. 81 82 Results: 83 A truncated version of Ph, “Mini-Ph” forms phase separated condensates with DNA or chromatin: In 84 Drosophila melanogaster, the Ph gene is present as a tandem duplication in the genome; the two genes 85 (Ph-p and Ph-d) encode highly related proteins with largely redundant function 45. Drosophila Ph is a 86 large protein (1589 amino acids for Ph-p), the majority of which is disordered (Fig. 1A), and which is 87 difficult to work with in vitro. To focus on the function of the domains conserved in Ph orthologues, 88 particularly the SAM, and to facilitate biochemical analysis, we used a truncated version of Drosophila 89 Ph-p, termed “Mini-Ph” 5. Mini-Ph (aa1289-1577) contains the three conserved domains—from amino- 90 to carboxy-terminus: the HD1, the FCS Zinc finger that can bind nucleic acids 46, and the Ph SAM (Fig. 91 n1A). A unstructured linker connects the FCS to the SAM, and restricts Ph SAM polymerization 5. Thus, 92 while Ph SAM alone forms extensive helical polymers in vitro, Mini-Ph exists mainly as short polymers of 93 4-6 units (Fig. 1B-C), even at high concentrations 5. 94 We expressed Mini-Ph in E. coli, purified it (Supplementary Fig. 1A), and tested whether it can 95 form phase-separated condensates, alone or with chromatin. Chromatin was prepared on a circular 96 plasmid containing 40 copies of the Lytechinus 5S rDNA nucleosome positioning sequence 47 using 97 histones fluorescently labelled with Cy3 on histone H2A (Fig. S1C-E). Neither Mini-Ph alone, nor 98 chromatin alone formed condensates in buffer (Fig. 1D, E). When Mini-Ph is mixed with chromatin, or 99 plasmid DNA, large, round, phase bright drops are observed (Fig. 1F; Supplementary Fig. 2A). Drops 100 formed with either DNA or chromatin undergo fusion (Fig. 1G, Supplementary Fig. 2B; Supplementary 101 Movie 1, 2), and settle to the bottom of the imaging plate where they flatten and continue to fuse (Fig. 102 1H). Under phase separation conditions, Mini-Ph and DNA can be pelleted by centrifugation 103 (Supplementary Fig. 2C, D), consistent with them forming a denser phase. Mini-Ph-DNA solutions also 104 become turbid, as measured by OD340 (Supplementary Fig. 2E). To evaluate the relationship between the 105 concentration of chromatin or DNA and Mini-Ph, and phase separation, we titrated both Mini-Ph and 106 DNA or chromatin, and manually scored each point in the resulting matrix as “one phase” or “two bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

107 phases” (Fig. 1I, J; Supplementary Fig. 2F, G). This produces a limited coarse-grained delineation of the 108 boundary between one- and two-phase regimes. Phase separation is sensitive to the concentration of 109 both components, and the ratio between the two. This is most notable for Mini-Ph-DNA titrations, 110 where we are able to add high concentrations of DNA, which prevent phase separation (Supplementary 111 Fig. 2F, G). From similar titrations of NaCl and Mini-Ph at a fixed DNA concentration, we find that phase 112 separation is observed in NaCl concentrations up to 125 mM (Supplementary Fig. 3). We conclude that 113 Mini-Ph forms phase separated condensates with either DNA or chromatin. 114 The disordered linker connecting Ph SAM to the FCS domain was previously demonstrated to 115 restrict Ph SAM polymerization, possibly due to its ability to contact Ph SAM in trans 5. A scrambled 116 linker has the same effect, implicating amino acid composition rather than organization 5. The sequence 117 properties of linkers that connect structured domain play a central role in phase separation 48, by 118 restricting or promoting interactions between structured domains, and by contributing weak 119 interactions 49. We therefore analyzed the sequence properties of the linker (Supplementary Fig. 4), 120 both in Drosophila Ph, and in the three human homologues (PHC1-3). The Ph linker is acidic (pI 3.9), but 121 relatively uncharged (fraction charged residues (FCR) =0.15), and does not have strongly segregated 122 charge (Supplementary Fig. 4B, E, Supplementary Table 1). Overall, the Ph linker is expected to be 123 collapsed (Supplementary Fig. 4D). 124 The linker region is conserved between the two Drosophila Ph homologues (Supplementary Fig. 125 4F), but both the sequence and charge properties of the linker in mammalian PHCs are distinct 126 (Supplementary Fig. 4C, D, E, G; Supplementary Table 1). The human PHC linkers are basic (pI >10), more 127 charged (FCR 0.25-0.34), have more segregated charges, and have a higher fraction of expansion 128 promoting residues (Supplementary Fig. 4C, E). They occupy a distinct position on the Das-Pappu 129 diagram of states (Supplementary Fig. 4D), predicting context dependent collapse or expansion. 130 Previous analysis indicates that the PHC3 linker promotes polymerization of either PHC3 or Ph SAM, and 131 does not interact with the PHC3 SAM in trans 50. A synthetic linker designed to be unstructured (Rlink 5) 132 promotes polymerization of both Ph and PHC3 SAM, and shares properties with PHC linkers, including a 133 basic pI (Supplementary Table 1). Evolutionary tuning of the linker sequences is likely to affect phase 134 separation properties of PHCs, although this will need to be tested experimentally. 135 136 Chromatin is highly concentrated in Mini-Ph condensates. One potential function of phase separation is 137 to concentrate (compact) chromatin. To measure the concentration of chromatin in Mini-Ph-chromatin 138 condensates, we first prepared calibration curves using the same Cy3-labelled histone octamers 139 (labelled on H2A) that were used to assemble chromatin (Supplementary Fig. 5A). The concentration of 140 nucleosomes in Mini-Ph condensates, starting from a mixture of 150 nM nucleosomes, and 5 µM Mini- 141 Ph, was measured as 22.5 +/- 4.4 µM (Supplementary Fig. 5B). We note that this value is lower than the 142 reported concentration of chromatin in pure chromatin condensates induced by monovalent cations 143 (~340 µM 28). The reported measurements used free dye to prepare the calibration curve. When we 144 imaged calibration curves prepared from free Cy3, although the curves are linear, they predict at least a 145 60x higher concentration than curves prepared with labelled histone octamers using the same imaging 146 parameters. Because ladders prepared with free Cy3 do not accurately predict known concentrations of 147 Cy3-labelled histone octamers in our hands, we believe the chromatin concentrations measured using 148 the Cy3-labelled histone calibration curve (Supplementary Fig. 5) are correct for Mini-Ph-chromatin 149 condensates. 150 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

151 Mini-Ph is dynamic in condensates, but chromatin intermixes slowly. A characteristic of liquid 152 condensates is that the components are dynamic. We carried out fluorescent recovery after 153 photobleaching (FRAP) experiments with Mini-Ph-chromatin condensates. A fraction of Mini-Ph is 154 mobile and exchanges in condensates, so that bleached Mini-Ph drops partially recover fluorescence 155 within several minutes (Fig. 2A, B; Supplementary Fig. 6A-D). In contrast, when the histones (labelled 156 with H2A-Cy3) were bleached, less than 15% of the fluorescence is recovered after several minutes (Fig. 157 2B, Supplementary Fig. 6E, F). We quantified our FRAP data with user selected ROI for the bleach area 158 and background, and fit the data with a double exponential function. Recent work has drawn attention 159 to the complexity of FRAP measurements in phase-separated condensates, and in selecting and applying 160 the appropriate biophysical model to the data 51. Because of the complexities cited above, we interpret 161 the FRAP curves qualitatively. Although we have calculated the half-times of the fast and slow 162 populations and mobile fractions from our data (Supplementary Fig. 6A-D), we do not think these 163 numbers can be used to compare with other systems, or with the measured FRAP behavior of Ph in vivo 164 52. Nevertheless, they indicate that Mini-Ph and chromatin have very different kinetics in condensates. 165 Similar behavior has been dissected in a model system of lysine or argnine rich peptides and 166 homopolymers of RNA 53. In this case, slow kinetics for the RNAs could be explained by RNA-RNA 167 interactions 53. It is possible that nucleosome-nucleosome interactions contribute to the slow kinetics of 168 chromatin. However, it must also be emphasized that the chromatin templates used in these 169 experiments are large (11 kb of DNA, ~55 nucleosomes, ~13750 kDa). This system may partially mimic 170 chromatin in vivo, which also does not freely intermix (discussed in 54). Experiments with 12-nucleosome 171 linear arrays (more than 4X smaller than the templates used here) indicate that while chromatin alone 172 can form a liquid like state that shows (slow) recovery in FRAP experiments 28, in most conditions, 173 chromatin forms condensates that behave as solids and do not recover in FRAP experiments, similar to 174 chromaitn in vivo 55. 175 To further understand how chromatin intermixes in condensates, we used two-colour chromatin 176 experiments (Fig. 2C-H). Mini-Ph was incubated separately with chromatin labelled with Cy3 or Alexa 177 647. Once condensates had formed, the two sets were mixed together, and images collected as the 178 condensates fused (Fig. 2C-H). Although condensates of both colours fused, ultimately forming a fused 179 network at the bottom of the imaging plate (Fig. 2G, H), distinct Cy3 and Alexa-647 regions remained, 180 indicating that the chromatin in pre-formed condensates does not fully intermix when the condensates 181 fuse, at least over the 60 minutes that we monitored (Fig. 2H). This is in clear contrast to control 182 experiments in which the two chromatins are mixed prior to addition of Mini-Ph, where all structures 183 contain a uniform mix of both fluorophores (Fig. 2C, D). These experiments are consistent with the 184 coexistance of different dynamics in Mini-Ph-chromatin condensates. The persistence of unmixed 185 regions could also reflect dynamically arrested phase separation in the pre-formed condensates. We 186 note that in the mixtures shown in Fig. 2C-H, the Alexa-647 labelled chromatin (white in Fig. 2) has a 187 slightly lower nucleosome density than the Cy3 (red) labelled chromatin. The persistent unmixed regions 188 tend to be red regions at the junctions of fused drops. This raises the possibility that nucleosome density 189 affects chromatin dynamics in condensates, due to nucleosome-nucleosome interactions, or 190 nucleosome-Mini-Ph interactions. We conclude that although a fraction of Mini-Ph in Mini-Ph-chromatin 191 condensates is mobile, the chromatin polymers mix slowly and incompletely, a process that could 192 maintain partial compartmentalization of Mini-Ph bound chromatin over short (minutes to hours) time 193 scales. 194 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

195 Ph SAM, but not its polymerization activity, is required for formation of phase-separated condensates. 196 To test whether Ph SAM is important for condensate formation by Mini-Ph, we prepared Mini-Ph lacking 197 the SAM (Mini-PhΔSAM), or lacking the HD1/FCS domains (Mini-PhΔFCS) (Fig. 3A; Supplementary Fig. 198 1A). The structure of Ph SAM, including its two polymerization interfaces, termed “End Helix” (EH) and 199 “Mid Loop” (ML) is well characterized 18 (Fig. 3B). Mutation of these interfaces blocks SAM 200 polymerization in vitro and impairs Ph function in vivo 5,18,20. We therefore prepared Mini-Ph containing 201 a point mutation that disrupts the EH interface (L1565R) (“Mini-Ph EH”), or a single point mutation that 202 weakens but does not fully disrupt the ML interface (L1547R) (“Mini-Ph ML”) (Supplementary Fig. 1A). 203 Previous AUC experiments with these mutants indicate that Mini-Ph ML forms shorter polymers than 204 Mini-Ph, and Mini-Ph EH at most may form some dimers at high concentrations (see Fig. 3 of 5) We first 205 measured the DNA binding activity of each of these proteins using double filter binding with a 150 bp 206 DNA probe (Fig. 3C, D; Supplementary Fig. 7A). Mini-Ph binds DNA with an apparent Kd (Kdapp) of 37 nM. 207 Partial disruption of polymerization activity with the single ML mutation increases the Kdapp to 190 nM. 208 The more severe EH mutation further increases the Kdapp to 706 nM, similar to the Kdapp of Mini- 209 PhΔSAM (990 nM). DNA binding was not detected with Mini-PhΔFCS by filter binding or EMSA 210 (Supplementary Fig. 7B), indicating that Ph SAM does not bind DNA. Consistent with this conclusion, the 211 Kdapp of Mini-PhΔSAM is similar to that for Mini-Ph EH. The much lower Kdapp of Mini-Ph presumably 212 reflects cooperative binding by Mini-Ph oligomers. We do not know what the oligomeric state of Mini-Ph 213 is at the concentrations where DNA binding is observed. The Kdapp of the SAM-SAM interaction was 214 previously measured as ~200 nM using an immobilized SAM 18, but it is possible that Mini-Ph 215 oligomerization occurs at lower concentrations, which would be consistent with the observed high 216 affinity binding. We conclude that the polymerization activity of Ph SAM increases the affinity of Mini-Ph 217 for DNA. 218 Neither Mini-PhΔSAM nor Mini-PhΔFCS forms condensates with chromatin or with DNA (Fig. 3E, 219 F). A mixture of the two proteins also does not form condensates with DNA (Fig. 3F). Thus, both the 220 SAM, and the HD1/FCS domains are required for phase separation. We then tested the Mini-Ph 221 polymerization mutants (Mini-Ph ML and Mini-Ph EH). We find that both form phase separated 222 condensates with chromatin or DNA under the same conditions as Mini-Ph (Fig. 3G, Supplementary 223 Figure 8). While the concentration of nucleosomes in condensates is similar (Fig. 3H), condensates 224 formed with Mini-Ph EH are smaller (Fig. 3I, J). 225 To look more carefully at the effects of the Ph SAM mutations, we titrated Mini-Ph EH or Mini- 226 Ph ML with DNA over a range of NaCl concentrations, and scored each reaction as one-phase or two 227 phases (Supplementary Fig. 8). We find that both mutants are more sensitive to NaCl than Mini-Ph 228 (Supplementary Fig. 3A, B, 8A-C). ATP has been shown to dissolve many protein-RNA condensates, and 229 is hypothesized to have a physiological role in regulating phase separation 56. To test whether ATP might 230 also regulate Mini-Ph-chromatin condensates, we formed condensates with Mini-Ph, Mini-Ph ML, or 231 Mini-Ph EH, and challenged them with 2 mM ATP for 15 or 60 min. (Supplementary Fig. 9A). 232 Condensates are smaller after ATP treatment, and Mini-Ph EH is more sensitive than either Mini-Ph or 233 Mini-Ph ML (Supplementary Fig. 9 B-E). Treatment of Mini-Ph condensates with 8 mM ATP completely 234 dissolves them (Supplementary Fig. 10F). We conclude that the Ph SAM, and the HD1/FCS regions are 235 both required for condensate formation, while Ph SAM polymerization activity, which increases DNA 236 binding affinity and changes the oligomeric state of Mini-Ph, enhances condensate formation but is not 237 required for it. Although this result may seem surprising, it is consistent with Mini-Ph existing in a 238 limited oligomeric state prior to condensate formation that cannot be increased further. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

239 Mini-Ph EH and Mini-PhΔSAM have similar DNA binding activities (Fig. 3C), but different abilities 240 to form condensates (Fig. 3D-F). This indicates that the SAM imparts an activity (presumably protein- 241 protein interactions) that is distinct from the effect on DNA binding and polymerization, but essential for 242 condensate formation. The unstructured linker that connects the FCS/HD1 to the Ph SAM 243 (Supplementary Fig. 4A) was previously shown to interact with the SAM (in trans) by NMR 5. This linker- 244 SAM interaction may allow homotypic interactions between Mini-Ph molecules, even when SAM-SAM 245 interactions are disrupted (as in Mini-Ph EH) and contribute to phase separation. It is also possible that 246 weak SAM-SAM interactions can occur in the EH mutant 5 and contribute to phase separation. Ph SAM 247 polymerization may thus indirectly contribute to phase separation by clustering the DNA binding FCS 248 domains (increasing multivalency) and increasing the affinity for DNA/chromatin. Supplementary Fig. 10 249 summarizes the known and hypothesized interactions that may underlay phase separation by Mini-Ph 250 and DNA or chromatin. 251 252 DNA binding and phase separation modify lysine accessibility in Mini-Ph: To explore how Mini-Ph 253 interactions change on formation of phase separated condensates, and how SAM polymerization affects 254 them, we used a mass spectrometry based protein footprinting method to probe accessible lysines in 255 Mini-Ph (Fig. 4A). We incubated Mini-Ph alone, or with three different amounts of DNA. In the 1X DNA 256 condition (1 Mini-Ph per 10 bp) and 2X DNA conditions, phase separated condensates form, while 257 increasing the DNA amount to 16X prevents their formation (Fig. 4B). To display the data, we generated 258 a heat map of the average accessibility at each lysine under each condition (Fig. 4C). To compare these 259 values, we used student’s t-tests at each lysine position. Accessibility of lysines in the HD1 and FCS 260 domains of Mini-Ph are changed on binding DNA: K1302 and K1340 of the HD1 in Mini-Ph are less 261 accessible in the 2X DNA conditions (condensates present), while K1298 and K1302 are less accessible in 262 the 16X DNA condition (Fig. 4C). As the ratio of DNA to Mini-Ph increases, the accessibility of three 263 lysines in the FCS domain (K1370, K1376, K1380) decreases relative to Mini-Ph alone (Fig. 4C). These 264 decreases in accessibility are consistent with this region being protected by binding to DNA, and indeed, 265 K816 of PHC1 (equivalent to K1380 in Ph) was previously identified as a nucleic acid binding residue in 266 NMR experiments 46 (Supplementary Fig. 12A, B). Changes in accessibility could also reflect changes in 267 protein conformation, particularly in the HD1, which is not known to bind DNA. The accessibility of the 268 three lysines in the linker region is low, and does not significantly change with addition of DNA. This is 269 consistent with the linker being in a collapsed state (Supplementary Fig. 4), although the low number of 270 lysines in the linker limits the resolution of the analysis. The accessibility of lysines in the SAM is low 271 both with and without DNA, with no significant changes (Fig. 4C). 272 To validate global changes in accessibility, we also compared average accessibility of all lysines 273 in each domain under the different conditions (Supplementary Fig. 11C). This confirms the reduction in 274 accessibility of the HD1 and FCS under conditions where condensates form, and no change in the 275 accessibility of the SAM (Supplementary Fig. 11C). These data are consistent with the SAM maintaining 276 its folded structure and pre-existing polymeric state on binding DNA and in condensates. Prolonged 277 incubation in sulfo-NHS-acetate leads to dissolution of condensates (Suplementary Fig. 12 A-C), likely by 278 disrupting binding of Mini-Ph to DNA. Indeed, if Mini-Ph is fully acetylated with sulfo-NHS-acetate, it 279 does not bind DNA, and does not form condensates with DNA (Supplementary Fig. 12 D-F). 280 We then compared accessibility of lysines in Mini-Ph to that in Mini-Ph EH, which does not form 281 polymers. The pattern of lysine accessibility in Mini-Ph EH is distinct from that of Mini-Ph, and 282 differences are not restricted to the SAM (Fig. 4D). Three lysines in the HD1, one in the FCS, and one in bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

283 the SAM are significantly altered in Mini-Ph EH versus Mini-Ph. When differences are considered over 284 each domain, they are more striking (Fig. 4E). While the overall accessibility of the HD1 is the same 285 between the two, probably because both increases and decreases in accessibility are observed, the FCS 286 is less accessible in Mini-Ph EH than in Mini-Ph. while the linker and the SAM are more accessible (Fig. 287 4D-F). The accessibility of the SAM is consistent with the expected monomeric state of Mini-Ph EH and 288 the positions of the lysines in the SAM polymer structure (Fig. 4F). However, thedifferences in the other 289 domains of Mini-Ph EH versus Mini-Ph indicate that SAM polymerization likely affects the whole 290 conformation of Mini-Ph and the interactions available for phase separation. The changes in the HD1 291 both on condensate formation and between Mini-Ph and Mini-Ph EH also raise the possibility that this 292 domain contributes interactions to phase separation, which will need to be directly tested. Whether Ph 293 SAM would also affect the conformation of Ph in the context of the full length protein, or when it is in 294 PRC1 (an interaction mediated by the HD1) remains to be determined. Finally, we attempted to analyze 295 lysine accessibility in Mini-Ph EH condensates (Supplementary Fig. 13), but the condensates dissolved 296 within 5 min. of adding the acetylation reagent. 5 min. of acetylation results in most of the protein being 297 inaccessible (Supplementary Fig. 13 C, D). After 15 min. of acetylation, accessibility is similar with and 298 without DNA (Supplementary Fig. 13E), consistent with DNA binding being completely disrupted. 299 Comparison of Mini-Ph EH alone after 5 min. or 15 min. of acetylation indicates that the linker and SAM 300 are increasingly acetylated with time (Supplementary Fig. C, F). This is consistent with SAM-SAM and/or 301 linker-SAM interactions (Supplementary Fig. 10), although other explanations are possible. 302 303 Ph SAM Polymerization affects the mobility of Mini-Ph in condensates. The experiments presented 304 above indicate that Ph SAM polymerization increases the DNA binding affinity of Mini-Ph (Fig. 3C), 305 increases the driving forces for phase separation (Fig. 3F-J, Supplementary Fig. 8, 9), and changes the 306 accessibility of Mini-Ph (Fig. 4). To determine whether the polymeric state of Mini-Ph also affects the 307 material properties of condensates, we compared Mini-Ph and Mini-Ph EH mobility in condensates 308 formed with chromatin (Supplementary Fig. 14). In side-by-side experiments, recovery of fluorescence is 309 consistently faster with Mini-Ph EH (Supplementary Fig. 14 A, C, D). We fit FRAP data to a double 310 exponential function. The T1/2 for both slow populations is lower for Mini-Ph EH than for Mini-Ph the % 311 of molecules in the fast fraction is higher for Mini-Ph EH than for Mini-Ph, and the mobile fractions are 312 similar for both (Supplementary Fig. 14 G-J). To analyze chromatin mobility, we analyzed the Cy3 label 313 on H2A in same condensates used to collect FRAP traces for Alexa-647 labelled Mini-Ph or Mini-Ph EH 314 before and after bleaching (Supplementary Fig. 14 B, E, F). Less than 10% of the fluorescence is 315 recov ered over the 5 min. experiment for condensates formed with Mini-Ph and Mini-Ph EH 316 (Supplementary Fig. 13B). Thus, consistent with Fig. 2, chromatin and Mini-Ph or Mini-Ph EH have 317 distinct kinetics in condensates. The slow kinetics of chromatin may be intrinsic to the template since 318 the EH mutation in Mini-Ph does not affect them. We conclude that assembly of Mini-Ph into polymers 319 not only increases the driving force for phase separation, but influences the material properties of the 320 condensates that are formed. 321 322 Mini-Ph-chromatin condensates recruit PRC1 from nuclear extracts. One function of phase separation 323 is to create biochemical compartments that are enriched for specific components, and can stimulate or 324 inhibit biochemical reactions. To determine whether Mini-Ph-chromatin condensates can create unique 325 biochemical compartments, we asked whether condensates can recruit proteins from nuclear extracts 326 (Fig. 5A). We prepared nuclear extracts from Drosophila S2R+ cells, and used an anion exchange resin to bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

327 deplete nucleic acids from the extracts. Even after depletion, the nuclear extracts contain substantial 328 amounts of RNA (Supplementary Fig. 15A). Treatment of extracts with RNAseA resulted in precipitation 329 of most of the protein from the extracts, so that we used extracts containing RNA for our experiments. 330 Chromatin alone forms a few tiny structures in extracts (Fig. 5B, C, reaction 1). Mini-Ph does not form 331 condensates in buffer (e.g. Fig. 1C), but does form small condensates in extracts, likely by binding to 332 RNA, since the condensates stain with YOYO-1 (Fig. 5B, C, reaction 2). When Mini-Ph is incubated with 333 chromatin to form condensates, and then nuclear extracts are added, the condensates are preserved, 334 although they are smaller than condensates in equivalent reactions incubated in buffer (Fig. 5B, C, 335 compare reactions 3 and 4). Although the condensates are smaller, the concentration of chromatin in 336 them is similar to that in condensates incubated in buffer (Fig. 5D). We do not know why the 337 condensates are smaller after incubation in nuclear extracts. Post-translational modifications can 338 influence phase separation 57, but the small molecule substrates needed for enzymes that mediate them 339 should be depleted in our desalted extracts. The presence of nucleic acids, in the extracts could disrupt 340 condensates, analogous to what is observed at high concentrations of DNA (Supplementary Fig. 2F, G). 341 Alternatively, proteins in the extracts that bind to Mini-Ph and/or chromatin may disrupt interactions 342 required for condensates. 343 We used low speed centrifugation (2 min. @ 2500*g) to isolate condensates and analyzed their 344 nucleic acid content on agarose gels. When Mini-Ph is incubated with extract in the absence of 345 chromatin, the pelleted condensates contain RNA (Fig. 5E). When Mini-Ph is incubated with chromatin, 346 and extract added subsequently, the isolated condensates contain both chromatin and RNA (Fig. 5E). 347 Since the amount of RNA that is pelleted with Mini-Ph is similar with and without chromatin, we infer 348 that Mini-Ph condensates in extracts can contain both RNA and chromatin (Fig. 5E-G). To confirm this, 349 we analyzed co-localization of fluorescently labelled Mini-Ph with chromatin after incubation in buffer, 350 or in nuclear extracts (Supplementary Fig. 15 B-D). Most Mini-Ph-containing structures also contain 351 chromatin. This is consistent with chromatin condensates recruiting RNA from the extracts, rather than 352 formation of a separate class of Mini-Ph-RNA condensates. 353 To analyze the protein components of Mini-Ph condensates in nuclear extracts, we used 354 Western blotting. PRC1 components are enriched in condensates formed in extracts with or without 355 chromatin, while the PRC2 subunits Su(Z)12 and p55, the single strand DNA binding protein RPA70, and 356 the chromatin remodeling complex subunit ACF1 are not enriched (Fig. 5H, I). Thus, Mini-Ph 357 condensates can concentrate endogenous PRC1 provided by nuclear extracts. 358 359 Mini-Ph-chromatin condensates enhance ubiquitylation of histone H2A. To determine whether the 360 PRC1 recruited to Mini-Ph condensates is active, we tested whether chromatin present in condensates is 361 ubiquitylated on histone H2A. When extracts were supplied with ATP and ubiquitin, very low levels of 362 ubiquitylated H2A (H2A-Ub) were detected. Addition of the E1 ubiquitin activating enzyme and E2 363 ubiquitin conjugating enzyme along with ATP and ubiquitin resulted in detectable H2A-Ub in extracts 364 (Fig. 6A, B). Formation of Mini-Ph-chromatin condensates prior to incubation in extracts increased H2A- 365 Ub by about two-fold. This suggests the PRC1 recruited to condensates is functional, and that Mini-Ph- 366 chromatin condensates enhance the ubiquitylation reaction (Fig. 6B, C). 367 To determine if the Ph SAM polymerization state can influence condensate formation in the 368 more physiological environment of nuclear extracts, we prepared condensates with Mini-Ph ML, or 369 Mini-Ph EH, and added nuclear extracts to them. Mini-Ph ML condensates behave similar to those 370 formed with Mini-Ph in extracts (Supplementary Fig. 16). In contrast, incubation of Mini-Ph EH bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

371 condensates in extracts transforms them into diffuse structures that occupy a larger area but have a 372 reduced chromatin concentration relative to condensates incubated in buffer (Supplementary Fig. 16). 373 We tested histone ubiquitylation in extracts in the presence of Mini-Ph ML or Mini-Ph EH, and find that 374 neither mutant stimulates histone ubiquitylation (Fig. 6B, C). We do not know if this is because the 375 condensates formed by the polymerization mutants have different properties (e.g. Supplementary Fig. 376 14), or because they recruit less PRC1, as would be expected if SAM-SAM interactions (between Mini-Ph 377 and Ph in PRC1) are directly involved in recruiting PRC1 to chromatin. 378 The observation that Mini-Ph condensates increase histone ubiquitylation might reflect the 379 increased concentration of PRC1 in condensates (Fig. 5H, I). It is not necessarily predicted, however, that 380 the environment of condensates, in which chromatin is compacted, would enhance enzyme activity. 381 Thus to determine whether Mini-Ph-chromatin condensates enhance PRC1 activity under optimal 382 conditions, we reconstituted the ubiquitylation reaction in vitro, using chromatin alone or Mini-Ph- 383 chromatin condensates as the substrate (Supplementary Fig. 17). We used PRC1ΔPh for these 384 experiments (Supplementary Fig. 1B), which can interact with Mini-Ph via the HD1 domain (but unlike 385 PRC1 found in extracts, not via SAM-SAM interactions), and is fully active as an E3 ligase. PRC1ΔPh 386 catalyzes formation of H2A-Ub on chromatin in a dose dependent manner (Fig. 6D; Supplementary Fig. 387 17C, D). When Mini-Ph-chromatin condensates are used as the substrate, the activity of PRC1ΔPh is 388 increased by about two-fold over the entire titration, indicating that condensates stimulate PRC1ΔPh 389 activity (Fig. 6D, E). We also analyzed condensates at the end of the reactions to confirm that they 390 persist under reaction conditions (Fig. 6F, G). Because a high fraction of the histones is ubiquitylated in 391 these experiments (Fig. 6D, E), these results indicate that H2A-Ub does not disrupt condensates. 392 393 Ph SAM affects ubiquitylation of H2A in vivo. To test whether the activity of Ph SAM is important for 394 histone ubiquitylation in vivo, we used Drosophila S2 cell lines that express Ph or Ph with the strong ML 395 mutation (L1547R/H1552R), which disrupts Ph SAM polymerization as effectively as the EH mutant used in 396 our in vitro studies, under control of an inducible promoter 14. We isolated histones from control S2 cells 397 and cells induced to overexpress Ph or Ph-ML, and measured levels of H2A-Ub (Fig. 7A-D). Cells 398 overexpressing Ph have an approximately two-fold increase in overall H2A-Ub relative to control cells 399 (Fig. 7B). Cells overexpressing Ph-ML have increased H2A-Ub in some experiments, but this difference 400 was not significant, even though Ph-ML is expressed at higher levels than Ph (Fig. 7A, C). 401 Because we find that Ph SAM polymerization activity is not strictly required for phase separation 402 in vitro, we wondered if Ph-ML might be able to phase separate in vivo, particularly when present at 403 high concentrations. Formation of highly concentrated foci in cells is consistent with phase separation, 404 although it can arise through other mechanisms, as has been pointed out 35. To test whether Ph-ML can 405 form foci in cells, we transiently transfected Drosophila S2 cells with Venus-tagged Ph, Ph-ML, or 406 PhΔSAM under control of the heat shock promoter. After heat shock induction, Venus-Ph forms large, 407 round, bright foci. These foci are mainly (although not exclusively) nuclear, and little Venus signal is 408 observed in the nucleoplasm outside the foci (Fig. 7E). In contrast, Venus-PhΔSAM is uniformly 409 distributed in the nucleus, and does not form foci (Fig. 7F). Venus-Ph-ML forms foci but is also 410 distributed throughout the nucleus (Fig. 7G). Thus, foci formation in vivo and phase separation in vitro 411 are correlated with each other and with enhanced histone ubiquitylation. We tested Venus-Mini-Ph in 412 Drosophila S2 R+ cells, and find that, unlike Venus-Ph, it does not form foci in most cells. In about 7% of 413 the cells, it forms a single focus, which can be quite large (Supplementary Fig. 18 A, B, D); these unusual 414 foci are not observed with Venus-Mini-PhΔSAM (Supplementary Fig. 18C, D). Thus, although Ph SAM is bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

415 required for foci formation in cells, the other disordered regions of Ph shape its behavior in cells as been 416 observed for other proteins that can undergo LLPS 57. 417 418 Discussion: We have identified phase separation as a new activity of the Ph SAM, the domain that is 419 most clearly implicated in large-scale chromatin organization by PcG proteins. Our data are consistent 420 with two possible functions of Ph SAM-dependent phase separation: 1) formation of a compacted yet 421 fluid chromatin state; 2) creating unique biochemical compartments that enhance PRC1-mediated 422 histone modification. 423 424 Phase separation by Ph SAM does not strictly require its polymerization activity. In developing 425 Drosophila embryos, Ph lacking the SAM cannot rescue any Ph functions, while Ph with a polymerization 426 interface mutated can partially rescue Ph function, although with defects in transcriptional repression 20. 427 In vitro, Mini-Ph lacking the SAM does not form phase-separated condensates, while Mini-Ph with the 428 polymerization interface mutated (Mini-Ph EH) does form condensates although they are smaller. In 429 Drosophila tissue culture cells, Ph lacking the SAM does not form foci, while polymerization defective Ph 430 (Ph-ML) can form foci when overexpressed (Fig. 7). Thus, foci formation in vivo, and phase separation in 431 vitro are correlated with full Ph function, and the LLPS activity of Ph SAM may be the critical function of 432 the SAM that remains even when polymerization is disrupted. 433 434 Previous work, implicates Ph polymerization in both transcription repression and chromatin 435 organization 5,13,14,20,21. Stochastic Optical Reconstruction Microcsopy (STORM) analysis of PcG proteins 436 in normal Drosophila tissue culture cells or those that mildly overexpress Ph or Ph-ML showed that 437 normal Drosophila tissue culture cells contain hundreds of nanoscale PcG clusters, although only a few 438 large PcG bodies are visible by conventional microscopy 14. Mild overexpression of Ph increased the 439 number but not the size of clusters, and increased long-range contacts, while overexpression of the 440 strong Ph-ML mutant disrupted clusters and reduced long-range contacts. This work and work in 441 mammalian cells 13 directly implicates Ph SAM polymerization in the nanoscale organization of PcG 442 proteins and large-scale organization of chromatin.

443 Although Ph SAM alone can form open-ended polymers, the extent to which long SAM polymers 444 occur in the context of the full protein is unclear. In vitro, the oligomeric state of Mini-Ph is limited to 445 four to six units 5; this can be explained by the action of the unstructured linker that separates Ph SAM 446 from the FCS in conjunction with the helical configuration of SAM polymers 18. Steric considerations 447 suggest Ph SAM polymerization may be even further restricted in the context of PRC1. Thus, the 448 contribution of polymerization to LLPS may be much subtler than would occur with an actual open- 449 ended Ph SAM polymer. The linker connecting the SAM to the FCS is not conserved in Ph homologues 450 (Supplementary Fig. 4). The linker of PHC3, unlike the Drosophila Ph linker 5, does not bind the PHC3 451 SAM in trans 50, and allows much more extensive SAM polymerization than that of Ph 50. It is therefore 452 possible that the linker has been tuned across evolution to control polymerization and its interplay with 453 phase separation. This is consistent with modeling based analysis indicating that the properties of 454 linkers connecting interacting domains tune phase separation properties 48. Two other PcG proteins, 455 SCM and Sfmbt, also have SAMs, and the three SAMS have been shown to co-assemble 58; joining of 456 SAM-mediated polymers of these three proteins could allow formation of large and diverse polymers. 457 Evaluating the phase separation activity of these other PcG SAMs, alone or in combination, and of Ph 458 homologues, will be an important future goal. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

459 The phase separation activity of Ph SAM is also likely subject to negative regulation. A 460 disordered, serine/threonine rich sequence adjacent to the HD1 undergoes O-linked glycosylation 461 mediated by the PcG protein Sxc 20,59. This region, and Sxc, are both important for Ph function in 462 regulation of some genes 20,59. In the absence of glycosylation, Ph undergoes SAM-dependent “non- 463 productive aggregation”, which is not alleviated by mutating the Ph SAM polymerization interfaces 20. It 464 is possible that “non-productive aggregation” in fact reflects SAM-dependent phase separation (or 465 maturation of phase-separated protein into stable, insoluble aggregates) 23. The glycosylated sequence 466 is not part of Mini-Ph. Mini-Ph is produced in E. coli, and is not glycosylated, yet Mini-Ph is soluble. It 467 therefore seems likely that the effect of glycosylation, although dependent on Ph SAM, also involves 468 other sequences in Ph. We speculate that the glycosylated region may restrict Ph SAM-mediated phase 469 separation, and preliminary in vitro data support this idea (unpublished observation). 470 471 A hallmark of LLPS is that it depends on weak, multivalent interactions that allow rapid 472 reorganization and unrestricted stoichiometry. The polymerization activity of Ph SAM may contribute 473 multivalent interactions. However, additional interactions are required, which (at least in Mini-Ph) 474 involve the HD1 and/or the FCS. Based on the comparison between Mini-Ph and Mini-Ph EH, linker-SAM 475 and (possibly) SAM-SAM interactions that do not require an intact polymerization interface likely also 476 contribute (Supplementary Fig. 10). In vitro, dynamic SAM polymerization is not likely to directly drive 477 phase separation by Mini-Ph because the Kd for polymerization is so much lower than the saturation 478 concentration at which phase separation occurs. However, in the polymerization mutants, and in vivo 479 where the concentration of Ph is lower 52,60, dynamic polymerization of Ph SAM could control phase 480 separation. In LLPS of Mini-Ph with chromatin or DNA, the role of the FCS is likely nucleic acid binding; 481 however, the HD1 and/or the FCS may form additional protein-protein interactions (Fig. 4). It is 482 interesting to note that Sfmbt and SCM, the other two SAM containing proteins also contain an FCS, 483 although the distance and additional motifs separating the FCS from the SAM varies. The combination of 484 an FCS (i.e. a nucleic acid binding domain) and a SAM could allow these proteins also to undergo phase 485 separation. In support of this idea, the C. elegans SOP-2 protein functions as a PcG protein 61, and forms 486 large nuclear bodies 62. Although it is not a clear sequence homologue of Ph, SOP-2 consists of an RNA 487 binding motif, an intrinsically disordered region (IDR), and a SAM 63. Recently, the IDR of SOP-2 was 488 shown to undergo LLPS in vitro, induced by crowding agents or RNA 41. Addition of the SAM to the IDR 489 still allowed LLPS, but resulted in formation of smaller condensates that showed lower recovery in FRAP 490 experiments 41. 491 492 A model for the function of Ph SAM that can reconcile the seemingly different requirements for 493 the SAM and its polymerization activity in different contexts is that Ph SAM drives at least three 494 different states. First, Ph SAM polymerization activity may drive formation of tiny PcG clusters that 495 mediate local repression of transcription simply through cooperative binding interactions. This is 496 consistent with our finding that Ph SAM and its polymerization activity increases the DNA binding 497 affinity of Mini-Ph, at concentrations well below the range where phase separation occurs (Fig. 3). It is 498 also consistent with the dependence of Ph repressive activity when targeted to a reporter gene on Ph 499 SAM polymerization activity 5. Second, bridging of nucleosomes mediated by the polymerization 500 interfaces of Ph SAM associated with chromatin bound PRC1 may drive collapse of the chromatin 501 polymer over larger regions of PRC1 bound chromatin 14,44,64,65. Indeed, a model of this process could 502 explain the observed effects of overexpressing Ph with the strong ML mutation or wild-type Ph, which bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

503 increases the number but not size of Ph clusters 14. In cases where the local concentration of Ph is very 504 high, Ph may undergo LLPS mediated by multivalent interactions among Ph molecules and between Ph 505 and chromatin (or Ph and RNA), as captured by our in vitro assays, and possibly, in the foci observed 506 when Venus-Ph is overexpressed in cells (Fig. 7). Which mechanism dominates in any situation could be 507 modulated by the local concentration of PcG proteins (i.e. how strong a PcG recruitment site is, or the 508 density of recruitment sites). This could be analogous to the distinction between enhancers and super- 509 enhancers, which recruit higher levels of transcription factors and co-factors and where LLPS is believed 510 to occur 34,66. There is also no reason at this time to exclude hybrid models 54. For example, LLPS could be 511 a mechanism to create biochemical compartments, and within these domains, strict SAM-SAM 512 interactions could establish precise chromatin contacts required for gene repression. LLPS may also 513 represent an extreme and transient state, used to silence large chromatin domains rapidly during 514 development 12,67, or as a step in re-establishing gene expression patterns during the cell cycle. All of 515 these possibilities remain to be tested, but the separation of phase separation and polymerization 516 activity revealed by our simple in vitro assays may provide a means to do so. 517 518 Many proteins with diverse localizations and functions have SAMs. Some SAMs have been 519 shown to polymerize in a concentration dependent manner, while others require additional recruitment 520 mechanisms to induce polymerization. The SAMS of a subset of proteins, including Ets1, Fli1, and p63 68, 521 have not been observed to polymerize. It is therefore possible that phase separation is a property of the 522 SAM that is distinct from polymerization, a hypothesis that is testable by measuring the phase 523 separation activity of proteins with monomeric SAMs. 524 525 Ph SAM and histone ubiquitylation. We find that Ph SAM driven chromatin condensates can enhance 526 PRC1-mediated histone ubiquitylation. We do not know what the mechanism of stimulation of H2A-Ub 527 is. It is unlikely to be concentration of the reaction components in condensates because all of the 528 components (except PRC1ΔPh) are present at saturating concentrations in these reactions. PRC1ΔPh 529 binds chromatin tightly (Kd for 150 bp DNA is <=1 nM 69) so that Mini-Ph is also not needed to recruit 530 PRC1ΔPh to chromatin. Although further experiments will be needed to determine the mechanism, the 531 environment of condensates may stimulate steps in the reaction subsequent to substrate binding, which 532 could include the actual ubiquitin transfer or steps affecting processivity 70. It has recently been shown 533 that H2A-Ub mediated by PRC1 is stimulated by chromatin compaction 71, and that spreading of H2B-Ub 534 along chromatin is facilitated by formation of structured, phase-separated compartments by the 535 ubiquitylation machinery 72, which may be relevant to our observations. Formation of protein-chromatin 536 condensates with the heterochromatin protein HP1 alters the conformation of the nucleosome, 537 rendering specific regions of the histone proteins more accessible 73. It is possible that nucleosome 538 conformation is also changed in Mini-Ph condensates, and that these changes facilitate histone 539 ubiquitylation. Detailed characterization of chromatin in condensates will be an important future goal. 540 Stimulation of H2A-Ub is unlikely to be the essential function of the Ph SAM in Drosophila, since 541 the modification is not required for PRC1-dependent gene repression in vivo, including repression of 542 genes that depend on Ph SAM 74,75. However, H2A-Ub is required for full development 74,75. Drosophila 543 cPRC1 also does not seem to mediate most H2A-Ub in tissue culture cells, and it is likely that another 544 ncPRC1 containing L3(73)Ah, a homologue of mammalian Pcgf3, in place of PSC, is present in these cells 545 76. This also means that in our experiments with nuclear extracts, although we observe PRC1 recruitment bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

546 to condensates, we cannot be certain that it is responsible for the ubiquitylation activity we observe 547 (Fig. 6). 548 Histone ubiquitylation by PRC1 has been most intensively studied in mouse embryonic stem 549 cells (mESCs), where systematic analysis of the effect of disrupting PRC1 subunits implicates ncPRC1 (i.e. 550 non PHC-containing) in creation of most H2A-Ub 7-9. However, using an artificial tethering system that 551 allows PcG proteins to be reversibly targeted to a reporter gene so that persistent effects on chromatin 552 and gene expression (i.e. memory) can be measured, Moussa et al. 77 found that heritable gene 553 repression and propagation of H2A-Ub depend on cPRC1. Recent work indicates a central role for H2A- 554 Ub in PcG-dependent gene regulation in mESCs 78,79, in seeming contrast with observations in 555 Drosophila; it will be interesting to determine how Ph SAM contributes to H2A-Ub activity in mammals. 556 The ability of Ph SAM to condense chromatin and to promote H2A-Ub could be important for rapidly 557 building PcG chromatin domains, or restoring them at the end of mitosis. H2A-Ub is not detected on 558 mitotic chromosomes in mammalian cells 80,81, suggesting it is re-acquired after cells exit mitosis. 559 Finally, Cbx2, a member of some mammalian canonical (PHC-containing) PRC1s, which has a 560 strong chromatin compacting activity 82, has also been shown to form phase separated condensates with 561 chromatin in vitro, and to form 1,6-hexanediol-sensitive foci in ES cells 40,42. This phase separation 562 activity is mediated by a charged IDR in Cbx2 that is important for the developmental function of Cbx2 563 83. Further, as shown in Supplementary Fig. 17, Mini-Ph does not form foci in cells, indicating that other 564 sequences in Ph, all of which are predicted to be disordered, can regulate the activity of the Ph SAM. 565 How the activity of Ph SAM is regulated by other sequences in Ph and coordinated with that of other 566 components of PRC1, particularly that of PSC which has a powerful chromatin compacting activity 567 analogous to that of Cbx2 84, is an important question for future study. 568 569 Methods: 570 Cloning: Cloning of Mini-Ph and the polymerization mutants was described previously 5. Mini-Ph∆SAM 571 (residues 1291 – 1507) and Mini-PhΔFCS (residues 1397 – 1577) were cloned into a modified pET-3c 572 vector expressing a leader sequence containing a hexahistidine tag followed by a TEV cleavage site. To 573 express Venus-tagged proteins in S2 cells, Ph, Ph-ML, or PhΔSAM were first cloned into a house- 574 modified gateway donor vector and full sequences confirmed. LR recombination was used with pHVW 575 from the DGRC (stock # 1089) to produce the final expression plasmids. 576 577 Protein purification: 578 Mini-Ph: His-tagged Mini-Ph, Mini-Ph-EH, and Mini-Ph-ML were expressed in Rosetta (DE3) E. coli. 579 Cultures were grown at 37°C to an OD of 0.8-1.0, and then shifted to 15°C for overnight induction with 580 1mM IPTG. Cells were pelleted, flash frozen, and stored at -80°C. Cells were resuspended in 2 ml/g lysis 581 buffer (50 mM Tris, pH 8.5, 200 mM NaCl, 10 mM β-ME, 100 µM ZnCl2, 0.2 mM PMSF, 0.5 mM 582 Benzamidine). Cells were incubated on ice for 10 min, flash frozen in liquid nitrogen, thawed at 37°C, 583 and sonicated 6*30 sec. at 30% intensity. Freeze-thaw and sonication were repeated, and the lysate 584 centrifuged for 1 hour at 100,000*g and 4°C. Cleared lysate was sonicated 6*30” at 40% intensity, and 585 filtered through a 22 µm filter. Lysate (from 1 L) was applied to a 1 ml His-Trap column using an AKTA 586 FPLC, and eluted with a gradient of imidazole (from 10-300 mM) in lysis buffer. Fractions with Mini-Ph 587 were dialyzed overnight against 1 L of 20 mM Tris, pH 8.5, 50 mM NaCl, 100 µM ZnCl2, and 10 mM β-ME. 588 Dialyzed fractions were centrifuged for 10 min. at 20,800*g, and loaded on a 1 ml HiTrapQ-HP column 589 and eluted with a gradient from 50 mM to 1 M NaCl in binding buffer. Fractions were pooled and bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

590 dialyzed overnight into 20 mM Tris, pH8, 50 mM NaCl, 10 µM ZnCl2, 1 mM βME, aliquotted and stored at 591 -80. In some cases, Mini-Ph was further purified by size exclusion chromatography on a Superose 12 size 592 exclusion column. 593 Mini-Ph∆SAM and Mini-Ph∆FCS: Both proteins were expressed in BL21 (DE3) Gold cells pre- 594 transformed with the pRARE plasmid. The transformed cells were grown at 37°C in LB media to an OD600 595 of ~0.7 – 0.8 and induced overnight at 15°C. Cells harvested from 1 L of culture were resuspended with 596 10 ml of lysis buffer (50 mM Tris pH 8.0, 200 mM NaCl, 5 mM βME, 30 mM imidazole pH 7.5, 1 mM 597 PMSF) and lysed by sonication. The soluble lysates were introduced onto am Ni-NTA column, washed 598 with lysis buffer (without PMSF), and bound proteins eluted using 300 mM imidazole, 200 mM NaCl, 5 599 mM βME. The leader sequence was cleaved using TEV protease, and the cleaved sequence and 600 uncleaved proteins removed by passing through a Ni-NTA column. Further purification was performed 601 using a HiTrap Q-HP column. Fractions containing protein were pooled, buffer exchanged into 50 mM 602 Tris pH 8.0, 100 mM NaCl, 5 mM βME, and concentrated. Mini-PhΔSAM was further purified on a 603 Superdex 200 size exclusion column in 50 mM Tris pH 8.0, 100 mM NaCl, 5 mM βME. Purified, 604 concentrated proteins were stored at -80°C 605 E1, E2, and His-Ub: The following plasmids were used: human 6X-His-UBA1 (E1) (pET21d-Ube1, 606 addgene #34965), Human UbcH5c (E2) (pET28a-UbcH5c, addgene # 12643), 6XHis-Ubiquitin (pET15b- 607 His-Ub) (kind gift of B. Schulman). Proteins were expressed in E.coli and purified essentially as described 608 85,86. His-Ube1 was purified by Ni-NTA affinity followed by Superdex 200 chromatography 85. UbcH5c 609 was purified on a HiTrap SP-XL column followed by Superdex 200 86. 6X-His-Ub was purified by Ni-NTA 610 chromatography. 611 Histone purification: Xenopus laevis histones, including H2B-122C mutant were expressed in and 612 purified from E. coli, as described 87,88. All experiments were carried out with histone H3 with Cys110 613 (the only cysteine natively present in the histones) mutated to Ala. 614 Fluorescent labeling of histone H2A with NHS-Cy3 was carried out under conditions favouring 615 labeling of the N-terminal amine. Lyophilized H2A was resuspended in labeling buffer (20 mM Hepes, pH 616 6.2, 7 M Guanidium-HCl, 5 mM EDTA) to a concentration of 0.1 mM. NHS-Cy3 stock (in DMF) was added 617 to a final ratio of 0.5:1 (dye to histone) and incubated at room temp. for 90 min. Free dye was removed 618 with Amicon concentrators, after diluting with labeling buffer without Guanidium to reduce the Gu-HCl 619 concentration to 6 M. In some cases, Zeba columns were used instead to remove free dye. To label H2B- 620 122C with maleimide-Alexa 647, lyophilized histone was reconstituted in denaturing labeling buffer (20 621 mM Tris-HCl, pH 7.0, 7 M guanidium HCl, 5 mM EDTA) to a final concentration of 0.1 mM followed by 622 treatment with a 100-fold excess of TCEP for 30 minutes. Maleimide-Alexa 647 was added to a final ratio 623 of 3:1 (dye:histone) and incubated for 3 hours at room temp. The labeling reaction was quenched with 624 β-ME (final concentration 80 mM), and free dye removed as above. Octamer reconstitutions and 625 purification on a Superdex 200 size exclusion column were carried out as described 87,88. Concentrated 626 octamers were dialyzed into octamer refolding buffer (2 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA, 5 mM 627 β-ME) with 50% glycerol and stored at -80°C. 628 PRC1ΔPh: PRC1ΔPh was purified from nuclear extracts of baculovirus-infected Sf9 cells essentially as 629 described previously 69, with the following changes. Nuclear extracts were prepared from Sf9 cells 630 infected with viruses for the three subunits (Flag-PSC, Pc, dRING) but nuclei were purified through a 631 sucrose cushion prior to nuclear extraction. During the purification, the 2 M KCl wash in the published 632 protocol was replaced with a wash consisting of BC2000N + 1 M Urea (20 mM Hepes, pH 7.9, 2 0.4 mM 633 EDTA, 2 M KCl, 1 M deionized urea, 0.05% NP40, no glycerol). Additionally, prior to eluting the protein, bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

634 anti-FLAG beads were incubated 3-5 volumes of BC300N with 4 mM ATP + 4 mM MgCl2 for 30 min. at 635 room temperature. This step reduces the amount of HSC-70 that co-purifies with PRC1ΔPh. Protein was 636 eluted with 0.4 mg/ml FLAG in BC300 without NP40, concentrated to ~1 mg/ml and stored in BC300N 637 (20 mM Hepes, pH 7.9, 300 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.05% NP40). 638 639 Fluorescent labelling and acetylation of Mini-Ph and other proteins: To fluorescently label proteins, 640 NHS-ester-Cy3 or Alexa-647 were used to randomly label lysines. A Zeba column (Thermo Fisher) was 641 used to buffer exchange the protein into 20 mM Hepes, pH 7.9, 200 mM NaCl for Mini-Ph, or BC300N for 642 proteins expressed in Sf9 cells; labeling was carried out with a 0.5:1 (dye:protein) ratio for 15 min. at 643 room temp. Labelling was quenched by addition of Lysine to 10 mM. Free dye was removed using two 644 Zeba columns, which were equilibrated in the labeling buffer with 200 mM NaCl. Labelled protein was 645 mixed with unlabelled at a ratio of 1 to 25 for imaging experiments. Acetylation of Mini-Ph was carried 646 out exactly as for fluorescent labelling except that a ratio of 8:1 sulfo-NHS-acetate:lysine residues in 647 Mini-Ph was used and labeling was carried out for 1 hour at room temp. 648 649 Preparation of Nuclear Extracts from Drosophila S2R+ cells: S2R+ cells were grown in M3-BYPE media 650 with 10% FBS. 20*15 cm dishes were used to prepare nuclear extracts as described 89, except that nuclei 651 were purified through a sucrose cushion prior to extraction. Cells lysed in hypotonic buffer were layered 652 over two volumes of 30% sucrose in hypotonic buffer, and centrifuged 18’ @ 1400*g. Nuclei were 653 washed once in hypotonic buffer, and extracted as described. The high salt extraction buffer was 1.2 M 654 KCl, and extracts were not dialyzed. To use the extracts to treat condensates, up to 100 µl of extract was 655 buffer exchanged into 20 mM Tris, pH 8, 50 mM NaCl using a Zeba column. Extracts were centrifuged 2’ 656 @ 20,000*g and incubated for 15’ on ice with 60% volume of Q-sepharose. Extracts were spun through 657 an empty column (2’ @ 10,000*g), and then centrifuged 2’ @20,000 *g. All procedures were carried out 658 on ice or at 4°C and contained protease inhibitors and 0.4X PhosStop phosphatase inhibitor. 659 660 Chromatin preparation: Most experiments were carried out with the plasmid p5S*8, which contains 5 661 blocks of 8-5S nucleosome positioning sequences (repeat length 208 base pairs). Plasmids were 662 assembled by salt gradient dialysis as described 90. Chromatin was finally dialyzed into HEN (10 mM 663 Hepes, pH 7.9, 0.25 mM EDTA, 10 mM NaCl) buffer and stored at 4˚C. To measure chromatin assembly, 664 100 ng of each assembly was digested overnight with 10 U of EcoRI in NEB buffer 2.1, and loaded on a 665 0.5X TBE, 5% acrylamide native gel. Gels were stained with Ethidium bromide and imaged on a Typhoon 666 imager. For quantification, the nucleosomal signal is multiplied by 2.5 to account for the quenching 667 effect of bound protein on ethidium bromide 91. For Micrococcal nuclease analysis, 800-1000 ng of 668 chromatin was diluted into 40 µl of the following buffer: 12 mM Hepes, pH 7.9, 0.12 mM EDTA, 60 mM 669 KCl, 2 mM MgCl2 and split into 4 tubes. Micrococcal nuclease (Sigma, #N3755) (0.5 U/µl in 50 mM Tris, 670 pH 8, 0.05 mM CaCl2, 50% glycerol) was diluted 1:18, 1:54, 1:162, and 1:486 in MNase dilution buffer 671 (50 mM Tris, pH8, 10 mM NaCl, 126 mM CaCl2, 5% glycerol). 1 µl of each dilution was used to digest 672 chromatin for 7 min. at room temp. Reactions were stopped with DSB-PK (10X stock: 50 mM Tris, pH 673 8.0, 0.1 M EDTA, 1% SDS, 25% glycerol + 10 mg/ml Proteinase K), digested overnight at 50˚C, and 674 analyzed on 1X TBE-1.5% agarose (SeaKem) gels which were stained with Ethidium bromide and imaged 675 on a Typhoon Imager. 676 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

677 Phase separation assays: Proteins and templates were routinely centrifuged full speed in a microfuge 678 for 2-5 min. at 4°C to remove aggregates before setting up phase separation assays. For phase 679 separation assays, reactions (10-20µl) were assembled in a 384-well glass-bottom imaging dish 680 (SensoPlate, Greiner Bio-One). Wells were not pre-treated; pre-coating with BSA did not influence phase 681 separation by Mini-Ph. Phase separation was initiated by addition of the protein or the DNA, and mixing 682 the reaction by gentle pipetting, with care taken not to introduce air. Reactions were incubated in the 683 dark for 15 min. or up to several hours. For reactions where YOYO-1 (Thermo Fisher) was used, it was 684 added at the beginning of the reaction to a final dilution of 1:3000. Typical reaction conditions are 50 685 mM NaCl or 50 mM KCl, 20 mM Tris pH8. Reactions were set up on ice, and transferred to room temp. 686 for 15 min. Turbidity measurements were made in duplicate using a NanoDrop spectrophotometer. 687 Phase separated condensates were pelleted by centrifugation at 14,000*g for 2 min. at 4°C, and 688 supernatants removed to fresh tubes. Pellets were resuspended in 12 µl 1.5X SDS-Sample buffer, and 6X 689 SDS-Sample Buffer was added to the supernatant. 10% of the pellet and supernatant were removed and 690 digested in DSB-PK for 2 hours at 50°C for DNA analysis. The remainder of the sample was boiled and 691 analyzed by SDS-PAGE. 692 693 Imaging of condensates: All images were collected on a Zeiss microscope, equipped with a Yokogawa 694 CSU-1 spinning disc confocal head. Zen 2012 software was used for image acquisition with a 63X oil 695 objective, or a 100X oil objective (for movies and FRAP) and evolve EMCCD camera from Photometrics. 696 The excitation wave length for YOYO/Venus, Cy3/RFP and Alexa 647 were 488, 561 and 639 nm 697 respectively

698 Measuring nucleosome concentration in condensates: Images were collected at 25% laser power, 200 699 msec exposure for buffer, chromatin alone, a titration of labelled histone octamers (in octamer refolding 700 buffer, which contains 2M NaCl, and in which histone octamers remain assembled), and Mini-Ph 701 chromatin condensates. Histones are the same histones used to prepare the chromatin; 43% of the 702 histone octamers are labelled (measured both using the NanoDrop and by loading histones and free dye 703 on SDS-PAGE gels), corresponding to a 21.5% labeling efficiency on H2A (since there are two copies of 704 H2A in each octamer). Image J “measure” was used to measure the mean grey intensity for each of 9 705 images for each point. Images were manually checked and images with bright artifacts removed, 706 although these had little impact on the measured intensities. A linear regression was fit to the 707 calibration curve and used to convert measured intensities to nucleosome concentrations. To measure 708 intensities in condensates, Image J was used to threshold the images (AutoThreshold-->Li); Analyze 709 Particles was used to measure the mean grey intensity in each thresholded structure. Particle size was 710 set as 100-infinity pixels. The mean grey intensity from the buffer image was subtracted from all 711 measurements, which were converted to nucleosome concentrations using the calibration curve.

712 FRAP: FRAP experiments were carried out with Alexa-647 labelled Mini-Ph or Mini-Ph EH. Bleaching was 713 done with a 595 nm laser, for 1500 msec. This effectively bleaches both Alexa-647 Mini-Ph, and Cy3- 714 H2A, although we were only able to record FRAP images from one channel. Two pre-bleach images were 715 collected, followed by an image every 5 or 10 sec. All FRAP analysis of Mini-Ph was done by bleaching 716 single complete structures. Images were analyzed in Image J (Fiji). An ROI was selected for the bleach 717 area, background, and a non-bleached structure. Background subtracted, normalized data were fit with 718 a double exponential fit using GraphPad Prism 8. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

719 Y=Y0+ SpanFast*(1-exp(-KFast*X)) + SpanSlow*(1-exp(-KSlow*X)). We excluded data sets that could not 720 be fit, and obvious technical artifacts (e.g. if a drop fuses with the bleached condensate during the 721 experiment). 722 723 Image analysis of condensates: Images for display were prepared using Zen2 (blue edition). For 724 quantification, images were exported as TIFs from Zen (original data). ImageJ (Fiji) was used to threshold 725 the images (Li algorithm); thresholds were manually checked and images with too few structures to 726 threshold were removed. Areas of thresholded structures were measured using ImageJ (“Analyze 727 Particles”, size=10-infinity pixel), and intensities using Analyze Particles. For colocalization analysis, the 728 GDSC-->Colocalization-->Particle Overlap was used. Masks were created in the Alexa 647 (Mini-Ph) and 729 Cy3 (chromatin) channels, and overlap of Cy3 with Mini-Ph structures measured. 730 Movies were created from .czi files in ImageJ (Fiji). Movies were saved as .avi files at 1, 2, or 3 731 frames per second, and using PNG compression. 732 733 Filter binding: Filter binding was carried out as described 69,92. Briefly, a 150 bp internally labelled DNA 734 probe was prepared by PCR and gel purified. The probe was used at 0.02 nM. Reaction conditions were 735 60 mM KCl, 12 mM Hepes, pH7.9, 0.24 mM EDTA, 4% glycerol, in a 20 µl volume. Proteins were 736 centrifuged 2 min. at full speed in a microfuge before preparing the dilution series. Binding reactions 737 were incubated 1 hour at room temperature. Hybond-XL was used as the bottom membrane (binds 738 DNA), and was pre-equilibrated in 0.4 M Tris, pH 8. Nitrocellulose was used as the top membrane (binds 739 protein + DNA), and was pre-treated with 0.4 M KOH for 10 min., neutralized by washing through 740 several changes of Milli-Q water, and equilibrated for at least 1 hour in binding buffer. Filters were 741 assembled in a 48-well slot-blot apparatus, and each well washed with 100 µl binding buffer. The 742 vacuum was turned off, and reactions loaded on the filters. Slots were immediately washed with 2*100 743 µl binding buffer. Filters were air dried, exposed to a phosphoimager screen, and scanned on a Typhoon. 744 ImageQuant was used to quantify top (bound) and bottom (unbound) filters, and fraction bound 745 calculated in Excel. Curve fitting was done in GraphPad Prism 8, using the following equation: 746 Y=ABmax*X/(X+Kd)+b 747 748 Protein footprinting assay: The acetylation footprinting assay is described in detail in Kang et al. 6. Phase 749 separation reactions were directly scaled up to use 4 µg of protein for each sample. Condensates were 750 allowed to form at room temp. for 15 min.; an aliquot of each sample was removed to confirm phase 751 separation using microscopy. Sulfo-NHS-acetate was dissolved immediately before use, and added to a 752 final concentration of 0.5 mM. An aliquot of each sample was removed to monitor phase separation by 753 microscopy, and reactions were stopped after 15 min. by addition of Trifluoroacetic acid to a final 754 concentration of 1%. For Mini-Ph EH, acetylation of condensates was restricted to 5 min. because these 755 condensates dissolved rapidly on exposure to Sulfo-NHS-acetate. We therefore analyzed Mini-Ph EH 756 alone, and bound to DNA (16X DNA, Fig. 4) after both 5 and 15 min. of acetylation. Samples were TCA 757 precipitated, denatured with 8 M urea, reduced with DTT (45 mM final concentration), treated with a 758 final concentration of 10 mM Iodoacetamide, and diluted 1:2 with H2O before treating with Propionic 759 anhydride twice. Samples were dried, treated with Propionic anhydride again, dried, resuspended and 760 digested sequentially with Trypsin and Chymotrypsin. Samples were purified with a ZipTip and analyzed 761 by LC-MS/MS on an Orbitrap-Fusion mass spectrometer. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

762 Mass Spectrometry data were analyzed using Maxquant (v 1.6.10.43) with Acetyl(K) and 763 Propionylation(K) as variable modifications. 10 missed cleavages were allowed since lysine modification 764 will block trypsin digest. All data files were analyzed together, with the “match between runs” option. 765 Intensities for identified Acetyl and Propionyl sites were used for quantification. Accessibility was 766 calculated (in Excel) as (intensity acetylated)/(intensity acetylated+intensity prop +0.5) for each site. To 767 compare accessibility between samples, GraphPad Prism 8 was used to conduct student’s t-test, 768 assuming equal variance across samples, and with the Holm-Sidak method of correction for multiple 769 comparisons, with alpha=0.05 (unpaired, 2-tailed test). Heat maps were prepared from averaged 770 accessibilities using Morpheus (https://software.broadinstitute.org/morpheus). 771 772 Analysis of condensates after incubation in nuclear extracts: Phase separation reactions were set up in 773 40 µl with 80 nM nucleosomes, 7.5 µM Mini-Ph, in 20 mM Tris pH 8.0 and 50 mM NaCl. After incubating 774 10 min. at room temp., 12 µl of nuclear extracts were added, and reactions mixed by gently pipetting up 775 and down. 7.5 µl were removed and diluted to 10 µl for imaging, and 7.5 µl mixed with the 776 uibiquitylation machinery to assay histone ubiquitylation. After 60 min. of total incubation, samples 777 were pelleted by centrifugation for 2 min. at 2500*g, 4°C. Supernatants were removed and SDS-sample 778 buffer added to 1X. Pellets were resuspended in 2X SDS sample buffer. 2 µl of each pellet and 779 supernatant were removed and digested with Proteinase K for at least 1 hour at 55°C before analysis on 780 1.2% agarose, 1X TAE gels, which were stained with SYBR Gold to visualize nucleic acids. The remainder 781 of the samples were boiled and loaded on 8% SDS-PAGE gels, transferred to nitrocellulose, and used for 782 Western blotting. Membranes were blocked with 5% nonfat dry milk in PBST (PBS + 0.3% Tween-20), 783 and incubated with primary antibodies diluted in 5% milk-PBST overnight at 4°C. Membranes were 784 washed 3*10 min. in PBST, incubated in secondary antibody diluted in 5% milk-PBST for 1-2 hours, 785 washed 3*10 min. in PBST, and visualized using a Li-Cor Odyssey imaging system. Image J (Fiji) was used 786 to quantify band intensities. 787 788 Histone Ubiquitylation assays: For ubiquitylation assays, 125 ng chromatin per 5 µl was pre-incubated 789 with 5 µM Mini-Ph (or buffer) for 15 min. at room temp. to induce phase separation, followed by 790 addition of the ubiquitylation machinery and PRC1ΔPh. Final reaction conditions are 40 nM

791 nucleosomes, 20 mM Hepes, pH 7.9, 0.25 mM MgCl2, 0.25 mM ATP, 0.6 mM DTT, 60 mM KCl, 25mM 792 NaCl, 700 nM E1, 800 nM E2, and 500 ng Ub. Titrations of the E1, E2, and His-Ub indicate that none are 793 limiting under these conditions. Reactions were further incubated for 45 min. at room temp. Aliquots 794 were removed for imaging, and the remainder of the reaction stopped by addition of SDS-Sample buffer. 795 Boiled samples were loaded on 16% SDS-PAGE gels, which were scanned for Cy3 to detect H2A, and 796 then stained with SYPRO Ruby. Histone ubiquitylation assays in nuclear extracts were carried out under 797 the same conditions except that the pre-incubation of chromatin with Mini-Ph was 10 min., nuclear 798 extracts were added just before the ubiquitylation components, and reactions were incubated for 80 799 min. at room temp. 800

801 Cell culture: Wild type S2 cells (from Expression Systems) and S2 cell lines harbouring stable Ph or Ph-ML 802 14transgenes were grown in suspension in ESF-921 media with 5% FBS. Protein expression was induced 803 with 0.5 µM CuSO4 for 4 days. For whole cell extracts, cells were resuspended in 2X-SDS sample buffer 804 and boiled. For histone extraction, we followed the protocol of Abcam 805 (https://www.abcam.com/protocols/histone-extraction-protocol-for-western-blot); HDAC inhibitors bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

806 were not included. Western blots were carried out as described above, and ImageQuant was used to 807 quantify bands.

808 Live cell imaging: For live cell imaging, S2 (Fig. 7), or S2R+ (Supplementary Fig. 17) cells were plated at 809 106 cells per well in 6-well plates the night before transfection. Transfection was carried out using Trans- 810 IT lipid (Mirus), according to the manufacturer’s protocol. 2 µg of each Venus-Ph construct was used 811 along with 0.5 µg of pAct5C-H2A-RFP 93. One to two days after transfection, cells were replated on ConA- 812 coated imaging dishes (Ibidi). Heat shock was for 8 min. (S2R+) or 12 min. (S2) at 37°C, and cells were 813 analyzed within 24 hours of protein induction. Confocal stacks of thick slices (3 µm) were collected on 814 the spinning disc microscope described above using the 63X objective to capture foci throughout the 815 cell.

816 Image analysis of live cells: The .czi files of image stacks were opened in Image J (Fiji), converted to 817 maximum intensity projections, and the channels split. The red channel (H2A-RFP) was used to segment 818 nuclei as follows. Images were thresholded with the Li algorithm, followed by removing outliers less 819 than 5 pixels, and 3 rounds of erosion. Thresholded images were converted to masks, processed with a 820 watershed algorithm, and “Analyze Particles” used with a size threshold of 200-inifinity pixels to select 821 nuclei. The green channel (YFP fusion proteins) was then processed with “Find maxima” with the 822 following parameters: Prominence: 20000; strict; exclude edge maxima; output type: single points. The 823 nuclei selected from the red channel were used as ROIs, and the # maxima per ROI (i.e. # foci/nucleus) 824 obtained using Measure in the ROI tool, followed by dividing the raw integrated density by 255. This 825 entire pipeline is explained here: https://microscopy.duke.edu/guides/count-nuclear-foci-ImageJ. To 826 compare the # foci per cell, cells with zero foci were excluded; since Venus-PhΔSAM does not form foci, 827 the majority of cells were excluded. 828

829

830

831 Data Availability: Mass spectrometry raw files will be uploaded to MassIVE. The Source Data file 832 includes data for FRAP traces (Fig. 2, Supplementary Fig. 14) and MaxQuant output (intensities) for 833 acetylation footprinting experiments (Fig. 4), filter binding data (Fig. 3C), nucleosome and condensate 834 measurements (Fig. 3H, I, J), western blot quantification (Fig. 5I, 7C, D), ubiquitylation activity (Fig. 6E), 835 foci measurements (Fig. 7H). All other raw data are available on reasonable request.

836

837

838 839 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

840 References:

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1062 Figure Legends: 1063 1064 Figure 1 Mini-Ph forms phase separated condensates with chromatin. A. Schematic of Polyhomeotic- 1065 proximal and Mini-Ph, which spans aa 1389-1577 and includes the 3 conserved domains and an 1066 unstructured linker. Grey line indicates predicted disordered sequence (using PONDR-VSL2)94. Note that 1067 91.9% of the sequence is predicted to be disordered (disregarding segments less than 30 amino acids), 1068 with only the SAM predicted to be ordered. B. Schematic depicting the oligomeric state of Mini-Ph, 1069 which forms limited polymers of 4-6 units (6 are shown) 5. C. Structure of nine units of the Ph SAM 1070 polymer demonstrating its helical architecture. The N-terminus, from which the linker extends, is shown 1071 in cyan. D, E. Neither chromatin (D), nor Mini-Ph (E) form condensates in buffer. F. Mini-Ph forms phase 1072 separated condensates with chromatin. G. Time-lapse of droplet fusion of Mini-Ph-chromatin 1073 condensates, visualized with Alexa 647-labelled Mini-Ph. H. 3D-reconstruction of confocal stack of 1074 images demonstrating that Mini-Ph-chromatin condensates form a fused layer on the bottom of the 1075 imaging plate. Scale is in microns. I. Representative images from a matrix of Mini-Ph and chromatin 1076 showing the relationship between protein and chromatin concentration and condensate formation. 1077 [nucleosomes] assumes 8 fmol nucleosomes per 1 ng DNA. J. Graph depicting conditions where one- 1078 phase and two-phases were scored in two experiments like the one shown in I. See also Supplementary 1079 Figures 1-3 and Supplementary Movies 1-3. 1080 1081 1082 Figure 2 Mini-Ph chromatin condensates intermix slowly in vitro although Mini-Ph is dynamic. A. FRAP 1083 experiment demonstrating that Mini-Ph-chromatin condensates exchange Mini-Ph. Structure indicated 1084 with the arrow was bleached at t=0. B. Representative FRAP traces for Mini-Ph or chromatin (after 28 or 1085 27 min. of condensate formation, respectively). Data were fit with a double exponential function. For

1086 Mini-Ph, % fast=25; T1/2Fast=30 sec; T1/2Slow=199 sec; mobile fraction (plateau)=0.79. For chromatin, % 1087 fast=19; T1/2Fast=21 sec; T1/2Slow=289 sec; mobile fraction =0.14. C. Scheme for mixing chromatin 1088 labelled with different colours before adding Mini-Ph. D. Mini-Ph-chromatin condensates formed with 1089 an equal mix of Cy3 and Alexa-647-labelled chromatin. E. Mini-Ph-chromatin condensates formed with 1090 either Cy3 or Alexa-647-labelled chromatin. F. Scheme for mixing Mini-Ph-chromatin condensates 1091 formed with differently labelled chromatins. G. Mini-Ph-chromatin condensates were formed with Cy3- 1092 or Alexa 647- labelled chromatin and mixed together. H. Time lapse of fusion of condensates in the 1093 mixing experiment. See also Supplementary Fig. 6. 1094 1095 Figure 3 Ph SAM, but not its polymerization activity, is essential for formation of phase-separated 1096 condensates in vitro. A. Schematic diagram of Mini-Ph truncations. B. Structure of the Ph SAM-SAM 1097 interface indicating the position of the ML and EH mutations that impair SAM polymerization. The EH 1098 mutation (Leu 1565 Arg) has a stronger effect on polymerization than the single ML mutation (Leu 1547 1099 Arg). The figure was prepared from the structure of the ML mutant, PDB 1D 1KW4. C. Summary of filter 1100 binding experiments to measure DNA binding. Points show the mean +/- SEM. D. Kdapp for each protein. 1101 E. Both the SAM and the FCS/HD1 region are required for formation of phase-separated condensates 1102 with chromatin. F. Both the SAM and the FCS/HD1 region are required for formation of phase-separated 1103 condensates with DNA or induced by crowding agents. G. Representative images of condensates 1104 formed by Mini-Ph or the polymerization mutants (ML and EH) in the presence of chromatin (1 hour 1105 incubation). H. The concentration of nucleosomes in condensates formed by wild type (WT) and bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1106 polymerization mutants (ML and EH) are similar. I, J. EH forms smaller condensates with chromatin than 1107 WT Mini-Ph, as determined by measuring the average size of the condensates (I, not significant), or the 1108 % area covered by condensates (J). p-values for H-J are for one-way ANOVA comparing each sample to 1109 the WT control, with Dunnett’s correction for multiple comparisons. Bars show the mean + SEM of 3 1110 experiments; 9 images were analyzed for each experiment. See also Supplementary Fig. 7. 1111 1112 Figure 4 Lysine accessibility in Mini-Ph-DNA condensates. A. Schematic of lysine footprinting assay. 1113 Mini-Ph or Mini-Ph EH alone, or in the presence of 1X, 2X, or 16X DNA, is treated with sulfo-NHS acetate 1114 to acetylate accessible lysines. The protein is denatured and treated with propionic acid to propionylate 1115 unacetylated lysines. Samples are processed for mass spectrometry, and accessibility is quantified as 1116 fraction acetylated for each lysine position. B. Mini-Ph-DNA condensates before and after 15 min. 1117 acetylation reaction. C. Heat map showing accessibility for Mini-Ph alone or with the indicated DNA 1118 amounts (n=3-6). D. Heat map comparing lysine accessibility in Mini-Ph versus Mini-Ph EH (Mini-Ph n=6; 1119 Mini-Ph EH n=3). Heat maps are not scaled so that accessibility can be compared across rows and 1120 column. Asterisks indicate significant differences between samples with and without DNA by two-tailed 1121 student’s t-test with Holm-Sidak correction for multiple comparisons (green=2X DNA vs. no DNA, black 1122 =16X DNA vs. no DNA; grey=Mini-Ph versus Mini-Ph EH). E. Average accessibility of lysines in each Mini- 1123 Ph region compared between Mini-Ph and Mini-Ph EH. Accessibility of all residues in each region was 1124 averaged for each replicate and the averages compared across conditions by student’t t-test with Holm- 1125 Sidak correction for multiple comparisons. F. Structure of the Ph-p SAM polymer (PDB 1D 1KW4) with 1126 lysine side chains shown and labeled for the central SAM unit. Red highlights residue with significantly 1127 changed accessibility in Mini-Ph versus Mini-Ph EH. Structural data are not available for the HD1 1128 residues studied in the footprinting assay. See also Supplementary Fig. 11-13. 1129 1130 1131 Figure 5 Mini-Ph condensates recruit PRC1 from extracts. A. Scheme for isolating Mini-Ph-chromatin 1132 condensates from nuclear extracts. B. Representative images of condensates formed in each of the 4 1133 indicated reactions. C. Quantification of phase separated condensates (% area covered by condensates, 1134 9 images analyzed for each of 3 experiments using YOYO-1 staining). p-values are for one-way ANOVA 1135 with Tukey’s correction for multiple comparisons. D. Ratio of average intensity in condensates formed 1136 by Mini-Ph + chromatin + nuclear extracts (reaction 3) versus Mini-Ph + chromatin (reaction 4) for 3 1137 experiments. p-value is for one sample t-test comparing the ratio to the expected value of 1. E. SYBR 1138 Gold stained gel of nucleic acid content of pelleted reactions. Reactions 1-4 are as indicated in panel B 1139 for C-I. Summary of three experiments quantifying the fraction of the chromatin (F) and RNA (G) in the 1140 pellet. p-values are for paired t-test between reactions 3 and 4. H. Representative Western blots of one 1141 experiment analyzing the content of pelleted condensates. Equal amounts of pellet and supernatants 1142 were loaded. I. Summary of three experiments analyzing the content of condensates formed in extracts. 1143 Su(Z)12 was only analyzed in two experiments. One-way ANOVA was used to compare all three samples 1144 for each antibody with Tukey’s multiple comparisons test. p<0.05 *; p<0.01**. All bar graphs show 1145 mean, and error bars are SEM. See also Supplementary Fig. 15. 1146 1147 Figure 6 Mini-Ph condensates facilitate histone ubiquitylation by PRC1. A. Scheme for carrying out 1148 ubiquitylation assays in nuclear extracts. B. Representative SDS-PAGE of Cy3-labelled H2A showing 1149 ubiquitylation of chromatin in nuclear extracts in the presence or absence of Mini-Ph (wild-type), Mini- bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1150 Ph ML or Mini-Ph EH. Note that the condensates formed in buffer (reaction 4) were poorly recovered in 1151 this experiment. C. Quantification of three ubiquitylation assays. Bars are mean + SEM. p-values are for 1152 one-sample t-test comparing values to expected value of 1. D. Representative SDS-PAGE of Cy3-labelled 1153 H2A showing ubiquitylation reaction with PRC1ΔPh and chromatin in the presence or absence of Mini- 1154 Ph. E. Quantification of ubiquitylation reactions; n=3. Points are the mean +/- SEM and data were fit 1155 with an exponential function. F. Microscopy of Mini-Ph-chromatin condensates at the end of fully 1156 reconstituted in vitro ubiquitylation reactions. G. Fibrous condensates are formed by high 1157 concentrations of PRC1ΔPh in the absence of Mini-Ph; at lower concentrations, no structures are visible. 1158 See also Supplementary Fig. 16, 17. 1159 1160 Figure 7 Ph with an intact SAM increases H2A-ub in vivo. A. Representative western blot of Ph levels in 1161 induced cell lines. Note that Ph ML is the strong double mutant (L1547R/H1552R), which was previously 1162 shown to disrupt Ph clustering in cells 14. B. Representative Western blot of histone H2A-Ub levels in 1163 induced cell lines. Blots were re-probed with ant-H3 to normalize loading. 125 and 250 ng of acid 1164 extracted histones were loaded for each sample. C, D. Quantification of Ph (C) and H2A-Ub (D) for 3 1165 experiments. p-values are for one-way ANOVA comparing Ph-WT and Ph-ML cells to control (S2) for the 1166 250 ng point. E-G. Representative images of cells overexpressing YFP-Ph (E), YFP-PhΔSAM (F), or YFP-Ph- 1167 ML (G). H. Graph of the number of foci per cell for two independent experiments. Note that only cells 1168 with >zero foci were included. p-values are for Kruskal-Wallis tests with Dunn’s multiple comparisons 1169 correction. Supplementary Fig. 18. 1170 1171 1172 1173 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.HD1/FCS It is made available under aCC-BY-NC 4.0 International license. Polyhomeotic-proximal (Ph-p) linker A B SAM

YFP-Mini-Ph Mini-Ph HD1 FCS linker SAM Mini-Ph polymer C D chromatin E Mini-Ph (Cy3-H2A) (Cy3)

20 µm 20 µm 0 sec.

Ph SAM polymer

F chromatin Mini-Ph (YOYO-1) (Cy3) merge DIC 60 sec.

120 sec. 20 µm 2 µm

H I [Mini-Ph]

10 µM 50

bottom 5 µM 100 of dish 50

2.5 µM two phases J 16 one phase 8 1.25 µM 4

2 [Mini-Ph], µ M 0.63 µM 1 10 µm 0.5 [nucleosomes] 40 nM 80 nM 160 nM 320 nM 32 64 128 256 512 [nucleosomes], nM

Seif et al. Figure 1 A B bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted 1.August0 21, 2020.Mini-Ph The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to displaychromatin the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International0.8 license.

0.6 pre-bleach 5 sec. 25 sec. 125 sec. 0.4

0.2 Fractional recovery 10 µm 0.0 0 120 240 360 480 600 225 sec. 275 sec. 325 sec. 600 sec. time, seconds

C D Cy3 Alexa 647 mix

20 µm

E Cy3 Alexa 647 F

G 0 sec. before mix, Cy3 before mix, Alexa 647 0 min. 5 min.

50 sec.

100 sec.

10 min. 15 min. 30 min. 60 min. 150 sec.

200 sec.

20 µm 250 sec.

5 µm

Seif et al. Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A E Mini-Ph chromatin HD1 FCS SAM (Cy3) (YOYO-1) merge Mini-Ph linker

∆ Mini-Ph SAM Mini-Ph Mini-Ph∆FCS

B Mini-Ph∆SAM

Leu 1565

Mini-Ph∆FCS Leu 1547 Arg 20 µm

F DNA + 10 µM protein Mini-Ph∆FCS C Mini-Ph Mini-Ph EH + Mini-Ph ML Mini-Ph∆SAM Mini-Ph Mini-Ph∆SAM Mini-Ph∆FCS Mini-Ph∆SAM 1.00

0.75 5 µm

0.50 G Mini-Ph Mini-Ph ML Mini-Ph EH chromatin

fraction bound (Cy3-H2A) 0.25 200 nM nuc. 5 µM protein 5 µm 0.00 20 22 24 26 28 210 212 214 216 H I J [protein], nM 40 0.4 60 p=0.32 p=0.35 D 30 0.3 p=0.405 p=0.96 40 Protein Kdapp, nM 95% CI, nM 20 0.2 p=0.006

Mini-Ph 37 25-55 % area 20 Mini-Ph ML 190 158-229 10 0.1 p=0.10 average size, AU

Mini-Ph EH 706 547-910 [nucleosomes], µ M 0 0.0 Mini-Ph∆SAM 990 549-1806 0 WTML EH WTML EH WTML EH

Seif et al. Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A C D no DNA 1X DNA 2X DNA 16X DNA No DNA 1X DNA 2X DNA 16X DNA Mini-Ph Mini-Ph EH

sulfo-NHS acetate 1288 acetylation of accessible lysines (15’) 1294 1298 * 1302 * * urea denaturation 1332 ** HD1 1340 propionic acid 1341 * propionylation of unacetylated lysines 1342 * 1350 LC-MS 1370 1374 ** 1376 accessibility= FCS ** acet./(acet.+prop.) 1378 1380 1392 ** B pre-acetylation post-acetylation 1431 * 1439 linker 1498 1x DNA 1549 1551 EH 1560 16X DNA 20µm SAM 1566 ML * (post-acetylation) 1570 2X DNA 1575

accessibility 0.00 0.50 1.00

E F Mini-Ph Mini-Ph EH 0.8 p=0.016 p=0.009 1551 0.6 p=0.038 1549 0.4 1560 1575 0.2 1566 0.0 average accessibility 1570 HD1 FCSLinker SAM

SAM (Ph-p)

Seif et al. Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. A B chromatin Mini-Ph YOYO-1 (Cy3) (Alexa 647) merge

form Mini-Ph-chromatin chromatin condensates + extract 10’ (1)

add nuclear extract Mini-Ph 45’ +extract (2) centrifuge image @ 2500*g Mini-Ph 2’ +chromatin pellet supernatant +extract (3)

Mini-Ph +chromatin C 8 p=0.006 (4) 6 5 µm p=0.056 E F 4 p=0.044 % area pellet sup. 1.0 2 2-log

0 1 2 3 4 0.5 in pellet D chrom.

fraction chromatin 0 1.00 p=0.18 G 1 3 4 p=0.654 0.75 0.4 0.50 RNA 0.2

0.25 in pellet intensity, rx3/rx4 intensity, fraction RNA 0.00 reaction 1 2 3 4 1 2 3 4 0 % chrom. 4 0 82 100 1 2 3 % RNA 8 3744

H I pellet supernatant 0.6 Ph * PSC Ph * Pc ** Su(Z)12 Pc 0.4 ** p55 PSC Acf1 RPA-70 Su(Z)12 0.2

p55 fraction in pellet Acf1 RPA-70 0 reaction 1 2 3 4 1 2 3 4 chromatin Mini-Ph Min-Ph + extract + extract +chromatin (1) (2) +extract (3)

Seif et al. Figure 5 A

formbioRxiv Mini-Ph-chromatin preprint doi: https://doi.org/10.1101/2020.08.20.259994 add nuclear; this extract version posted+ August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license toSDS-PAGE display the preprint in perpetuity. It is made condensates 10 min.availableE1, under E2, aCC-BY-NC Ub, ATP 4.0 International80 min. license.

B C wild-type ML EH 12341234 1234 2.5 p=0.004 2.0 1 chromatin + extract p=0.010 2 Mini-Ph + extract 1.5 p=0.108 3 Mini-Ph + chromatin Ub H2A + extract 1.0 unmod. 4 Mini-Ph + chromatin H2A 0.5 fold/chromatin alone 0.0 Mini-Ph wild- ML EH % ubiqu. 0.6 1.3 0.8 1.0 0.6 0.8 type

F D DNA H2A (YOYO-1) (Cy3) merge [PRC1∆Ph] 2.5 [PRC1∆Ph], nM 0 0.63 1.3 5 10 20 40 80

0 nM 2-Ub - Mini-Ph 1-Ub unmod. H2A 2.5 nM

2-Ub + Mini-Ph 1-Ub unmod. H2A 20 nM

E 80 nM 10µm 100 - Mini-Ph + Mini-Ph G 80

60 20 nM

40 % ubiquitylated 20 80 nM 0 0.5 124816 32 64 128 10µm [PRC1∆Ph], nM

Seif et al. Figure 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A C D 0.8 S2 Ph Ph ML 1.00 p=0.0009 WCE anti-Ph 0.75 0.6 p=0.0804 anti-tubulin 0.50 0.4 Ph/tubulin B S2 Ph Ph ML H2A-Ub/H3 histones 0.25 0.2 anti-H2A-Ub 0.00 anti-H3 0.0 S2 Ph MLPh S2 Ph MLPh

E F Venus- H2A-RFP Venus-Ph merge H2A-RFP Ph∆SAM merge

5 µm

G H Venus- 20 H2A-RFP Ph ML merge

15 p=0.0011 p<0.0001 10

# foci per nucleus p<0.0001 p<0.0001 0 WT ML ∆SAM WT ML ∆SAM replicate 1 replicate 2

Seif et al. Figure 7 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Phase separation by the Sterile Alpha Motif of Polyhomeotic compartmentalizes Polycomb Group proteins and enhances their activity

Supplemental information:

Supplementary Figures 1-18

Supplemental Movie Legends 1-3

Supplementary Table 1

A B

bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 InternationalE license. 2-log ladder MWM, kDa Mini-Ph Mini-h ∆ FCS Mini-Ph ∆ SAM Mini-Ph ML Mini-Ph EH MNase MWM, kDa PRC1 ∆ Ph 250 150 250 Flag-PSC 150 100 100 75 75 Pc 50 dRING 50 10kb 37 5kb 37 4kb 17N 3kb 25 15N 25 13N 12N 15 15 11N 2kb 10N 9N C 8N 1.5kb

5S nucleosome EcoRI 7N 1.2kb positioning sequences EcoRI native PAGE 6N 1kb EcoRI digest 5N 900bp 800bp 4N 700bp plasmid backbone 600bp 3N 500bp

D 400bp chromatin DNA MWM 2N 300bp

400bp 200bp 300bp 1N 200bp 100bp

100bp

% assembled 93 DNA (EtBr) Cy3 Cy3 (Histone H2A)

Supplementary Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 1 Proteins and Chromatin used in these experiments. A. SYPRO Ruby stained SDS-PAGE of Mini-Ph and its derivatives that were prepared in E. coli. Equimolar amounts of each protein were loaded. B. Ruby stained gel of 3-subunit PRC1 consisting of Flag-PSC, Pc, and dRING (PRC1ΔPh). C. Schematic of plasmid used for chromatin assembly. The plasmid used for most experiments consists of 40*5S nucleosome positioning sequences (208 base pair repeat) (blue), and the plasmid backbone (green). Each 5S sequence is flanked by EcoRI sites. To estimate nucleosome assembly, chromatinized plasmids are digested with EcoRI, and the naked and nucleosomal 5S repeats resolved by native PAGE. D. Representative gel of EcoRI digest. Left shows DNA stain used to quantify naked and nucleosomal repeats, and right side shows the Cy3 label on histone H2A. E. Micrococcal nuclease analysis of chromatin used for condensate formation. Numbers on the right indicate fragments representing nucleosomal increments.

bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint A (which was not certifiedDNA by peer review) is the author/funder,Mini-Ph who has granted bioRxiv a license to display the preprint in perpetuity. It is made (YOYO-1) available(Cy3) under aCC-BY-NC 4.0 Internationalmerge license. DIC

20 µm

B 0 sec. 20 sec. 40 sec. 60 sec. 80 sec.

5 µm C D * E DNA (SYBR Gold) Mini-Ph (Alexa-647) pellet sup. pellet sup. 1.0 0.020 * 1 DNA 1 2 3 1 2 3 1 2 3 1 2 3 0.015 2 Mini-Ph 340 0.010 0.5 3 DNA+ OD Mini-Ph 0.005

fraction in pellet 0.000 0.0 Mini-Ph DNA Mini-Ph Mini-Ph Mini-Ph DNA Mini-Ph +DNA +DNA +DNA protein DNA

% in pellet 6.4 100 5 16

F G

[Mini-Ph] one phase two phases 32

20 µM 16

10 µM 4

[Mini-Ph], µ M 2

5 µM 1

0.5 0.5 1 2 4 8 16 32 64 2.5 µM [DNA], nM

1.3 µM

0.63 µM 50 µm [DNA] 0.7 nM 1.5 nM 2.9 nM 5.8 nM 11.5 nM 23 nM 46 nM

Supplementary Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 2 Mini-Ph forms phase separated condensates with DNA. A. Mini-Ph forms phase separated condensates with DNA. B. Time-lapse of droplet fusion of Mini-Ph-chromatin condensates, visualized with Alexa 647-labelled Mini-Ph. C-E. Mini-Ph-DNA condensates can be pelleted by brief centrifugation. Representative gels (C) of DNA (left) and protein (fluorescent scan of Alexa 647- labelled Mini-Ph). Condensates were allowed to form for 15 min. at room temperature. D. Quantification of pelleting experiments. E. Mini-Ph-DNA condensates increase turbidity measured by OD340. * indicates p<0.05, student’s t-test. F. Matrix of Mini-Ph and DNA. G. Graph of one-phase versus two-phase regions of the Mini-Ph-DNA matrix. Note that high concentrations of DNA disrupt condensate formation (e.g. 46 nM DNA with 5 µM Mini-Ph). [DNA] refers to the concentration of plasmids.

A B [NaCl] 25 mM 50 mM 75 mM 100 mM 125 mM 150 mM one phase two phases [Mini-Ph] 128 80 µM 64 32 40 µM 16 8 4 20 µM [Mini-Ph], µ M 2 1 10 µM 0.5 0 50 100 150 [NaCl], mM 5 µM C 0.08

2.5 mM 0.06 340 1.25 µM 0.04 OD

0.02 0.63 µM 50 µm 0.00 0 50 100 150 200 [NaCl], mM

Supplementary Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 3 Mini-Ph-DNA condensates are sensitive to NaCl concentration. A, B. Matrix of of Mini-Ph-DNA condensates and across different concentrations of NaCl. A shows representative images, and B the graph of one-phase versus two-phase points. C. Increasing [NaCl] disrupts condensates as measured by OD340. Mean +/- SD of three titrations.

bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made A available under aCC-BY-NC 4.0 International license. Mini-Ph linker positively charged polar GVGSGETNGL GTGGIVGVDA MALVDRLDEA MAEEKMQTEA TPKLSESFPI negatively charged aliphatic LGASTEVPPM SLPVQAAISA PSPLAMPLGS PLSVALPTLA PLSVVTSGAA aromatic proline PKSSEVNGTD R

B NCPR distribution (blob 5) D 1.0 Ph-p (Mini-Ph)

0.8 NCPR 0.6

0.4 C Blob index 0.2 PHC3 Ph-p PHC3 PHC1 Ph-d PHC2 fraction of negatively charged residues 0.0 0.0 0.2 0.4 0.6 0.8 1.0 fraction of positively charged residues NCPR

E Blob index NCPR, fr. expanding, fr. disord. protein FCR, pH 8 pH 8 PI fr. Neg fr. Pos pH 8 pro. kappa Ph-p 0.153 -0.063 3.9 0.108 0.045 0.261 0.703 0.115 Ph-d 0.121 -0.069 3.6 0.095 0.026 0.233 0.647 0.128 PHC1 0.281 0.088 10.9 0.096 0.184 0.368 0.746 0.291 PHC2 0.254 0.063 10.1 0.095 0.159 0.325 0.762 0.233 PHC3 0.339 0.046 10.5 0.147 0.193 0.422 0.734 0.220 F Ph-p GV GS GE T NGL GT GGI V GV DAMAL V DR L DE AMAE E K MQT E AT P K L S E S F P I L GAS T E V P P M Ph-d GV GS GE T NGL GT GGI V GV DAMAL V DR L DE AMAE E K MQT E S Y QT V S DAL P I QAAT P E V P P I

Ph-p S L P V QAAI S AP S P L AMP L GS P L S V AL P T L AP L S V V T S GA- - - - - AP K S S E V NGT DR Ph-d S MP V L AAMS T S S P L S L P L T L P L P I AI AP T V S L P V V S AGV V AP V L AI P S S NI NGS DR G PHC1 R Y NV S CS HQF R L K R K K MK E F QE ANY AR - V R R R GP R R S S S DI ------AR AK I QGK PHC2 R Y NV GCT K R V GL F HS DR S K L QK AGAAT HNR R R AS K AS L P P L T K DT K K QP T GT V P L S V T AA PHC3 R Y NV S CS K K F AL S R WNR K P DNQS L - - - - - GHR GR R P S GP D------GAAR E HI L R Q

PHC1 - - CHR GQE DS S R GS DNS S Y DE AL S P T S P GP L S V R AGHGE R DL GNP NT AP P T P E L HGI NP V PHC2 L QL T HS QE DS S R CS DNS S Y E E P L S P I S AS S S T S R R R QGQR DL E L P DMH- - MR DL V GMGHH PHC3 L P I T Y P S AE E DL AS HE DS V P S AMT T R L R R QS E R E R E R E L R DV R I R K MP E - NS DL L P V AQ-

PHC1 F L S S NP S - R PHC2 F L P S E P T - K PHC3 - - - T E P S I W

Supplementary Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 4 Sequence properties of Mini Ph linker. A. Colour coded Mini-Ph linker sequence. Gray and red lines indicate linker residues that may interact with Ph SAM based on NMR results with the linker and the SAM in trans. Red line indicates residues that had altered NMR signals when mixed with 0.4 molar equivalents of Ph SAM, and gray lines those with altered signals with 1.6 molar equivalents of Ph SAM. See Robinson et al. (2012, JBC) for details. B. Plot of net charge per residue (NCPR, pH 8.0) of Mini-Ph linker indicating charge distribution in the linker. C. Plot of NCPR (pH 8.0) for PHC3, indicating distinct charge patterning compared with Ph-p. D. Das-Pappu diagram of states for both Drosophila and all three human Ph linkers, showing that the Drosophila and human linkers have distinct properties. E. Summary of properties of Ph linkers calculated with http://pappulab.github.io/localCIDER/. See also Supplementary Table 1 for all sequences and parameters (FCR=fraction charged residues). F. Alignment of Ph-p and Ph-d linkers. G. Alignment of PHC1-3 (human Ph homologues) linkers.

A B 50 40 Y = 5.269*X - 1.607 40 R2=0.99 30 30

intensity 20 20

10 10 0 [nucleosomes], µ M 2 4 6 8 -1 0 0 [octamers], µM chromatin 1 chromatin 2 C D

20 µm

E 50

20 [nucleosomes], µ M 10

0 1x 0.1x

200ng 20ng

180ng

Supplementary Figure 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 5 Quantification of nucleosome concentration in condensates. A. Standard curve prepared from serial dilution of Cy3-labelled (on H2A) histone octamers. Buffer alone was used to collect a background intensity, which was subtracted, from all images. B. [nucleosomes] in individual structures measured from two reactions carried out with different chromatin assemblies. Chromatin 2 was assembled at higher nucleosome density, presumably explaining the higher nucleosome concentration in condensates. C. Example image used for quantification of nucleosome concentration. D. Outlines of thresholded structures selected for quantification for the image shown in C. E. Graph of experiments comparing concentrations of structures measured with a 1:9 mixture of Cy3-labelled (0.1X) to unlabelled (1X) chromatin versus all Cy3-labelled. The calculated concentrations are similar (21+/-4 µM and 26+/-5 µM) in both reactions, suggesting the measurements in the dense phase with all labelled chromatin are in the linear range.

A bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994B C ; this version postedD August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made 40 0.8 available under aCC-BY-NC40 4.0 International license300. n 30 0.6 30 sec.

sec. 200 , , fractio ast 20 0.4 20 FAST %f SLOW 100

10 t1/2 0.2 10 t1/2 mobile

0 0.0 0 0 E DNA bleach (YOYO1)

F chromatin bleach (Cy3-H2A)

Supplementary Figure 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 6 Kinetics of chromatin and Mini-Ph in condensates are distinct. A-D. Summary of parameters from double exponential fits of FRAP data. All bars show mean +/- SEM. E. Time lapse of recovery of chromatin in a condensate. The DNA component of chromatin was visualized (with YOYO1) and bleached. F. Time laps of recovery of chromatin in a condensate after photobleaching. H2A-Cy3 was visualized.

A Mini-Ph Mini-Ph bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. TheEH copyright holder for this preprint [protein],(which wasnM not0 certified by peer review) is the author/funder,1024 who has[protein], granted nMbioRxiv0 a license to display the preprint in perpetuity.4096 It is made available under aCC-BY-NC 4.0 International license. bound bound

unbound unbound Mini-Ph Mini-Ph ML ∆SAM 4096 7000 [protein], nM 0 [protein], nM 0 bound bound

unbound unbound

Mini-Ph Mini-Ph∆FCS Mini-Ph∆SAM

[protein], µM 0 0.05 0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8 25.6 0 0.05 0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8 25.6 0 0.05 0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8 25.6

bound

unbound

Supplementary Figure 7 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 7 Ph SAM polymerization increases DNA binding affinity of Mini-Ph. A. Representative filters from filter binding experiments to measure DNA binding affinity. B. EMSA demonstrating that Mini-PhΔFCS does not bind DNA. Plasmid DNA was used for EMSA; using these large substrates at high concentrations (25 ng plasmid per reaction) underestimates Mini-Ph DNA binding affinity.

A C [NaCl] 25 mM 50 mM 75 mM 100 mM 125 mM 150 mM [Mini-Ph ML] WT+ML+EH WT+EH none WT+ML WT only [80 µM] 64 32

M 16 [40 µM] µ , 8 4 [20 µM]

[Mini-Ph] 2 1 [10 µM] 0.5 050 100 150 [NaCl], mM [5 µM]

[2.5 µM]

50 µm

B [NaCl] 25 mM 50 mM 75 mM 100 mM 125 mM 150 mM [Mini-Ph EH] [40 µM]

[20 µM]

[10 µM]

[5 µM]

[2.5 µM]

[1.25 µM]

[0.63 µM] 50 µm

Supplementary Figure 8 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 8 Mini-Ph SAM polymerization mutants are more sensitive to NaCl than wild- type. A, B. Matrix of of Mini-Ph ML (A), and Mini-Ph EH (B)-DNA condensates and across different concentrations of NaCl. C. Plot of one-phase versus two-phase regimens for Mini-Ph, Mini-Ph ML, and Mini-Ph EH. The difference between green and yellow symbols indicates that condensate formation is more sensitive to NaCl for both mutants. See also Supplementary Figure 3 for the matrix of Mini-Ph versus NaCl.

A ATP, or ? bu er + 15’ 15’, 60’ Mini-Ph Mini-Ph ML Mini-Ph EH

B C 15 min. [ATP] 15 min. 15

0 mM 10 AU

area, 5

2 mM 0 [ATP], mM 0 2 8 0 2 0 2 Mini-Ph Mini-Ph ML Mini-Ph EH Mini-Ph Mini- Mini- Ph ML Ph EH D E 60 min. 60 min. [ATP] 25

20 0 mM

AU 15

10 area, 5 2 mM 0 [ATP], mM 0 2 8 0 2 0 2 Mini-Ph Mini-Ph ML Mini-Ph EH Mini-Ph Mini- Mini- Ph ML Ph EH F 15 min. 60 min

20µm

Mini-Ph, 8 mM ATP

Supplementary Figure 9 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 9 Mini-Ph SAM polymerization mutants are more sensitive to ATP than wild- type Mini-Ph. A. Schematic of experiments to test effect of adding ATP (or buffer as a control) to condensates formed by Mini-Ph, Mini-Ph ML, or Mini-Ph EH with chromatin. B-E. Representative images (B, D) and quantification (C, E) of condensates after 15 min. incubation with ATP or buffer. F. Effect of 8 mM ATP on condensates formed with Mini-Ph and chromatin.

A B Phase separation No phase separation

Mini-Ph Mini-Ph∆FCS

SAM-SAM SAM-SAM SAM SAM

linker linker-

SAM linker linker- HD1 FCS SAM

chromatin DNA chromatin DNA

HD1 C No phase separation D Phase separation

Mini-Ph∆SAM Mini-Ph-EH ? SAM-SAM SAM

linker linker-

linker SAM HD1 FCS HD1 FCSFCS

chromatin DNA chromatin DNA

Supplementary Figure 10 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 10 Models for the interactions of Ph SAM in condensate formation. A. Schematic of all known interactions that may occur in Mini-Ph-chromatin or Mini-Ph DNA condensates, including interactions among nucleosomes. B-D. Schematic indicating which interactions are missing when different mutated or truncated proteins that do or do not form condensates are used. The FCS- DNA/chromatin interaction is weaker in the absence of the SAM or when the polymerization interface is disrupted. The residual SAM-SAM interaction in the EH mutant is hypothesized based on the acetylation footprinting results (Supplementary Fig. 13F). The ML mutation, which weakens but does not eliminate SAM-SAM interactions may behave similar to the schematic in A (i.e. the SAM-SAM interaction is dynamic under phase separation conditions, unlike wild-type Mini-Ph which forms short, limited polymers before phase separation). It is important to point out that this is the simplest scheme; it is possible that there are other interactions in the system that have not been characterized. These could include interactions involving the HD1, hinted at by the difference in accessibility measured for Mini-Ph and Mini-Ph EH (Fig. 4D, E). We also do not know how the structure of Mini-Ph polymers influences binding to large chromatin or DNA templates, and whether Mini-Ph binding influences nucleosome- nucleosome interactions (as suggested in the diagrams).

A 1392

1380

1378 1370 1376

1374

FCS (PHC1)

no DNA 2X DNA 0.8 16X DNA p=0.048 0.6 p=0.002 p=0.001 0.4

0.2 average accessibility

0.0 HD1 FCSLinker SAM

Supplementary Figure 11 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 11 Chemical footprinting analysis of lysine accessibility of Mini-Ph alone and with DNA. A. Structure of the FCS of human PHC1 (PDB 2L8E), with the positions of the equivalent lysine residues in Ph-p indicated. Red numbers indicate positions where accessibility is significantly changed. B. Clustal alignment of human PHCs with Ph-p indicating the relationship between lysines in Ph-p and the sequence of the FCS. C. Average accessibility of lysines in each Mini-Ph region compared across conditions. Accessibility of all residues in each region was averaged for each replicate and the averages compared across conditions by student’t t-test with Holm-Sidak correction for multiple comparisons. Bars show the error +/- SEM. n=6.

A B 15 min. 30 min. 45 min. 60 min. 70 min. bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holderhigh for this DNA preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

control 5 min.

sulfo-NHS- 60 min. acetate 25 µm 25 µm

C D control

MWM, kDa control acetylated acetylated 30 75 50 37 20 25

AU 20

10 15 Area, 10 0

time, min. 5 10 15 30 60 70455 10 15 30 60 7045

E F control acetylated [Mini-Ph], µM 5 10

µ [Mini-Ph, M) 0 0.05 0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8 0 0.05 0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8 control

unbound

acetylated

20µm

Supplementary Figure 12 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 12 Acetylation of Mini-Ph-DNA blocks condensate formation and DNA binding. Time course of effect of sulfo-NHS acetate on Mini-Ph-DNA condensates. Mass spectrometry samples were collected after 15 min. B. Images of high DNA reaction (without acetylation) in which condensates do not form even after prolonged incubation. C. Quantification of time course shown in A. A similar time course was observed in three experiments. D. SYPRO Ruby stained SDS-PAGE of control Mini-Ph and Mini-Ph after acetylation with sulfo-NHS-Acetate. E. EMSA comparing binding of control and acetylated Mini-Ph to DNA; representative of two experiments. Main unbound band is supercoiled plasmid, and faint band (grey arrow) is nicked. F. Acetylated Mini-Ph does not form condensates with chromatin (representative of three experiments). Images were taken after 60 min. incubation at room temperature.

sulfo-NHS no DNA 2X DNA 16X DNA no DNA 16X DNA acetate 1288 acetylation of accessible lysines 1294 * 1298 * 1302 * 5 min. 15 min. 1332

B HD1 1340 * Mini-Ph EH + 2X DNA 1341 * pre-acetylation post acetylation (5 min.) 1342 * 1350 1370 1374 1376 FCS 1378 1380 D 1392 1431 no DNA vs. 16X 5 min. 1439 DNA, 5 min. 0.6 linker 1498 2X DNA vs. 16X no DNA 2X DNA 16X DNA 1549 DNA, 5 min. 1551 0.4 p=0.002 * no DNA vs. 2X 1560 DNA, 5 min. p=0.029 SAM 1566 no DNA 5 min. 0.2 1570 1575 vs. 15 min. average accessibility

0.0 0.00 0.50 1.00 accessibility E HD1 FCSLinker SAM F

Mini-Ph EH alone 15 min. 5 min. 15 min. 1.0 no DNA 16X DNA 0.8 p<0.001 0.8 0.6 p=0.001 0.6 0.4 0.4 0.2

0.2 average accessibility average accessibility

0.0 0.0 HD1 FCSLinker SAM HD1 FCSLinker SAM

Supplementary Figure 13 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 13 Chemical footprinting analysis of lysine accessibility of polymerization mutant Mini-Ph EH alone and with DNA. A. Schematic of chemical acetylation (abbreviated from Figure 4A). Acetylation was carried out for 5 or 15 min. with Mini-Ph EH because protein-DNA condensates dissolve rapidly after addition of sulfo-NHS acetate. B. Mini-Ph EH-DNA condensates before (left) and after 5 min. of acetylation (right). C. Heat maps of Mini-Ph EH alone, with 2X DNA (condensates form) or 16X DNA (no condensates). Acetylation was carried out for 5 (n=3) or 15 (n=2) min. Asterisks indicate lysines with significantly different accessibility in the indicated comparisons. D-F. Accessibility averaged over Mini-Ph regions for different conditions after 5 min. (D), 15 min. (E), or 5 versus 15 min. for Mini-Ph EH alone (F). Accessibility of all residues in each region was averaged for each replicate and the averages compared across conditions by student’t t-test with Holm-Sidak correction for multiple comparisons. Bars show the error +/- SEM.

0.4 0.4 ecover re-bleach lr np 0.2 0.2 fractiona fractio

0.0 0.0

060 120 180 240 300 H h E time, sec i-P h- in -P M i C in M Mini-Ph pre 5 sec. 15 sec. 45 sec. 135 sec. 285 sec.

D Mini-Ph-EH

E chromatin, Mini-Ph F chromatin, Mini-Ph-EH pre post pre post

G H I J 80 1.2 30 400 p=0.004 p<0.001 1.0 60 300 sec.

0.8 sec. 20 , , ast 40 0.6 200 FAST %f 0.4 10 SLOW t1/2

20 t1/2 100 mobile fraction 0.2 0 0.0 0 0 WT EH WT EH WT EH WT EH

Supplementary Figure 14 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 14 Mini-Ph EH that lacks polymerization activity is more mobile in condensates than wild-type Mini-Ph. A. FRAP traces for Mini-Ph and Mini-Ph EH with chromatin. Representative traces with R2 for fits >0.99 were pooled for condensates formed at different times (22-41 min. for Mini- Ph; 28-73 min. for Mini-Ph-EH; n=5). Graph shows the mean +/- SEM. Data were fit with a double exponential equation. B. Fluorescence of chromatin (H2A-Cy3) before and after FRAP experiments. The same regions of interest (ROI) used to measure FRAP, acquisition induced bleaching, and background were used to analyze chromatin as for Mini-Ph. We were unable to collect FRAP traces in both channels simultaneously. C, D. Representative bleaching and recovery of single condensate for Alexa-647 labelled Mini-Ph (C) and Mini-Ph-EH. Condensates were formed for 25 min. for Mini-Ph and 28 min. for Mini-Ph- EH. E, F. Fluorescence of chromatin (H2A-Cy3) for the same condensates shown in C and D immediately before and after the FRAP experiment. G-I Summary of parameters from double exponential fits. All graphs show the mean +/-SEM. Mini-Ph and Mini-PH EH were compared by Mann-Whitney test. All fits have R2>0.99.

A B

100 2-log extract desalted Q-sepharose

% overlap 25

0 1 2 3 nuc. ext. - +

C chromatin Mini-Ph merge (Cy3-H2A) (Alexa 647) % overlap

buffer 99%

nuclear 94% extract

D 5 µm

buffer 99%

nuclear 61% extract

5 µm

Supplementary Figure 15 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 15 Colocalization analysis of Mini-Ph and chromatin in nuclear extracts. A. SYBR Gold stained gel of nuclear extract (lane 1), extract after desalting (lane 2), and after incubation with Q- sepharose to deplete nucleic acids (lane 3). B. Summary of overlap between Mini-Ph condensates and chromatin incubated in buffer or in nuclear extract from three experiments. C, D. Representative images from two of the three experiments; two resembled panel C, while in one experiment, Mini-Ph structures that are not positive for Cy3-H2A are observed. These structures frequently appear to be connected to chromatin condensates.

A B chromatin Mini-Ph Mini-Ph (Cy3-H2A) (Alexa 647) merge 0.4 p=0.0015 0.3 p=0.0032 buffer 0.2

p=0.523 0.1 average size, AU

0.0 NE NE NE nuclear B B B Mini-Ph WT ML EH extract B=buffer NE=nuclear extract

Mini-Ph ML C 60 p<0.0001

p<0.0001 buffer 40 p=0.078

% area 20

0 nuclear B NE B NE B NE extract Mini-Ph WT ML EH B=buffer NE=nuclear extract

Mini-Ph EH D p=0.031 p<0.0001 100 p=0.704 buffer 75

mean intensity, AU mean intensity, 25

0 nuclear B NE B NE B NE extract Mini-Ph WT ML EH 5 µm B=buffer NE=nuclear extract

Supplementary Figure 16 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 16 Effect of nuclear extracts on condensates formed by Mini-Ph, or Mini-Ph polymerization mutants with chromatin. A. Representative images of condensates incubated in buffer (top panel in each set), or nuclear extracts (bottom panel in each set). Image intensities were adjusted to facilitate visualization of the structures. The structures formed by Mini-Ph EH with chromatin in nuclear extract are much fainter than those formed in buffer are. B-D. Average size of structures (B), % area covered by condensates (C), or mean intensity of structures (D) after incubation in buffer or extract from three experiments. Bars are the mean +SEM and p-values are for ANOVA comparing buffer to nuclear extract for each protein with Sidak correction for multiple comparisons.

Cy3 SYPRO Ruby bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint A (which was not certified by peer review) is the author/funder, who hasB granted bioRxiv a license to display the preprint in perpetuity. It is made E1 + - + +available + under a+CC-BY-NC 4.0 International+ license - . + + + + E2 + + - + + + + + - + + + His-Ub + + + - + + + + + - + + ATP + + + + - + + + + + - + PRC1∆Ph + + + + + - + + + + + - PSC E1 Pc dRING 2-Ub * 1-Ub * unmod. H2A E2 His-Ub

time, min. 15 60 15 60 15 60 15 60 15 60 15 60 15 60 15 60 15 60 15 60 15 60 15 60

∆

[PRC1 Ph], nM 0 0.63 1.3 2.5 5 10 20 40 80 PRC1∆Ph E1

- Mini-Ph E2

His-Ub

PRC1∆Ph E1

Mini-Ph

+ Mini-Ph E2

His-Ub

Supplementary Figure 17 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 17 Histone ubiquitylation assay. A, B. SDS-PAGE of histone ubiquitylation assay. A shows Cy3-labelled histone H2A and B is same gel after SYPRO-Ruby staining. The E1 and E2, ATP, Ubiquitin, and PRC1ΔPh are all required for ubiquitylation. *contaminants present in one gel lane. C. SYPRO Ruby stain of gels shown in Figure 6.

A B Venus-Mini-Ph H2A-RFP merge Venus-Mini-Ph H2A-RFP merge

C D Venus-Mini- Ph∆SAM H2A-RFP merge

one focus

800 no foci

600

cells 400 #

200

0 Mini-Ph Mini-Ph ∆SAM

5µm

Supplementary Figure 18 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.20.259994; this version posted August 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

Supplementary Figure 18 Mini-Ph forms a single, SAM-dependent focus or no foci in cells. A-C. Representative images of S2R+ cells transfected with Venus-Mini-Ph (A, B) or Venus-Mini-PhΔSAM (C) and H2A-RFP. Cells expressing Venus-Mini-Ph either have a single focus, as in A, or no foci, as in B. We do not observe multiple Mini-Ph foci in cells. D. Quantification of cells with foci. 7% of Venus-Mini-Ph (39/570) and 0.5% of Venus-Mini-PhΔSAM (4/730) cells were identified as having a single maxima (focus). In the case of Mini-PhDSAM, the small number of identified foci represent cells with very high expression rather than actual foci.