In Silico APC/C Substrate Discovery Reveals Cell Cycle Degradation of Chromatin Regulators 3 Including UHRF1 4

bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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.

1 Title

2 In silico APC/C substrate discovery reveals cell cycle degradation of chromatin regulators 3 including UHRF1 4

5 6 Author list

7 Jennifer L. Kernan1,2 8 Raquel C. Martinez-Chacin1 9 Xianxi Wang2 10 Rochelle L. Tiedemann3 11 Thomas Bonacci2 12 Rajarshi Choudhury2 13 Derek L. Bolhuis1 14 Jeffrey S. Damrauer2,4 15 Feng Yan2 16 Joseph S. Harrison5 17 Michael Ben Major2,6 18 Katherine Hoadley2,4 19 Aussie Suzuki7 20 Scott B. Rothbart3 21 Nicholas G. Brown1,2,8 22 Michael J. Emanuele1,2,8* 23 24 Affiliations

25 1- Department of Pharmacology. The University of North Carolina at Chapel Hill. Chapel 26 Hill, NC 27599, USA.

27 2- Lineberger Comprehensive Cancer Center. The University of North Carolina at Chapel 28 Hill. Chapel Hill, NC 27599, USA.

29 3- Center for Epigenetics, Van Andel Research Institute. Grand Rapids, MI 49503, USA.

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30 4- Department of Genetics. The University of North Carolina at Chapel Hill. Chapel Hill, 31 NC 27599, USA.

32 5- Department of Chemistry, University of the Pacific. Stockton, CA 95211, USA. 33 6- Current address: Department of Cell Biology and Physiology, Department of 34 Otolaryngology, Washington University in St. Louis. St. Louis, MO 63110, USA.

35 7- McArdle Laboratory for Cancer Research, Department of Oncology, University of 36 Wisconsin, Madison. Madison, WI 53706, USA.

37 8- Correspondence should be addressed to NGB ([email protected]) or MJE 38 ([email protected]); * lead contact: [email protected]

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40 Short title 41 Chromatin regulation by APC/C 42 43 44 Keywords 45 APC/C; chromatin; cell cycle; ubiquitin; DNA methylation; UHRF1 46 47 48

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49 Abstract

50 The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase and 51 critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to 52 globally map APC/C substrates. By combining orthogonal features of known substrates, we 53 predicted APC/C substrates in silico. This analysis identified many known substrates and 54 suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among 55 putative substrates and we show that several chromatin proteins bind APC/C, oscillate during the 56 cell cycle and are degraded following APC/C activation, consistent with being direct APC/C

57 substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key 58 chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. 59 Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and 60 perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates 61 crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences 62 in normal cell physiology, where the chromatin environment changes depending on proliferative 63 state, as well as in disease. 64

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65 Introduction

66 Regulated protein degradation is central to cell and organismal physiology and plays a

67 particularly important role in proliferation. In eukaryotes, protein degradation is controlled largely

68 by the ubiquitin (Ub) system. E3 Ub ligases provide substrate specificity and facilitate the transfer

69 of Ub onto substrates. The formation of poly-Ub chains on substrates provides a signal that often

70 targets substrates to the proteasome for degradation (1).

71 The Anaphase-Promoting Complex/Cyclosome (APC/C) is a 1.2 megadalton, multi-

72 subunit E3 ligase and essential cell cycle regulator. APC/C utilizes two coactivators, Cdc20 and

73 Cdh1, which directly bind substrates, recruiting them to the E3 complex (2). APC/CCdc20 becomes

74 active in mid-mitosis and promotes the metaphase to anaphase transition. APC/CCdh1 becomes

75 active in late mitosis and remains active until the end of G1, during which time it prevents S-phase

76 entry (3). Thus, APC/CCdc20 and APC/CCdh1 play opposing roles, the former promoting cell cycle

77 progression in mitosis and the latter inhibiting cell cycle progression in G1.

78 In addition to its role in normal cell cycles, APC/C dysfunction has been implicated in

79 disease. Cdh1 is a haploinsufficient tumor suppressor in mice and cooperates with the

80 retinoblastoma protein to restrain proliferation (4–8). Several oncogenic kinase cascades impinge

81 on Cdh1 function, further supporting a role for APC/CCdh1 in tumor suppression (9–11). In

82 addition, the APC/C subunit Cdc27 is mutated in cancer and associated with aneuploidy (12).

83 APC/C is also linked to inherited disorders that give a range of disease phenotypes, including

84 microcephaly, cancer predisposition, and skeletal abnormalities (13,14).

85 Cdh1 and Cdc20 bind substrates through short, linear sequence motifs termed degrons. The

86 most well-defined APC/C degron motifs are the KEN-box and D-box (15,16). In addition, binding

87 of Cdc20 and Cdh1 to APC/C promotes a conformational change in the E3 that stimulates ligase

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88 activity (17). This results in substrate poly-ubiquitylation by its two cognate E2 enzymes.

89 UBE2C/UbcH10 deposits the first Ub monomers onto substrates and forms short Ub chains,

90 whereas UBE2S elongates poly-Ub chains (18–21).

91 Most known APC/C substrates are linked to cell cycle processes, including mitotic

92 progression, spindle function and DNA replication. The paramount importance of APC/C in cell

93 cycle and non-cell cycle processes, and its dysfunction in disease, highlight the importance of

94 systematically defining substrates, whose regulation (or dysregulation), will likely contribute to

95 proliferation and disease phenotypes. Nevertheless, barriers exist to the identification of APC/C

96 substrates, as well as most other E3s. E3-substrate interactions are dynamic and binding often

97 triggers substrate proteolysis. Additionally, the abundance of most substrates is low, and for

98 APC/C, most targets are cell cycle regulated. Furthermore, since APC/C is a massive complex

99 with many substrates, the relative binding stoichiometry to each individual substrate is low.

100 Finally, degron sequences are short and occur vastly across proteomes, making it difficult to

101 predict substrates.

102 We developed a simple in silico approach to identify potential APC/C targets. We took

103 advantage of common features among known substrates, namely their transcriptional regulation

104 during cell cycle and the presence of a degron motif. Super-imposing these features onto the

105 proteome enriched for substrates and suggested previously undescribed targets.

106 This analysis revealed a role for APC/C in chromatin biology. We validate several

107 substrates involved in chromatin dynamics, highlighting a previously underappreciated role for

108 APC/C in chromatin regulation. We further define the mechanisms of ubiquitylation for UHRF1

109 (Ubiquitin-like with PHD and RING finger domains 1), a multivalent chromatin binding protein

110 and itself an E3 ligase that can ubiquitylate histone H3 (22–25). UHRF1 plays an important role

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111 in DNA methylation and has been implicated in other DNA templated processes, including DNA

112 repair (26–28). Additionally, UHRF1 is suggested to be an oncogene, whose expression correlates

113 with high tumor grade and poor prognosis (29–31).

114 Altogether, these results reveal a role for APC/C-dependent UHRF1 degradation in cell

115 cycle progression and shaping the DNA methylation landscape. More broadly, our data suggest

116 that cell cycle regulated protein degradation helps organize the epigenetic landscape during

117 proliferation. This suggests a potential mechanistic link contributing to changes in the chromatin

118 landscape observed between proliferating and non-proliferating cells (32,33). We predict that

119 altering APC/C function could promote changes in the histone and DNA modification landscape,

120 and that these effects could contribute to the biochemical and phenotypic features of diseases,

121 including cancer and neurological disorders.

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122 Results

123 Identification of APC/C substrates

124 To identify human APC/C substrates, we first performed FLAG immunoprecipitations (IP) 125 from HEK-293T cells expressing amino-terminal tagged FLAG-Cdh1 or an empty vector and 126 analyzed precipitated proteins by mass spectrometry (Table S1). Several APC/C complex 127 components and known substrates, including Rrm2, Kif11, Claspin, and cyclin A were enriched 128 in Cdh1 pulldowns. Compared to a previously established dataset (34), we identified 15 out of 53 129 known substrates. However, hundreds of proteins were enriched over controls and many known

130 substrates scored weakly, confounding our ability to prioritize candidates. For example, a single 131 spectral count was observed for the substrate Kif22/KID (35,36). 132 We considered computationally identifying substrates based on features common among 133 substrates. APC/C binds substrates most often through D- and KEN-box degron motifs. The 134 minimal D-box motif (R-x-x-L) is present in most human proteins and insufficient as a prediction 135 tool. The KEN-motif is found in approximately 10% of human proteins (2,206; Table S2), and 136 several D-box regulated substrates also contain a KEN-motif, including Securin and Cdc6 (37,38). 137 In addition, the gene expression of most APC/C substrates oscillates during the cell cycle (39). We 138 cross-referenced the KEN-motif containing proteins against a set of 651 proteins whose mRNAs 139 scored in at last two cell cycle mRNA profiling studies (40–43). Overlapping the 2,206 KEN-motif 140 containing proteins with 651 transcriptionally controlled genes produced a set of 145 proteins, 141 which represent known and putative APC/C substrates (Fig. 1A, Table S2). 142 We compared our in silico analysis with two previously curated datasets, one containing 143 53 known APC/C targets (34), and a second containing 33 specifically KEN-dependent APC/C 144 substrates (16). When compared to these lists of 53 and 33 substrates, our dataset captured 26 and 145 22 of them, respectively, the latter representing an enrichment of more than 140-fold, compared to 146 what would be expected by chance (Fig. 1B). We also compared our data to other studies that 147 identified APC/C substrates, interactors, proteins degraded at mitotic exit, or proteins ubiquitylated

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148 in mitosis (Table S3) (34,44–49). Our in silico analysis identified the most KEN-dependent 149 substrates relative to these studies (Figure 1C; Table S3). When compared to the set of 53 150 substrates, which includes both D- and KEN-box dependent substrates, our dataset captured 26 out 151 of 53 known substrates, despite not focusing on D-box substrates. Combining the in silico 152 predictions with our Cdh1-pulldown proteomics data, we captured 31 out of 53 substrates. 153 Among the 145 computationally identified known and potential substrates, gene ontology 154 (GO) analysis showed a strong enrichment for processes linked to various aspects of cell division 155 (Fig. 1D). Manual curation demonstrated that nearly half of the proteins we identified (70 of 145) 156 have well-established roles in cell cycle. These were sub-classified into the sub-categories 157 cytoskeleton and motors, centromere-kinetochore, APC/C and spindle checkpoint, cytokinesis, 158 mitotic entry, cell cycle transcription, cohesion and condensation, and DNA replication (Fig. 1E). 159 Among these 70, 50% have literature evidence for regulation by APC/C, highlighting our 160 enrichment for APC/C substrates (Fig. 1E; shown in magenta). All 145 proteins, their known 161 function, sub-category, KEN-box sequence motif with flanking sequence, aliases, and citations 162 describing regulation by APC/C are detailed in Table S2. 163

164 Regulated degradation of chromatin factors

165 Unexpectedly, our dataset revealed several proteins involved in chromatin regulation (Fig. 166 2A) and an enrichment for GO processes related to chromatin (Fig. 2B). The dataset includes 167 readers and writers of histone post-translational modifications, including the lysine 168 acetyltransferases, PCAF/KAT2B and NCOA3/KAT13B, the lysine methyl-transferase 169 MLL2/KMT2D, the chromatin reader and histone Ub ligase UHRF1, and the mitotic histone H3 170 kinase Aurora B (Fig. 2A and 1E). We identified proteins involved in chromatin assembly and 171 structure, including: CHAF1B, a component of the CAF-1 nucleosome assembly complex; TTF2, 172 a Swi2/Snf2 family member and DNA-dependent ATPase; KI-67, which prevents chromosome 173 aggregation in mitosis and regulates histone post-translational modifications; and proteins

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174 associated with cohesion and condensation, including SMC4 and NIPBL (Fig. 1E). We also 175 identified proteins involved in DNA damage repair. 176 To validate potential substrates, we developed an in vivo APC/C activation assay that is 177 amenable to analysis of endogenous or exogenously expressed proteins, and which is similar to 178 approaches described elsewhere (50). U2OS cells were synchronized in mitosis with the 179 microtubule poison nocodazole. After harvesting cells by mitotic shake-off, CDK1 was inactivated 180 with either the CDK1-specific inhibitor RO-3306 or pan-CDK inhibitor Roscovitine, driving cells 181 out of mitosis and triggering APC/C activation and destruction of substrates, including FoxM1, 182 NUSAP1, and Cyclin B (Fig. 2C, Fig. S1) (51). 183 Using a combination of exogenous expression and endogenous protein analysis, we 184 examined the levels of chromatin related proteins not previously shown to be APC/C substrates. 185 Using this assay, there was a decrease in the levels of several writers of histone modifications, 186 including UHRF1, PCAF, TTF2, and NCOA3 (Fig. 2C, S1A, S1B). We observed a decrease in 187 the levels of the chromatin assembly factors NASP and CHAF1B as well as the RNA processing 188 proteins LARP1 and LARP7 (Fig. 2C, S1A, S1B). All of these have been previously identified as 189 ubiquitylated in proteomics studies by an unknown E3 ligase (52–56). 190 Since the role of APC/C in chromatin regulation is not well established, we focused our 191 attention on the potential regulation of chromatin proteins by APC/C. We determined the ability 192 of a subset to bind Cdh1 by coIP. CHAF1B, PCAF, NCOA3, and TTF2 interact with Cdh1 by 193 coIP in 293T cells (Fig. 2D-2G). Accordingly, the levels of endogenous CHAF1B, TTF2, and 194 NCOA3 oscillate during the cell cycle in U2OS, analyzed following a nocodazole-induced block 195 in mitosis and then release into the cell cycle (Fig. 2H). PCAF levels did not decrease at mitotic 196 exit in U2OS (Fig. S1C) but do decrease at mitotic exit in HeLa cells (Fig. S1C), suggesting a 197 potentially complex regulation. Finally, we purified recombinant TTF2 and found that APC/C 198 could trigger its ubiquitylation in vitro (Fig. S2). A table of all proteins tested in these assays and 199 their validation is shown in Table S4. Taken together, this analysis uncovered new APC/C 200 substrates and a role for APC/C in controlling chromatin regulators.

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201

202 UHRF1 regulation by APC/CCdh1

203 To further understand the function of APC/C in chromatin biology, we pursued UHRF1, a

204 key chromatin regulator that reads and writes histone modifications. UHRF1 associates with the

205 DNA methyltransferase DNMT1 and is required for DNA methylation (26). UHRF1 has also been

206 implicated in replisome assembly (57,58) and its phosphorylation oscillates during the cell cycle

207 (59).

208 We examined UHRF1 protein levels following a mitotic block and release. 209 Immunoblotting for UHRF1 and other cell cycle markers showed that UHRF1 protein levels 210 decrease during mitotic exit in HeLa S3, HeLa, and U2OS cell lines (Fig. 3A, S3A-B). In each cell 211 line, UHRF1 levels remain low in G1 and then re-accumulate starting around G1/S, based on the 212 expression of other cell cycle markers, including cyclin E and cyclin A, and then further increasing 213 throughout the subsequent G2/M phase. 214 We performed several assays to assess whether UHRF1 is regulated by APC/C. We 215 analyzed UHRF1 in the aforementioned in vivo APC/C activation assay. U2OS cells were arrested 216 in mitosis and then treated with RO-3306. We observed a decrease in UHRF1 that was partially 217 mitigated by the proteasome inhibitor, MG-132, indicating that the reduction is dependent on the 218 proteasome (Fig. 3B). In addition, transient siRNA depletion of Cdh1 (Fzr1 mRNA transcript) 219 augmented UHRF1 protein levels (Fig. 3C). Conversely, ectopic expression of increasing 220 concentrations of FLAG-Cdh1 led to a dose-dependent decrease in both exogenous and 221 endogenous UHRF1 protein levels (Fig. 3D). We examined UHRF1 levels in cells that were first 222 synchronized in G1 by a mitotic block and release, and then treated with the pharmacological 223 APC/C inhibitor proTAME for 90 minutes (Fig. S3C). This led to an increase in UHRF1 levels. 224 Together, these data suggest that APC/C controls UHRF1 in vivo. 225

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226 UHRF1 ubiquitylation by APC/CCdh1 227 UHRF1 is a multi-domain protein (Fig. 4A) that exhibits multivalent binding with 228 chromatin through histone and DNA binding domains (24,60,61). Additionally, UHRF1 is a RING 229 domain E3 that ubiquitylates histone H3 (22,23,25). To determine whether UHRF1 is a direct 230 APC/CCdh1 substrate, we tested its binding to Cdh1 by expressing HA-Cdh1 and Myc-UHRF1 in 231 293T cells. Cells were treated with the proteasome inhibitor MG-132 prior to harvesting to prevent 232 UHRF1 degradation. Myc-UHRF1 was enriched in the HA-Cdh1 pull-down, and HA-Cdh1 was 233 enriched in the Myc-UHRF1 pull-down (Fig. 4B, 4C). 234 Next, we purified and fluorescently labeled recombinant, bacterially expressed, full-length 235 (FL) UHRF1 (FL-UHRF1*, where the * denotes fluorescently labelled protein). We found that 236 FL-UHRF1* was ubiquitylated in an APC/C- and Cdh1-dependent manner using an entirely in 237 vitro recombinant system (Fig. 4D). Multiple, high molecular weight ubiquitylated forms are 238 observed using either wild-type Ub or methylated-Ub, the latter of which cannot form poly-Ub 239 chains. This indicates that APC/C ubiquitylates multiple lysines in UHRF1 (Fig. 4D, S4A-B). 240 Since UHRF1 can auto-ubiquitylate itself through its RING domain, we confirmed that its 241 ubiquitylation is APC/C dependent. First, we purified a version of APC/C selectively missing the 242 APC2 WHB domain and the APC11 RING domain, which are required to recruit its initiating E2 243 UBE2C (designated ∆RING∆WHB) (62,63). This version of APC/C was unable to ubiquitylate 244 UHRF1 (Fig. 4E). 245 Next, we purified and fluorescently labeled a truncated version of UHRF1 that contains the 246 Linker, PHD, and SRA domains (termed LPS), spanning amino acids 287-715 (Fig. 4A). The LPS 247 fragment omits three potential APC/C D-box degron motifs, as well as the RING domain, 248 precluding auto-ubiquitylation. A D-box motif remains in the highly structured SRA domain but 249 is unlikely to be accessible as a degron motif (64). 250 Significantly, LPS-UHRF1* is more robustly ubiquitylated in an APC/C- and Cdh1- 251 dependent manner compared to FL-UHRF1* (Fig. 4D-E). Moreover, UHRF1 ubiquitylation is 252 fully inhibited by the APC/C inhibitor Emi1 (Fig. 4F). Ubiquitylation of UHRF1 is initiated by

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253 APC/CCdh1-UBE2C while APC/CCdh1-UBE2S elongates Ub chains, indicating that UHRF1 254 ubiquitylation is similar to that of other substrates tested in this in vitro system (Fig. 4F). We 255 conclude that UHRF1 is a bona fide APC/C substrate. 256 The ubiquitylation of truncated LPS-UHRF1* (Fig. 4D, 4E, 4F) strongly suggests the 257 importance of the KEN-motif, located in an unstructured region at amino acids 622-624 (Fig. 4A). 258 Alanine substitutions were introduced into the KEN sequence (UHRF1KEN:AAA). The KEN mutant 259 version (Myc-UHRF1KEN:AAA) showed reduced, although not completely abolished, binding to 260 HA-Cdh1 by coIP, compared to Myc-UHRF1WT (Fig 4G). Additionally, the KEN mutant versions 261 of FL-UHRF1* and LPS-UHRF1* were completely resistant to ubiquitylation by APC/C (Fig. 262 4H). We conclude that UHRF1 ubiquitylation by APC/CCdh1 is dependent on its KEN-box motif. 263 APC/C substrates are recruited by Cdc20 and Cdh1, and many substrates can be controlled 264 by both. To test if UHRF1 is controlled by APC/CCdc20, in addition to APC/CCdh1, we used a 265 phosphomimetic version of APC/C (termed pE-APC/C) that can utilize either Cdc20 or Cdh1, 266 since Cdc20 cannot bind to unphosphorylated APC/C (62). Surprisingly, unlike other, well- 267 established APC/C substrates, including Cyclin B (CycBNTD, amino acids 1-95) and Securin, the 268 FL-UHRF1* and LPS-UHRF1* were ubiquitylated by APC/CCdh1 but not by APC/CCdc20 (Fig. 4I, 269 S4C). 270 We transiently expressed FLAG-Cdh1 in HEK-293T cells in combination with either Myc- 271 UHRF1WT or mutant versions harboring alanine mutations in either the KEN-box (Myc- 272 UHRF1KEN:AAA) or the fourth D-box motif (Myc-UHRF1D4). Ectopic FLAG-Cdh1 overexpression 273 triggers the degradation of Myc-UHRF1WT and Myc-UHRF1D4, whereas Myc-UHRF1KEN is 274 resistant to degradation (Fig. 5A), further supporting the importance of the KEN-motif in UHRF1 275 degradation. 276 Next, we generated cell lines constitutively expressing GFP-tagged UHRF1WT or

277 UHRF1KEN:AAA using lentiviral transduction and examined UHRF1 stability upon mitotic exit. 278 Exogenous UHRF1 levels were only moderately overexpressed compared to endogenous levels 279 (Fig. 5B). Following synchronization with nocodazole, GFP-UHRF1WT levels decrease at mitotic

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280 exit. Conversely, GFP-UHRF1KEN:AAA levels remain stable through mitotic exit and G1 phase (Fig. 281 5B). Cells expressing GFP-UHRF1KEN:AAA exit mitosis normally based on immunoblotting for the 282 APC/C substrates cyclin A, cyclin B, cyclin F and Aurora A, which are degraded with normal 283 kinetics (Fig. 5B). Thus, the KEN box regulates UHRF1 ubiquitylation in vitro and degradation in 284 vivo. In addition, the mild over-expression of UHRF1 in these cells does not affect overall APC/C 285 activity. 286

287 UHRF1 degradation and cell cycle progression

288 Since many APC/C substrates are linked to proliferative control, we examined the 289 contribution of UHRF1, and its degradation by APC/C, to cell cycle. Consistent with prior reports,

290 UHRF1 depletion increased the fraction of cells in G1-phase ((65); data not shown). To further 291 investigate the role of UHRF1 in cell cycle, we examined mitotic cells following UHRF1 292 depletion. We observed an approximately three-fold increase in cells with mis-aligned 293 chromosomes in metaphase and anaphase in UHRF1 depleted cells using two independent siRNA 294 oligonucleotides (Fig. S5A). Surprisingly, there was no statistically significant difference in the 295 overall percent of mitotic cells. 296 To determine the role of UHRF1 degradation in cell cycle, we examined cell cycle markers 297 in cells expressing UHRF1WT or UHRF1KEN:AAA. In Hela cells traversing the cell cycle after 298 synchronization at G1/S, following a double thymidine block and release, we found that the GFP- 299 UHRF1KEN:AAA cells contain more of the G1/S regulator cyclin E (Fig. S6A). This was also evident 300 in cells that had been synchronized in mitosis and released into G1 (Fig. 5B). This suggested that 301 an inability to degrade UHRF1 in G1 alters cyclin E expression, a key driver of S-phase entry. 302 These data suggested that UHRF1 might promote progression into S-phase and that a 303 failure to degrade UHRF1 could shorten the duration of G1. To better address this possibility, we 304 depleted endogenous UHRF1 with an shRNA targeting the UHRF1 3’UTR (66). Cells expressing 305 GFP-UHRF1WT or GFP-UHRF1KEN:AAA were synchronized in mitosis, released into the cell cycle,

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306 and analyzed by immunoblot. Several markers of S-phase entry accumulate early in cells 307 expressing GFP-UHRF1KEN:AAA compared to GFP-UHRF1WT. Both cyclin E and the G1/S 308 transcription factor E2F1 are elevated at early time points following release from mitosis (Fig. 309 6A). Elevated levels of cyclin E and E2F1 are evident in asynchronous RPE1-hTRET cells, and to 310 a lesser extent in asynchronous HeLa S3 cells, where cell cycle transcription is perturbed due to 311 HPV oncoproteins (Fig. S6B, S6C). 312 To analyze G1 duration, cells were release from a mitotic block and pulsed with EdU prior 313 to harvesting for flow cytometry, to determine the percent of cells that were in S-phase. GFP- 314 UHRF1KEN:AAA expressing cells begin S-phase earlier than control cells (Fig. 6B). Six hours after 315 release into the cell cycle, 3.6% of control cells had entered S-phase, whereas 9.6% of GFP- 316 UHRF1KEN:AAA expressing cells had started S-phase. Thus, a failure to degrade UHRF1 accelerates 317 G1, indicating a key role for UHRF1 destruction in determining timing between the end of mitosis 318 and start of DNA synthesis. 319

320 UHRF1 degradation and DNA methylation homeostasis

321 UHRF1 is required for DNA methylation maintenance (26). To determine if stabilizing 322 UHRF1 in G1 affects DNA methylation, we performed base-resolution DNA methylation analysis 323 at approximately 850,000 unique human CpG loci spanning all genomic annotations and 324 regulatory regions using the Infinium MethylationEPIC BeadChip (EPIC arrays) (67,68). We 325 compared parental U2OS cells and those expressing GFP-UHRF1WT or GFP-UHRF1KEN:AAA. 326 Considering all probes, DNA methylation changes between parental, GFP-UHRF1WT, and GFP- 327 UHRF1KEN:AAA were insignificant (Fig. 7A). However, multidimensional scaling (MDS) of the top 328 50,000 variable CpG probes among all samples/replicates (agnostic of sample group) clustered 329 experimental conditions (Fig. 7B), indicating a unique and reproducible profile of methylation 330 patterning.

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331 We queried the GFP-UHRF1WT and GFP- UHRF1KEN:AAA samples for differentially 332 methylated CpGs relative to the parental controls. Consistent with a previous report (29), 333 expression of GFP-UHRF1WT and GFP-UHRF1KEN:AAA induced a comparable number of 334 hypomethylation events (Fig. 7C). Altrenatively, GFP-UHRF1KEN:AAA induced approximately 335 two-fold more hypermethylated CpGs compared to GFP-UHRF1WT (Fig. 7C). Analysis of 336 differentially methylated CpG probes between GFP-UHRF1WT and GFP-UHRF1KEN:AAA revealed 337 a 32% overlap in hypomethylated probes and a 17% overlap in hypermethylated probes (Fig. 7D). 338 Significantly, hypermethylated CpG probes in the GFP-UHRF1KEN:AAA expressing cells were 2.5- 339 fold more abundant compared to GFP-UHRF1WT, despite no significant change in hypomethylated 340 CpG probes. Thus, the non-degradable form of UHRF1 induces site-specific DNA 341 hypermethylation (Fig. 7D). 342 The CpGs that were hypermethylated in GFP-UHRF1KEN:AAA -expressing cells started with 343 a higher methylation level than other categories and gained methylation due to expression of non- 344 degradable mutant (Fig. 7E). Enrichment analysis of the differentially methylated CpGs revealed 345 that gene body annotations, including exons, introns, and transcription termination sites (TTS), 346 were positively enriched for hypermethylation in GFP-UHRF1KEN:AAA -expressing cells (Fig. 7F, 347 left panel). We next queried enrichment of differential methylation events in regions of early and 348 late replication (69). Hypermethylation events in GFP-UHRF1KEN:AAA, but not GFP-UHRF1WT, 349 were positively enriched in early replicating regions of the genome, while hypomethylation events 350 by both GFP-UHRF1WT and GFP-UHRF1KEN:AAA (alone or shared in common) were enriched in 351 late replicating DNA (Fig. 7F). The enrichment of these hypermethylated features was consistent 352 with known DNA methylation patterns that occur across gene bodies and early replicating DNA 353 (Fig. 7E), as CpG loci in these regions typically demonstrate high levels of methylation (70,71) . 354 Taken together, these results demonstrate that expression of non-degradable UHRF1 enhances 355 methylation at gene-rich, early replicating regions of the genome. 356

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357 Discussion 358 Identification of new E3 ligase substrates 359 APC/C is a core component of the cell cycle oscillator and mounting evidence points to its 360 dysfunction in cancer and neurological disease. Here we provide a comprehensive, unencumbered, 361 annotated list of known and candidate APC/C substrates. Our data highlights the importance of 362 APC/C in various aspects of proliferative control and points to its potentially broader impact on 363 unanticipated cellular processes, including chromatin organization. 364 Identifying E3 substrates remains technically challenging. Since E3-substrate interactions 365 exhibit low stoichiometry, mapping substrates by defining interactors is difficult. In addition, Ub 366 ligase substrates are often in low abundance. APC/C is inhibited throughout the cell cycle by 367 myriad mechanisms (72) and the time when it binds substrates coincides with when targets are 368 being degraded and their abundance is lowest. This complicates many proteomics-based 369 approaches. Alternative techniques for identifying E3 ligase substrates, including Global Protein 370 Stability Profiling (GPS) and in vitro expression cloning, circumvent these challenges by 371 measuring changes in substrate stability using fluorescent reporters or metabolic labeling with 372 radioisotopes. These represent powerful tools for mapping E3 substrates (56,73). However, both 373 approaches are laborious and time intensive, require significant technical expertise, and depend on 374 gene expression libraries, which are neither complete nor available to most laboratories. 375 We bypass these challenges using a simple in silico approach based on publicly available 376 information, which is simple, inexpensive, and easily repeated with different variables. While our 377 approach shares some similarities with previous approaches, it improves upon those in its 378 simplicity, expanded use of multiple cell cycle mRNA datasets, and inclusion of a degron motif in 379 the search criteria (35,39,74). Its success stems from the use of orthogonal filtering criteria, that 380 is, unlinked features between mRNA and proteins. We predict that similar uses of unrelated 381 properties could be leveraged for mapping targets of other enzymes, such as kinases, where 382 defining substrates has proven similarly challenging. It is notable that degron sequences remain

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383 unknown for most Ub ligases, highlighting the importance of mechanistic studies in enabling 384 systems-level discoveries. 385 386 Involvement of APC/C in chromatin regulation 387 Determining the enzymes and substrates in kinase signaling cascades has been instrumental 388 in determining proliferative controls in normal cells, their responses to stress and damage, and 389 disease phenotypes and treatments. Relatedly, decoding Ub signaling pathways involved in 390 proliferation is likely to provide insight into enzyme function in normal cell physiology as well as 391 in disease. 392 A major finding of this work is that numerous chromatin regulators are controlled 393 temporally during proliferation by APC/C. Impairing the degradation of one such substrate, 394 UHRF1, altered the timing of cell cycle events and changed global patterns of DNA methylation. 395 Since numerous chromatin regulators are controlled by APC/C, we anticipate widespread, 396 pleiotropic effects on chromatin in cells where APC/C activity is impaired, either physiologically 397 or pathologically. 398 Our observations raise the possibility that dysregulation of the cell cycle machinery, as is 399 seen in diseases such as cancer, could alter the chromatin environment. The discovery that many 400 chromatin regulators are mutated in cancer, a disease of uncontrolled proliferation, together with 401 our data, imply a bidirectional relationship between the chromatin landscape and the cell cycle 402 oscillator. Consistent with the notion that dysregulation of APC/C controlled proteins could play 403 important roles in determining the chromatin environment in disease, the mRNA expression of our 404 145 known and putative substrates strongly predict breast cancer aneuploidies and copy number 405 variations (Fig. S7). This observation is not due solely to the selection of specific breast cancer 406 subtypes, since our gene signature is elevated in multiple breast cancer subtypes. Interestingly, the 407 expression of this signature correlates with the CIN70 signature, which was previously developed 408 based on gene expression in chromosomally unstable cancers (75). We observed an extraordinary 409 correlation between the CIN70 and our 145 gene signature in breast cancer (Fig. S7). This is

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410 remarkable since our signature was generated completely independent of gene expression in cancer 411 and was instead derived, in part, by short sequence motifs on proteins. 412 APC/CCdh1, but not APC/CCdc20, ubiquitylates UHRF1. This is notable because the Cdh1- 413 bound form of APC/C is active both G1 and quiescent cells and is critical for restraining S-phase 414 entry. Our findings suggest that impaired UHRF1 degradation promotes a premature G1/S 415 transition. We propose that the proper degradation of UHRF1, and other chromatin regulators, 416 serves to integrate growth factor dependent proliferative decisions with the chromatin regulatory 417 environment. This could help explain the complex chromatin rearrangements observed in 418 quiescent cells, where APC/CCdh1 is active (32,33,76). Further, APC/C controls key cell cycle 419 transcriptional regulators, including the G2/M transcription factor FoxM1 and the repressor E2F 420 proteins, E2F7 and E2F8 (77,78). Thus, our data point to a higher order role regulatory role for 421 APC/C in gene regulation, by controlling transcription factors (i.e. FoxM1), transcriptional 422 repressors (i.e. E2F7, E2F8,) and chromatin modifiers. 423 Aberrant DNA methylation is a hallmark of cancer (79). UHRF1 promotes DNA 424 methylation maintenance, and too much or too little UHRF1 expression is detrimental to 425 methylation stasis (26,29). It is interesting to speculate that the redistribution of DNA methylation 426 in disease could be caused, in part, by the aberrant stabilization of UHRF1, resulting from 427 APC/CCdh1 inactivation. It will be important, in the future, to determine if oncogene activation acts 428 through the APC/C to re-organize the chromatin landscape. Furthermore, determining ubiquitin 429 ligase substrates, like UHRF1, that might be dysregulated in pathological settings via altered 430 degradative mechanisms could suggest therapeutic strategies to reverse their effects.

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431 Materials and Methods

432 Computational identification of putative APC/C substrates 433 Human proteins containing a KEN-box sequence (amino acid sequence K-E-N) were

434 identified using the “Find a Sequence Match” feature on the Scansite web search platform

435 (currently https://scansite4.mit.edu/4.0/#home). Proteins with cell cycle regulated mRNA were

436 curated from four independent cell cycle transcriptional studies (42,43,80,81). The genes which

437 scored in two or more of these screens was previously compiled in the supplemental data of Grant

438 et al., 2013. Gene and protein name conversions were performed using the DAVID online tool

439 (https://david.ncifcrf.gov/conversion.jsp). The overlapping set 145 proteins, which contain a KEN

440 sequence and exhibit oscillating cell cycle regulated mRNA expression, were identified. For all

441 145 proteins, we manually curated information on their alias, function, sequence flanking the KEN

442 motif, and evidence for regulation by APC/C from various online databases and repositories,

443 including UNIPROT, Pubmed, and Genecards.

444 The set of 33 well-validated, KEN-containing, human APC/C substrates was derived from 445 (16). Our own FLAG-Cdh1 IPs were compared to other APC/C substrate discovery efforts (47,82). 446 Singh et al. identified “clusters” of proteins whose levels changed at mitotic exit. For each cluster, 447 they reported a top percentile, and for the clusters that most accurately revealed APC/C substrates 448 (1, 2, and 3), we compile their data in Supplemental Table 3 in terms of which KEN-dependent 449 substrates were identified. Their data from Cluster 1, which identified the most KEN-containing 450 APC/C substrates, is shown in Figure 1C. Lafranchi et al. rank ordered proteins based on the degree 451 of change from mitosis to G1, analyzed by proteomics. We curated their data to identify the cut- 452 off point where the last KEN-dependent APC/C substrate was identified among their rank ordered 453 list. Since they provided no cut-off point, the data comparison in Figure 1C represents the best 454 estimate of their ability to capture APC/C substrates. 455

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456 Cell Culture 457 HeLa, HeLa S3, U2OS, RPE-1, and HCT116 cells were grown in 10% FBS with high 458 glucose DMEM without antibiotics. Cell culturing utilized standard laboratory practices whereby

459 cells were grown and incubated at 37°C containing 5% CO2. Frozen cell stocks were stored under 460 liquid nitrogen in 10% DMSO/90% FBS. 461 GFP-UHRF1WT and GFP-UHRF1KEN:AAA stable overexpression cells were generated by 462 transducing HeLa S3, U2OS, and RPE-1-hTERT cell lines with pHAGE-GFP lentivirus that had 463 been produced in HEK293T cells. Infections were performed in the presence of 8µg/mL polybrene 464 for 48 hours prior to antibiotic selection. Cells were selected for 5-7 days with 8ug/mL (HeLa S3 465 and U2OS) or 10ug/mL (RPE-1) Blasticidin. Lentiviral particles were produced by transfecting 466 HEK293T cells with Tet, VSVg, Gag/pol, and Rev viral packaging vectors together with the 467 pHAGE-GFP lentiviral vectors using TransIT® MIRUS. Viral particles were collected 48 and 72 468 hours after transfection and stored at -80°C prior to transduction. 469 To generate the rescue cell lines, the U2OS and HeLa S3 stable GFP-UHRF1WT and GFP- 470 UHRF1KEN:AAA expression cell lines were transduced with previously described and validated 471 pLKO.1 lentiviral vectors encoding either shControl or 3’UTR targeting shUHRF1 (66), using 472 8ug/mL polybrene to aid infection. After 48 hours, cells were selected with 2µg/mL Puromycin 473 for 3-5 days. Viral particles were produced by transfecting HEK293T cells with the pLKO.1 474 constructs and psPAX2 and pMD2.G packaging vectors using TransIT® MIRUS (cat no. MIR 475 2700), collecting after 48 and 72 hours as mentioned previously. 476 Mitotic block was induced by treating 25% confluent HeLa S3 cells with 2mM thymidine 477 for 24 hours. After washing the plates three-four times with warm media and incubating in drug- 478 free media for 3-4 hours, cells were treated with 100 ng/mL nocodazole for 10-11 hours prior to 479 harvesting by mitotic shake-off. Samples were washed three or four times with warm media, 480 counted, and re-plated for indicated timepoints. 481 To synchronize cells in G1/S, HeLa S3 were plated at 20% confluency prior to addition of 482 2mM thymidine. After 16 hours, cells were washed three times with warm media and left to

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483 incubate for 8 hours before the second block in 2mM thymidine for another 16 hours. Cells were 484 washed three times in warm media and collected at specific timepoints as they progress through 485 the cell cycle. 486 To transiently inactivate the APC/C, HCT116 or U2OS cells were treated with 15µM 487 proTAME (Thermo Fisher cat no. I-440-01M), a pan-APC/C inhibitor (83), for 90 minutes prior 488 to harvest and immunoblotting. Cells had been released from nocodazole-induced mitotic block 489 for 90 minutes in drug-free media prior to addition of drug. 490 491 In vivo APC/C Activation assay 492 70-80% confluent U2OS cells were transfected with the indicated plasmids for 24 hours 493 and then exchanged into fresh media. Alternatively, untransfected cells were used to analyze 494 endogenous proteins. After an eight-hour incubation in fresh media following transfection, cells 495 were treated with 250ng/mL nocodazole for 16 hours. Mitotic cells were isolated by shake-off, 496 washed once in pre-warmed media, counted, and divided equally among 15mL conical tubes. Cells 497 in suspension were treated with DMSO, RO-3306 (10 µM), Roscovitine (10 µM), or MG-132 498 (20µM) for the indicated amount of time at 37ºC. Identical volumes of cells were removed from 499 cell suspensions by pipetting, isolated by centrifugation, and frozen at -20°C prior to processing 500 for immunoblot. 501 502 Molecular Biology 503 Plasmid transfection of HEK293T, U2OS, and HCT116 was performed with either MIRUS 504 or PolyJet (cat no. SL100688) at 1:3 or 1:4 DNA:plasmid ratio on cells with 50-60% confluency. 505 After 24 hours, the media was changed, and cells were expanded to larger dishes as needed. 506 Samples were collected 24-48 hours after siRNA transfection was performed using a 1:3 ratio of 507 RNAi oligonucleotide to RNAiMAX (cat no. 13778-030). UHRF1 was cloned into the indicated 508 lentiviral vectors mentioned previously using standard gateway recombination cloning. Other 509 APC/C substrates tested for binding to Cdh1 or degradation in the APC/C activation assay were

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510 obtained from either the ORFeome collection and cloned into the indicated vectors using gateway 511 recombination cloning or from addgene (see supplemental table) (84).

512 513 Cell lysis and immunoblotting

514 Cells were lysed on ice for 20 minutes in Phosphatase Lysis buffer (50 mM NaH2PO4, 150 515 mM NaCl, 1% Tween-20, 5% Glycerol, pH 8.0, filtered) or NETN (20 mM Tris pH 8.0, 100 mM 516 NaCl, 0.5 mM EDTA, 0.5% NP40) supplemented with 10µg/mL each of aprotonin, pepstatin A, 517 and leupeptin, 1mM sodium orthovanadate, 1mM NaF, and 1mM AEBSF. Following incubation

518 on ice, cell lysates were centrifuged at (20,000 x g) in a benchtop microcentrifuge at 4°C for 20 519 minutes. Protein concentration was estimated by BCA assay (Thermofisher cat no. PI-23227) 520 according to manufacturer’s protocol. Cell extracts were diluted with SDS-PAGE Gel Loading 521 Buffer (Laemmli Buffer) prior to analysis by SDS-PAGE. Typically, 20-40 µg of protein were 522 loaded on SDS gels (either BioRad 4-12% Bis-Tris or homemade SDS-PAGE gels) and separated 523 at 140-200V for approximately 1 hour. Proteins were transferred by wet-transfer methods onto 524 nitrocellulose membrane, typically at 100V for 1 hour or 10-17V overnight at 4°C. Nitrocellulose 525 membranes were then incubated with TBST (137mM NaCl, 2.7mM KCl, 25mM Tris pH 7.4, 0.5% 526 Tween-20) supplemented with either 5% bovine serum albumin or non-fat dry milk for at least one 527 hour or overnight at 4°C. Blocked membranes were incubated overnight with primary antibodies 528 at 4°C, washed in TBST, incubated in appropriate secondary antibodies for 1 hour at room 529 temperature, and then developed by chemiluminescence using Pierce ECL (ThermoFisher) or 530 Clarity ECL (Bio-Rad). See reagent list in supplement for detailed primary and secondary antibody 531 information. 532

533 Immunoprecipitation

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534 For co-immunoprecipitation (coIP) experiments, cells were lysed in NETN for 20 minutes 535 on ice and then centrifuged in a benchtop centrifuge on maximum speed (20,000 x g) for 20 536 minutes at 4°C, prior to determining protein concentration by either Bradford or BCA assay. 537 A master mix of 1-2 mg/mL protein concentration was calculated, 10% of which was 538 retained as input while the remaining 90% was used for coIP. Prior to coIP, antibody coated beads 539 were prewashed with 1X TBST three times prior to incubation with lysis buffer. Cell lysates were 540 also pre-cleared by incubation with the same volume of empty Protein A/G agarose beads. 541 Clarified cell lysates were immunoprecipitated for 2-4 hr at 4°C with 25-50uL of EzView M2- or

542 Myc-antibody beads (F2426-1ML or E6654-1ML). After coIP, beads were pelleted at low speed 543 centrifugation, washed twice with wash buffer, and one time with lysis buffer to remove unbound 544 proteins. Buffers were removed from beads using a 27 gauge needle to avoid the aspiration of 545 beads between washes. Washed beads were resuspended in 2X SDS-PAGE Gel Loading Buffer 546 (Laemmli Buffer) and boiled 5-10 minutes at 95°C. Samples were removed from the beads using 547 a 27-gauge needle to avoid the aspiration of beads after boiling. Typically, 20µL of coIP was 548 loaded alongside 1% of the input volume. Samples were analyzed by immunoblotting as described. 549

550 Protein Purification 551 Substrates for in vitro ubiquitylation assays were expressed as N-terminal GST-TEV- 552 fusion (TTF2) or His-MBP-TEV-fusions (FL-UHRF1WT, LPS-UHRF1WT, FL-UHRF1KEN:AAA, 553 LPS-UHRF1KEN:AAA) in BL21 (DE3) codon plus RIL cells. TTF2 was purified by glutathione- 554 affinity chromatography, treated with TEV protease to liberate GST, and further purified by ion 555 exchange chromatography. UHRF1 wild-type and variants were purified by amylose-affinity 556 chromatography, treated with TEV, and followed by ion exchange chromatography. Fluorescently 557 labeled substrates were generated by incubating 1 µM Sortase, 20x 5-carboxyfluorescein (5- 558 FAM)-PEG-LPETGG peptide, and substrates in 10 mM HEPES pH 8, 50 mM NaCl, and 10 mM

559 CaCl2. After 2 hours of incubation at 4°C, reactions were stopped by removing the His6-tagged

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560 Sortase by nickel affinity chromatography. Then, excess 5-FAM-LPETGG was removed by size 561 exclusion chromatography. 562 Expression and purification of UBA1, UBE2C, UBE2S, recombinant APC/C and pE- 563 APC/C, Cdh1, Cdc20, Emi1, ubiquitin, and methylated ubiquitin were performed as described 564 previously in Brown et al. 2016 (85–89). 565 566 APC/C Ubiquitylation assays 567 Qualitative assays to monitor APC/C-dependent ubiquitylation were performed as 568 previously described (89). In brief, reactions were mixed on ice, equilibrated to room temperature 569 before the reactions are initiated with Ub or meUb, and quenched at the indicated time points with 570 SDS. TTF2 ubiquitylation was monitored by mixing 100 nM APC/C, 1 µM Cdh1, 5 µM UBE2C, 571 5 µM UBE2S (when indicated), 1 µM UBA1, 5 µM TTF2, 5 mM Mg-ATP, and 150 µM Ub or 572 meUb (Fig. S2). Ubiquitylation of UHRF1 wild-type or its variants by APC/C were performed 573 with 100 nM APC/C or pE-APC/C, 1 µM Cdh1 or Cdc20, 0.4 µM UBE2C, 0.4 µM UBE2S (when 574 indicated), 1 µM UBA1, 0.4 µM UHRF1, 5 mM Mg-ATP, and Ub or meUb (Fig. 4 and Fig. S4). 575 Following SDS-PAGE, ubiquitylation products of the fluorescently labeled substrates were 576 resolved by SDS-PAGE and imaged with the Amersham Typhoon 5. 577 578 Flow cytometry cell cycle analysis 579 HeLa S3 GFP-UHRF1WT and GFP-UHRF1KEN:AAA (shUHRF1) cells were synchronized in 580 mitosis by sequential thymidine-nocodazole treatment as described above, using 2mM thymidine 581 and 100ng/mL nocodazole. After release, cells were pulsed with 10µM EdU thirty minutes prior 582 to collection at specific timepoints. After counting the cells, 2 million cells were retained for 583 Western blotting (WB) analysis and 1 million cells were fixed for flow cytometry. For WB, cells 584 we pelleted and washed once with cold PBS prior to freezing at -20°C. For flow cytometry, cells 585 were fixed in 4% formaldehyde/PBS for 15 minutes at room temperature. Cells were pelleted and 586 resuspended in 1% BSA/PBS and stored overnight at 4°C. The next day, cells were pelleted and

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587 resuspended in 1% BSA/PBS/0.5% Triton X-100 for 15 minutes at room temperature. Cells were

588 pelleted, resuspended with labelling solution (100mM ascorbic acid, 1mM CuSO4, 2µM Alexa 589 Fluor 488 azide in PBS), and incubated for thirty minutes in the dark at room temperature. After 590 addition of 1% BSA/PBS/0.5% Triton X-100, cells were pelleted and stained with 1µg/mL DAPI 591 in 1% BSA/PBS/0.5% Triton X-100 for one hour in the dark at room temperature. Flow cytometry 592 was performed on an AttuneTM Nxt Flow Cytometer (Thermo Fisher Scientific). Channel BL1 was 593 used for Azide 488 dye. Channel VL1 was used for DAPI dye. Following acquisition, data were 594 analyzed using FlowJo software. 595 596 Immunofluorescence imaging 597 HeLa cells were plated on poly-L-lysine-coated #1.5 coverslips. Next day, cells were 598 treated with siRNA (control siFF and siUHRF1) and RNAi Max according to manufacturer’s 599 protocol (Invitrogen). After 48 hours of siRNA treatments, cells were fixed in 3%

600 paraformaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, 601 pH 7.0) for 15 minutes at 37 °C. Then, cells were washed with PHEM buffer and permeabilized 602 using 0.5% of Nonidet P-40 in PHEM buffer for 15 minutes at room temperature. Cells were 603 washed and then blocked with 5% BSA in PHEM. Primary antibodies used were: α-CENP-C 604 (MBL:1:1000) as a kinetochore marker and α-tubulin (Sigma: 1:500). Samples were incubated in 605 primary antibody solution for 1 hour at 37 °C. All fluorescently labeled secondary antibodies (anti- 606 mouse Alexa 488, anti-guinea pig 564) were diluted 1:200 dilution, and cells were incubated for 1 607 hour at 37 °C. DNA was counterstained with DAPI for 15 minutes at room temperature after 608 washing out secondary antibodies. All samples were mounted onto glass slides in Prolong Gold 609 antifade (Invitrogen). For image acquisition, three-dimensional stacked images were obtained 610 sequentially at 200 nm steps along the z axis through the cell using MetaMorph 7.8 software 611 (Molecular Devices) and a Nikon Ti-inverted microscope equipped with the spinning disc confocal 612 head (Yokogawa), the Orca-ER cooled CCD camera (Nikon), and an ×100/1.4 NA PlanApo 613 objective (Nikon).

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614 615 Genomic DNA isolation for methylation analysis 616 Genomic DNA was isolated from Parental U2OS cells and U2OS cells overexpressing 617 either GFP-UHRF1WT or GFP-UHRF1KEN:AAA. All samples groups were processed in biological 618 triplicates. Briefly, cells were lysed overnight at 37°C in 2 mL of TE-SDS buffer (10 mM Tris- 619 HCl pH 8.0, 0.1 mM EDTA, 0.5% SDS), supplemented with 100 μl of 20 mg/ml proteinase K. 620 DNA was purified by phenol:chloroform extraction in three phases: (1) 100% phenol, (2) 621 phenol:chloroform:isoamyl alcohol (25:24:1), and (3) chloroform:isoamyl alcohol (24:1). For each 622 phase, the aqueous layer was combined with the organic layer in a 1:1 ratio. Samples were quickly 623 shaken, allowed to sit on ice for approximately 5 minutes, and then separated by centrifugation at 624 1,693 RCF for 5 minutes at 4°C. The top aqueous layer was then transferred to a new tube for the 625 next organic phase. Following extraction, DNA was precipitated with 1/10 volume 3M sodium 626 acetate pH 4.8 and 2.5 volumes 100% ethanol and stored overnight at -20°C. Precipitated DNA 627 was pelleted by centrifugation at 17,090 RCF for 30 minutes at 4°C. The pelleted DNA was 628 washed twice with 70% ethanol, allowed to dry for 15 minutes, and resuspended in TE buffer (10 629 mM Tris-HCl pH 8.0, 0.1 mM EDTA). Samples were then treated with 1 mg/ml RNAse A at 37°C 630 for 30 minutes and then re-purified by ethanol precipitation as described above. 631 632 Infinium Methylation EPIC BeadChip (EPIC array) 633 Genomic DNA was quantified by High Sensitivity Qubit Fluorometric Quantification 634 (Invitrogen), and 1.5 ug of genomic DNA was submitted to the Van Andel Institute Genomics 635 Core for quality control analysis, bisulfite conversion, and DNA methylation quantification using 636 the Infinium Methylation EPIC BeadChIP (Illumina) processed on an Illumina iScan system 637 following the manufacturer’s standard protocol (67,68). 638

639 EPIC array data processing

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640 All analyses were conducted in the R statistical software (Version 3.6.1) (R Core Team). 641 R script for data processing and analysis is available in Supplemental Code File 1. 642 Raw IDAT files for each sample were processed using the Bioconductor package 643 “SeSAMe” (Version 1.2.0) for extraction of probe signal intensity values, normalization of probe 644 signal intensity values, and calculation of β-values from the normalized probe signal intensity 645 values (90–92). The β-value is the measure of DNA methylation for each individual CpG probe, 646 where a minimum value of 0 indicates a fully unmethylated CpG and a maximum value of 1 647 indicates a fully methylated CpG in the population. CpG probes with a detection p-value > 0.05 in

648 any one sample were excluded from the analysis. 649 650 Genomic and Replication Timing annotation 651 CpG probes were mapped to their genomic coordinate (hg38) and were then annotated to 652 their genomic annotation relationship (promoter-TSS, exon, etc.) using HOMER (Version 4.10.3) 653 (93). 654 Repli-seq data for U2OS cells used for determining CpG probe localization relative to 655 replication timing was generated by Dr. David Gilbert’s lab (Florida State University) as part of 656 the 4D Nucleome project (Experiment #4DNEXWNB33S2)(69). Genomic regions were 657 considered early-replicating if the replication timing value was > 0 and late-replicating if < 0. CpG 658 probes were annotated for replication timing domains by intersecting the Repli-seq genomic 659 coordinates with CpG probe coordinates using BEDTools (Version 2.16.2) (94). 660 661 Identification of differentially methylated CpG probes 662 The Bioconductor package “limma” (Version 3.40.6) was used to determine differential 663 methylation among sample groups and perform multidimensional scaling (MDS) analysis (95–97).

664 For statistical testing of significance, β-values were logit transformed to M-values: =

665 . M-values were then used for standard limma workflow contrasts to determine𝑀𝑀 β WT KEN:AAA 666 𝑙𝑙𝑙𝑙𝑙𝑙differential2 �1−β� methylation of U2OS GFP-UHRF1 or GFP-UHRF1 overexpression to

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667 Parental U2OS cells (98,99). CpG probes with an adjusted p-value 0.05 were considered

668 significant, and log fold-change of M-value was used to determine hypermethylation≤ (logFC > 0) 669 or hypomethylation (logFC < 0) relative to U2OS parental cells. 670 671 Enrichment Bias Calculation and Hypergeometic Distribution Testing 672 Enrichment Bias Calculations were done by first determining the following values for each 673 feature (e.g. Genomic Annotation, Replication Timing): 674 q = Number of CpGs that are differentially methylated in feature (e.g. exon) 675 m = Total number of CpGs on the EPIC array that match feature (e.g. exon) 676 n = Total number CpGs on the EPIC array that do not match feature (e.g. everything that 677 is not an exon) 678 k = Total number of all differentially methylated CpGs 679 680 Next, the expected number of CpGs that would be differentially methylated in that feature 681 by random chance was determined with the following equation: 682 = + 𝑚𝑚 683 𝑒𝑒 � � 𝑘𝑘 𝑚𝑚 𝑛𝑛 684 Finally, percent enrichment bias was calculated with the following equation: 685 % = × 100 𝑞𝑞 −𝑒𝑒 686 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒Whereℎ𝑚𝑚𝑚𝑚𝑚𝑚 positive𝑚𝑚 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 or� negative� enrichment values indicate more or less enrichment for a feature 𝑘𝑘 687 than would be expected by random chance, respectively. 688 Hypergeometric distribution testing for determining significance of enrichment bias was 689 performed using the phyper() function in R with the following values: q,m,n,k. 690

691 Data access 692 EPIC array data can be found under GEO Accession # GSE137913. 693 To review GEO accession GSE137913:

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694 Go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE137913 695 The following secure token has been created to allow review of record GSE137913 while it 696 remains in private status: eletaomyfnqrlun 697 698 Signature evaluation in TCGA BRCA samples 699 Upper quartile normalized RSEM gene expression data for TCGA BRCA (n=1201) was 700 downloaded from the GDC legacy archive (https://portal.gdc.cancer.gov). The data was log2 701 transformed and median centered. To determine the per sample UB signature score, the samples 702 were ranked by the median expression of the 145 UB gene signature. Sample were then divided at 703 the median and grouped as high or low based on rank. Copy number burden, aneuploidy, and 704 homologous recombination deficiency data were extracted from Thorsoon et. al. (100) and plotted 705 by UB signature group and PAM50 subtype (101). Significance was calculated by t-test. The 706 CIN70 score was determined as previously described in Fan et. al. (102). The CIN70 was plotted 707 against the UB, colored by PAM50 subtype, and r2 and Pearson correlation were calculated. All 708 analysis were performed in R (v3.5.2). 709 710 Cdh1 pulldown for analysis of interactors by mass spectrometry 711 FLAG-tagged Cdh1 was expressed in HEK293T cells for 24 hours by transient 712 transfection. Transfections were performed on 150 mm dishes (8 per condition) using Mirus 713 TransIT®-LT1 Transfection Reagent (Mirus Bio) and Lipofectamine 2000 (Life Technologies). 714 Cells were treated with MG-132 (10 μM for 4 hours) in culture prior to lysis, dislodged by 715 trypsinization, washed with PBS, and lysed in NETN supplemented with 2 μg/ml pepstatin, 716 2 μg/ml apoprotinin, 10 μg/ml leupeptin, 1 mM AEBSF (4-[2 Aminoethyl] benzenesulfonyl

717 fluoride), 1 mM Na3VO4, and 1 mM NaF on ice for 20 minutes. Cell lysates were then clarified by 718 centrifugation at 15,000 rpm for 15 minutes. 719 Anti-FLAG M2 agarose (Sigma, catalog no. F2426) was used for precipitation (6 hours at 720 4°C). The beads were washed with NETN three times and eluted twice with 150 µl of 0.1 M

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721 Glycine-HCl, pH 2.3 and then neutralized with Tris 1M (pH 10.0). The total eluted protein was 722 reduced (5 mM DTT) and alkylated using iodoacetamide (1.25 mM) for 30 minutes in the dark. 723 The resultant protein was then digested overnight with sequencing grade trypsin (Promega). The 724 trypsin: protein ratio was maintained at 1:100. Total peptides were purified on Pierce C18 spin 725 columns (Cat 89870) using the manufacturer’s protocol. Peptides were eluted using 70% 726 acetonitrile and 0.1% TFA solution in 50 μl volumes twice, dried on a SpeedVac at room 727 temperature, and processed by mass spectrometry proteomic analysis. 728 729 Mass Spectrometry 730 Peptides were separated by reversed-phase nano-high-performance liquid chromatography 731 using a nanoAquity UPLC system (Waters Corp.). Peptides were first trapped in a 2 cm trapping 732 column (Acclaim® PepMap 100, C18 beads of 3.0 μm particle size, 100 Å pore size) and a 25 cm 733 EASY-spray analytical column (75 μm inner diameter, C18 beads of 2.0 μm particle size, 100 Å 734 pore size) at 35°C. The flow rate was 250 nL/minute over a gradient of 1% buffer B (0.1% formic 735 acid in acetonitrile) to 30% buffer B in 150 minutes, and an in-line Orbitrap Elite mass 736 spectrometer (Thermo Scientific) performed mass spectral analysis. The ion source was operated 737 at 2.6 kV with the ion transfer tube temperature set at 300°C. A full MS scan (300–2000 m/z) was 738 acquired in Orbitrap with a 120,000 resolution setting, and data-dependent MS2 spectra were 739 acquired in the linear ion trap by collision-induced dissociation using a 2.0 m/z wide isolation 740 window on the 15 most intense ions. Precursor ions were selected based on charge states (+2, +3) 741 and intensity thresholds (above 1e5) from the full scan; dynamic exclusion (one repeat during 30 742 seconds, a 60 seconds exclusion time window) was also used. The polysiloxane lock mass of 743 445.120030 was used throughout spectral acquisition. 744 Raw mass spectrometry data files were searched using SorcererTM-SEQUEST® (build 745 5.0.1, Sage N Research), the Transproteomic Pipeline (TPP v4.7.1), and Scaffold (v4.4.1.1) with 746 the UniProtKB/Swiss-Prot human canonical sequence database (20,263 entries; release 07/2013). 747 The search parameters used were a precursor mass between 400 and 4500 amu, zero missed

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748 cleavages, a precursor ion tolerance of 3 amu, accurate mass binning within PeptideProphet, fully 749 tryptic digestion, a static carbamidomethyl cysteine modification (+57.021465), variable 750 methionine oxidation (+15.99492), and variable serine, threonine and tyrosine (STY) 751 phosphorylation (79.966331). A 1% protein-level FDR was determined by Scaffold. 752 753

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754 755 Acknowledgements

756 We thank Brian Strahl (UNC) and members of the Emanuele and Brown labs for helpful 757 discussions throughout this project. We thank Marie Adams and Julie Koeman from the Van Andel 758 Institute Genomics Core for their assistance with Illumina Infinium MethylationEPIC BeadChip 759 processing. We acknowledge the UNC Flow Cytometry Core Facility (supported in part by P30 760 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer 761 Center). The Emanuele lab (JLK, MJE, XW, TB, and RC) is supported by the UNC University 762 Cancer Research Fund (UCRF), the National Institutes of Health (R01GM120309), the America 763 Cancer Society (Research Scholar Grant; RSG-18-220-01-TBG). The Hoadley lab (KAH and JD) 764 is supported by a Komen Career Catalyst Award (CCR16376756). The Brown lab (RCMC, DLB, 765 and NGB) is supported by UCRF and National Institutes of Health (R35GM128855). The Suzuki 766 lab is supported by startup funds from the University of Wisconsin-Madison School of Medicine, 767 Graduate School, McArdle Laboratory for Cancer Research, the UW Carbone Cancer Center, and 768 by the New Investigator Program grant from Wisconsin Partnership Program (WPP). The Rothbart 769 lab (SBR and RLT) are supported by grants from the NIH (R35GM124736) and the American 770 Cancer Society – Michigan Cancer Research Fund (PF-16-245-01-DMC). 771 772 773

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774 Author Contributions

775 JLK performed most cell biological experiments. RCMC and DLB purified recombinant 776 proteins and performed in vitro ubiquitylation assays. TB and XW performed cell-based assays. 777 RC, FY, and MBM performed proteomics experiments. JSD and KAH performed bioinformatic 778 analysis of TCGA datasets. AS performed imaging analysis of mitotic cells. SBR and RLT 779 performed DNA methylation analysis. JSH provided UHRF1 constructs and technical advice on 780 protein purification. MJE conceived of the computational screen and analyzed data. NGB and MJE 781 designed experiments. JLK, RCMC, RLT, SBR, NGB, and MJE wrote and revised the manuscript.

782

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783 Declaration of Interest

784 The authors declare no conflicts of interest. 785 786

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1097 RING E3 architectures regulate multiubiquitination and ubiquitin chain elongation by 1098 APC/C. Cell [Internet]. 2016;165(6):1440–53. Available from: 1099 http://dx.doi.org/10.1016/j.cell.2016.05.037 1100 90. Zhou W, Triche TJ, Laird PW, Shen H. SeSAMe: reducing artifactual detection of DNA 1101 methylation by Infinium BeadChips in genomic deletions. Nucleic Acids Res [Internet]. 1102 2018;46(20):e123. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30085201 1103 91. Huber W, Carey VJ, Gentleman R, Anders S, Carlson M, Carvalho BS, et al. Orchestrating 1104 high-throughput genomic analysis with Bioconductor. Nat Methods [Internet]. 2015 1105 Feb;12(2):115–21. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25633503 1106 92. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, et al. Bioconductor: 1107 open software development for computational biology and bioinformatics. Genome Biol 1108 [Internet]. 2004;5(10):R80. Available from: 1109 http://www.ncbi.nlm.nih.gov/pubmed/15461798 1110 93. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, et al. Simple Combinations of 1111 Lineage-Determining Transcription Factors Prime cis-Regulatory Elements Required for 1112 Macrophage and B Cell Identities. Mol Cell. 2010;38(4):576–89. 1113 94. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic 1114 features. Bioinformatics [Internet]. 2010 Mar 15;26(6):841–2. Available from: 1115 http://www.ncbi.nlm.nih.gov/pubmed/20110278 1116 95. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential 1117 expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 1118 [Internet]. 2015 Apr 20;43(7):e47. Available from: 1119 http://www.ncbi.nlm.nih.gov/pubmed/25605792 1120 96. Huber W, Carey VJ, Gentleman R, Anders S, Carlson M, Carvalho BS, et al. Orchestrating 1121 high-throughput genomic analysis with Bioconductor. Nat Methods. 2015 Feb;12(2):115– 1122 21. 1123 97. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, et al. Bioconductor: 1124 open software development for computational biology and bioinformatics. Genome Biol. 1125 2004;5(10):R80. 1126 98. Maksimovic J, Phipson B, Oshlack A. A cross-package Bioconductor workflow for 1127 analysing methylation array data. F1000Research [Internet]. 2016;5:1281. Available from:

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1128 http://www.ncbi.nlm.nih.gov/pubmed/27347385 1129 99. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential 1130 expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015 1131 Apr;43(7):e47. 1132 100. Thorsson V, Gibbs DL, Brown SD, Wolf D, Bortone DS, Ou Yang TH, et al. The Immune 1133 Landscape of Cancer. Immunity. 2018; 1134 101. Hoadley KA, Yau C, Hinoue T, Wolf DM, Lazar AJ, Drill E, et al. Cell-of-Origin Patterns 1135 Dominate the Molecular Classification of 10,000 Tumors from 33 Types of Cancer. Cell. 1136 2018; 1137 102. Fan C, Prat A, Parker JS, Liu Y, Carey LA, Troester MA, et al. Building prognostic models 1138 for breast cancer patients using clinical variables and hundreds of gene expression 1139 signatures. BMC Med Genomics. 2011; 1140

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1141 Main Figure Legends

1142 Fig. 1. In silico analysis reveals a high confidence set of APC/C substrates involved in mitosis. 1143 (A) KEN-box containing human proteins were identified and cross-referenced against a set 1144 of 651 genes whose expression is cell cycle regulated based on multiple, independent 1145 studies. This revealed a set of 145 KEN-box containing proteins whose mRNA 1146 expression is cell cycle regulated. 1147 (B) Analysis of the enrichment of bona fide KEN-dependent substrates among these three 1148 datasets (blue- KEN box only set (2206); black- cell cycle regulated mRNAs (651); red- 1149 the overlapping set of 145 proteins) compared against a curated set of bona fide, KEN- 1150 dependent APC/C substrates (Davey and Morgan, Mol Cell, 2016). Enrichment was 1151 calculated based on the expected number of substrates which would be captured by 1152 chance based on the size of the dataset. 1153 (C) Analysis of putative substrates recovered in the indicated studies. 1154 (D) Gene ontology (GO) analysis for indicated studies (blue- KEN box only set (2206); 1155 black- cell cycle regulated mRNAs (651); red-the overlapping set of 145 proteins). 1156 (E) The set of 145 putative substrates was manually curated and analyzed for roles in various 1157 aspects of cell cycle progression. Seventy proteins, involved in cell cycle activities, are 1158 shown. The ones labelled in magenta signify that there is evidence in the literature of 1159 their regulation by APC/C. (Note that AURORA B, a mitotic kinase that phosphorylates 1160 histone H3, is listed here and in Figure 2A)

1161

1162 Fig. 2. Putative APC/C substrates are enriched for roles in chromatin regulation. 1163 (A) The set of 145 known and putative APC/C substrates is enriched for proteins involved in 1164 various chromatin related process. This includes chromatin readers and writers, 1165 chaperones, RNA regulation and processing, DNA damage repair, and others. (Note that 1166 AURORA B, a mitotic kinase that phosphorylates histone H3, is listed here and in Figure 1167 1E) 1168 (B) Gene ontology (GO) analysis of the overlapping KEN-box containing cell cycle regulated 1169 transcripts. This set is enriched for the indicated biological process, including DNA 1170 metabolism, protein-DNA complex assembly, DNA packaging, and DNA conformation. 1171 (C) APC/C activation assay to monitor substrate degradation. Following synchronization in 1172 mitosis, cells were washed one time and treated with CDK inhibitors to remove inhibitory 1173 phosphorylation marks that hinder the formation of APC/CCdh1 needed for the M/G1 1174 phase transition. Protein degradation was monitored by immunoblot. CHAF1B and 1175 PCAF are putative APC/C substrates, and FoxM1 and Cyclin B are known targets. 1176 (D) coIP of HA-Cdh1 with Myc-CHAF1B in transiently transfected 293T cells treated with 1177 proteasome inhibitors prior to harvesting. The underline indicates which protein or tag 1178 was blotted for in a particular panel (here and below). . Input equal to 1% of IP, here and 1179 below.

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1180 (E) coIP of HA-Cdh1 with FLAG-PCAF in transiently transfected 293T cells treated with 1181 proteasome inhibitors prior to harvesting. 1182 (F) coIP of HA-Cdh1 with FLAG-NCOA3 in transiently transfected 293T cells treated with 1183 proteasome inhibitors prior to harvesting 1184 (G) coIP of HA-Cdh1 with FLAG-TTF2 in transiently transfected 293T cells treated with 1185 proteasome inhibitors prior to harvesting. 1186 (H) Mitotic shake-off of synchronized U2OS cells collected after release at the indicated 1187 timepoints. Immunoblotting for select endogenous proteins that are putative APC/C 1188 substrates or the positive control Cyclin B. 1189 1190 Fig. 3. UHRF1 levels are controlled by APC/CCdh1. 1191 (A) HeLa S3 cells were synchronized in mitosis and released into the cell cycle. Timepoints 1192 were taken at the indicated time points and analyzed by immunoblot. 1193 (B) U2OS cells were synchronized in prometaphase with 250ng/mL nocodazole for 16hr 1194 prior to mitotic shake-off. Cells were released into fresh media containing 10µM RO- 1195 3306 CDK inhibitor (used as described in Fig. 2C) with or without addition of 20µM of 1196 proteasomal inhibitor MG-132 and harvested 1hr later. Cyclin B is a positive control for 1197 a known APC/C substrate that is degraded at mitotic exit. 1198 (C) HCT116 cells were transfected with siRNA targeting Cdh1 (Fzr1 mRNA) or firefly 1199 luciferase as a control and harvested after 24 hr for immunoblotting. 1200 (D) Myc-UHRF1 was transiently expressed in 293T cells with increasing concentrations of 1201 FLAG-Cdh1 for 24hr before analysis by immunoblot.

1202 1203 Fig. 4. UHRF1 binding and ubiquitylation by APC/CCdh1 depends on KEN degron.

1204 (A) Schematic of UHRF1 domain structure with location of KEN degron in both full-length 1205 (FL) and truncated LPS UHRF1. 1206 (B) coIP of HA-Cdh1 with Myc-UHRF1 in transiently transfected 293T cells treated with 1207 proteasome inhibitors prior to harvesting and α-Myc IP. Input equal to 1% of IP, here 1208 and below. 1209 (C) coIP of Myc-UHRF1 with HA-Cdh1 in transiently transfected 293T cells treated with 1210 proteasome inhibitors prior to harvesting and α-HA IP. 1211 (D) Ubiquitylation reactions with APC/CCdh1, UBE2C, FL UHRF1* or LPS UHRF1*, and 1212 wild-type ubiquitin. UHRF1 was detected by fluorescence scanning (* indicates 1213 fluorescently labeled protein). 1214 (E) Ubiquitylation reactions similar as in (D) but using two variants of APC/C: WT and 1215 catalytically dead APC/C∆RING∆WHB, a version of APC/C that can neither recruit nor

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1216 activate its E2, UBE2C. UHRF1 was detected by fluorescence scanning. Samples were 1217 collected at 30 min. 1218 (F) Representative in vitro ubiquitylation reactions showing UBE2S-dependent chain 1219 elongation reactions of LPS UHRF1*. Titration of UBE2S: 0 µM, 0.1 µM (+), 0.5 µM 1220 (++). The addition of Emi1 completely inhibited the reaction. UHRF1 was detected by 1221 fluorescence scanning. Samples were collected at 30 min. 1222 (G) coIP of HA-Cdh1 with Myc-UHRF1WT or Myc-UHRF1KEN:AAA in transiently transfected 1223 293T cells treated with proteasome inhibitors prior to harvesting and α-Myc IP. 1224 (H) Polyubiquitylation reactions of FL-UHRF1* and LPS-UHRF1* by APC/CCdh1, UBE2C, 1225 and UBE2S. UHRF1 ubiquitylation by APC/CCdh1 is dependent on the KEN degron motif 1226 (lane 4 in both gels). UHRF1 was detected by fluorescence scanning. Samples were 1227 collected at 30 min. 1228 (I) Dependence of UHRF1 ubiquitylation on phosphorylation state of the APC/C (referred 1229 to as pE-APC/C) and subsequent coactivator recruitment. The well-established APC/C 1230 substrates, CycBNTD* and Securin*, are ubiquitylated by either APC/CCdc20 or 1231 APC/CCdh1, whereas UHRF1 is only ubiquitylated by APC/CCdh1. Reactions were run in 1232 parallel. Collections taken at 1hr (for FL and LPS UHRF1*) and 30 min (for CycBNTD* 1233 and Securin*). Ubiquitylated proteins were detected by fluorescence scanning. 1234 1235 Fig. 5. UHRF1 non-degradable mutant protein is stable at mitotic exit. 1236 (A) Myc-UHRF1WT or mutant versions harboring alanine substitutions in either its KEN-box 1237 (KEN) or the fourth putative D-box motif (D4) (see Fig 4A for location of sequences) 1238 were transiently expressed in 293T cells with or without FLAG-Cdh1 for 24hr before 1239 analysis by immunoblot. 1240 (B) HeLa S3 stably expressing GFP-UHRF1WT or GFP-UHRF1KEN:AAA were synchronized 1241 in mitosis, released into the cell cycle, and collected for immunoblot analysis at the 1242 indicated timepoints. 1243 1244 Fig. 6. UHRF1 degradation restrains S-phase entry. 1245 (A) HeLa S3 stably expressing GFP-UHRF1WT or GFP-UHRF1KEN:AAA along with 3’UTR 1246 targeting shUHRF1 were synchronized in mitosis as described previously, released into 1247 the cell cycle, and collected for immunoblot analysis at the indicated timepoints, probing 1248 for cell cycle proteins as shown. 1249 (B) HeLa S3 stably expressing GFP-UHRF1WT or GFP-UHRF1KEN:AAA along with 3’UTR 1250 targeting shUHRF1 were synchronized in mitosis, released into the cell cycle, and pulsed 1251 with 10µM EdU for thirty minutes prior to harvest and analysis by flow cytometry. A 1252 representative experiment (n=3) is shown. 1253 Fig. 7. A non-degradable form of UHRF1 induces DNA hypermethylation of gene bodies and 1254 early replicating regions of the genome. 1255 (A) Global DNA methylation analysis for Parental U2OS and U2OS cells overexpressing 1256 GFP-UHRF1WT or GFP-UHRF1KEN:AAA with the Infinium MethylationEPIC BeadChip

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1257 (Illumina) platform. Each sample group is represented in biological triplicate. All CpG 1258 probes that passed quality control analysis (n = 724,622 CpGs) are plotted as β-values 1259 population averages from 0 (fully unmethylated) to 1 (fully methylated). The midlines of 1260 each box plot represent the median DNA methylation value for all CpG probes in a 1261 sample. 1262 (B) Multidimensional scaling (MDS) of the top 50,000 variable CpG probes among samples. 1263 (C) Number of CpG probes that were differentially hypermethylated or hypomethylated in 1264 the GFP-UHRF1WT and GFP-UHRF1KEN:AAA groups relative to the Parental samples 1265 adjusted p-value 0.05).

1266 (D) Overlap analysis ≤of significantly hypermethylated (left) or hypomethylated (right) CpG 1267 probes between GFP-UHRF1WT and GFP-UHRF1KEN:AAA sample groups. 1268 (E) DNA methylation levels of significantly hypermethylated (left) or hypomethylated 1269 (right) probes from (D) that are common between GFP-UHRF1WT and GFP- 1270 UHRF1KEN:AAA sample groups, unique to GFP-UHRF1KEN:AAA (KEN only), or unique to 1271 GFP-UHRF1WT (WT only). Color code from Fig. 7A applies. Outliers removed to 1272 simplify visualization. 1273 (F) Enrichment bias analysis of significantly hypermethylated (left) or hypomethylated 1274 (right) CpG probes among genomic annotations and U2OS replication timing data. *p- 1275 value 1E-300 for positive enrichment of the feature by hypergeometric testing. 1276 ≤ 1277

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1278 Supplemental Figure Legends 1279 1280 Fig. S1. Analysis of putative APC/C substrates. 1281 (A) U2OS cells were arrested in mitosis with nocodazole, collected by shake-off, treated with 1282 the CDK1 inhibitor RO-3306, and harvested for immunoblot at the indicated timepoints. 1283 Cyclin B and NUSAP1 serve as positive APC/C controls. 1284 (B) U2OS cells were transiently transfected with the indicated plasmids, arrested in mitosis 1285 with nocodazole, collected by shake-off, treated with the CDK1 inhibitor RO-3306, and 1286 harvested for immunoblot after 2 hr. FoxM1 serves as a positive control for APC/C 1287 activation. 1288 (C) HeLa and U2OS cells were synchronized in mitosis by nocodazole and released by 1289 mitotic shake-off. Timepoints were collected as shown and analyzed by immunoblot. 1290 FoxM1 serves as positive APC/C control that is degraded at M/G1 phases. 1291 1292 Fig. S2. TTF2 is ubiquitylated by APC/C in vitro. 1293 (A) Ubiquitylation reactions of TTF2* by UBE2C using methylated Ub or wild-type Ub 1294 (lanes 1-6) in combination with APC/CCdh1, APC/C alone, or Cdh1 alone. Ubiquitylation 1295 reactions of TTF2* by both E2s, UBE2C and UBE2S, (lanes 7-9) in combination with 1296 APC/CCdh1, APC/C alone, or Cdh1 alone. Ubiquitylation was detected by fluorescence 1297 scanning at 60 minute timepoints. 1298 1299 Fig. S3. UHRF1 protein levels are cell cycle regulated and sensitive to APC/C inhibition with 1300 the small-molecule inhibitor proTAME. 1301 (A) HeLa cells were synchronized in mitosis, collected by shake-off, released into the cell 1302 cycle, and analyzed by immunoblot at the indicated timepoints. 1303 (B) U2OS cells were synchronized in mitosis, collected by shake-off, released into the cell 1304 cycle, and analyzed by immunoblot at the indicated timepoints. Line indicates samples 1305 that were run on separate gels, with appropriate corresponding loading controls for each 1306 gel. 1307 (C) HCT116 and U2OS cells were released into G1 from a mitotic block for 1.5hr and then 1308 were subsequently treated with proTAME for 1.5 hr. Endogenous UHRF1 and Cdh1 were 1309 analyzed by immunoblot. 1310 1311 Fig. S4. UHRF1 ubiquitylation by APC/C. 1312 (A) Ubiquitylation reactions of FL-UHRF1* by UBE2C with either methylated Ub or wild- 1313 type Ub. Reactions were performed using UHRF1WT or a variant harboring alanine 1314 substitution in the KEN-box (KEN:AAA ). KEN degron motif mutants in UHRF1 are 1315 shown in lanes 4 and 8. Ubiquitylation was detected by fluorescence scanning at 30 1316 minute timepoints.

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1317 (B) Ubiquitylation reactions of LPS-UHRF1* by UBE2C with either methylated Ub or 1318 wild-type Ub. Reactions were performed using UHRF1WT or a variant harboring 1319 alanine substitution in the KEN-box (KEN:AAA ). KEN degron motif mutants in 1320 UHRF1 are shown in lanes 4 and 8. Ubiquitylation was detected by fluorescence 1321 scanning at 30 minute timepoints. 1322 (C) Ubiquitylation reactions of FL-UHRF1* and LPS-UHRF1* are exclusive to Cdh1 as 1323 the coactivator. Ubiquitylation reactions were performed using wild-type APC/CCdh1 1324 which can only utilize Cdh1, but not Cdc20, as well as pE-APC/CCdh1, which mimics 1325 the APC/C phosphorylated state and can therefore use either Cdc20 or Cdh1. In 1326 parallel, we analyzed ubiquitylation of CycBNTD* and Securin*, which can be 1327 ubiquitylated by both APC/CCdc20 and APC/CCdh1. 1328 1329 Fig. S5. UHRF1 depletion impairs chromosome alignment. 1330 (A) HCT116 cells were depleted of UHRF1 using two independent siRNA 1331 oligonucleotides. Cells were fixed and stained with antibodies to the kinetochore protein 1332 CENP-C and microtubules. 1333 1334 Fig. S6. Progression through S/G2 phases in cells expressing non-degradable UHRF1. 1335 (A) HeLa S3 cells stably expressing GFP-UHRF1WT or GFP-UHRF1KEN:AAA were 1336 synchronized at G1/S by double thymidine block, released in the cell cycle, and analyzed 1337 by immunoblot at the indicated time points. Cells progressed through S/G2 phases with 1338 minimal differences except for an increase in cyclin E levels. 1339 (B) Asynchronous RPE-1 cells stably expressing GFP-UHRF1WT or GFP-UHRF1KEN:AAA 1340 were harvested for immunoblotting for cell cycle markers as shown. 1341 (C) Asynchronous HeLa S3 cells stably expressing GFP-UHRF1WT or GFP-UHRF1KEN:AAA 1342 along with 3’UTR targeting shUHRF1 were harvested for immunoblotting for cell 1343 cycle markers as shown. 1344 1345 Fig. S7. A 145 gene signature derived from KEN-containing proteins which have cell cycle 1346 dependent gene transcription is associated with makers of chromosome instability in breast 1347 cancer. 1348 1349 (A) TCGA BRCA samples (n=1201) were assigned to High or Low based on the ranked 1350 median value of the 145 gene signature score. Samples were then plotted for the given 1351 genomic feature based on Thorsson et. al. by both gene signature group and PAM50 1352 subtype. Significant was determined by t-test or ANOVA where appropriate. The median 1353 145 gene signature score was plotted against the chromosome instability score (CIN70) 1354 (r2=0.72, Pearson correlation p<0.001). Colors indicate PAM50 subtypes. 1355

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A. B. KEN-box containing Enrichment bona fide KEN substrates (2206) 150- Cell cycle regulated (CCR) genes (651) 100- KEN only 50- CCR Fold enrichment KEN + CCR

Substrates captured vs. C. 25- other studies D. GO Biological Process

20- 15-

15- 10-

10- 5- Fold enrichment Fold enrichment

# KEN substrates 5-

Min 2014

Merbl 2013 Merbl 2009 Singh 2014 CCR + KEN Spindle org. Huttlin 2017 Cdh1 IP-MS Bakos 2018 Cell division (this study) (this study)

Lafranchi 2014 G2/M transition DNA replication Mitotic cell cycle E. Chromosome seg.

Cytoskeleton and motors Centromere-Kinetochore APC/C and spindle checkpoint CKAP2 KIF18A AURORA B CEP350 KIF20B c14orf106 (KNL2) BUB1 CKAP5 (TOG) KIF22 (KID) CENP-E Cohesion and BUBR1 BUB3 DLG7 (HURP) NDE1 CENP-F condensation GTSE1 NUSAP1 CENP-U CDC20 SMC4L (SMC4) CDC27 HAUS5 ODF2 (Cenexin) CDCA2 (REPO-MAN) IDN3 (NIPBL) HMMR SIL SGO2 EMI1 KIF5B SNK/PLK2 SECURIN KIF11 (EG5) TACC3 SEPARASE KIF15 TPX2 Cytokinesis Mitotic entry ARHGAP19 CEP1 MASTL (Greatwall) ECT2 MELK KIF4A CDC25A MPHOSPH1 (KIF20B) CDC25B M KIF23 (MKLP1) CDC25C G2 G1 MID1 NEK2 PRC1 CDCA3 (TOME1) S DNA replication Cell cycle transcription ATAD5 MCM4 CASP8A2 (FLASH) CDC6 MCM10 CDR2 CDC45 POLD3 E2F7 CDT2 PAF15 E2F8 RFC4 NPAT DBF4 (ASK) GEMININ RRM2 FOXM1 MCM2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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. Figure 2 Chromatin readers and writers DNA damage repair A. BRCA1 NEIL3 Chromatin chaperone AURORA B MLL2 (KMT2D) CLASPIN TOPBP1 and organization NCOA3 (KAT13B) DCLRE1C (ARTEMIS) POLQ CHAF1B (CAF-1) PCAF (KAT2B) ELG1 RBBP8 (CtIP) NASP UHRF1 EXO1 USP1 HELLS MSH2 WDHD1 (AND1) MKI67 (KI-67) TLK2 RNA regulation and processing TOP1 CLK4 H3 H4 TOP2A DZIP3 TTF2 LARP1 ZBTB11 HDCMA18P (LARP7) H2A PCF11 H2B PNN RNPS1

KEN + CCR: GO Biological Process B. 10- C. RO-3306 Roscovitine 8- 0 1 2 0 1 2 Time (hours)

6- CHAF1B

4- PCAF (KAT2B) Fold enrichment 2- FoxM1

Cyclin B

DNA repair GAPDH

DNA packaging

Chromosome org. DNA conformation change Protein-DNA cmplex D. DNA metabolic process E. F. Input HA IP Input HA IP Input HA IP Myc-CHAF1B FLAG-PCAF FLAG-NCOA3 HA-Cdh1 HA-Cdh1 HA-Cdh1 Myc-CHAF1B FLAG-PCAF NCOA3 (long exp.) (long exp.) (long exp.)

HA-Cdh1 HA-Cdh1 HA-Cdh1

GAPDH GAPDH GAPDH

Mitotic block and release G. Input HA IP H. FLAG-TTF2 AS 0 1 2 3 4 6 8 10 12 14 16 Time (hours) HA-Cdh1 CHAF1B

TTF2 TTF2 HA-Cdh1 NCOA3 GAPDH Cyclin B

GAPDH bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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. FigureAS 03 2 3 4 5 6 7 8 10 12 14 16

APC/C Activation Assay U2OS 250ng/uL NBR, 16hr then add Cdk1i for 1hr -/+MG132 HeLaS3 APC/C Activation Assay U2OS 250ng/uL NBR, 16hr then add Cdk1i for 1hr -/+MG132 A.T.B. CC Endogenous Regulation B. T.B. CC EndogenousMitotic blockRegulation and release AS 0 2 3 4 5 6 7 8 10 12 14 16 APC/C Activation Assay U2OS 250ng/uL NBR, 16hr then add Cdk1i for 1hr -/+MG132 AS 0 2 3 4 5 6 7 8 10 12 14 16 Time (hours) MG132 T.B. ASCC Endogenous0 2 3 4 Regulation 5 6 7 8 10 12 14 16 CCNE1 RO-3306 (CDK1 inh.) UHRF1 AS 0 2 3 4 5 6 7 8 10 12 14 16 1110 Time with drug (hr) CCNE1 Cdh1 UHRF1 CCNE1 Cyclin E Cyclin B GAPDH T.B. CC Endogenous Regulation CyclinGAPDH A GAPDH AS 0 2 3 4 5 6 7 8 10 12 14 16 CyclinGAPDH F CCNE1 pH3-S10 CCNE1 GAPDH CCNE1

C. D. Myc-UHRF1 FLAG-Cdh1

UHRF1 siFF siFZR1 UHRF1 Myc-UHRF1 CCNE1UHRF1 UHRF1 Cdh1 UHRF1 Cdh1 Cdh1 Cdh1 FLAG-Cdh1 HRP-Flag Tubulin Tubulin Tubulin Tubulin GAPDH Order:siFF vs siFZR1 (+Vec) and siFF vs siFZR1 (Myc-UHRF1) Order:siFF vs siFZR1 (+Vec) and siFF vs siFZR1 (Myc-UHRF1) Order:siFF vs siFZR1 (+Vec) and siFF vs siFZR1 (Myc-UHRF1) bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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. Figure 4 D-Box D-Box A. D-Box D-Box KEN-Box

Full length (FL) 1 793 UBL TTD PHD SRA PBR RING Myc PD Myc PD Linker-PHD-SRAMyc PD (LPS) 287 715 Myc B. Myc C. 1% Input Myc IP Myc 1% Input HA IP Myc (short exp) WT - - Myc-UHRF1Myc (shortWT exp) -- Myc-UHRF1 Myc (short exp) HA-Cdh1 HA-Cdh1 HA (short exp) HA-Cdh1HA (short (short exp) exp.) Myc-UHRF1 HA (short exp) Myc PD HA HA-Cdh1HA (long exp.) HA HA-Cdh1 Myc-UHRF1Myc GAPDH GAPDH GAPDH GAPDHGAPDHMyc (short exp)

HA (short exp) D. FL UHRF1* LPS UHRF1* E. FL-UHRF1* LPS-UHRF1* APC/C HA WT-APC/C ΔRINGΔWHB-APC/C Cdh1 GAPDH Cdh1

5 30 5 30 5 30 5 30 5 30 5 30 Time (min)

F. LPS-UHRF1* G. 1% Input Myc IP APC/C HA-Cdh1 UBE2C Myc-UHRF1KEN:AAA UBE2S ++ + ++ ++ Myc-UHRF1WT Emi1 Myc-UHRF1

HA-Cdh1

GAPDH

H. I. NTD* * APC/C APC/C FL-UHRF1* LPS-UHRF1* CycB Securin Cdh1 Cdh1 APC/CCDH1 KEN: KEN: WT WT WT WT WT WT CDC20 FL-UHRF1* AAA LPS-UHRF1* AAA pE APC/C

UBE2C+UBE2S

UBE2C+UBE2S bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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.

Figure 5

A. EV WT D4 KEN Myc-UHRF1 FLAG-Cdh1

Myc-UHRF1

FLAG-Cdh1

GAPDH

B. GFP-UHRF1WT GFP-UHRF1KEN:AAA AS 8643210 10 12 14 16 AS 8643210 10 12 14 16 Time post mitotic release (hours)

UHRF1 (short exp.)

UHRF1 (long exp.)

Cdh1Cdh1 LE LE Cdh1

Aurora A

CCNE1CCNE1 Cyclin B

Cyclin D

CCNE1CCNE1 Cyclin E

Cyclin A

Cyclin F

GPADHCCNE1GPADHCCNE1 GAPDH bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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. Cyclin E (long) Figure 6 UHRF1 Cyclin E A. GFP-UHRF1WT GFP-UHRF1KEN:AAA B. GFP-UHRF1WT GFP-UHRF1KEN:AAA GFP-UHRF1 WT GFP-UHRF1 KEN GFP-UHRF1(shUHRF1) WT GFP-UHRF1(shUHRF1) KEN Cyclin E (long) (shUHRF1) (shUHRF1) 0 1 2 4630 1 2 463 NBR GFP-UHRF1GFP-UHRF11 2 4 WT WT 630643210 GFP-UHRF1GFP-UHRF1 1 2 4 63 KEN 643210 KENTimeNBR postCyclin mitotic E release (hours) 0.52% EdU + 0.50% EdU + 00 1 1 2 2 4630 4630 1 1 2 2 463 463 NBRNBRCyclin E UHRF1CyclinUHRF1UHRF1 (short E (long) exp.) 4hr GFP-UHRF1 WT GFP-UHRF1 KENUHRF1UHRF1UHRF1 (long exp.) 1 2 4 630 1 2 4 63 NBR Cdh1Cyclin E UHRF1 CyclinCyclinCyclinCyclin E (short E EE (long) exp.) Cyclin E 3.6% EdU + 9.64% EdU + CyclinCyclinCyclin E (long EE(long)(long) exp.) Cyclin E (long) E2F1 6hr EdU Cyclin E FoxM1 (short exp.)

FoxM1Cyclin (long E exp.(long)) 21.8% EdU + 37.6% EdU + pH3-S10Cyclin E

GAPDH 8hr

DNA Content

40- GFP-UHRF1KEN:AAA (shUHRF1) 30-

20- GFP-UHRF1WT (shUHRF1) 10- Percent EdU Pos. Cells 2 4 6 8 Time post mitotic release (hours) bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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. Figure 7

A. B. C. 60000 1.00 Parental Hypermethylation KEN:AAA GFP-UHRF1 Hypomethylation GFP-UHRF1WT 0.75 40000 pG s 0.50 0.0 0.1 0.2 #C 20000 0.25 Leading LogFC dim2 DNA methylation ( β -value) 0.00 −0.2 −0.1 0 −0.3 −0.2 −0.1 0.0 0.1 0.2 GFP-UHRF1WT GFP-UHRF1KEN:AAA Parental UHRF1 UHRF1 Leading LogFC dim1 KEN:AAA WT vs Parental

D. Hypermethylation Hypomethylation E. Hypermethylation Hypomethylation 1.00

KEN:AAA KEN:AAA GFP-UHRF1 GFP-UHRF1 28,203 CpGs 0.75 11,666 CpGs 5015 0.50 23,804 CpGs 3,490 CpGs

0.25 GFP-UHRF1WT

WT DNA methylation ( β -value) 4,532 CpGs GFP-UHRF1 21,519 CpGs 0.00 Common KEN WT Common KEN WT only only only only F. Hypermethylation Hypomethylation

non-coding Common CpGs GFP-UHRF1KEN:AAA only CpGs Intergenic * WT ** GFP-UHRF1 only CpGs TTS * 3'UTR Genomic intron * Annotation ** exon * 5'UTR

promoter-TSS

Late * Replication * * Timing Early *

0 5 0 -10 -5 10 -20 -10 10 20 Enrichment Bias (%) bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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. Figure S1

A. B. Endogenous Exogenous

RO-3306

0 1 2 Time (hours) LARP7 LARP1 NASP NCOA3 TTF2 UHRF1 FoxM1 UHRF1 - + - + - + - + - + - + - + RO-3306

NCOA3

PCAF Anti-Myc

CHAF1B

TTF2 GAPDH

MSH2

EGFP CHAF1B LARP7 LARP1 NCOA3 MSH2 WDR76 NUSAP1 - + - + - + - + - + - + - + Anti-Myc Cyclin B

GAPDH GAPDH

C. HeLa U2OS AS 0 2 5 AS 0 2 5 Time after release from mitosis (hours)

PCAF (short exp.)

PCAF (long exp.)

FoxM1

Figure S2

A. APC/C CDH1

TTF2*-

UBE2C UBE2C UBE2C+UBE2S meUB UB UB bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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. Figure S3

AS 0 2 4 6 8 10 12 14 16 18 24 Time (h) after A. Mitotic block and release B. AS 0 Mitotic 2 4 6 block 8 10 and 12 14 release 16 18 24 Time (h) after CycF AS 0 2 4 6 8 10 12 14 16 18 24 30 36 Time (hours) AS 00 22 4 4 6 6 8 8 10 12 1414 16 16 18 18 24 24 TimeTime (hours) (h) after CycF UHRF1 AS 0 2 4 6 8 10 12 14 16 18 24 UHRF1Time (h) after Fzr1CycF Cdh1 GAPDHFzr1CycF

AS 0 2 4 6 8 10 12 14 16 18 24 Time (h) after Cyclin F Cdh1CycAFzr1 Fzr1CycA CyclinCycF F Cyclin A CycBCycA CycB CyclinCycA A pH3-S10 Fzr1p-HistoneH3 U2OS pH3-S10p-HistoneH3CycB U2OS CycB GAPDH GAPDHGAPDH p-HistoneH3 CycAGAPDH U2OS p-HistoneH3 HeLa U2OS U2OS GAPDH CycB GAPDH

C. HCT116 U2OS p-HistoneH3 proTAME U2OS GAPDH UHRF1 (short exp.)

UHRF1 (long exp.)

Cdh1

Figure S4

A. B. APC/C APC/C Cdh1 Cdh1 KEN/ KEN/ KEN/ KEN/ WT WT WT WT WT WT WT WT WT WT WT WT FL-UHRF1* AAA AAA LPS-UHRF1* AAA AAA

meUB UB UBE2C UBE2C

meUB UB UBE2C UBE2C C. pE APC/C WT APC/C

Cdh1 Cdc20

FL UHRF1*-

LPS UHRF1*-

CycBNTD*-

Sec*- E2: UBE2C bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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. Figure S5

A. siFF Control siUHRF1 Metaphase Anaphase Metaphase Anaphase DNA / KT MT

Note: KT = kinetochore labbeled by CENP-C

5 % of Mitotic Defects

Control siUHRF1 siUHRF1 (siRNA1) (siRNA2) bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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. S3 UHRF1 WT vs. UHRF1 KEN 0-12hr DTBR FigureS3 UHRF1 S6 WT vs. UHRF1 KEN 0-12hr DTBR S3S3 UHRF1 UHRF1 WT WT vs. vs. UHRF1 UHRF1 KEN KEN 0-12hr 0-12hr DTBR DTBR S3 UHRF1 WT vs. UHRF1WT KEN 0-12hr DTBRKEN:AAA A. GFP-UHRF1 GFP-UHRF1 p21 AS 121086420 AS 121086420 Time post double thymidine release (hours) pH3 UHRF1

FoxM1 FoxM1

RPE-1pChk1 GFP WT UHRF1 vs. KEN UHRF1 (asynchronousCycin A 09-13-19) Still need Cdc6 and CCNA,CCNA could not get Rb or FoxM1 to work? E2F1 Cyclin B RPE-1p21 GFP WT UHRF1 vs.Dimensions: KEN UHRF1 5 (2nd (asynchronous tick) to 5.5 inches09-13-19) Height CCNE1 Cyclin E Still need Cdc6 3.5 and inches CCNA, to 4 could (3rd tick) not Lengthget Rb or FoxM1 to work? pH3S3 UHRF1E2F1 WT vs. UHRF1 KEN 0-12hr DTBR Skp2 Cyclin F RPE-1 GFP WT UHRF1 vs.Dimensions: KEN UHRF1 5 (2nd (asynchronous tick) to 5.5 inches 09-13-19) Height FoxM1p21CCNE1 p21 p21 p27 Still need Cdc6 3.5 and inches CCNA to 4 (3rd tick) Length p21p21E2F1 pChk1pH3p21Skp2 pChk1 RPE-1pH3 GFPp21 WT UHRF1 vs. KEN UHRF1 (asynchronous 09-13-19) pH3pH3CCNE1 pH3p27 GAPDH FoxM1 Still need Cdc6 and CCNA RPE-1FoxM1 GFPE2F1Skp2 WT UHRF1 vs. KEN UHRF1 (asynchronous 09-13-19) FoxM1FoxM1 pChk1FoxM1 p21 Cdc6 pChk1FoxM1 Still need Cdc6 and CCNA pChk1CCNE1E2F1 Cdc6 pChk1pChk1 p27 pChk1B. RPE-1, asynchronous C. HeLa S3, asynchronous RPE-1CCNE1 GFPSkp2p21 WT UHRF1 vs. KEN UHRF1 (asynchronous 09-13-19) WT KEN GFP-UHRF1Still need Cdc6 and CCNA WT KEN GFP-UHRF1 (shUHRF1) E2F1Skp2p27 UHRF1 UHRF1 RPE-1CCNE1 GFPp21 WT UHRF1 vs. KEN UHRF1 (asynchronous 09-13-19) E2F1 p27 E2F1 E2F1 p21 Still need Cdc6 and CCNA Skp2E2F1p21 Cyclin D Cyclin D RPE-1 GFP WT UHRF1 vs. KEN UHRF1 (asynchronousCdc6 09-13-19) pH3CCNE1 Cyclin E Cyclin E p27 Still need Cdc6 and CCNA E2F1 Cyclin A Cyclin A RPE-1CCNA GFP WT UHRF1 vs. KEN UHRF1 (asynchronous 09-13-19) RPE-1FoxM1 GFPSkp2p21 WT UHRF1 vs. KEN UHRF1 (asynchronous 09-13-19) RPE-1 GFP WT UHRF1 vs. KEN UHRF1 (asynchronous 09-13-19)Cyclin F CCNE1 CyclinStill F need Cdc6 and CCNA pChk1Cdc6E2F1 Still need Cdc6 and CCNA E2F1p27 Still need Cdc6 and CCNA CCNAE2F1 Cyclin B Skp2 Cyclin B CCNE1p21 CCNE1CCNE1 Cdh1 Cdc6 Cdc6 p27 Cdc6 Skp2 Cdc6 Cdc6 Skp2Skp2 Skp2 Cdc6 p21 p27 Skp2 p27 p27 p27 Cdc6 p27 p21 p21 p21p21 p21 pRb FoxM1 GAPDH Cdc6 GAPDH bioRxiv preprint doi: https://doi.org/10.1101/2020.04.09.033621; this version posted April 10, 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.

Figure S7 A. Copy Number Burden Copy Number Burden (Number of Segments) (Fraction of Genome Altered)

p = 4.88e−30 p = 1.98e−35 400 500 600 value value 300 100 200 0.0 0.2 0.4 0.6 0.8 1.0 High Low High Low Aneuploidy Homologous Recombination Deficiency p = 1.82e−14 p = 4.54e−47 30 value value 0 5 10 15 20 25 0 20 40 60 80 High Low High Low Copy Number Burden Copy Number Burden (Number of Segments) (Fraction of Genome Altered) p = 7.35e−50 p = 1.52e−67 400 500 600 value value 300 100 200 0.0 0.2 0.4 0.6 0.8 1.0 Basal Her2 LumA LumB Normal Basal Her2 LumA LumB Normal

Aneuploidy Homologous Recombination Deficiency

30 p = 6.12e−29 p = 4.14e−109 value value 0 5 10 15 20 25 0 20 40 60 80 Basal Her2 LumA LumB Normal Basal Her2 LumA LumB Normal

CIN70 vs UB Signature Score Basal Her2 LumA LumB Normal 0.0 0.5 1.0 1.5 CIN70 Score

p= 4.648e−305 −1.5 −1.0 −0.5 r2= 0.7208

−2 −1 0 1 UB Signature Score