Kinome-Wide Rnai Screen Uncovers Role of Ballchen in Maintenance Of

Home , MRPS35, OCRL, PFDN4, RANBP9, RPLP0, SMC2

bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license.

1 Title: Kinome-wide RNAi screen uncovers role of Ballchen in maintenance

2 of gene activation by trithorax group in Drosophila

3 Authors: Muhammad Haider Farooq Khan1†, Jawad Akhtar1†, Zain Umer1†, Najma Shaheen1,

4 Ammad Shaukat1, Aziz Mithani1, Saima Anwar1,2, Muhammad Tariq1*

5 1Syed Babar Ali School of Science and Engineering, Lahore University of Management

6 Sciences, 54792 Lahore, Pakistan

7 2Present address: Biomedical Engineering Centre, University of Engineering and Technology

8 Lahore, KSK Campus, Lahore, Pakistan

9 †These authors contributed equally.

10 *Corresponding author: Muhammad Tariq, Email: [email protected]

11 Abstract

12 Polycomb group (PcG) and trithorax group (trxG) proteins are evolutionary conserved factors

13 that contribute to cell fate determination and maintenance of cellular identities during

14 development of multicellular organisms. The PcG behaves as repressors to maintain heritable

15 patterns of gene silencing and trxG act as anti-silencing factors by maintaining activation of

16 cell type specific genes. Genetic and molecular analysis has revealed extensive details about

17 how different PcG and trxG complexes antagonize each other to maintain cell fates, however

18 the cellular signaling components that contribute to maintenance of gene expression by

19 PcG/trxG remain elusive. Here, we report an ex vivo kinome-wide RNAi screen in

20 Drosophila aimed to identify cell signaling genes that facilitate trxG to counteract PcG

21 mediated repression. From the list of trxG candidates, Ballchen (BALL), a histone kinase,

22 known to phosphorylate histone H2A at threonine 119 (H2AT119p), was characterized as a

23 trxG regulator. The ball mutant exhibit strong genetic interaction with Polycomb (Pc) and

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24 trithorax (trx) mutants and loss of BALL also affects expressions of trxG target genes in ball

25 mutant embryos. BALL co-localizes with Trithorax on chromatin and depletion of BALL

26 results in increased H2AK118 ubiquitination, a histone mark central to PcG mediated gene

27 silencing. Moreover, analysis of genome-wide binding profile of BALL shows an overlap

28 with 85% known binding sites of TRX across the genome. Both BALL and TRX are highly

29 enriched at actively transcribed genes, which also correlate with presence of H3K4me3 and

30 H3K27ac. We propose that BALL mediated signal positively contributes to the maintenance

31 of gene activation by trxG by counteracting the repressive effect of PcG.

32 Keywords: trithorax group, Polycomb group, kinases, Ballchen, NHK-1, transcriptional

33 memory, histone modifications

34 Introduction

35 In metazoans, specialization of cell types that make up an organism is linked to cell type

36 specific gene expression patterns established during early development. In order to maintain a

37 specialized state, the particular expression profiles of genes need to be transmitted to

38 daughter cells through successive mitotic divisions in all cell lineages, the phenomenon

39 termed as transcriptional cellular memory. Maintenance of transcriptional cellular memory

40 and consequent cellular identity involves a combinatorial act of various epigenetic

41 mechanisms, such as DNA methylation (Holliday and Pugh, 1975), histone modifications

42 (Allfrey et al., 1964), non-coding RNAs (Jones et al., 1999; Matzke et al., 2001; Volpe et al.,

43 2002) and chromatin remodeling (Sudarsanam and Winston, 2000; Narlikar et al., 2002).

44 Genetic analysis in Drosophila uncovered two groups of evolutionarily conserved genes,

45 namely Polycomb group (PcG) and trithorax group (trxG), responsible for maintaining stable

46 and heritable states of gene repression and activation, respectively (Lewis, 1978; Capdevila

47 and García-Bellido, 1981). Molecular analysis revealed that proteins encoded by the PcG and

48 trxG act in large multi-protein complexes, and modify the local properties of chromatin to

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49 maintain expression patterns of their target genes. Both groups exert their functions by

50 binding to particular chromosomal elements known as PREs (Polycomb Response Elements)

51 and by interacting with the histones and transcription machinery (Kassis et al., 2017; Cavalli

52 and Heard, 2019). The PcG complexes, PRC1 and PRC2 (Polycomb Repressive Complex 1

53 and 2), are known to maintain repression by generating ubiquitination of histone H2A at

54 lysine 118 (H2AK118ub1) (Wang et al., 2004) and methylation of histone H3 at lysine 27

55 (H3K27me) (Francis et al., 2001; Cao et al., 2002; Czermin et al., 2002), respectively. In

56 contrast to PcG, trxG is quite heterogeneous and comprises of proteins that activate

57 transcription by modifying histone tails and ATP dependent chromatin remodeling (Piunti

58 and Shilatifard, 2016). One cellular function that unifies this diverse group of proteins is their

59 role in counteracting PcG mediated gene silencing.

60 The fact that trxG and PcG coexist at the chromatin regardless of gene expression states of

61 their target genes suggests that PcG and trxG not only compete but also associate with their

62 target genes as dynamic complexes (Breiling et al., 2001; Dellino et al., 2004; Klymenko and

63 Müller, 2004; Papp and Müller, 2006; Beisel et al., 2007). Although, the chromatin structure

64 and modifications appear to play a fundamental role in the maintenance of transcriptional

65 cellular memory, the signal that favors PcG or trxG to either repress or activate remains

66 elusive. It is plausible to assume that cell signaling pathways can be of prime importance due

67 to their ability to respond to intra and extracellular changes as well as their capacity to

68 influence nuclear factors involved in gene repression or activation. Cell signaling

69 components, especially the protein kinases, control a repertoire of cellular processes by

70 modifying more than two-third of cellular proteins function (Ardito et al., 2017), but both the

71 PcG and trxG complexes lack kinases. In Drosophila, FSH is the only kinase present in

72 canonical trxG members. Being an atypical kinase with no known kinase domain (Chang et

73 al., 2007), FSH too performs its known cellular functions via its bromodomain and

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74 interaction with ASH1 (Kockmann et al., 2013). Although different cellular processes linked

75 to epigenetic inheritance, such as maintenance of chromosomal architecture to transcription

76 (Stadhouders et al., 2019) are regulated by protein kinases (Nowak and Corces, 2000, 2004),

77 the role of cell signaling components in maintaining gene activation by trxG or repression by

78 PcG remains elusive.

79 Here, we report an RNA interference (RNAi) based reverse genetics screen to identify cell

80 signaling proteins that contribute to the maintenance of gene activation by trxG. An ex vivo

81 kinome-wide RNAi screen was carried out using a well-characterized reporter in Drosophila

82 cells (Umer et al., 2019). The primary RNAi screen led to the identification of 27 cell

83 signaling genes that impaired expression of reporter similar to trx and ash1, two known trxG

84 genes. The majority of the candidates were protein kinases, but regulatory subunits of kinase

85 complexes, kinase inhibitors, nucleotide kinases and a few lipid kinases, were also present in

86 the list. Remarkably, presence of FSH, the only trxG member with predicted kinase activity,

87 in the list of candidates validated functionality of the screen. From the list of candidates,

88 selected serine-threonine kinases were further confirmed in a secondary screen which

89 affected reporter system similar to the effect of TRX and ASH1 depletion.

90 Next, we performed genetic and molecular analysis of Ballchen (BALL), a histone kinase in

91 the list of candidate genes, and showed that BALL is required to maintain gene activation by

92 trxG. BALL mutant exhibit trxG like behavior by strongly suppressing extra sex comb

93 phenotype caused by Pc mutations as well as by enhancing the homeotic phenotype in trx

94 mutants. This strong genetic interaction between ball and trxG system corroborates with a

95 drastic reduction in expression of homeotic and non-homeotic targets of trxG in ball mutant

96 embryos. Importantly, reduced expression of trxG targets due to depletion of BALL also

97 correlates with enhanced levels of H2AK118ub1, a histone modification central to PcG

98 mediated gene repression (Blackledge et al., 2014, 2020; Kalb et al., 2014; Tamburri et al.,

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99 2020). Finally, the genome-wide binding sites of BALL identified in Drosophila S2 cells by

100 ChIP-seq revealed BALL occupancy at transcription start sites and it binds at 85% of known

101 TRX binding sites. Since BALL is known to phosphorylate H2A threonine 119 (H2AT119p)

102 (Aihara et al., 2004), our data supports the notion that BALL contributes to maintenance of

103 gene activation by counteracting PRC1 mediated H2AK118ub1.

104 Results

105 Kinome-wide RNAi screen discovered kinases affecting cell memory maintenance 106 To identify the role of cell signaling genes in the maintenance of gene activation by trxG, we

107 used a previously characterized cell-based reporter PBX-bxd-IDE-F.Luc (hereafter referred to

108 as PRE-F.Luc). In this reporter, the Firefly luciferase gene is under the control of Drosophila

109 Ubx (Ultrabithorax) promoter and bxd PRE along with PBX (postbithorax) and IDE

110 (Imaginal Disc Enhancer) enhancers of Ubx. The sensitivity and specificity of this reporter to

111 the changing levels of PcG and trxG are already described (Umer et al., 2019). Using this

112 reporter, we performed an ex vivo RNAi screen, covering all known and predicted kinases

113 and their associated proteins from the HD2 dsRNA library (Horn et al., 2010). Each gene was

114 knocked down in triplicates and the entire experiment was performed twice. dsRNAs against

115 known trxG members (trx, ash1) and the reporter gene (F.Luc) were used as positive

116 controls, whereas dsRNA against LacZ or GFP was used as a negative control in all plates.

117 Drosophila cells treated with dsRNAs were transfected with PRE-F.Luc reporter along with

118 actin promoter-driven Renilla luciferase (R.Luc), used as a normalization control (Figure 1A,

119 S1). Since R.Luc is driven by a constitutive promoter, it also served as a control to exclude

120 genes that may affect general transcription of genes. After five days of transfection, the

121 activity of both F.Luc and R.Luc was determined and Z-scores were calculated. Based on the

122 Z-scores obtained from positive controls (trx, ash1), cut-offs were defined and a list of

123 potential trxG regulators was generated (Table 1). Of 400 genes that were screened, 27

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124 specifically resulted in reduced PRE-F.Luc expression, an effect similar to the knockdown of

125 trx and ash1. The only trxG member with predicted kinase activity, fs(1)h was also found

126 among top hits of the screen. Moreover, proteins having known genetic and molecular

127 interactions with trxG also appeared in the list of candidates. For instance, human CDK1, a

128 candidate gene in our list, is known to phosphorylate EZH2, enzymatic subunit of PRC2, at

129 threonine 487 and as a consequence decreases its methyltransferase activity in mesenchymal

130 stem cells (Wei et al., 2011). In addition to protein kinases, lipid kinases, nucleotide kinases,

131 kinase inhibitors and regulatory subunits of kinase complexes were also present in the list of

132 candidates. Gene ontological analysis using STRING database revealed that most candidates

133 reside in nucleus and many interact with each other in cell cycle regulation (Figure 1B). For

134 example, CDK2 along with associated Cyclin E are in cell cycle regulators cluster that also

135 physically interact with trxG proteins, Brahma (BRM) and Moira (MOR), the core proteins of

136 Brahma associated protein complex (Brumby et al., 2002).

137 Next, we selected serine/threonine protein kinases from the list of candidates and performed a

138 secondary screen using PRE-F.Luc (Figure 1C). Cells treated with dsRNA against trx and

139 ash1 were used as positive controls while cells treated with dsRNA against LacZ served as a

140 negative control. Depletion of nine of thirteen selected kinases in secondary screen resulted in

141 significantly decreased activity of F.Luc. Since dsRNA amplicons used to knockdown target

142 genes in the secondary screen were different from the ones used in the primary screen, it

143 further validated our results from the primary screen. Finally, we chose ballchen (ball) from

144 the list of candidate genes to explore its genetic and molecular link with trxG because it is a

145 known histone kinase which phosphorylates histone H2AT119 (Aihara et al., 2004).

146 Depletion of ball drastically reduced expression of the reporter gene, relative F.Luc activity,

147 when compared with other candidates in the secondary screen. Moreover, it is also known to

148 be involved in both cell cycle (Fiona Cullen et al., 2005) and signal transduction pathways

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149 (Herzig et al., 2014; Yakulov et al., 2014), making it the most suitable representative gene of

150 the candidates list.

151 ball exhibits trxG like behavior 152 To investigate whether ball genetically interacts with the PcG/trxG system, ball mutant flies

153 were crossed with two different mutant alleles of Pc (Pc1, PcXL5). Pc heterozygous mutants

154 display a strong extra sex comb phenotype in males. The ball mutant (ball2) strongly

155 suppressed this extra sex comb phenotype (Figure 2A, B) which supports the role of ball as a

156 trxG-like factor controlling homeotic phenotype. Next, we examined the genetic interaction

157 of ball with trithorax (trx) by crossing ball2 with two different alleles of trx (trx1 or trxE2)

158 mutants. As compared to wild-type flies, trx heterozygous males show loss of pigmentation

159 on 5th abdominal segment referred to as A5 to A4 transformation (Figure 2C) and ball2/trx

160 trans-heterozygotes exhibit significantly more A5 to A4 transformations (Figure 2C) as

161 compared to trx heterozygotes indicating strong enhancement of trx phenotype by ball. The

162 suppression of extra sex combs phenotype and enhancement of trx phenotype by ball

163 mutation indicate that ball exhibits a trxG like behavior.

164 Next, we investigated the effect of ball mutation on expression of homeotic and non-

165 homeotic targets of PcG/trxG. Since homozygous ball mutants do not develop into adults

166 (Herzig et al., 2014), we used homozygous ball embryos to analyze mRNA levels of abd-A,

167 Abd-B, and pnt through real-time PCR (Figure 2D). As compared to w1118 embryos, a

168 significant reduction in expression of abd-A, Abd-B and pnt was observed. Since Abd-B is the

169 gene responsible for pigmentation in A5 and A6 abdominal segments in males (Jeong et al.,

170 2006), its depletion in ball mutant embryos corroborates with loss of pigmentation in ball2/trx

171 trans-heterozygotes. It was further validated by immunostaining of stage 15 homozygous

172 ball2 embryos with anti-Abd-B antibody (Figure 2E-H). In wild-type embryos at this stage of

173 development, Abd-B expression progressively increases from PS10-14 (Figure 2F). However,

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174 in ball2 mutants, Abd-B expression is drastically reduced (Figure 2H) which correlates with

175 significantly diminished Abd-B mRNA levels in homozygous ball2 embryos (Figure 2D).

176 Together with genetic evidence, these results suggest that trxG require BALL for

177 maintenance of gene activation during development.

178 BALL co-localizes with TRX and inhibits PRC1 179 BALL is known to bind chromatin during mitosis, however its association with chromatin

180 during interphase is not yet established. We show that immunostaining of polytene

181 chromosomes from third instar larvae with anti-BALL antibody revealed association of

182 BALL on a number of sites on chromosomes (Figure 3A-C). Next, to investigate if BALL

183 and TRX co-localize on chromatin, UAS-ball-EGFP transgenic flies were crossed to a

184 salivary gland specific GAL4 driver line (sgs-gal4). Immunostaining of polytene

185 chromosomes from salivary glands of third instar larvae expressing ball-EGFP revealed a

186 substantial but partial overlap between BALL and TRX (Figure 3D-F). Besides co-localizing

187 with TRX, BALL was seen to associate with couple of hundred sites across the polytene

188 chromosomes (Figure 3B, D). Chromatin association of BALL at TRX binding sites was

189 validated by ChIP analysis from stable cells expressing the ball coding sequence fused with

190 FLAG epitope under copper inducible promoter (Figure 3G). BALL was found at the

191 transcription start sites (TSS) of pipsqueak (psq), pannier (pnr), pointed (pnt) and

192 disconnected (disco) which are known binding sites of PcG/trxG. Moreover, an association of

193 BALL was also observed at iab-7PRE, bxd and Dfd regulatory regions of homeotic genes

194 (Figure 3G). In contrast, ChIP from empty vector control cells using anti-FLAG antibody

195 resulted in negligible enrichment at all the regions analyzed. Additionally, chromatin

196 association of BALL on TRX binding sites was also analyzed with antibody against

197 endogenous BALL using wild-type Drosophila S2 cells. A stronger enrichment of BALL, but

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198 with a similar pattern, was seen on all the non-homeotic and homeotic targets analyzed

199 (Figure 3G).

200 Since BALL is known to phosphorylate histone H2AT119, a residue adjacent to H2AK118,

201 we questioned whether the H2AT119ph mark is inhibitory for H2AK118ub1, an established

202 hallmark of PcG mediated gene repression (Blackledge et al., 2014, 2020; Kalb et al., 2014;

203 Tamburri et al., 2020). To this end, we performed ChIP from cells where ball was depleted

204 using dsRNA against ball. The BALL depleted cells exhibited an increased H2AK118ub1 at

205 both transcriptionally active (pnt, pnr) and repressed (bxd, Dfd) targets of trxG as compared

206 to cells treated with dsRNA against LacZ which served as a control (Figure 3H). Depletion of

207 ball and the efficiency of knockdown was confirmed through Western blotting using anti-

208 BALL antibody (Figure 3H). As the increase in H2AK118ub1 should also reflect on

209 expression levels of the target genes, we analyzed transcriptionally active genes (pnt and pnr)

210 mRNA levels through qRT-PCR in BALL depleted cells. The depletion of BALL indeed

211 resulted in decreased expression of pnt and pnr compared to cells treated with dsRNA against

212 LacZ (Figure 3I). These results suggest that BALL is required by trxG to maintain gene

213 activation and it may counteract PcG by inhibiting H2AK118ub1, a histone modification

214 catalyzed by PRC1 subunit dRING, and in turn shift the balance in favor of trxG.

215 Genome-wide binding profile of BALL correlates with TRX and gene activation

216 The association of BALL with chromatin and its co-localization with TRX on polytene

217 chromosomes intrigued us to determine genome-wide binding sites of BALL. For this

218 purpose, ChIP with antibody against endogenous BALL was performed using formaldehyde

219 fixed chromatin from Drosophila S2 cells and purified DNA was sequenced using BGISEQ-

220 500. BALL showed very similar binding profile to that of TRX as it was found to associate

221 with 85% known binding sites of TRX across the genome (Figure 4A), which further

222 signifies the involvement of BALL in transcriptional cellular memory governed by PcG/trxG

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223 system. Further analysis of ChIP-seq data revealed that BALL is present within ±1kb of TSSs

224 (Transcription Start Sites) of genes (Figure 4A). BALL binds to total 6195 sites, details of

225 which are given in Figure S2 and Table S1. Besides an overwhelming overlap between

226 BALL and TRX binding sites, BALL association with chromatin also correlates with

227 H3K4me3 and H3K27ac marks linked to gene activation (Figure 4B, C). Since expression of

228 pnt and pnr genes is reduced after depletion of BALL or TRX, ChIP-seq data was specifically

229 analyzed for association of BALL and TRX across genomic regions of pnt and pnr. High

230 levels of both the BALL and TRX at the pnt and pnr genomic regions coincide with high

231 levels of H3K27ac and H3K4me3 and little to no PC is present at these sites (Figure 4B).

232 Additionally, analysis of BALL binding sites across the bithorax complex (BX-C) genomic

233 region revealed that BALL binding profile mimics known TRX binding sites in BX-C. Since

234 genes within BX-C are silent in S2 cells, their repression correlates with high prevalence of

235 of PC enrichment throughout BX-C whereas both BALL and TRX are present on few

236 overlapping binding sites across the BX-C. Importantly, an actively transcribed gene,

237 CG14609, just downstream of Ubx in BX-C show highly enriched BALL and TRX

238 association that correlates with both H3K4me3 and H3K27ac. All these results indicate a

239 close association between BALL and TRX which suggests an important role for BALL in

240 maintenance of gene activation governed by trxG.

241 Discussion

242 Our results demonstrate success of reverse genetics approach to identify cell signaling genes

243 that impact trxG mediated gene activation. With the help of a robust cell based reporter, we

244 identified 27 genes from the kinome-wide RNAi screen in Drosophila. This list of candidate

245 genes includes cell signaling kinases and associated genes which were finalized using a

246 stringent criterion based on the Z-scores of trx and ash1 knockdown. Presence of fs(1)h, the

247 only trxG member with predicted kinase activity, among the candidate genes validated the

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248 robustness of our screen in identifying kinases regulating trxG dependent gene activation.

249 FSH protein facilitates transcription by recognizing acetylated histone marks through its

250 bromodomain and interaction with ASH1 (Kockmann et al., 2013). Other interactors of trxG

251 including skittles (sktl), Cyclin E (CycE) and Cdk2 were also present in the list of candidate

252 genes. SKTL, a nuclear phosphatidylinositol 4- phosphate 5-kinase, is known to interact with

253 trxG member ASH2 (Cheng and Shearn, 2004) and its catalytic product, phosphoinositol-4,5-

254 bisphosphate (PIP4,5), also stabilizes mammalian Brahma associated factors (BAF) complex

255 on the chromatin (Zhao et al., 1998). CycE and its associated kinase CDK2 are also known to

256 interact with BRM and MOR, two known members of trxG, which form core of chromatin

257 remodeling Brahma associated protein (BAP) complex (Brumby et al., 2002).

258 Protein kinases can also affect epigenetic cellular memory by causing phosphorylation of a

259 repertoire of epigenetic regulators including PcG and trxG proteins. For instance, CDK1, a

260 candidate gene in our list is reported to phosphorylate several proteins involved in histone

261 modifications and transcription regulation. For example, MLL2, LSD1, G9a, SUV39H2,

262 SETD2, DOT1L, p300, KDM2A, HDAC6 are some important histone modifiers included in

263 the list of CDK1 substrates among many others. Additionally, DNA maintenance

264 methyltransferase (DNMT1) as well as Tet1 and Tet2 proteins that reverse DNA methylation

265 are also phosphorylated by CDK1 (Michowski et al., 2020). Cell cycle regulation is a

266 unifying feature of many of our candidates as suggested by the STRING analysis (Figure

267 1B). Such an involvement of cell cycle regulators in maintenance of epigenetic cellular

268 memory corroborates with the gradual decrease in PRC2 mediated H3K27me3 levels with

269 each round of cell division in flies (Laprell et al., 2017). Similarly differential recruitment of

270 PRC2 subunits to cell lineage-specific promoters is reported across cell cycle in mouse

271 embryonic stem cells (mESCs) (Asenjo et al., 2020). Identification of cell cycle regulators in

272 our screen supports the notion that PcG/trxG system is regulated in a cell cycle dependent

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273 manner (Laprell et al., 2017; Asenjo et al., 2020), which may play a role in faithful

274 inheritance of epigenetic states.

275 Genetic and molecular characterization of BALL, one of the candidate genes from the

276 kinome-wide RNAi screen, described here has revealed novel role for BALL in

277 transcriptional cellular memory. Although, BALL is a known histone kinase that modifies

278 histone H2AT119 (Aihara et al., 2004), it was not associated with the maintenance of gene

279 activation by trxG prior to our kinome-wide RNAi screen. Our results revealed a strong

280 genetic interaction of ball mutant with Pc and trx mutations. We demonstrated that ball

281 mutant exhibit trxG like behavior as it strongly suppresses extra sex comb phenotype of Pc

282 mutants. Moreover, mutation in ball enhances trx mutant phenotype, which corroborates with

283 strong suppression of extra sex comb phenotype. This trxG like behavior of ball is

284 substantiated by the fact that expression of trxG target genes is reduced in ball homozygous

285 mutant embryos. Presence of BALL together with TRX at chromatin suggests that trxG

286 requires BALL for maintenance of gene activation. Since BALL phosphorylates histone

287 H2AT119, a residue adjacent to H2AK118 which is ubiquitinated by dRING in PRC1, it is

288 plausible to assume that BALL mediated phosphorylation may counteract H2AK118ub1 by

289 PRC1 and facilitate trxG. Additionally, enhanced levels of H2AK118ub1 were found at trxG

290 targets when function of BALL was compromised in cells which corroborates with

291 downregulation of trxG target genes. All the results presented here support a notion that

292 BALL mediated phosphorylation of H2AT119 counteracts H2AK118ub1 (Aihara et al.,

293 2004, 2016) and plays a role in gene activation. Interplay between phosphorylation of

294 specific amino acids with covalent modifications of neighboring lysine residues in histones is

295 reported to play a role in epigenetic gene regulation. For example, H3S28 phosphorylation is

296 known to counteract H3K27me3 and promote H3K27ac in maintenance of gene activation by

297 trxG (Gehani et al., 2010). Similarly, H3S10 phosphorylation is proposed to promote

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298 H3K14ac and counteract H3K9me3 and contribute to gene activation (Lo et al., 2000; Fischle

299 et al., 2003). Since genome-wide binding profile of BALL also correlates with presence of

300 H3K27ac and H3K4me3 on active genes, it is important to investigate if BALL mediated

301 phosphorylation impacts H3K27ac or H3K4me3. Notably, phosphorylated histone H3

302 threonine 3 (H3T3p), a mitosis specific mark catalyzed by Haspin kinase (Dai et al., 2005)

303 and mammalian VRK1 (homolog of BALL) (Kang et al., 2007), is reported to inhibit

304 demethylation of H3K4me3 (Su et al., 2016). Interestingly, H3K4me3 is also a known

305 boundary feature of topologically associated domains (TADs) (Dixon et al., 2012), thus such

306 interactions may explain maintenance of active TAD boundaries and epigenetic inheritance

307 across cell division. Moreover, depletion of mammalian VRK1 was shown to result in loss of

308 H3K14 acetylation and H4 acetylation (Salzano et al., 2015). Such a wide-ranging effect of

309 VRK1 on different covalent modifications of histones may well be an indirect consequence

310 of phosphorylation of chromatin binding proteins by VRK1. For example, CREB is also

311 phosphorylated by VRK1 at serine 133 (Kang et al., 2008) and this specific phosphorylation

312 is known to enhance CREB interaction with CBP involved in H3K27ac. This interplay of

313 VRK1, CREB and CBP is known to increase CREB target genes transcription

314 (Radhakrishnan et al., 1998; Dyson and Wright, 2005). Recently, VRK1 is shown to

315 positively regulate DNA damage repair by phosphorylating Tip60 (García-González et al.,

316 2020). However, discovery of mechanistic basis of BALL mediated phosphorylation in

317 maintenance of gene activation by trxG requires further scrutiny at molecular and

318 biochemical levels. Since BALL plays a role in chromosome condensation during mitosis, it

319 will be interesting to probe if phosphorylation mediated signaling events involving BALL

320 may help survival of chromatin structures and modifications involved in the maintenance of

321 transcriptional cellular memory during processes of DNA replication and cell division. Based

322 on the discovery of a predominantly large number of cell cycle associated genes in our

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323 screen, it is also interesting to scrutinize if phosphorylation could be the central epigenetic

324 mark that acts as a stable anchor for epigenetic inheritance and help restore dynamic

325 interactions between PcG and trxG proteins and their target genes after cell division.

326 Materials and methods

327 RNAi screen 328 HD2 kinome-wide sub-library was used for primary RNAi screen (Horn et al., 2010), details

329 of which can be obtained from http://rnai.dkfz.de. D.Mel-2 cells were incubated with

330 dsRNAs against all known and predicted kinases and associated proteins. In 384 well plates,

331 dsRNAs against each gene was present in triplicates and the entire experiment was performed

332 twice in primary screen. Six 384 well plates were used for each experiment and a final

333 concentration of 50ng/µl dsRNA was used in each well. The trx, ash1 and F.Luc specific

334 dsRNAs were used as positive controls whereas dsRNAs against LacZ and GFP were used as

335 negative control in each plate. 8000 cells in 30µl were dispensed per well with a multidrop

336 dispenser. The cells were spun down for 10s at 900 rpm and the plates were sealed and

337 incubated at 25oC. Next day, PRE-F.Luc and Actin-R.Luc were transfected in these cells

338 where R.Luc served as a normalization control. After 5 days of transfections, F.Luc and

339 R.Luc values were recorded and ratios of the experimental reporter (F.Luc) to the invariant

340 co-reporter (R.Luc) values were calculated to exclude possible artifacts and rule out genes

341 that may affect general transcription. Knockdown of genes that affected both F.Luc and

342 R.Luc were removed from further analysis. Relative F.Luc expressions were averaged for

343 both replicates. Instead of using 99.99% confidence values as cut-offs, more stringent cut-

344 offs were defined based on the Z-scores of trx and ash1 knockdown. In secondary screen,

345 primers for synthesizing dsRNA were chosen from DRSC library instead of HD2 library that

346 can be found at https://fgr.hms.harvard.edu/fly-cell-based-rnai. Instead of 384 well plates, 96

347 well plates were used with each well having 2µg dsRNA. 50000 cells in 100µl were

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348 dispensed in each well and same experimental procedure described above for primary screen

349 was performed.

350 Fly strains and genetic analysis 351 The following fly strains were obtained from Bloomington Drosophila Stock Center:

352 PcXL5/TM3Ser,Sb, Pc1/TM3Ser, trx1 (BN 2114), trxE2 (BN 24160). ball2 was a gift from A.

353 Herzig (Herzig et al., 2014). The strain used for gene expression analysis in embryos was:

354 P{neoFRT}82B e ball2/TM3, P{w[+mC]=ActGFP}JMR2, Ser[1] (Referred to as ball2/GFP).

355 For immunostaining of polytene chromosomes P{w+mCUASp-ball.T:Avic/EGFP = pballE}2.1

356 (gift from A. Herzig) was crossed with P{Sgs3-GAL4.PD} (BN 6870) and third instar larvae

357 from the progeny were used. For extra sex combs analysis, mutant ball2 and w1118 were

358 crossed to Pc alleles (Pc1 and PcXL5) at 25°C. Males in the progeny of these crosses were

359 scored for extra sex comb analysis as described previously (Tariq et al., 2009). ball2 and w1118

360 were also crossed to trx alleles (trx1 and trxE2) and with each other as control at 29°C. The

361 progeny of these crosses were scored for trx mutant phenotypes. For analyzing expression of

362 homeotic genes in vivo, embryos homozygous for ball2 and w1118 were used to stain with

363 antibodies as described previously (Umer et al., 2019).

364 Drosophila cell culture 365 Schneider’s Drosophila medium (Gibco, ThermoFisher Scientific), supplemented with 10%

366 fetal bovine serum (Gibco, ThermoFisher Scientific) and 1% penicillin–streptomycin (Gibco,

367 ThermoFisher Scientific) was used to culture Drosophila S2 cells. Express Five SFM (Gibco,

368 ThermoFisher Scientific) supplemented with 20mM GlutaMAX (Gibco, ThermoFisher

369 Scientific) and 1% penicillin–streptomycin was used to grow D.Mel-2 cells.

370 Generation of stable cells, RNA isolation, Chromatin immunoprecipitation 371 To generate stable cell line with inducible expression vector of FLAG tagged BALL, w1118

372 embryos were used to prepare cDNA and ball CDS was amplified. The ball CDS was first

Page 15 of 31

373 cloned in pENTR-d-TOPOTM entry vector (Thermo Fisher Scientific) followed by cloning in

374 pMTWHF destination vector from DGVC (Drosophila Gateway Vector Collection) by setting

375 up LR Clonase reaction following manufacturer’s protocol (ThermoFisher Scientific). The

376 resulting pMT-ball-FLAG vector was transfected into S2 cells using Effectene transfection

377 reagent (Qiagen) and stable cell line was generated by following the manufacturer’s

378 instructions. The procedures employed for RNA isolation followed by expression analysis

379 through qPCR as well as for ChIP and Western blotting using Drosophila cells are described

380 previously (Umer et al., 2019). Genomic localization was determined by high throughput

381 sequencing of libraries generated with BGISEQ-500. The data was filtered and the adaptor

382 sequences were trimmed using BGI software (Drmanac et al., 2010; Huang et al., 2017). The

383 filtered data was mapped to Drosophila genome (dm3 assembly) using bowtie tool of Galaxy

384 version (Langmead et al., 2009). To obtain input normalized profiles, the callpeak function of

385 MACS version 2 of Galaxy was used (Zhang et al., 2008). LogM-value profiles were

386 obtained from GEO for H3K4me3 (GSE20787), H3K27ac (GSE20779), PC (GSE104059)

387 and TRX (GSM2175519).

388 Immunostaining 389 Larvae expressing GFP tagged BALL in salivary glands were dissected to isolate the salivary

390 glands. Polytene squashes were prepared and immunostainings were performed using

391 standard protocol (Sullivan et al., 2000). Stage 15 embryos were dechorionated and GFP-

392 negative embryos were selected for immunostaining using standard protocol (Sullivan et al.,

393 2000).

394 Microscopy 395 For observing extra sex comb phenotype, Olympus SZ51 stereomicroscope was used. For

396 imaging loss of abdominal pigmentation phenotype, male flies of the desired genotype were

397 transferred to 70% ethanol to dehydrate. The dehydrated flies were dissected under Olympus

Page 16 of 31

398 SZ51 stereomicroscope on a dissection slide. The abdominal portion of the fly was isolated

399 and rehydrated in water for 5-10 minutes. After rehydration, abdomens were mounted on a

400 glass slide in Hoyer’s medium. A coverslip was placed over the specimen and was incubated

401 at 65oC for 50 minutes. The final imaging was done using Nikon C-DSS230 epifluorescent

402 stereomicroscope at 3.5× magnification. The same epifluorescent stereomicroscope was used

403 for the selection of GFP negative embryos (homozygous ball2 mutant embryos) in

404 experiments where immunostaining and real-time PCR analysis of embryos was performed.

405 Imaging of embryos was done at 20× magnification on Nikon C2 confocal microscope. NIS

406 elements image acquisition software was utilized for imaging and analysis. All larval

407 dissections for polytene chromosomes were performed using LABOMED CZM6 stereo zoom

408 microscope and Nikon C2 confocal microscope was used for imaging. Zooming to 60× was

409 used for visualization of polytene chromosomes.

410 Antibodies 411 Following antibodies were used during this study: mouse anti-Abd-B (DSHB, 1A2E9, IF:

412 1:40), rabbit anti-TRX (gift from R. Paro, IF: 1:20), rabbit anti-PC (Santa Cruz, D220, IF:

413 1:20), rabbit anti-BALL (Gift from A. Herzig, IF 1:20, ChIP: 2µl) mouse anti-GFP (Roche,

414 11814460001, IF:1:50), mouse anti-Tubulin (Abcam, ab44928, WB: 1:2000), mouse anti-

415 FLAG M2 (Sigma Aldrich, WB: 1:2000, ChIP: 5µl), mouse anti-H2A-ubi (Millipore, 05-678,

416 ChIP: 5µl). HRP conjugated secondary antibodies (Abcam) were used at 1:10,000 dilution

417 for western blotting while cy3 and Alexa Fluor 488 conjugated secondary antibodies (Thermo

418 Fisher Scientific) were used at 1:100 dilution.

419 Supplementary Materials

420 Figure S1 Schematics and data normalization of kinome-wide screen.

421 Figure S2. ChIP peak coverage of BALL across genome.

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422 Table S1. List of peaks generated using ChIPseeker and presented along with their

423 coordinates.

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617

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618 Table 1 List of candidate genes along with their respective Z-scores, annotation 619 symbols, human orthologs and a summary of their known function. General description 620 of each gene is based on information obtained from Uniprot and Flybase databases. Candidate genes Z- Annotation Summary of known functions/General Human Orthologs score Symbol Description Cyclin-dependent 16.45 CG7597 Hyperphosphorylates the C- terminal CDK12, CDK13 kinase 12 heptapeptide domain of RNA Pol-II Sphingosine kinase 1 15.27 CG1747 Converts sphingosine to sphingosine 1- SPHK1, SPHK2 phosphate CG13369 13.17 CG13369 Ribokinase RBKS female sterile (1) 10.72 CG2252 Involved in pattern formation by regulating BRD2, BRD3, homeotic homeotic genes expression BRD4, BRDT S6 Kinase Like 10.62 CG7001 Promotes proteasomal degradation of BMP RSKR receptor tkv thus inhibits BMP signaling Polo 9.75 CG12306 Control different aspects of cell division PLK1, PLK2, PLK3 abnormal wing discs 9.7 CG2210 A nucleotide diphosphate kinase, that is NME1, NME1- involved in the biosynthesis of nucleotide NME2, NME2, triphosphates NME3, NME4 Dacapo 8.49 CG1772 CDK inhibitor from the CIP/KIP family that CDKN1C inhibits the CycE-CDK2 complex crossveinless 2 8.48 CG15671 A secreted protein that can bind BMPs and their BMPER receptor tkv to either inhibit or promote BMP signaling Cyclin-dependent 8.08 CG5363 Regulates cell cycle progression by CDK1 kinase 1 phosphorylating hundreds of target proteins Death-associated 8.06 CG32666 Involved in the development of epithelial tissues STK17B, STK17A protein kinase- related Bub1-related kinase 7.93 CG7838 Important for spindle assembly checkpoint BUB1, BUB1B during the cell cycle aurora B 7.39 CG6620 Known to phosphorylate Histone H3 at serine AURKB, AURKC 10. Cell cycle-related roles include chromosome condensation, kinetochore assembly and cytokinesis Adenylyl cyclase 78C 7.37 CG10564 Catalyzes the synthesis of 3',5'-cyclic AMP from ADCY8 adenosine triphosphate in response to G-protein coupled receptor signaling Not1 7.32 CG34407 Poly(A)-specific ribonuclease that is involved in CNOT1 mRNA degradation

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Continued from previous page Salt-inducible kinase 7.17 CG4290 Important for lipid storage and energy SIK2, SIK1B, SIK1 2 homeostasis Cyclin-dependent 7.13 CG10498 Catalytic subunit of CycE-Cdk2 complex that CDK2, CDK3 kinase 2 acts during progression from G1 and S phase of mitosis Cyclin E 6.96 CG3938 A member of the cyclin group of proteins that CCNE1, CCNE2 act as a regulatory subunit of CycE-Cdk2 complex Cdk5 activator-like 6.63 CG5387 Regulatory subunit of Cdk5-Cdk5α complex that CDK5R1, CDK5R2 protein is active in neurons TBP-associated 6.55 CG17603 Binds with initiator elements at transcription TAF1, TAF1L factor 1 start sites. Part of TFIID, an evolutionary conserved multimeric protein complex involved in general transcription Downstream of raf1 6.55 CG15793 Dual specificity kinase that acts as MAPKK. It MAP2K1, MAP2K2 is activated by Raf Arginine kinase 6.51 CG32031 Belongs to the ATP: guanidino CKM, CKB, phosphotransferase family CKMT1A, CKMT2 Ballchen 6.44 CG6386 A nucleosomal histone kinase that VRK1, VRK2, phosphorylates Histone H2A at threonine 119 VRK3 Cyclin-dependent 6.32 CG5072 Essential for cell cycle progression and CDK6, CDK4 kinase 4 promotes cellular growth wishful thinking 6.28 CG10776 BMP type II receptor that regulates BMPR2, AMHR2 neurotransmission at the neuromuscular junction and synaptic homeostasis Homeodomain 6.27 CG17090 A member of the DYRK family of kinases that HIPK1, HIPK2, interacting protein contributes to several different signaling HIPK3, HIPK4 kinase pathways including Wingless, Notch, Hippo, JNK and cell death Skittles 6.15 CG9985 Converts phosphatidylinositol 4-Phosphate (PIP) PIP5K1A into phosphoinositol-4,5-bisphosphate(PIP4,5)

621 Figure captions:

622 Figure 1. Kinome-wide RNAi screen data analysis and validation. (A) Scatterplots of the

623 plate median corrected intensity values for Firefly luciferase (F.Luc) against the plate median

624 corrected intensity values for Renilla luciferase (R.Luc). To generate a list of trxG candidate

625 genes that affect gene activation, Z-scores of trx (red) and ash1 (maroon) knockdown were

Page 27 of 31

626 used to set cut-off value which is represented by a dashed line. Knockdown of trx (red) and

627 ash1 (maroon) as well as knockdown of F.Luc (blue) were used as positive controls. Cells

628 treated with dsRNA against LacZ (green) or GFP (orange) served as negative controls. Genes

629 affecting activity of both F.Luc and R.Luc were masked (gray) and were not investigated

630 further. Data shown represents two independent experiments of the kinome-wide RNAi

631 screen. (B) Protein-protein interaction network of candidate genes, generated using STRING

632 database (Snel, 2000; Szklarczyk et al., 2019). Nodes depict proteins that are connected by

633 lines of varying thickness. Thickness of lines demonstrates the degree of confidence for the

634 interaction between connected nodes. The yellow circle is marking candidates that are known

635 to associate with cell cycle regulation. (C) Knockdown of selected serine-threonine kinases

636 from list of candidate genes from primary screen resulted in significantly lower relative

637 Firefly luciferase activity when compared with cells treated with dsRNA against LacZ. Cells

638 treated with dsRNA against trx and ash1 were used as positive control. Experiment was

639 performed in triplicate and independent t-tests were done for analysis (** p ≤ 0.01 or **** p≤

640 0.0001).

641 Figure 2. ballchen mutation exhibits trxG like behavior. (A, B) Ballchen mutant flies

642 (ball2) were crossed with two different alleles of Pc (Pc1 and PcXL5), while Pc alleles (Pc1 and

643 PcXL5) crossed with w1118 flies were used as control. Heterozygous Pc/+ males, Pc1/+ (A) and

644 PcXL5/+ (B), from control crosses exhibit strong extra sex combs phenotype. In contrast, ball2

645 strongly suppressed both the Pc alleles to none or few extra sex combs in ball2/Pc1 (A) as

646 well as ball2/PcXL5 (B) flies. 200 male flies were analyzed for each cross and data shown

647 represents two independent experiments. Based on strength of phenotype, male flies were

648 categorized according to the severity of extra sex combs phenotype. These categories are: -,

649 no extra sex combs; +, 1-2 hairs on 2nd leg; ++, more than 3 hairs on 2nd leg; +++, more than

650 3 hairs on 2nd leg and 1-2 hairs on 3rd leg; ++++, strong sex combs on both 2nd and 3rd pairs of

Page 28 of 31

651 legs. (C) ball2 mutant flies were crossed to two different alleles of trx (trx1 and trxE2), and

652 ball2/trx males in F1 were scored for loss of pigmentation in A5 abdominal segment (A5 to

653 A4 transformation, marked with asterisk). The trx alleles (trx1 and trxE2) crossed with w1118

654 flies i.e. trx1/+ and trxE2/+, show A5 to A4 transformation when compared to wild type flies.

655 Strong enhancement of A5 to A4 transformation can be seen in ball2/trx double mutants when

656 compared to trx/+. Representative images for wild-type phenotype (top right) and A5 to A4

657 transformation (marked by white asterisk) are presented for trx/ball male flies (bottom right).

658 All crosses were done in triplicates and independent t-tests were performed for analyzing

659 each category (* p ≤ 0.05, ** p ≤ 0.01, *** p≤ 0.001 or **** p≤ 0.0001). (D) Significantly

660 low levels of abd-A, Abd-B and pnt expression was detected through qRT-PCR in

661 homozygous ball2 embryos when compared with w1118 embryos. Independent t-tests were

662 performed for each gene analysis (* p ≤ 0.05, ** p ≤ 0.01, *** p≤ 0.001 or **** p≤ 0.0001).

663 (E-H) Immunostaining of stage 15 embryos with anti-Abd-B is shown in w1118 (E, F) as well

664 as homozygous ball2 (G, H) embryos. As compared to w1118 (F), ball2 embryos showed

665 strongly diminished Abd-B (H) expression.

666 Figure 3. BALL co-localizes with TRX at the chromatin and antagonizes PcG. (A-C)

667 Polytene chromosomes from third instar larvae stained with DAPI (A) and anti-BALL (B)

668 antibody. (D-F) Third instar larvae expressing GFP-tagged BALL were stained with anti-GFP

669 (D) and anti-TRX (E) antibodies. Co-localization of BALL and TRX is clearly seen at several

670 loci (F, white arrows). (G, top panel) Western blot analysis of Drosophila S2 stable cells,

671 expressing FLAG-tagged BALL (ball-HF) protein only in the induced sample (I) as

672 compared to un-induced (U) cells. (G, bottom panel) Strong enrichment of BALL at trxG

673 targets was observed by ChIP using anti-FLAG antibody in Drosophila S2 cells expressing

674 FLAG-tagged BALL (BALL-HF). ChIP from empty-vector control cell line using anti-FLAG

675 antibody was used as control. ChIP from wild-type S2 cells using anti-BALL antibody also

Page 29 of 31

676 showed similar but stronger enrichment. (H, top panel) Knockdown of ball drastically

677 decreased the amount of BALL protein expressed when compared to cells treated with LacZ

678 dsRNA. (H, bottom panel) ChIP from BALL depleted cells showed enhanced H2AK118ub1

679 levels at non-homeotic (pnt, pnr) and homeotic (bxd, Dfd) targets of trxG, as compared to

680 cells treated with dsRNA against LacZ.

681 Figure 4. Genome-wide binding profile of BALL analyzed by ChIP-seq from Drosophila

682 S2 cells. (A) Venn diagram analysis showed 85% co-occupancy of BALL on known TRX

683 binding sites. Heat map illustration of BALL binding sites across genome show enrichment

684 of BALL within ± 1kb of TSSs of genes. (B) Genome browser view of BALL association

685 compared with patterns of TRX, PC, H3K27ac and H3K4me3 association at pnt and pnr

686 genes. (C) Genome browser view of ChIP-seq data showing binding profile of BALL and its

687 comparison with TRX, PC, H3K27ac and H3K4me3 association across the bithorax complex

688 (BX-C). Highly enriched BALL and TRX correlates with high levels of both H3K27ac and

689 H3K4me3 and absence of PC at CG14906 present just downstream of Ubx. In contrast, BX-C

690 genes i.e. Ubx, abd-A and Abd-B which are silent in S2 correlates with highly enriched PC all

691 across BX-C. Both BALL and TRX are present at few overlapping binding sites in BX-C.

692 Supplementary Figure legends

693 Figure S1. Schematics of ex vivo kinome-wide RNAi screen and data analysis. (A) 384-

694 well plates containing dsRNAs against different genes were loaded with equal number of

695 cells in each well. 24 hours after seeding cells, PRE-F.Luc and Actin-R.Luc were co-

696 transfected and luciferase values were determined five days later. (B) Box plots for plate

697 median normalized data for kinome-wide RNAi screen, replicate 1 (left) and replicate 2

698 (right).

699

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700 Figure S2. ChIP peak coverage of BALL across genome. ChIPseeker is utilized to

701 generate a pie chart that represent distribution of BALL across different regions of genome.

702 BALL is enriched predominantly in promoter (<=1kb) regions followed by promoter (1−2kb)

703 regions, distal intergenic regions and promoter (2−3kb) regions (Yu et al., 2015).

704 Table S1. List of peaks generated using ChIPseeker and presented along with their

705 coordinates.

706 Declarations

707 Acknowledgements: We would like to thank Michael Boutros at DKFZ, Germany for

708 providing access to all the facilities for RNAi screen and Alf Herzig for providing us

709 Ballchen flies and anti-BALL antibodies. Funding: This work is supported by Higher

710 Education Commission of Pakistan, Grant 5908/Punjab/NRPU/HEC and Lahore University

711 of Management Sciences (LUMS) Faculty Initiative Fund (FIF), Grant LUMS FIF 165.

712 Author contributions: MHFK, JA, ZU, NS, SA and MT designed research and MHFK, JA,

713 ZU, NS, AS and SA performed experiments. MHFK, JA, ZU, NS and MT wrote the

714 manuscript. AM analyzed the screen data. All authors approved the final version of the

715 manuscript. Competing interests: The authors have no competing interests. Data and

716 materials availability: All data generated and analyzed in the study are available in the main

717 or additional files provided.

Page 31 of 31 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Figure 4 A B

BALL TRX 85% PC H3K27ac H3K4me3 Genes BALL TRX pnt pnr

-1kb TSS 1Kb 0 1 2 C BALL TRX

PC H3K27ac H3K4me3

CG14906 Ubx bxd Glut3 Abd-A Iab-4 CG10349 Abd-B bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Figure S2

Promoter (<=1kb) (80.21%) Promoter (1−2kb) (5.78%) Promoter (2−3kb) (3.34%) 5' UTR (0.11%) 3' UTR (0.69%) 1st Exon (0.79%) Other Exon (0.44%) 1st Intron (1.13%) Other Intron (2.84%) Downstream (<=300) (0.66%) Distal Intergenic (4%) bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

128up AGO3 Appl asRNA:CR44093 ATPsynB 14-3-3epsilon Agpat1 Aprt asRNA:CR44106 ATPsynbeta 14-3-3zeta Agpat2 Aps asRNA:CR44114 ATPsynC 26-29-p Agpat4 apt asRNA:CR44169 ATPsynCF6 2mit agt AQP asRNA:CR44179 ATPsynD 312 Ahcy aqz asRNA:CR44375 ATPsyndelta 4E-T AhcyL1 Arc1 asRNA:CR44390 ATPsynE 5PtaseI AIMP1 Arc2 asRNA:CR44599 ATPsynF 5SrRNA:CR33452 AIMP2 Archease asRNA:CR44690 ATPsyngamma 7SLRNA:CR32864 AIMP3 Arf102F asRNA:CR44724 ATPsynO a Ak6 Arf79F asRNA:CR44873 Atu a6 Akt1 ArfGAP1 asRNA:CR44969 Atx2 AANATL3 Aladin ArfGAP3 asRNA:CR44981 Atxn7 Aasdh AlaRS Arfrp1 asRNA:CR44987 aub Aatf Alas Argk asRNA:CR45009 aux ab alc ArgRS asRNA:CR45024 awd abd-A Ald1 ArgRS-m asRNA:CR45124 Axn Abd-B Aldh ari-1 asRNA:CR45126 Axs Abl Aldh-III ari-2 asRNA:CR45131 Axud1 Abp1 Alg-2 Arl2 asRNA:CR45139 B-H1 abs Alg1 Arl4 asRNA:CR45141 B52 Ac76E Alg10 Arl5 asRNA:CR45281 bab1 Ac78C Alg11 Arl6IP1 asRNA:CR45367 bab2 Acbp1 Alg14 Arl8 asRNA:CR45371 babo Acbp5 Alg2 arm asRNA:CR45525 baf Acf Alg3 Arp1 asRNA:CR45612 Baldspot achi Alg9 Arp10 asRNA:CR45663 bam Ack Alh Arp2 asRNA:CR45802 bap Acn ALiX Arp53D asRNA:CR45806 Bap170 Acsl AlkB Arpc1 asRNA:CR45891 Bap55 Act42A Alp12 Arpc2 asRNA:CR45912 Bap60 Act57B Alp13 Arpc3B asRNA:CR45928 bark Act88F Alp4 arr asRNA:CR46050 baz Actbeta alph Ars2 asRNA:CR46058 bbc Actn alpha-Cat Art1 asRNA:CR46093 bbg Actn3 alpha-Est10 Art4 asRNA:CR46105 BBS4 Acyp alpha-Est9 Art7 AstC bbx Acyp2 alpha-Man-Ia Art8 asun bc10 Ada alpha-Man-IIa Arv1 Asx bcd Ada2a alpha-Man-IIb Asap Atac1 bchs Ada2b alphaKap4 Asator Atac2 BCL7-like AdamTS-A alphaSnap Asciz Atac3 bcn92 Adar alphaTub84B asf1 ATbp Bdbt Adat1 Alr Ask1 Ate1 bdg Adck AMPdeam asl Atet be AdenoK Amph AsnRS Atf-2 BEAF-32 Adf1 Amy-p AsnRS-m Atf6 beg Adgf-A ana3 asp Atg12 bel Adgf-A2 Ance-4 Asph Atg13 Bem46 Adi1 anchor ASPP Atg14 Best1 AdipoR Ank AspRS Atg16 Best2 Adk1 Ankle2 AspRS-m Atg17 Best4 Adk2 anne asrij Atg18a Bet1 AdoR Ant2 asRNA:CR17025 Atg2 Bet3 AdSL Antp asRNA:CR32252 Atg3 beta-Man AdSS AnxB11 asRNA:CR43629 Atg5 beta-PheRS Aduk AnxB9 asRNA:CR43916 Atg8a beta4GalNAcTA Ady43A aos asRNA:CR43957 atms beta4GalT7 Aef1 AOX3 asRNA:CR43959 Atox1 betaCOP aft AP-1sigma asRNA:CR44019 ATP6AP2 betaGlu AgmNAT Apc asRNA:CR44030 ATP7 betaTub56D ago Apc2 asRNA:CR44053 ATP8A betaTub60D AGO1 Apf asRNA:CR44069 Atpalpha Bgb AGO2 aPKC asRNA:CR44071 ATPCL bgcn bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

bgm CAHbeta Cfp1 CG10495 CG11103 BHD CalpA cg CG10508 CG11122 bi Calr CG10011 CG10512 CG11123 BI-1 Cals CG10014 CG10527 CG11125 BicD Calx CG10019 CG10543 CG11137 bif Cam CG10038 CG10550 CG11149 Bin1 CaMKI CG10051 CG10561 CG1116 bip1 CaMKII CG10068 CG10565 CG11168 bip2 cana CG10077 CG10576 CG11178 Bka CanA-14F CG10103 CG10600 CG11180 blanks CanB CG10126 CG10602 CG11200 Blos1 CanB2 CG10137 CG10616 CG11210 blot Cand1 CG10139 CG10631 CG1124 blow CAP CG10147 CG10651 CG11247 blw Cap-H2 CG10151 CG10660 CG11257 bnl Caper CG10171 CG10669 CG11263 BOD1 Capr CG10177 CG10700 CG11267 boi caps CG10183 CG10737 CG11279 bol capu CG10184 CG10738 CG1129 bond CASK CG10185 CG1074 CG11317 bor cass CG10188 CG10747 CG11333 bora Cat CG10189 CG10778 CG11342 borr cathD CG10194 CG10795 CG11357 botv Catsup CG10211 CG10802 CG11360 Br140 caup CG10214 CG10804 CG11362 brat cav CG10222 CG10809 CG11367 Brca2 Cbl CG10232 CG10853 CG11377 Brd8 Cbp80 CG1024 CG10863 CG11378 Bre1 cbs CG10254 CG10866 CG11391 brk cbt CG10264 CG10874 CG11396 bru2 CCDC53 CG10265 CG10889 CG11398 brv1 Ccdc58 CG10268 CG10898 CG11399 BRWD3 CCHa1 CG10283 CG1090 CG11403 bs Ccm3 CG10286 CG10903 CG11414 Bsg Ccp84Aa CG10324 CG10904 CG11444 Bsg25D CCT1 CG10332 CG10907 CG11453 bsk CCT2 CG10333 CG10914 CG11454 BtbVII CCT3 CG10336 CG10915 CG11474 BthD CCT4 CG10338 CG10916 CG11486 Btk29A CCT5 CG10340 CG10918 CG11504 btsz CCT6 CG10344 CG1092 CG11523 Bub1 CCT7 CG10348 CG10939 CG11537 Bub3 CCT8 CG10353 CG10947 CG11550 BubR1 CD98hc CG10357 CG10948 CG11555 Buffy Cda5 CG10366 CG10949 CG11560 BuGZ cdc14 CG10413 CG10953 CG11562 bun Cdc27 CG10417 CG10959 CG11563 bwa Cdc2rk CG10418 CG10960 CG11586 Bx Cdc37 CG10428 CG10962 CG11588 bys CDC50 CG10433 CG10973 CG11596 c11.1 Cdc7 CG10435 CG11007 CG11597 c12.1 Cdep CG10444 CG1103 CG11601 C1GalTA cdi CG10445 CG11030 CG11608 C3G Cdk2 CG10462 CG11034 CG11617 ca CDK2AP1 CG10463 CG11035 CG11629 Ca-beta Cdk4 CG10465 CG11044 CG11638 CaBP1 Cdk9 CG10466 CG1105 CG11658 cact cdm CG10467 CG11050 CG11674 Cad87A CenG1A CG10470 CG11052 CG11686 CadN Cenp-C CG10477 CG11070 CG11700 Cadps Cep135 CG10479 CG11076 CG1172 Caf1-180 Cep97 CG10481 CG11077 CG11722 CAH15 Cerk CG10492 CG11089 CG11737 CAH5 cert CG10494 CG11095 CG11753 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

CG11755 CG12213 CG13123 CG13810 CG14512 CG11777 CG12219 CG13148 CG13813 CG14544 CG11779 CG12236 CG13151 CG13827 CG14545 CG11781 CG12253 CG1316 CG13868 CG14561 CG11790 CG12299 CG1317 CG13876 CG14609 CG11791 CG12301 CG13170 CG13891 CG14621 CG11802 CG1231 CG13185 CG13893 CG14635 CG11807 CG12316 CG13192 CG13896 CG14641 CG11808 CG1233 CG13198 CG13907 CG14646 CG11811 CG12333 CG13216 CG13912 CG14650 CG11815 CG12375 CG13248 CG13917 CG14657 CG11825 CG12379 CG13252 CG13937 CG14659 CG11828 CG1239 CG13280 CG13949 CG14667 CG11837 CG12391 CG13284 CG13970 CG1468 CG11839 CG12393 CG13285 CG13982 CG14683 CG11857 CG12395 CG13306 CG14042 CG14696 CG11873 CG12402 CG13310 CG14072 CG14710 CG11882 CG12512 CG13312 CG14074 CG14712 CG11885 CG12520 CG13315 CG14082 CG14715 CG11899 CG12607 CG13319 CG14100 CG14721 CG11906 CG12608 CG13321 CG14102 CG14762 CG11920 CG12617 CG13323 CG14104 CG14764 CG11927 CG1265 CG13326 CG14117 CG14795 CG11929 CG12659 CG13366 CG14130 CG14805 CG11961 CG12702 CG13392 CG14135 CG14806 CG11964 CG12713 CG13408 CG1416 CG14851 CG11970 CG12717 CG13430 CG14184 CG14852 CG11980 CG12725 CG13445 CG14186 CG14857 CG11983 CG12728 CG13454 CG14194 CG1486 CG11986 CG12730 CG13465 CG14200 CG14860 CG12004 CG12744 CG13484 CG14207 CG14864 CG12007 CG12769 CG13501 CG14210 CG14877 CG12009 CG12772 CG13531 CG14212 CG14879 CG12010 CG12784 CG1354 CG14220 CG14883 CG12012 CG12788 CG13551 CG14223 CG14903 CG12016 CG12824 CG13577 CG14252 CG14905 CG12017 CG12836 CG1358 CG14253 CG14906 CG12025 CG12851 CG13585 CG14270 CG14907 CG12054 CG12895 CG1360 CG14273 CG1492 CG12065 CG12896 CG13601 CG14274 CG14931 CG12075 CG12909 CG13603 CG14275 CG14964 CG12081 CG12935 CG13604 CG14286 CG14965 CG12091 CG12948 CG13605 CG14302 CG14966 CG12099 CG12974 CG13609 CG14304 CG14969 CG12104 CG12975 CG13623 CG14312 CG14971 CG12107 CG12986 CG13625 CG14314 CG14982 CG12111 CG12991 CG13658 CG14322 CG14985 CG12112 CG13001 CG13663 CG14324 CG14990 CG12118 CG13005 CG13667 CG14327 CG14995 CG12121 CG13014 CG13689 CG14341 CG14997 CG12123 CG13023 CG13698 CG14401 CG15019 CG12128 CG13025 CG13699 CG14407 CG15021 CG12129 CG13045 CG13707 CG14408 CG15024 CG12134 CG13056 CG1371 CG14411 CG15025 CG12155 CG1307 CG13720 CG14414 CG15027 CG12159 CG13070 CG13737 CG14416 CG15073 CG12163 CG13077 CG13741 CG14435 CG15084 CG12164 CG13088 CG13742 CG14441 CG15093 CG12171 CG1309 CG13743 CG14442 CG15096 CG12173 CG13096 CG13751 CG14459 CG15097 CG12179 CG13097 CG13766 CG14478 CG15099 CG1218 CG13108 CG13775 CG14483 CG15107 CG12182 CG13116 CG13784 CG14502 CG15111 CG12194 CG13117 CG13786 CG14506 CG15117 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

CG1513 CG1646 CG17375 CG18858 CG2938 CG15141 CG1647 CG17486 CG18870 CG2990 CG15160 CG1648 CG1750 CG1888 CG2991 CG15168 CG1662 CG17508 CG1890 CG30001 CG15269 CG1663 CG17514 CG1902 CG30010 CG15279 CG16704 CG17528 CG1910 CG30015 CG15282 CG1671 CG17544 CG1927 CG30020 CG1529 CG16711 CG17556 CG1943 CG30022 CG15317 CG16717 CG17568 CG1951 CG30026 CG1532 CG16721 CG17574 CG1998 CG30059 CG15356 CG16734 CG17597 CG2003 CG30065 CG15386 CG1674 CG17612 CG2006 CG30069 CG15387 CG16753 CG17648 CG2016 CG30076 CG15390 CG16758 CG17650 CG2017 CG3008 CG15403 CG1677 CG17652 CG2023 CG30089 CG1542 CG16781 CG17734 CG2034 CG3009 CG15432 CG16787 CG17746 CG2059 CG30094 CG15436 CG16791 CG17751 CG2061 CG30095 CG15439 CG16812 CG17752 CG2064 CG30096 CG1544 CG16868 CG17754 CG2065 CG30103 CG15440 CG16890 CG17760 CG2076 CG30105 CG15445 CG16894 CG17765 CG2091 CG30108 CG15468 CG16898 CG17768 CG2116 CG30109 CG15473 CG16903 CG17776 CG2124 CG30116 CG15482 CG16952 CG17834 CG2126 CG30120 CG15517 CG16964 CG17841 CG2147 CG30122 CG15529 CG16986 CG1785 CG2162 CG30157 CG15531 CG16989 CG17855 CG2182 CG30159 CG15536 CG17002 CG1789 CG2186 CG30161 CG15545 CG17010 CG17931 CG2199 CG30278 CG15550 CG17019 CG17977 CG2200 CG30283 CG15554 CG1703 CG17985 CG2201 CG30286 CG15561 CG17047 CG18011 CG2202 CG30291 CG15563 CG17065 CG18081 CG2224 CG30323 CG15601 CG17068 CG18095 CG2225 CG30343 CG15611 CG17078 CG1814 CG2277 CG30349 CG15615 CG17104 CG1815 CG2310 CG3036 CG15649 CG17105 CG18178 CG2493 CG30373 CG15653 CG17107 CG18190 CG2519 CG30377 CG15695 CG17108 CG18213 CG2608 CG30379 CG15715 CG17110 CG1824 CG2614 CG3038 CG15717 CG17119 CG18259 CG2617 CG30382 CG1572 CG17124 CG18278 CG2641 CG30383 CG15742 CG17127 CG18324 CG2656 CG30389 CG15743 CG17129 CG18343 CG2685 CG30392 CG15744 CG17141 CG1840 CG2698 CG30394 CG15747 CG17145 CG18418 CG2765 CG3040 CG15765 CG17162 CG18480 CG2767 CG30423 CG15773 CG17163 CG18508 CG2794 CG30428 CG15784 CG17180 CG18528 CG2811 CG30456 CG15814 CG17186 CG18542 CG2818 CG30457 CG15824 CG17202 CG18549 CG2846 CG30460 CG15879 CG17219 CG18596 CG2852 CG30466 CG15890 CG17224 CG18600 CG2862 CG30467 CG15916 CG17230 CG18605 CG2865 CG3085 CG15930 CG17233 CG18622 CG2875 CG3097 CG1598 CG17249 CG18659 CG2901 CG31021 CG1603 CG17265 CG18661 CG2909 CG31036 CG1607 CG17266 CG18731 CG2911 CG31038 CG1622 CG17292 CG18747 CG2915 CG3104 CG1628 CG17294 CG18749 CG2918 CG31064 CG1636 CG17333 CG18809 CG2924 CG3107 CG1637 CG17337 CG18815 CG2926 CG31075 CG1640 CG17343 CG1882 CG2931 CG31098 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

CG31109 CG31869 CG3262 CG33668 CG34457 CG31122 CG31875 CG32647 CG33671 CG34458 CG31126 CG31886 CG32687 CG3368 CG34462 CG31145 CG31898 CG32695 CG33695 CG3457 CG31195 CG31908 CG32700 CG3376 CG3505 CG31213 CG31909 CG32706 CG33774 CG3520 CG31220 CG31915 CG32708 CG33775 CG3529 CG31224 CG31917 CG32736 CG33777 CG3530 CG31229 CG31922 CG32756 CG33783 CG3542 CG31249 CG31928 CG32758 CG3386 CG3552 CG31251 CG31957 CG32767 CG33926 CG3556 CG31262 CG31960 CG3277 CG33941 CG3558 CG31278 CG3198 CG32772 CG33958 CG3566 CG31279 CG3199 CG32783 CG33970 CG3603 CG31301 CG31997 CG32797 CG33977 CG3618 CG31324 CG31998 CG3281 CG33981 CG3626 CG31342 CG31999 CG32812 CG34001 CG3631 CG31344 CG32017 CG32850 CG34015 CG3632 CG31368 CG32037 CG32856 CG34038 CG3634 CG31370 CG32066 CG3294 CG34039 CG3635 CG31388 CG32071 CG3295 CG34054 CG3638 CG31436 CG32091 CG32982 CG3407 CG3645 CG31441 CG32095 CG3301 CG3408 CG3651 CG31446 CG32099 CG33017 CG34116 CG3652 CG31457 CG32104 CG33054 CG34132 CG3663 CG31460 CG32107 CG33062 CG34133 CG3678 CG3149 CG32112 CG3308 CG34136 CG3702 CG31493 CG32115 CG33080 CG34140 CG3703 CG31495 CG32137 CG3309 CG34148 CG3726 CG31510 CG32170 CG33090 CG34149 CG3732 CG31523 CG32187 CG33093 CG34159 CG3735 CG31559 CG32195 CG33095 CG34164 CG3740 CG31601 CG32196 CG33099 CG34172 CG3744 CG31612 CG32204 CG33111 CG34174 CG3748 CG31626 CG32221 CG33116 CG34183 CG3756 CG3163 CG3224 CG33129 CG34195 CG3760 CG31637 CG32243 CG33144 CG3420 CG3764 CG3164 CG32249 CG33155 CG34200 CG3776 CG31648 CG3225 CG33156 CG34205 CG3777 CG3165 CG32262 CG33169 CG34213 CG3781 CG31668 CG32263 CG33199 CG34229 CG3792 CG31674 CG32264 CG33217 CG34232 CG3808 CG31677 CG32276 CG33225 CG34244 CG3817 CG31678 CG32295 CG33228 CG34250 CG3847 CG3168 CG32301 CG33260 CG34253 CG3860 CG31688 CG32344 CG33281 CG34260 CG3862 CG31689 CG32365 CG33290 CG34261 CG3894 CG31694 CG32369 CG33299 CG34265 CG3902 CG31698 CG32373 CG33303 CG34280 CG3907 CG31710 CG32409 CG33307 CG34292 CG3918 CG31715 CG32428 CG3335 CG34299 CG3939 CG31717 CG32440 CG3337 CG3434 CG3961 CG31743 CG32454 CG33453 CG34342 CG3995 CG31751 CG32457 CG33465 CG34345 CG40045 CG31759 CG32473 CG33474 CG34347 CG40178 CG31776 CG32500 CG3348 CG34348 CG40191 CG31793 CG3251 CG33483 CG34355 CG4022 CG31800 CG32512 CG33494 CG3436 CG40228 CG31804 CG3253 CG33506 CG34370 CG4036 CG31809 CG32554 CG33509 CG34376 CG4038 CG31810 CG32573 CG3356 CG34383 CG4042 CG31812 CG32576 CG3358 CG34401 CG4074 CG31813 CG32579 CG33626 CG34404 CG4080 CG31816 CG32590 CG33632 CG34435 CG4098 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

CG4101 CG42797 CG4438 CG4853 CG5510 CG4119 CG42806 CG44439 CG4860 CG5521 CG41242 CG4281 CG4452 CG4880 CG5522 CG41378 CG42813 CG44532 CG4882 CG5532 CG4174 CG42833 CG4459 CG4887 CG5535 CG4210 CG42837 CG4462 CG4896 CG5550 CG42231 CG4291 CG4476 CG4927 CG5555 CG42232 CG4293 CG44774 CG4935 CG5568 CG42238 CG4294 CG4502 CG4942 CG5569 CG42245 CG4297 CG45049 CG4949 CG5577 CG42258 CG43066 CG45050 CG4957 CG5589 CG42260 CG43071 CG45060 CG4968 CG5590 CG42268 CG43072 CG45065 CG4970 CG5626 CG42271 CG43102 CG45069 CG4972 CG5646 CG4230 CG43121 CG45072 CG4982 CG5664 CG42306 CG4313 CG45075 CG5004 CG5674 CG42324 CG43149 CG45080 CG5009 CG5676 CG42329 CG43163 CG45085 CG5013 CG5694 CG42336 CG43175 CG45086 CG5021 CG5707 CG42353 CG4318 CG45087 CG5023 CG5708 CG42354 CG43181 CG45089 CG5033 CG5721 CG42359 CG43206 CG45092 CG5045 CG5726 CG42360 CG43218 CG45099 CG5056 CG5728 CG4238 CG43229 CG45100 CG5065 CG5734 CG4239 CG43235 CG4511 CG5068 CG5745 CG42390 CG43245 CG4538 CG5087 CG5758 CG42394 CG43313 CG45413 CG5098 CG5789 CG42402 CG43316 CG45428 CG5114 CG5790 CG42458 CG43322 CG4552 CG5131 CG5791 CG42488 CG43332 CG4553 CG5144 CG5793 CG42495 CG43333 CG4554 CG5149 CG5800 CG42497 CG43338 CG4557 CG5151 CG5805 CG42500 CG43341 CG4558 CG5157 CG5808 CG42514 CG43342 CG4565 CG5174 CG5815 CG42516 CG43355 CG4587 CG5180 CG5823 CG42518 CG43367 CG4592 CG5196 CG5828 CG42526 CG43402 CG4593 CG5199 CG5835 CG42542 CG43448 CG4598 CG5213 CG5853 CG42553 CG4360 CG4603 CG5220 CG5854 CG42568 CG4364 CG4607 CG5235 CG5861 CG42613 CG43658 CG4610 CG5270 CG5877 CG42637 CG4367 CG4615 CG5273 CG5880 CG42638 CG43678 CG4622 CG5274 CG5885 CG4266 CG43693 CG46280 CG5276 CG5895 CG42662 CG43703 CG4629 CG5290 CG5903 CG42663 CG43707 CG46301 CG5292 CG5913 CG42668 CG43736 CG46306 CG5316 CG5946 CG42673 CG43737 CG46309 CG5323 CG5955 CG42674 CG43739 CG46311 CG5339 CG5966 CG42675 CG43778 CG46312 CG5346 CG5973 CG42680 CG43779 CG46319 CG5355 CG5989 CG42684 CG43780 CG46339 CG5359 CG6005 CG4269 CG43844 CG46385 CG5377 CG6006 CG42699 CG43886 CG46387 CG5380 CG6023 CG42700 CG4390 CG4645 CG5381 CG6026 CG42709 CG43924 CG4662 CG5382 CG6040 CG42711 CG43954 CG4702 CG5399 CG6051 CG42714 CG43968 CG4733 CG5418 CG6067 CG42724 CG43980 CG4751 CG5445 CG6071 CG42741 CG4407 CG4788 CG5447 CG6073 CG42749 CG44098 CG4806 CG5455 CG6084 CG42784 CG4424 CG4813 CG5478 CG6094 CG42788 CG44245 CG4820 CG5500 CG6115 CG42795 CG44286 CG4839 CG5504 CG6126 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

CG6142 CG6836 CG7483 CG8080 CG8728 CG6144 CG6841 CG7484 CG8097 CG8745 CG6145 CG6843 CG7488 CG8108 CG8765 CG6163 CG6845 CG7492 CG8119 CG8768 CG6171 CG6847 CG7506 CG8128 CG8774 CG6204 CG6878 CG7509 CG8149 CG8778 CG6225 CG6885 CG7518 CG8152 CG8786 CG6227 CG6888 CG7519 CG8176 CG8833 CG6230 CG6907 CG7530 CG8177 CG8838 CG6236 CG6908 CG7544 CG8180 CG8839 CG6244 CG6923 CG7546 CG8184 CG8841 CG6254 CG6928 CG7564 CG8188 CG8891 CG6271 CG6933 CG7587 CG8191 CG8927 CG6276 CG6961 CG7600 CG8195 CG8929 CG6277 CG6966 CG7627 CG8197 CG8939 CG6280 CG6967 CG7630 CG8199 CG8944 CG6287 CG6983 CG7637 CG8209 CG8963 CG6293 CG6984 CG7639 CG8223 CG8974 CG6294 CG6999 CG7646 CG8230 CG9003 CG6300 CG7006 CG7650 CG8272 CG9005 CG6310 CG7028 CG7655 CG8281 CG9008 CG6324 CG7029 CG7656 CG8298 CG9018 CG6325 CG7031 CG7692 CG8306 CG9034 CG6330 CG7044 CG7718 CG8311 CG9044 CG6340 CG7049 CG7720 CG8312 CG9065 CG6345 CG7065 CG7728 CG8319 CG9114 CG6356 CG7071 CG7730 CG8321 CG9132 CG6379 CG7101 CG7739 CG8326 CG9135 CG6405 CG7110 CG7741 CG8336 CG9143 CG6420 CG7115 CG7744 CG8366 CG9171 CG6421 CG7120 CG7745 CG8370 CG9175 CG6425 CG7133 CG7755 CG8388 CG9205 CG6426 CG7137 CG7768 CG8397 CG9213 CG6428 CG7139 CG7772 CG8399 CG9220 CG6431 CG7149 CG7785 CG8405 CG9253 CG6441 CG7168 CG7789 CG8412 CG9300 CG6443 CG7182 CG7791 CG8414 CG9302 CG6465 CG7194 CG7800 CG8435 CG9304 CG6479 CG7206 CG7824 CG8441 CG9328 CG6481 CG7218 CG7840 CG8461 CG9331 CG6512 CG7220 CG7857 CG8468 CG9336 CG6602 CG7246 CG7872 CG8475 CG9338 CG6607 CG7265 CG7878 CG8476 CG9344 CG6617 CG7272 CG7903 CG8478 CG9346 CG6621 CG7275 CG7920 CG8485 CG9380 CG6650 CG7289 CG7943 CG8490 CG9393 CG6659 CG7324 CG7945 CG8500 CG9399 CG6664 CG7330 CG7946 CG8516 CG9406 CG6665 CG7332 CG7956 CG8519 CG9410 CG6678 CG7369 CG7971 CG8520 CG9425 CG6683 CG7372 CG7985 CG8526 CG9426 CG6685 CG7379 CG7987 CG8531 CG9436 CG6693 CG7382 CG8001 CG8547 CG9449 CG6695 CG7394 CG8003 CG8549 CG9485 CG6700 CG7402 CG8005 CG8568 CG9500 CG6701 CG7408 CG8010 CG8611 CG9512 CG6724 CG7409 CG8032 CG8613 CG9515 CG6729 CG7414 CG8036 CG8617 CG9518 CG6766 CG7420 CG8038 CG8635 CG9519 CG6767 CG7430 CG8042 CG8661 CG9522 CG6791 CG7457 CG8046 CG8671 CG9541 CG6805 CG7458 CG8060 CG8675 CG9581 CG6808 CG7460 CG8066 CG8677 CG9609 CG6833 CG7461 CG8079 CG8726 CG9628 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

CG9630 Cka CRAT Cyp9f3 dikar CG9643 CkIalpha crc CysRS Dim1 CG9650 CkIIalpha-i1 CrebB Cyt-b5 Dip-C CG9662 CkIIalpha-i3 crim Cyt-c-p DIP-epsilon CG9667 CkIIbeta Crk Cyt-c1 DIP-lambda CG9674 ckn CRMP Cyt-c1L DIP1 CG9691 Cks30A crn d DIP2 CG9701 Clamp croc D1 dj-1beta CG9706 Claspin crok D19B dl CG9723 Clbn crol D2hgdh dlg1 CG9737 Clc crp d4 Dlic CG9743 ClC-b crq da Dlip2 CG9776 Clic Crtc Dab Dlip3 CG9784 CLIP-190 Cse1 Dad Dlish CG9795 Cln7 CSN3 Dak1 dmpd CG9815 Clp CSN4 Dap160 dmt CG9849 CLS CSN5 Dark Dmtn CG9853 cmet CSN6 dati DnaJ-1 CG9886 cN-IIIB CSN7 Daxx DNAlig3 CG9896 Cnb CSN8 Dbp73D DNApol-alpha180 CG9902 CNBP Csp dbr DNApol-alpha60 CG9915 cnc csw Dbx DNApol-epsilon58 CG9920 cnk cta DCAF12 DNApol-gamma35 CG9921 CNMa CtBP Dci DNApol-iota CG9932 cnn CTCF dco dnr1 CG9934 cno Ctl1 Dcp-1 dnt CG9941 Cnot4 Ctl2 DCP1 Doa CG9945 Cnx14D ctp DCP2 Doc2 CG9967 Cnx99A ctrip DCTN1-p150 dock CG9986 Coa7 Cul2 DCTN2-p50 dod chas Cog1 Cul3 DCTN3-p24 dop Chc Col4a1 Cul4 DCTN4-p62 dor Chchd2 comm2 Cul6 DCTN5-p25 dos Chchd3 comm3 cutlet Dd Dp Chd1 Cont cv-2 Deaf1 Dp1 Chd3 Coq2 cv-c Debcl Dpck Chd64 Coq5 cyc Dek DPCoAC CheB42c Coq6 CycA del Dph1 CheB93a Coq7 CycB Den1 Dph5 CheB93b cora CycB3 Der-1 DppIII cher CoRest CycC Der-2 dpr14 CHES-1-like corto CycD Dera dpr2 Chi COX4 CycE Desat1 Dpy-30L1 chic COX5A CycG Det Dr chico COX7C CycH Df31 dre4 chif COX8 CycK Dfd Dredd chinmo Cp1 CycT Dg Dref chm Cp190 CycY Dgat2 Drep2 Chmp1 cpa CYLD Dgkepsilon Drgx chn cpb Cyp1 Dgp-1 Drice CHOp24 cpo Cyp12b2 dgt1 drl CHORD Cpr Cyp12c1 dgt3 drongo Chrac-16 Cpr47Ea Cyp12e1 dgt4 Drp1 chrb Cpr47Ee Cyp312a1 Dhap-at Drs Chro Cpr49Ad Cyp318a1 Dhc64C Drsl4 CIAPIN1 Cpr49Ae cyp33 dhd Drsl5 cib Cpr62Ba Cyp4s3 Dhit dsb Cib2 Cpr62Bc Cyp6a13 DhpD Dscam1 cic Cpr67Fb Cyp6a16 Dhpr dsd cid Cpr76Ba Cyp6a2 Dhx15 dsh cindr Cpr97Ea Cyp6a21 Diap1 Dsk Cip4 Cpsf6 Cyp6a22 Diap2 Dsp1 Cirl CPT2 Cyp6v1 Diedel3 Dtg ck Cralbp Cyp9c1 Dif dtr bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

Duba eIF4A Fas1 Fur1 GLaz dve eIF4B FASN1 fus GlcAT-I dwg eIF4E1 Fatp1 fusl GlcAT-S Dyb eIF4E5 Fatp3 fuss GlcT Dyrk2 eIF4EHP fax fwd glec Dys eIF4G1 fbl fwe Glg1 dysc eIF5B Fbl6 fz Gli e Eip55E FBXO11 fz2 GlnRS E(bx) Eip63E Fdh fz3 Glo1 E(Pc) Eip63F-1 fdl fz4 glob1 e(r) Eip74EF Fdx1 fzr glu E(spl)m3-HLH Eip75B FDY g GluRIA E(var)3-9 Eip78C FeCH GAA1 Glut4EF e(y)1 EloB Fem-1 GABPI Glyat e(y)2b Elp2 feo galectin GlyP e(y)3 emb FER galla-2 GlyS E23 emc Fer3 Galphai GM130 E2f1 EMC1 fest Galphaq Gmap E2f2 EMC8-9 Fhos Galphas GMF E5 emp Fic GalT1 Gnf1 EAChm ems FIG4 gammaCOP gny Eaf en Fili gammaSnap1 godzilla Eaf6 ena Fim gammaSnap2 goe eag enc Fip1 gammaTub37C gol eas EndoA Fis1 GAPcenA Golgin245 east EndoB Fit1 Gapdh1 Golgin84 Eato EndoGI Fit2 GAPsec Gorab Eb1 endos fiz Gapvd1 Got1 ebd1 EndoU Fkbp12 Gas41 Got2 ebo Eno Fkbp14 Gasz Gp150 ec ens Fkbp39 GATAd Gp93 ecd Ent1 Fkbp59 GatB Gpat4 Echs1 Eo fl(2)d gbb Gpdh1 eco Eogt Flacc Gbeta13F gpp EcR Epg5 FLASH GCC185 Gprk1 Ect4 Eph flfl gce Gr22a Edem1 Ephrin fliI GckIII Gr61a Edg78E epsilonCOP Flo2 gcl Gr89a Edg84A eRF3 flw Gclc Gr92a edl ergic53 flz Gclm Gr98d EDTP Ero1L Fmr1 gcm Graf eEF1alpha1 ERp60 Fnta Gcn2 GramD1B eEF1alpha2 Es2 foi Gdap2 grh eEF1delta esc for Gdh Grip eEF1gamma Etf-QO form3 Gdi Grip75 eEF2 ETHR foxo gdl grk eEF5 Ets21C fra Gdn1 grn Efa6 Ets97D FRG1 Ge-1 gro eff Evi5 frj GEFmeso grp Efr ex frtz Gel Gs1 Egfr exd fru gem Gs1l egl Exd2 fry Gem2 Gss1 Eglp2 Exn Fs Gem3 GstD1 Egm exu fs(1)h geminin GstD11 Eh ey fs(1)K10 Gfat2 GstD3 Ehbp1 eya Fs(2)Ket Gfrl GstE1 eIF2alpha fabp fs(2)ltoPP43 Gga GstE2 eIF2beta Faf2 ft Ggamma1 GstE3 eIF3c Fak ftz Ggamma30A GstE6 eIF3d1 fal ftz-f1 ghi GstE8 eIF3f1 fan fu gig GstO2 eIF3i Fancd2 Fuca GILT1 GstO3 eIF3j FANCI FucT6 Gip GstS1 eIF3k Fancm Fum1 Gk2 GstT3 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

GstT4 holn1 Ing3 Kdm4B LanA GstZ1 hook Ing5 kdn LanB1 Gtp-bp how Inos Keap1 LanB2 Gug HP1D3csd INPP5E kek1 lap gukh Hph InR kek5 Lap1 gus hpo insv kel Lar gw hppy IntS14 ken larp gwl hpRNA:CR32205 IntS3 Khc-73 Larp4B GXIVsPLA2 hpRNA:CR32207 IntS4 kibra Larp7 Gyc76C hpRNA:CR33940 IntS6 kin17 Las Gycalpha99B HPS4 IntS8 kis lawc Gycbeta100B Hr38 inv klar ldbr Gyf Hr4 Invadolysin Klp31E Leash h Hr78 Inx2 Klp68D lectin-28C H15 Hrb27C Inx5 Klp98A lectin-46Cb Hacd1 Hrb87F Ip259 klu Lectin-galC1 Hacd2 Hrb98DE IP3K1 kn Letm1 Hacl hrg IP3K2 koi LeuRS-m hang Hrs Ir40a KP78b levy Haspin Hs6st Ir56d Kr lgs Hat1 Hsc20 Irbp Kr-h1 lic hbs Hsc70-3 Irc Kr-h2 lid Hcf Hsc70-4 Ire1 krimp lili hdm Hsc70Cb Irk1 krz Lim1 HEATR2 Hsepi Irk2 kst LIMK1 heix Hsf Irk3 kto lin Hel25E Hsp26 Irp-1A Ku80 lin-52 Hel89B Hsp60A Irp-1B kug Liprin-alpha Hem Hsp68 IRSp53 Kul Liprin-beta HemK1 Hsp70Bb IscU kune Liprin-gamma HemK2 hth isopeptidase-T-3 kuz Lis-1 hemo htk Iswi Kyat Lk Hen1 htl ITP l(1)G0007 Lk6 hep HtrA2 Itp-r83A l(1)G0020 LManII heph Hus1-like ix l(1)G0193 lmgA her Hyccin Jafrac1 l(1)G0289 Lmpt HERC2 hyd jagn l(1)G0469 lncRNA:bxd Herp Hydr1 jar l(2)05714 lncRNA:CR31044 Hers Hydr2 jdp l(2)09851 lncRNA:CR32636 Hexo2 hyx jet l(2)37Cc lncRNA:CR32730 hfp I-2 jhamt l(2)37Cd lncRNA:CR32835 HGTX IA-2 Jheh2 l(2)dtl lncRNA:CR34047 hid Ibf1 jigr1 l(2)gl lncRNA:CR40341 HIP Ice1 JIL-1 l(2)k01209 lncRNA:CR40469 HIP-R Ice2 jim l(2)k05911 lncRNA:CR42657 Hip1 ics jing l(2)k09022 lncRNA:CR42721 HIPP1 Idgf5 JMJD4 l(2)k09848 lncRNA:CR42736 Hira Idgf6 Jon74E l(2)k14505 lncRNA:CR42765 His2Av Idh Jon99Fii l(2)SH0834 lncRNA:CR42839 His3.3B Idh3b jp l(3)02640 lncRNA:CR42850 His4r ifc Jra l(3)05822 lncRNA:CR43144 HisRS IFT43 jtb l(3)72Ab lncRNA:CR43148 Hlc IFT52 jumu l(3)73Ah lncRNA:CR43241 Hmgs Ih Jupiter l(3)L1231 lncRNA:CR43242 HmgZ ihog Jwa l(3)mbn lncRNA:CR43252 Hml IleRS Kal1 l(3)neo38 lncRNA:CR43253 Hmt4-20 IleRS-m Kank l(3)neo43 lncRNA:CR43301 Hmu Ilp5 Kap-alpha3 l(3)psg2 lncRNA:CR43302 Hn Ilp8 Karybeta3 L2HGDH lncRNA:CR43421 Hnf4 Imp Kat60 La lncRNA:CR43422 hng3 IMPPP Kcmf1 Lac lncRNA:CR43431 HnRNP-K inaE KCNQ lace lncRNA:CR43432 hog inc KdelR lama lncRNA:CR43435 hoip Indy Kdm2 Lamp1 lncRNA:CR43437 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

lncRNA:CR43452 lncRNA:CR44518 lncRNA:CR45506 lncRNA:TS26 Mctp lncRNA:CR43496 lncRNA:CR44522 lncRNA:CR45517 lncRNA:TS6 MCTS1 lncRNA:CR43606 lncRNA:CR44531 lncRNA:CR45556 lncRNA:yar MCU lncRNA:CR43639 lncRNA:CR44539 lncRNA:CR45559 Lnk Mdh1 lncRNA:CR43650 lncRNA:CR44544 lncRNA:CR45565 Lnpk mdy lncRNA:CR43651 lncRNA:CR44598 lncRNA:CR45577 lobo me31B lncRNA:CR43653 lncRNA:CR44603 lncRNA:CR45582 loco MED1 lncRNA:CR43654 lncRNA:CR44615 lncRNA:CR45586 loj MED10 lncRNA:CR43656 lncRNA:CR44617 lncRNA:CR45618 lola MED11 lncRNA:CR43685 lncRNA:CR44641 lncRNA:CR45640 lolal MED14 lncRNA:CR43713 lncRNA:CR44662 lncRNA:CR45642 loqs MED17 lncRNA:CR43769 lncRNA:CR44679 lncRNA:CR45662 lost MED19 lncRNA:CR43804 lncRNA:CR44688 lncRNA:CR45667 Loxl1 MED20 lncRNA:CR43835 lncRNA:CR44697 lncRNA:CR45670 LPCAT MED21 lncRNA:CR43838 lncRNA:CR44727 lncRNA:CR45681 Lpin MED26 lncRNA:CR43840 lncRNA:CR44740 lncRNA:CR45684 LpR1 MED27 lncRNA:CR43849 lncRNA:CR44749 lncRNA:CR45689 LpR2 MED31 lncRNA:CR43855 lncRNA:CR44755 lncRNA:CR45692 lqf MED4 lncRNA:CR43857 lncRNA:CR44761 lncRNA:CR45695 Lrch MED6 lncRNA:CR43858 lncRNA:CR44769 lncRNA:CR45730 LRP1 MED8 lncRNA:CR43878 lncRNA:CR44800 lncRNA:CR45733 LRR Mef2 lncRNA:CR43892 lncRNA:CR44801 lncRNA:CR45737 Lrr47 mEFG1 lncRNA:CR43904 lncRNA:CR44829 lncRNA:CR45750 Lrrk mEFTs lncRNA:CR43942 lncRNA:CR44831 lncRNA:CR45758 Lsm10 Megf8 lncRNA:CR43963 lncRNA:CR44845 lncRNA:CR45810 Lsm12 mei-217 lncRNA:CR43967 lncRNA:CR44862 lncRNA:CR45820 lsn mei-P26 lncRNA:CR43987 lncRNA:CR44886 lncRNA:CR45826 Lst8 mei-W68 lncRNA:CR43995 lncRNA:CR44923 lncRNA:CR45898 lt Meics lncRNA:CR44026 lncRNA:CR44933 lncRNA:CR45914 LTV1 meigo lncRNA:CR44068 lncRNA:CR44947 lncRNA:CR45921 luna Mekk1 lncRNA:CR44127 lncRNA:CR44948 lncRNA:CR45922 lush Meltrin lncRNA:CR44138 lncRNA:CR44973 lncRNA:CR45937 lute Men lncRNA:CR44151 lncRNA:CR44974 lncRNA:CR45938 lva Men-b lncRNA:CR44183 lncRNA:CR44985 lncRNA:CR45939 lwr MEP-1 lncRNA:CR44197 lncRNA:CR44988 lncRNA:CR45958 LysB meru lncRNA:CR44206 lncRNA:CR45054 lncRNA:CR45963 mab-21 Mes4 lncRNA:CR44212 lncRNA:CR45102 lncRNA:CR45973 Mad Mesh1 lncRNA:CR44216 lncRNA:CR45169 lncRNA:CR45977 mad2 MESK2 lncRNA:CR44218 lncRNA:CR45187 lncRNA:CR45997 Madm MESR3 lncRNA:CR44222 lncRNA:CR45193 lncRNA:CR46002 MAGE MESR4 lncRNA:CR44229 lncRNA:CR45229 lncRNA:CR46005 magu MESR6 lncRNA:CR44265 lncRNA:CR45245 lncRNA:CR46006 mahe Met lncRNA:CR44274 lncRNA:CR45251 lncRNA:CR46042 mam metro lncRNA:CR44282 lncRNA:CR45252 lncRNA:CR46075 Manf MetRS-m lncRNA:CR44284 lncRNA:CR45267 lncRNA:CR46083 Map205 Mettl14 lncRNA:CR44302 lncRNA:CR45277 lncRNA:CR46090 Map60 mew lncRNA:CR44308 lncRNA:CR45297 lncRNA:CR46103 MAPk-Ak2 Mfap1 lncRNA:CR44315 lncRNA:CR45320 lncRNA:CR46118 Mapmodulin mfas lncRNA:CR44365 lncRNA:CR45321 lncRNA:CR46122 Marf1 Mfe2 lncRNA:CR44366 lncRNA:CR45328 lncRNA:CR46123 mask mfrn lncRNA:CR44392 lncRNA:CR45339 lncRNA:CR46127 Mat1 MFS10 lncRNA:CR44407 lncRNA:CR45352 lncRNA:CR46198 Mau2 MFS14 lncRNA:CR44415 lncRNA:CR45383 lncRNA:CR46215 MBD-R2 MFS15 lncRNA:CR44417 lncRNA:CR45386 lncRNA:CR46253 mbf1 MFS16 lncRNA:CR44418 lncRNA:CR45407 lncRNA:CR46254 mbl MFS3 lncRNA:CR44467 lncRNA:CR45421 lncRNA:CR46258 Mbs Mgat1 lncRNA:CR44468 lncRNA:CR45427 lncRNA:CR46267 mbt mge lncRNA:CR44471 lncRNA:CR45433 lncRNA:CR46271 Mcad mgl lncRNA:CR44478 lncRNA:CR45438 lncRNA:CR46322 Mccc2 mGluR lncRNA:CR44498 lncRNA:CR45456 lncRNA:CR46336 Mcm2 mh lncRNA:CR44499 lncRNA:CR45463 lncRNA:Hsromega Mcm6 Mhcl lncRNA:CR44503 lncRNA:CR45471 lncRNA:iab8 Mcm7 Mi-2 lncRNA:CR44508 lncRNA:CR45472 lncRNA:roX1 MCPH1 mia lncRNA:CR44517 lncRNA:CR45473 lncRNA:roX2 Mcr mib1 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

mib2 Mondo mts ND-B14.5B not Mical mop mtsh ND-B18 Not1 MICAL-like Mos mtSSB ND-MLRQ Not10 MICU1 Mp mTTF ND-MWFE Not3 mid MP1 mub ND-PDSW Np mIF2 Mpcp2 muc ND-SGDH Npc1a mIF3 Mpi mud Ndae1 Npc2b Miga Mppe Mul1 Ndc1 NPF milt mr Mulk Ndf Npl4 Mink mre11 Mur29B neb Nplp1 mip120 MRG15 mura Neb-cGP Nprl3 miple2 mri mus205 Nedd4 Nrd1 mir-10 mrj mus301 Nedd8 Nrg mir-1013 mRNA-cap muskelin nej Nrt mir-11 Mrp4 Mvb12 NELF-B nrv1 mir-14 mRpL1 Mvd Nelf-E nrv2 mir-184 mRpL10 mwh nemy nrv3 mir-2281 mRpL16 mxt Nep1 Nrx-IV mir-2493 mRpL19 Myb Nepl20 NSD mir-278 mRpL20 Myd88 Nepl3 Nse1 mir-279 mRpL22 myo Nepl8 Nse4 mir-282 mRpL3 Myo61F nes Nsf2 mir-317 mRpL30 Myo95E Neu2 nsl1 mir-34 mRpL32 MYPT-75D Nf-YC Ntan1 mir-4944 mRpL33 Myt1 Nf1 Ntf-2 mir-4978 mRpL4 N NFAT Ntf-2r mir-92b mRpL40 Naa15-16 Nhe1 NTPase mir-9375 mRpL41 Naa20B Nhe2 Nuak1 mir-9381 mRpL45 Naa30A Nhe3 nub mir-9385 mRpL49 Naa60 NHP2 NUCB1 mir-9388 mRpL9 Naam NijA nudC mir-989 mRpS18B Nab2 NijB nuf mir-9a mRpS23 Nacalpha NimA Nuf2 mir-9b mRpS25 nAChRalpha4 NimC3 numb mir-iab-8 mRpS29 nAChRalpha7 ninaA Nup133 Miro mRpS35 nAChRbeta1 Nipped-A Nup153 mirr mRpS5 NaCP60E Nipsnap Nup205 Mitf mRpS9 Nadsyn nito Nup214 Mitofilin Mrtf nahoda NK7.1 Nup35 Mkk4 msb1l Nak nkd Nup358 MKP-4 MSBP Nap1 NLaz Nup44A Mkp3 msi NaPi-III Nle Nup54 Mkrn1 msk Naprt Nlp Nup58 Mlc-c msl-1 NAT1 Nmda1 Nup62 mld msl-3 Naxd Nmdmc Nup93-2 mle msn Nbr nmdyn-D6 Nup98-96 Mlh1 Msp300 Nc73EF nmdyn-D7 Nurf-38 mlt msps Ncc69 Nmnat nxf2 MME1 mst nclb Nmt Nxf3 Mmp1 Mst85C ncm Nnf1b nxf4 Mmp2 MTA1-like Ncoa6 NO66 oaf mnb mtd Nct Noa36 Oat Mnn1 mtDNA-helicase ND-13A nod Oatp30B Mo25 MTF-1 ND-13B nolo Oatp58Dc Mob2 mtgo ND-15 Non1 Oatp74D Mob3 mthl1 ND-20 Non2 obe Mob4 mthl10 ND-23 Non3 Obp44a Moca-cyp mthl3 ND-24 nonA-l Obp56b Mocs1 mthl9 ND-39 nonC Obp56g mod mtm ND-49 Nop17l Obp99b mod(mdg4) Mtor ND-51 nop5 oc modSP Mtp ND-75 Nop56 Ocrl Moe Mtpalpha ND-B12 Nop60B Octbeta2R mof mtRNApol ND-B14.5A Nost Oda bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

Odj pav ph-p porin Ptpmeg2 OdsH Pax PH4alphaEFB Pp1-87B Ptth Oga Pbp95 Phb2 Pp1-Y1 Ptx1 Ogg1 Pc PheRS-m Pp1alpha-96A Pu olf186-F PCID2 Phf7 Pp2A-29B puc omd pcm PHGPx Pp2C1 pum ome Pcmt PhKgamma Pp4-19C Pur-alpha opa PCNA Phm Ppa Pura Opa1 pcs pho ppan Pus7 Opbp pcx phtf Ppat-Dpck Pvf2 Or2a Pcyt1 phyl ppk16 Pvf3 Or43a Pdcd4 PI31 ppk8 Pvr Or56a Pde11 Pi3K59F ppl px Or92a Pde1c Pi3K68D Ppm1 pxb orb2 Pde6 Pi3K92E Ppn Pxt Orc1 Pde8 pic Ppox pyd Orc2 Pde9 pico PPP1R15 pygo Orc4 Pdhb Picot PPP4R2r Pym Orc6 Pdi PIG-G Ppt2 pyr Orct2 Pdk1 PIG-H PpV QC osa pdm2 PIG-K PR-Set7 QIL1 Osbp pdm3 PIG-L prage qin Oscillin Pdp PIG-N PRAS40 qkr54B Oseg5 Pdp1 PIG-T Prat qkr58E-2 Osi1 Pdrg1 PIG-V Prat2 qless osp Pdss2 PIG-Wa pre-mod(mdg4)-AA r-l Ost48 PDZ-GEF Pink1 pre-mod(mdg4)-B Rab1 OstDelta Pect pinta Prestin Rab10 Ote PEK pip prg Rab11 out Pen PIP4K primo-1 Rab14 ovm Pep pit Prip Rab18 OXA1L Pepck2 pita prod Rab19 oys pes Pits prom Rab23 p120ctn Pex1 pk prominin-like Rab27 p130CAS Pex12 Pka-C1 Prosalpha2 Rab3-GAP p23 Pex13 Pka-C3 Prosalpha3 Rab32 p24-1 Pex16 Pka-R2 Prosalpha4 Rab35 P32 Pex19 Pkc53E Prosalpha5 Rab40 p38b Pex2 Pkc98E Prosap Rab6 p47 Pex23 Pkn Prosbeta1 Rab8 p53 Pex7 Plc21C Prosbeta2 Rab9 P58IPK Pfas Pld Prosbeta3 Rabex-5 P5CDh1 Pfdn1 Plekhm1 Prosbeta5 RabX1 Pa1 Pfdn4 PlexA Prosbeta6 RabX6 pAbp Pfdn5 PlexB Prosbeta7 Rac2 Paf-AHalpha Pfk Pli Prp38 Rack1 Paics Pfrx Plod Prp8 Rad1 pain Pgant1 Plp Prx3 Rad17 Paip2 Pgant2 plum Prx5 Rad23 Pak Pgant3 Plzf ps rad50 Pak3 Pgant4 PMCA Psa Rad51D pall Pgant6 Pmp70 Psf1 Rad60 pan Pgant7 Pms2 Psf2 Rae1 Papss PGAP1 Pngl Psf3 Raf par-1 PGAP2 Pnn psq RAF2 Parg PGAP5 pnt pst RagC-D Parp Pgd PNUTS Pten Rala pasi1 Pgi Poc1 Ptip Ran pasi2 Pgm2a poe PTP-ER RanBP3 Past1 PGRP-LA Pol32 Ptp10D Ranbp9 Pat1 PGRP-LC polo Ptp4E RanBPM path PGRP-LD poly Ptp69D RanGAP Patj PGRP-LF polybromo Ptp99A Rap1 Patronin ph-d Pomp Ptpmeg Rap2l bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

RapGAP1 RhoU RpL38 salm 5-Sep raptor rib RpL39 salr SERCA ras RIC-3 RpL5 Sam-S SerRS-m Ras64B ric8a RpL6 SamDC sesB Ras85D Rich RpL8 Samtor Sesn rasp Rif1 RpL9 Samuel Set RASSF8 Rift RpLP0 santa-maria Set1 raw Rilpl RpLP2 Sap-r Set2 Rb97D Rip11 Rpn1 Sap130 SF1 Rbbp5 Ripalpha Rpn10 Sap30 SF2 Rbcn-3A Rlip Rpn11 Sap47 Sf3a1 Rbf RluA-2 Rpn12 Sar1 Sf3a2 Rbfox1 Rm62 Rpn2 Sara Sf3b3 Rbm13 Rme-8 Rpn3 saturn Sf3b6 Rbp1 RN-tre Rpn6 sax sfl Rbp1-like RNASEK Rpp30 sba Sfmbt Rbp9 RNaseX25 RpS10a sbb Sfp79B Rbpn-5 Rnmt RpS10b Sbf Sfp84E Rca1 rno RpS11 sbr Sfxn1-3 Rcc1 Rnp4F RpS12 SC35 Sfxn2 Rcd2 RnrS RpS14b scaf sgg rdgA robl RpS15Aa SCaMC sgl rdog robo1 RpS17 SCAP sgll rdx robo2 RpS18 scaRNA:MeU5-C46 Sgs3 rec Roc1a RpS2 SCCRO Sgs4 Reck Roe1 RpS20 SCCRO3 Sgt1 RecQ4 Rok RpS23 Sce Shab RecQ5 rols RpS24 sced Shc red roq RpS26 scf shd ReepB Ror RpS27 schlank shep ref(2)P row RpS28b SclB shg REG Rpb10 RpS29 Sclp shi Reg-5 Rpb12 RpS3 Scm Shmt Rel Rpb5 RpS30 scny shn rempA Rpe RpS3A Scr shop Rep Rph RpS4 scramb1 shot Reph Rpi RpS5a scrib shps Reps RpI12 RpS6 Scsalpha1 Shrm retn RpI135 RpS7 ScsbetaA SiaT rev7 RpII15 RpS9 ScsbetaG sicily Rexo5 RpII18 Rpt1 Sdc sick RfC3 RpIII128 Rpt2 Sdhaf3 Sid RfC4 RpIIIC160 Rpt6 SdhB side-II RFeSP RpL10 Rrp4 SdhC side-IV Rfx RpL10Ab Rrp46 sds22 sif Rgl RpL11 Rsf1 sdt Sik3 rgn RpL12 RSG7 sea Sil1 Rh5 RpL13 Rtc1 Sec13 sim Rhau RpL15 RtcB Sec15 simj rhea RpL17 rtet Sec16 Sin1 rho RpL21 Rtf1 Sec24AB Sin3A rho-6 RpL22 Rtnl1 Sec24CD sina Rho1 RpL23 Rtnl2 Sec31 Sirt2 RhoBTB RpL23A ru Sec61beta Sirt7 RhoGAP100F RpL27 rump Sec61gamma Sirup RhoGAP15B RpL27A rut Sec63 sit RhoGAP18B RpL28 rux Sec8 siz RhoGAP19D RpL29 RYa Seipin Sk1 RhoGAP71E RpL30 RYBP sel skd RhoGAP93B RpL31 S SelD SKIP RhoGAPp190 RpL35 S2P SelR SkpA RhoGEF2 RpL35A S6kII Sem1 sktl RhoGEF3 RpL36 SA Sema5c sky RhoL RpL37a Saf-B 1-Sep sl bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

Slc45-1 snoRNA:Psi28S- Sps2 sun Tep4 sle 3385b Spt-I Sur-8 TER94 slgA snoRNA:Psi28S- Spt20 sut3 Tes sli 3436b spt4 Suv3 Tet slim snoRNA:U3:54Aa Spt6 Svil tex sll snRNA:7SK spz svp TfAP-2 Slmap snRNA:U1:21D spz6 sw Tfb1 SLO2 snRNA:U11 sqd SWIP Tfb4 slp1 snRNA:U2:34ABb sr sws TfIIA-L slp2 snRNA:U2:34ABc sra sxc TfIIA-S slpr snRNA:U3 Src42A Sxl TfIIA-S-2 slv snRNA:U4:39B Src64B Syb TfIIEalpha smash snRNA:U5:14B srl Syn1 TfIIEbeta SMC1 snRNA:U5:38ABb SRm160 Syn2 TfIIFbeta SMC2 snRNA:U5:63BC srp Synj TfIIS SMC3 snRNA:U6:96Aa Srp19 Syp Tgi SmD1 snRNA:U7 Srp68 Syx13 tgo SmD2 snRNP-U1-70K SRPK Syx17 TH1 SmD3 SNRPG SrpRbeta Syx18 THADA SmF snsl Sry-alpha Syx1A Thiolase Smg5 snu Sry-delta Syx6 tHMG2 Smg6 Snx1 ss Syx7 Thor Smn Snx3 Ssadh T3dh thr Sms Snx6 Ssb-c31a T48 ths SMSr snz Sse tacc Tie smt3 Socs16D Ssl1 tadr Tif-IA Smu1 Socs36E ssp Taf1 Tig Smurf Socs44A ssp3 Taf10 Tim17b Smyd4-1 sog SsRbeta Taf11 Tim17b1 Smyd4-4 solo Ssrp Taf13 Tim23 SmydA-2 Sos Ssu72 Taf2 Tim8 SmydA-8 sov Start1 Taf4 timeout snama sowah Stat92E Taf6 Timp Snap29 Sox102F stau Taf8 tin snf Sox15 stc tai Tip60 SNF4Agamma Sox21a stck Tak1 Tis11 snmRNA:419 SoxN step Taldo tko snmRNA:641 SP Sting Tango10 tkv sno SP1029 Stip1 Tango5 Tl snoRNA:185 SP1173 stj Tango9 Tlk snoRNA:229 Sp7 Stlk tant tll snoRNA:2R:944541 Spase12 stmA Tao Tm1 0 Spase22-23 Stoml2 tapas TM9SF2 snoRNA:CD-24 Spc105R Strumpellin Tapdelta TM9SF3 snoRNA:CG31191-a Spc25 Stt3A tara TM9SF4 snoRNA:CG43051-a spen STUB1 Task6 Tmem18 snoRNA:Me18S- Spf45 stwl Tasp1 Tmhs A1374 Spg7 sty tau TMS1 snoRNA:Me18S- spi Su(dx) tay Tmtc3 G1358a spin su(f) Taz tn snoRNA:Me28S- Spindly Su(H) TBC1D5 Tnks A2958 spir su(Hw) TBCB Tob snoRNA:Me28S- Spn su(r) TBCC toc A576 Spn27A Su(Ste):CR40820 Tbce tok snoRNA:Me28S- Spn28Db Su(Ste):CR42420 Tbp Toll-4 C1237 Spn42Da Su(var)2-10 TBPH Toll-7 snoRNA:Or-aca4 Spn75F Su(var)2-HP2 Tctp Tollo snoRNA:Or-CD1 Spn77Bb Su(var)3-3 Tdrd3 Tom20 snoRNA:Or-CD4 Spn88Ea su(w[a]) tef Tom70 snoRNA:Psi18S-301 SPoCk Su(z)2 Teh1 Tomosyn snoRNA:Psi18S-996 spok sub tej Top1 snoRNA:Psi28S- spoon subdued Tektin-C Top2 3342 Spp Sugb Ten-a Top3alpha spri Sulf1 Tep3 Top3beta bioRxiv preprint doi: https://doi.org/10.1101/2020.11.26.399824; this version posted November 26, 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-ND 4.0 International license.

Table S1: List of genes where BALL peaks are present in genome-wide binding profile. Gene names or annotation numbers are given.

Tor twf Usp2 wcd zormin tou twi Usp30 wcy Zw tow twit Usp39 wdb Zyx Tpc1 tws Usp47 wde Tpi twz Usp5 wdn TpnC25D tx Usp7 wdp Tpr2 TyrRS Utx Wdr59 tra tzn Uxs Wdr62 tra2 U2A uzip Wdr81 Traf4 U2af38 vap Wdr82 TRAM U3-55K Vap33 wech Trap1 Uba1 vari Wee1 Trax Uba3 Vav wek Trc8 Uba5 velo wge Tre1 Ubc2 vers wgn Treh Ubc4 Vha100-2 whd Trf4-1 Ubc6 Vha100-4 wisp Trf4-2 UbcE2H Vha13 wkd Trh Ubi-p5E Vha14-1 wnd Trim9 Ublcp1 Vha16-1 Wnk trio Ubqn Vha26 Wnt4 trol Ubr1 Vha36-3 Wnt5 Trp1 Ubr3 Vha68-1 woc Trpm Ubx Vha68-2 wol TrpRS Uck VhaAC39-2 wrd TrpRS-m Ufc1 VhaAC45 WRNexo Trs23 Ufd4 VhaM9.7-c wus Trs31 Ufm1 VhaPPA1-1 Wwox trus UGP VhaSFD X11L trx Ugt302C1 viaf Xbp1 Trx-2 Ugt303B2 vib xmas Trxr-1 Ugt303B3 Victoria Xpac Ts Ugt35A1 vig Xpd TSG101 Ugt36A1 vih Xport-A tsh Ugt36F1 Vinc Xport-B Tsp Ugt50B3 vir Xrp1 Tsp39D Uhg1 vir-1 Yip1d1 Tsp3A Uhg5 vis yki Tsp42Ec Uhg8 vkg Ykt6 Tsp42Ee UK114 vlc YME1L Tsp42Ef Ulp1 vls yps Tsp66E unc-104 Vm32E yrt Tsp74F Unc-115a Vps13 Ythdf Tsp96F unc-119 Vps15 ytr Tsp97E unc-4 Vps20 yuri Tspo unc-5 Vps24 ZAP3 tsr unc79 Vps28 Zasp52 Tsr1 unk Vps35 Zasp66 tsu Unr Vps39 Zcchc7 Ttc7 upd2 Vps4 zetaCOP Ttd14 Upf1 Vps51 zf30C tth Upf2 Vps53 zfh2 ttk upSET Vps8 Zif TTLL15 UQCR-11 vri zip TTLL4A UQCR-14 Vrp1 Zip48C TTLL4B UQCR-C2 vsg Zip71B ttv UQCR-Q Vsx2 Zip89B tub uri vtd ZIPIC tud Use1 Vti1a Zir Tudor-SN Usp1 Vti1b Ziz tun Usp10 wake Zn72D tup Usp14 wapl ZnT41F Tusp Usp15-31 WASp ZnT63C twe Usp16-45 wb ZnT86D