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6 Complex Orchestrates Developmental Signaling to Balance

7 Proliferation and Differentiation During Erythropoiesis

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10 Skye C. McIver1, Koichi R Katsumura1, Elsa Davids1, Peng Liu2, Yoon-A Kang1,

11 David Yang3 and Emery H. Bresnick1

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14 1Department of Cell and Regenerative Biology, UW-Madison Blood Research Program,

15 Carbone Center, University of Wisconsin School of Medicine and Public Health,

16 Madison, WI, 53705, USA; 2Department of Biostatistics and Medical Informatics, Carbone

17 Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison,

18 WI, 53705, USA; 3Department of Pathology, University of Wisconsin School of Medicine

19 and Public Health, Madison, WI, 53705, USA

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22 23 24 25 26 To whom correspondence should be addressed: Emery H. Bresnick, 1111 Highland Ave,

27 4009 WIMR, Madison, WI, 53705; email: [email protected]; TEL: 608-265-6446 28 SUMMARY

29 Since the highly conserved exosome complex mediates the degradation and

30 processing of multiple classes of , it almost certainly controls diverse biological

31 processes. How this post-transcriptional RNA-regulatory machine impacts cell fate

32 decisions and differentiation is poorly understood. Previously, we demonstrated that

33 exosome complex subunits confer an erythroid maturation barricade, and the erythroid

34 transcription factor GATA-1 dismantles the barricade by transcriptionally repressing the

35 cognate . While dissecting requirements for the maturation barricade in Mus

36 musculus, we discovered that the exosome complex is a vital determinant of a

37 developmental signaling transition that dictates proliferation and amplification versus

38 differentiation. Exosome complex integrity in erythroid precursor cells ensures Kit receptor

39 tyrosine kinase expression and stem cell factor/Kit signaling, while preventing

40 responsiveness to erythropoietin-instigated signals that promote differentiation.

41 Functioning as a gatekeeper of this developmental signaling transition, the exosome

42 complex controls the massive production of erythroid cells that ensures organismal

43 survival in homeostatic and stress contexts.

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2 52 INTRODUCTION

53 The highly conserved exosome complex, an RNA-degrading and processing

54 machine, is expressed in all eukaryotic cells (Januszyk and Lima, 2014; Kilchert et al.,

55 2016). The first subunit was discovered from an analysis of mechanisms controlling yeast

56 rRNA synthesis. Mutations of Exosc2 (Rrp4) disrupt 5.8S rRNA 3’-end processing (Mitchell

57 et al., 1996). Exosc2 assembles into a complex with components homologous to bacterial

58 3’ to 5’ (PNPase) (Mitchell et al., 1997). Nine exosome complex subunits

59 form a cylindrical core consisting of the RNA binding subunits Exosc1 (Csl4), Exosc2

60 (Rrp4) and Exosc3 (Rrp40), which cap a ring formed by Exosc4 (Rrp41), Exosc5 (Rrp46),

61 Exosc6 (Mtr3), Exosc7 (Rrp42), Exosc8 (Rrp43) and Exosc9 (Rrp45) (Liu et al., 2006;

62 Makino et al., 2013; Makino et al., 2015; Wasmuth et al., 2014) (Figure 1A). Despite

63 homology with bacterial PNPases, the vertebrate core subunits lack RNA-degrading

64 activity (Liu et al., 2006). Whereas Dis3 (Rrp44) (nuclear) and Dis3L (cytoplasmic) catalytic

65 subunits bind the same position of the core complex, adjacent to Exosc4 and Exosc7, the

66 predominantly nuclear catalytic subunit Exosc10 (Rrp6) binds the opposite site

67 (Dziembowski et al., 2007; Makino et al., 2015) (Figure 1A). The catalytic subunits, which

68 may function redundantly in certain contexts, mediate RNA degradation and/or processing

69 (Kilchert et al., 2016). Unlike Dis3L and Exosc10, which are strictly , Dis3

70 is also an (Lebreton et al., 2008; Tomecki and Dziembowski, 2010). The

71 core subunits, except Exosc1, are considered to confer structural integrity (Liu et al., 2006).

72 The apparent diversity of exosome complex-regulated RNAs (Schneider et al.,

73 2012) suggests the complex controls a plethora of cellular processes. Exosome complex

74 subunits regulate cell differentiation and are implicated in human pathologies. Exosc8,

75 Exosc9 and Dis3 suppress erythroid maturation of primary murine erythroid precursor cells

3 76 (McIver et al., 2014). EXOSC7, EXOSC9 and EXOSC10 maintain human epidermal

77 progenitor function (Mistry et al., 2012). In principle, the exosome complex might control

78 differentiation through complex RNA remodeling mechanisms or by targeting factors

79 mediating the balance between self-renewal and lineage commitment,

80 proliferation/amplification or terminal differentiation. DIS3 has been implicated as a tumor

81 suppressor mutated in multiple myeloma (Chapman et al., 2011) and is overexpressed in

82 colorectal cancer (de Groen et al., 2014). EXOSC8, EXOSC3 or EXOSC2 mutations cause

83 syndromes with complex phenotypes including neurological defects, cerebellar hypoplasia,

84 retinitis pigmentosum, progressive hearing loss and premature aging (Boczonadi et al.,

85 2014; Di Donato et al., 2016; Wan et al., 2012).

86 As downregulating exosome complex subunits can yield compelling phenotypes,

87 and exosome complex subunit mutations yield pathologies, it is instructive to consider

88 whether the phenotypes reflect complex disruption or subunit-specific activities. By

89 conferring exosome complex integrity, all exosome complex activities might require

90 structural subunits, or sub-complexes might have distinct functions (Kiss and Andrulis,

91 2011). The structural subunit requirement for complex stability in vitro (Liu et al., 2006),

92 and lethality due to loss of structural components in yeast (Allmang et al., 1999a; Allmang

93 et al., 1999b) support the importance of the intact complex. However, core subunit

94 downregulation revealed little overlap in the ensembles of regulated RNAs in Drosophila

95 (Kiss and Andrulis, 2010) and differentially influenced RNA processing in humans

96 (Tomecki et al., 2010). Moreover, Arabidopsis Exosc1, Exosc2 and Exosc4 knockouts

97 yielded distinct phenotypes (Chekanova et al., 2007).

98 While investigating these models in the context of erythroid maturation, we

99 discovered that Exosc8 or Exosc9 downregulation disrupted /protein interactions

4 100 within the complex and greatly decreased expression of the receptor tyrosine kinase Kit.

101 Loss of Stem Cell Factor (SCF)-induced Kit signaling occurred concomitant with

102 precocious acquisition of erythropoietin signaling, which drives erythroid maturation. As Kit

103 stimulates erythroid precursor cell proliferation, our results establish a paradigm in which

104 the exosome complex regulates a receptor tyrosine kinase to orchestrate a vital

105 developmental signaling transition dictating proliferation/amplification versus differentiation.

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5 124 RESULTS

125 Dismantling protein-protein interactions within the exosome complex

126 Previously, we demonstrated that downregulating exosome complex subunits

127 (Exosc8, Exosc9 or Dis3) in murine fetal liver erythroid precursor cells induced erythroid

128 maturation (McIver et al., 2014). Analogous to Exosc8 and Exosc9 (McIver et al., 2014),

129 experiments in which Exosc3 expression is impaired suggest that this protein also

130 suppresses maturation of primary murine fetal liver lineage-negative hematopoietic

131 precursor cells. In particular, Exosc3 downregulation using two distinct shRNAs increased

132 the R4 (late basophilic/orthochromatic erythroblasts) cell population 9 fold (p = 0.006 and p

133 = 0.01 for the two shRNAs, respectively) (Figure 1 - figure supplement 1). However, it

134 remains unclear whether the single subunit perturbations impact exosome complex

135 integrity. To address this, we developed a co- assay to test whether

136 individual components mediate complex integrity (Figure 1A). Using the X-ray crystal

137 structure of the exosome complex as a guide (Liu et al., 2006), the strategy involved

138 testing whether downregulating endogenous Exosc8 or Exosc9 alter interactions between

139 endogenous Exosc2 and Exosc3, subunits that do not interact directly in the complex

140 (Figure 1A). As Exosc2 and Exosc3 are only expected to co-immunoprecipitate when

141 residing in the complex or a sub-complex, the extent of co-immunoprecipitation constitutes

142 a metric of complex integrity.

143 We conducted the protein/protein interaction analysis in G1E cells stably expressing

144 a conditionally active GATA-1 allele (G1E-ER-GATA-1), which mimic a normal erythroid

145 precursor cell, the proerythroblast (Weiss et al., 1997). Estradiol activation of ER-GATA-1

146 induces an erythroid transcriptional program and recapitulates a physiological window of

147 erythroid maturation (Welch et al., 2004). G1E-ER-GATA-1 cells were infected with

6 148 retroviruses expressing control (luciferase) shRNA or shRNAs targeting Exosc8 or Exosc9.

149 Whole cell lysates prepared 48 hours post-infection were immunoprecipitated with anti-

150 Exosc3 or isotype-matched control antibody, and Western blotting was conducted with

151 anti-Exosc2 antibody. Whereas knocking down Exosc8 or Exosc9 by ~75% (Figure 1B) did

152 not alter Exosc2 levels in the input, the knockdowns reduced the amount of Exosc2

153 recovered with the anti-Exosc3 antibody (Figure 1C). Densitometric analysis indicated that

154 Exosc8 and Exosc9 knockdowns reduced the amount of Exosc2 co-immunoprecipitated

155 with Exosc3 by 58 (p = 0.007) and 87% (p = 1.3 x 10-4), respectively (Figure 1C, right). Of

156 relevance to this result, yeast Exosc8 (Rrp43) mutations decrease exosome complex

157 stability and RNA binding (Lourenco et al., 2013). As Exosc8 or Exosc9 downregulation

158 disrupted Exosc3-Exosc2 interactions that only occur in the complex, erythroid maturation

159 resulting from downregulating either of these subunits is associated with dismantling or

160 destabilizing intra-complex protein-protein interactions.

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162 Exosome complex-regulated signaling transition dictates proliferation versus

163 differentiation

164 Since Exosc8 or Exosc9 downregulation disrupts the exosome complex and

165 promotes erythroid maturation, we investigated how integrity of the complex creates an

166 erythroid maturation barricade. Although the parameters dictating the decision of whether

167 an erythroid precursor cell undergoes sustained proliferation or differentiates into an

168 erythrocyte are incompletely understood, cytokine signaling is a key determinant (Lodish et

169 al., 2010). It is instructive to consider the relationship between the exosome complex-

170 mediated erythroid maturation barricade and signaling mechanisms that orchestrate

171 proliferation versus differentiation. In principle, the complex might enhance signaling that

7 172 favors proliferation or oppose signaling mediating differentiation. Whereas SCF/Kit

173 signaling supports hematopoietic stem/progenitor cell (HSPC) and erythroid precursor cell

174 proliferation and survival (Lennartsson and Ronnstrand, 2012), erythropoietin (Epo)/Epo

175 receptor signaling uniquely promotes terminal differentiation (Munugalavadla and Kapur,

176 2005; Wu et al., 1995b). Epo and SCF/Kit can synergistically promote proliferation and

177 survival (Joneja et al., 1997; Muta et al., 1994; Sui et al., 1998; Wu et al., 1997).

178 To test whether the exosome complex and Epo signaling are functionally

179 interconnected, murine fetal liver erythroid precursor cells were infected with control or

180 Exosc8 shRNA-expressing retroviruses. After culturing cells for 48 hours in media

181 containing increasing amounts of Epo (0-0.5 U/ml), we conducted flow cytometric analysis

182 with Annexin V and the membrane-impermeable dye DRAQ7 to quantitate live cells

183 (DRAQ7-/Annexin V-), as well as late (DRAQ7+/Annexin V+) and early (DRAQ7-/Annexin

184 V+) apoptosis (Figures 2A, B). Without exogenous Epo, live cells decreased from 66 to

185 24% (control versus knockdown cells) (p = 2 x 10-5), and late apoptotic cells increased

186 proportionally (24 to 67%, p = 3 x 10-4). Exosc8 downregulation increased late apoptosis

187 two-fold (p = 0.02) when cells were cultured in media containing U / m l E p o .

188 Exosc8 downregulation did not significantly influence the percentage of live and late

189 apoptotic cells when cells were cultured with higher Epo concentrations. Surprisingly,

190 downregulating Exosc8 rendered cell integrity hypersensitive to limiting concentrations of

191 Epo.

192 As downregulating Exosc8 promotes erythroid maturation, the commencement

193 and/or progression of maturation when Epo is limiting might be incompatible with cell

194 survival. Alternatively, lowering Epo might impair a mechanism that supports erythroid

195 precursor proliferation, independent of enhanced maturation; thus, precursor cell survival

8 196 would be compromised. Using flow cytometry to quantitate the erythroid cell surface

197 markers Ter119 and CD71, we determined the impact of Exosc8 downregulation on

198 erythroid precursor cell maturation under normal and Epo-limiting conditions. In this

199 expansion culture, control cells were largely unaffected by limiting Epo. Whereas Exosc8

200 downregulation stimulated erythroid maturation (1.5 fold increase in R2 to R3 transition, p

201 = 0.003) in media containing 0.5 U/ml Epo, maturation of Exosc8-knockdown cells did not

202 proceed without exogenous Epo (Figures 3A, B). Without Epo, Exosc8 downregulation

203 also increased the percentage of immature erythroid precursors (R1). Morphological

204 analysis of DRAQ7- cells using Giemsa stain provided further evidence for the Epo-

205 dependent R2 to R3 transition resulting from Exosc8 knockdown (Figure 3C). The

206 exosome complex-dependent erythroid maturation barricade was also quantitated using an

207 alternative flow cytometric assay based on CD44 staining and cell size. Exosc8 or Exosc9

208 downregulation induced accumulation of more mature erythroid cells when cultured in 0.5

209 U/ml Epo (Figure 3 – figure supplement 1). Orthochromatic erythroblasts (gate IV)

210 increased 8 fold (p = 0.006) or 7 fold (p = 3.5 x 10-5) post-Exosc8 or -Exosc9

211 downregulation, respectively. Erythrocytes (gate VI) increased 2 fold (Exosc8, p = 0.005,

212 Exosc9, p = 0.005). Immature erythroid cells (proerythroblasts and basophilic erythroblasts,

213 gates I and II) decreased proportionally. Exosc8 downregulation reduced the number of

214 early SCF, IL-3 and Epo-dependent BFU-E (Burst Forming Unit – Erythroid) colonies by 10

215 fold (p = 4.7 x 10-10). However, the number of Epo dependent CFU-E (Colony Forming Unit

216 – Erythroid) colonies was unaffected (Figure 3D).

217 Erythroid maturation instigated by Exosc8 downregulation was Epo-dependent. We

218 tested whether Exosc8 downregulation corrupted a proliferation/amplification mechanism,

219 thereby promoting maturation. SCF signaling supports erythroid precursor proliferation

9 220 (Munugalavadla and Kapur, 2005), whereas Epo signaling promotes maturation (Wu et al.,

221 1995b). SCF and Epo can synergistically promote proliferation (Joneja et al., 1997; Sui et

222 al., 1998; Wu et al., 1997). Using a phospho-flow cytometry assay (Figure 4A) (Hewitt et

223 al., 2015), we quantitated the capacity of SCF (Figure 4B) or Epo (Figure 4C) to instigate

224 cell signaling using the shared downstream substrate Akt. SCF induced maximal Akt

225 phosphorylation in immature erythroblasts (Ter119-/CD71high) (5.5 fold, p = 3 x 10-6). As

226 erythroid maturation progressed to Ter119+/CD71high and Ter119+/CD71low stages, the

227 SCF response was diminished (Figure 4B). Exosc8 downregulation abrogated SCF-

228 mediated induction of phospho-Akt (Figure 4B). Epo induced maximal Akt phosphorylation

229 in Ter119+/CD71high erythroblasts, and Exosc8 downregulation accelerated acquisition of

230 this signaling response. Whereas Epo did not affect Akt phosphorylation in control Ter119-

231 /CD71high erythroblasts, Epo increased phospho-Akt 4 fold (p = 0.003) in Exosc8-

232 knockdown Ter119-/CD71high erythroblasts (Figure 4C). Similar results were obtained using

233 a phospho-flow cytometric assay to quantitate phosphorylation of ERK, an additional

234 shared downstream effector of SCF and Epo signaling, although phospho-ERK was higher

235 in the unstimulated Exosc8 condition in comparison with the control condition (Figure 4 –

236 figure supplement 1). Thus, downregulating an exosome complex subunit that dismantles

237 intra-complex protein-protein interactions abrogates SCF signaling that supports precursor

238 proliferation/amplification, while precociously inducing pro-differentiation Epo signaling. By

239 orchestrating this developmental signaling transition, the exosome complex ensures a

240 balance between proliferation/amplification and differentiation - a balance that shifts

241 physiologically towards differentiation as GATA-1 represses genes encoding exosome

242 complex subunits (McIver et al., 2014). Artificially, shRNA-mediated downregulation of

243 exosome complex subunits, which impairs exosome complex integrity, skews the balance.

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245 Mechanism governing exosome complex-regulated developmental signaling:

246 transcriptional induction of an essential receptor tyrosine kinase

247 As downregulating Exosc8 abrogated SCF-induced Kit signaling, we tested whether

248 Exosc8 is required for Kit expression in the erythroblast plasma membrane. Using flow

249 cytometry with primary fetal liver erythroblasts, R2 erythroblasts expressed the highest

250 levels of cell surface Kit, and Exosc8 downregulation reduced cell surface Kit to a nearly

251 undetectable level (Figure 5A). Similarly, Exosc9 downregulation reduced cell surface Kit 6

252 fold in R2 cells (Figure 5B). We tested whether exosome complex disruption reduced Kit

253 transcription, total Kit protein and/or Kit transit to the cell surface. Exosc8 downregulation

254 reduced Kit mRNA and primary transcript levels at all stages of erythroid maturation

255 (Figure 5C). By 24 h post-infection with shExosc8-expressing retrovirus, total Kit protein

256 declined 10 fold (p = 0.046) in Ter119- erythroid precursor cells (Figure 5C).

257 To further dissect the mechanism underlying exosome complex-dependent

258 expression of Kit and establishment of SCF-induced Kit signaling, we tested whether

259 exosome complex disruption influenced other genes downregulated during erythroid

260 maturation. Since GATA-2 directly activates Kit transcription in immature erythroblasts,

261 and GATA-1 directly represses Kit transcription during maturation (Jing et al., 2008;

262 Munugalavadla et al., 2005), we tested whether exosome complex disruption impacted

263 GATA-2 and GATA-1 levels. Exosc8 downregulation modestly increased Gata2 mRNA in

264 R3 cells, but GATA-2 protein levels were not significantly affected in Ter119- cells (Figure

265 5D). Previously, we demonstrated that Exosc8 knockdown did not influence Gata1 mRNA

266 levels (McIver et al., 2014). GATA-1 increased slightly in Exosc8-knockdown Ter119- cells

267 (1.4 fold, p = 0.02) (Figure 5D). GATA-2 activates transcription of Samd14, encoding a

11 268 facilitator of SCF/Kit signaling (Hewitt et al., 2015). In certain contexts, Kit signaling creates

269 a positive autoregulatory loop that increases Kit transcription (Zhu et al., 2011).

270 Downregulating Exosc8 had little to no effect on Samd14 expression (Figure 5D). To

271 assess whether Exosc8 downregulation reduced expression of genes resembling Kit in

272 being transcriptionally downregulated upon maturation, we quantitated expression of Vim

273 (encoding vimentin) (DeVilbiss et al., 2015) and the GATA-2 target Hdc (encoding

274 histidine decarboxylase) (Katsumura et al., 2014). Downregulating Exosc8 did not affect

275 the maturation-dependent reduction in Vim or Hdc mRNA levels (Figure 5D). The Exosc8

276 requirement for Kit transcription and expression of functional, cell surface Kit therefore

277 does not involve a major alteration in GATA factor levels nor a general Exosc8 activity to

278 sustain expression of genes destined for downregulation upon erythroid maturation.

279 Previously, we demonstrated that exosome complex disruption induces erythroid

280 precursor cells to arrest in the G1 phase of the cell cycle and is associated with increased

281 expression of genes promoting (or implicated in promoting) cell cycle arrest (McIver et al.,

282 2014). As erythroid maturation requires cell cycle progression (Pop et al., 2010), we asked

283 whether Kit downregulation precedes, is concomitant with, or a consequence of GATA-1

284 and Exosc8-mediated regulation of cell cycle arrest genes. At 24 h post-infection of fetal

285 liver erythroid precursor cells with shRNA-expressing retrovirus, Exosc8 mRNA declined

286 by 70% (p = 0.005). Whereas Kit mRNA decreased by 46% (p = 0.002), expression of the

287 GATA-1/Exosc8-regulated cell cycle-regulatory genes Cdkn1b (p27Kip1), Ddit3, Gadd45a,

288 Gas2l1 and Trp53inp1 was not significantly altered (Figure 5E). Analysis of the cell cycle

289 status of Kit-expressing Ter119- erythroid precursor cells 24 and 72 h post-infection

290 revealed only a slight increase in the percentage of G1 cells at 24 h (30% in control and

291 33.5% in Exosc8-knockdown cells (p = 2 x 10-6)). At 72 h post-infection, 49.5% of Exosc8-

12 292 knockdown Ter119- cells were in G1, in comparison with 36% of control cells (p = 1.6 x 10-

293 6) (Figure 5F). Thus, the prominent cell cycle arrest resulting from Exosc8 downregulation

294 occurs after Kit is repressed. To more definitively establish the relationship between Kit

295 downregulation and cell cycle arrest, we tested whether Exosc8 downregulation reduces

296 Kit expression in a system not competent for differentiation. shRNA-mediated Exosc8

297 downregulation in GATA-1-null G1E proerythroblast-like cells reduced Kit mRNA by 37%

298 (p = 0.002) (Figure 5G), and Kit cell surface expression by 44% in the GFP+ population (p

299 = 3.3 x 10-8). Importantly, cell surface Kit was unaffected in the GFP- population (Figure

300 5H). In aggregate, these analyses support a model in which exosome complex disruption

301 downregulates Kit expression independent of alterations in cell cycle status and is not a

302 consequence of cell cycle arrest or cellular maturation. Consistent with these results,

303 mining RNA-seq data from normal and Exosc3-/- ES cells (Pefanis et al., 2015) revealed

304 Kit expression 5.9 fold lower in mutant versus control cells (transcripts per million, false

305 discovery rate < 0.05).

306 We tested whether the Exosc8 requirement for Kit primary transcript, mRNA and

307 protein expression involved alterations in the distribution of transcriptionally-competent

308 serine 5-phosphorylated RNA polymerase II (Pol II) at Kit. Using quantitative ChIP analysis

309 with control and Exosc8-knockdown Ter119- cells, Exosc8 downregulation reduced

310 phospho-Ser5 Pol II occupancy within the coding region (+5 kb) and 3’ UTR, but not at the

311 promoter (Figure 5E). Exosc8 downregulation did not alter phospho-Ser5 Pol II occupancy

312 at the active Rpb1 gene or the inactive Krt5 gene (Figure 6A). These results provide

313 further evidence that exosome complex-mediated Kit expression involves a transcriptional

314 mechanism. As GATA-1 levels increased slightly after Exosc8 downregulation in Ter119-

315 cells, and GATA-1 represses Kit transcription, we tested whether GATA-1 occupancy at Kit

13 316 was altered. Exosc8 downregulation did not influence GATA-1 occupancy at -114, +5 and

317 +58 kb sites, relative to the promoter (Figure 6A). The nearly complete Kit repression upon

318 Exosc8 downregulation did not involve detectable changes in GATA-1 occupancy.

319 In Drosophila, a genome-wide analysis of Exosc10 (Rrp6) and Exosc3 (Rrp40)

320 chromatin occupancy revealed occupancy predominantly at chromatin insulators and

321 promoters of active genes (Lim et al., 2013). However, considering that the exosome

322 complex associates with elongating RNA polymerase II (Andrulis et al., 2002) and rapidly

323 degrades promoter upstream transcripts (PROMPTs), which are believed to be generated

324 at many genes (Lubas et al., 2011; Preker et al., 2011; Preker et al., 2008), one might

325 predict that the exosome complex has a broader distribution in and surrounding genes. To

326 investigate the mechanism by which the exosome complex confers Kit transcription and

327 suppresses Alas2, Hbb-b1 and Slc4a1 transcription (McIver et al., 2014), we asked

328 whether the exosome complex occupies these loci. Quantitative ChIP analysis with fetal

329 liver erythroid precursor cells expanded for 48 h revealed endogenous Exosc9 occupancy

330 at the Kit promoter, the coding region and the 3’-UTR, with occupancy higher at the

331 promoter and coding region sites versus the 3-UTR (Figure 6B). Exosc9 also occupied the

332 Alas2, Slc4a1 and Hbb-b1 promoters, with less occupancy at the MyoD promoter (Figure

333 6B). Exosc9 occupancy implies that the exosome complex directly regulates transcription

334 of these loci.

335 Although Kit downregulation correlated with erythroid maturation induced by

336 exosome complex disruption, whether this reflects causation was unclear. To establish

337 whether Kit downregulation is required for erythroid maturation induced by exosome

338 complex disruption, we asked whether enforced Kit expression in Exosc8-knockdown

339 Ter119- cells opposed differentiation. At 24 h post-infection, there was little to no difference

14 340 in the maturation state of control and Exosc8-knockdown cells (Figures 7A, B). By 48 h,

341 Exosc8 downregulation increased R3 cells from 35 to 63% (p = 4 x 10-7), while reducing

342 R2 cells 2-fold (p = 4 x 10-5). Kit expression prevented Exosc8 knockdown-dependent

343 maturation, reducing R3 cells from 63 to 27% (p = 1 x 10-7). Kit expression, concomitant

344 with Exosc8 downregulation, rescued R2 cells (increased from 21 to 42%, p = 8 x 10-4) to a

345 level indistinguishable from control cells (45%). At 48 h, Kit expression in control cells

346 increased R2 cells from 45 to 66% (p = 6 x 10-5). At 72 h, Exosc8-knockdown cells

347 matured further (18% R4, 56% R3 and 17% R2 versus 2% R4 (p = 4 x 10-5), 41% R3 (p =

348 3 x 10-5), and 47% R2 (p = 3 x 10-6) for control cells). Expressing Kit in Exosc8-knockdown

349 cells reduced R4 from 18 to 7% (p = 4 x 10-5), R3 from 56 to 28% (p = 9 x 10-7), while

350 increasing R2 from 17 to 38% (p = 5 x 10-5). At 72 h, Kit expression increased R2 cells

351 from 47 to 68% (p = 6 x 10-5).

352 We evaluated the relationship between the level of Kit required to oppose erythroid

353 maturation, caused by exosome complex disruption, and endogenous Kit expression.

354 Cells were infected with a range of retrovirus concentrations to establish the amount

355 required to restore Kit to the endogenous level after Exosc8 downregulation. Exosc8

356 downregulation reduced Kit MFI 6 fold (p = 0.001). At 48 h post-infection, 50 μl of Kit

357 retroviral supernatant (half of that used in the time-course) increased Kit cell surface

358 expression in Exosc8-knockdown cells to approximately the endogenous level (1.2 fold

359 higher than control). Under these conditions, infection of control cells increased Kit cell

360 surface expression 3 fold (p = 0.002). Exosc8 downregulation, without ectopic Kit

361 expression, increased the percentage of R3 cells ~2 fold (p = 0.003) 48 h post-infection.

362 The increased R3 population cells induced by Exosc8 downregulation was prevented by

363 enforced Kit expression at 1.2 fold higher than the endogenous level (control R3, 19.7%

15 364 versus Exosc8/Kit R3, 16.5%). A 3 fold increase in Kit cell surface expression in the control

365 condition decreased the R3 population cells from 19.7% to 6% (p = 0.001) (Figure 7C). As

366 enforced Kit expression negated erythroid maturation induced by exosome complex

367 disruption, the exosome complex activity to establish and/or maintain Kit expression is a

368 critical step in the exosome complex-dependent maturation process.

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389 DISCUSSION

390 Regulating the proliferation and differentiation balance of stem and progenitor cells

391 through intrinsic and extrinsic mechanisms ensures normal development and physiology.

392 Whereas exaggerated proliferation of stem and/or progenitor cells can underlie cancer (He

393 et al., 2004), a differentiation bias can exhaust the precursor cells. As the intestinal

394 epithelial layer is replaced every 4-5 days, the proliferation versus differentiation balance of

395 intestinal stem cells must be exquisitely regulated (Clevers et al., 2014). Wnt signaling

396 promotes intestinal stem cell self-renewal, while Bone Morphogenetic Protein (BMP)

397 signaling suppresses self-renewal and promotes differentiation (Clevers et al., 2014; He et

398 al., 2004).

399 In hematopoiesis, SCF signaling promotes erythroid precursor cell proliferation at

400 the expense of differentiation (Haas et al., 2015; Muta et al., 1995). Epo provides a pro-

401 differentiation stimulus (Wu et al., 1995b). During stress erythropoiesis, Epo, SCF and

402 glucocorticoids synergistically promote erythroid cell expansion (Kolbus et al., 2003;

403 Wessely et al., 1997) and Kit downregulation promotes maturation (Haas et al., 2015;

404 Munugalavadla et al., 2005). Epo and Kit can synergize (Joneja et al., 1997; Sui et al.,

405 1998; Wu et al., 1995a) or be antagonistic (Haas et al., 2015; Joneja et al., 1997;

406 Kosmider et al., 2009). Kit signaling induces Epo receptor tyrosine phosphorylation (Wu et

407 al., 1995a), although synergistic enhancement of proliferation may involve convergence of

408 SCF and Epo signals on ERK1/2 via distinct pathways (Sui et al., 1998). Constitutively

409 active Kit inhibits Epo-mediated Akt phosphorylation, increasing apoptosis in mature

410 erythroid cells (Haas et al., 2015). GATA-1 and the cooperating transcription factor

411 Scl/TAL1 repress Kit transcription as erythroblasts acquire Epo-dependence

17 412 (Munugalavadla et al., 2005; Vitelli et al., 2000).

413 While multiple pathways are implicated in controlling the proliferation and

414 differentiation balance of stem and progenitor cells, many questions remain regarding how

415 pathways intersect or function in parallel. Herein, we demonstrated that the exosome

416 complex is a critical determinant of the proliferation versus differentiation balance of

417 erythroid precursor cells, and many unanswered questions existed regarding how this

418 balance is controlled (Figure 8). The exosome complex establishes the balance by

419 ensuring expression of a receptor tyrosine kinase, Kit, which is essential for proliferation,

420 under conditions in which progenitors are not competent to transduce Epo pro-

421 differentiation signals. Disrupting exosome complex integrity downregulated Kit and SCF-

422 mediated proliferation signaling, while inducing Epo signaling (Figure 8). Physiologically,

423 GATA-1 represses expression of exosome complex subunits, instigating the

424 developmental signaling transition. Reduced SCF signaling upon exosome complex

425 disruption would be predicted to underlie or contribute to activation of Epo signaling in

426 erythroid precursor cells (Figures 4, 5) and promote erythroid differentiation (Figures 3, 7).

427 Our results establish a paradigm in which an RNA-regulatory machine orchestrates the

428 balance between opposing developmental signaling pathways. Considering the apparent

429 ubiquitous expression of the exosome complex, it is attractive to propose that the

430 paradigm may operate to control additional progenitor cell transitions.

431 Mice homozygous for the W mutation (white spotting) in Kit die perinatally from

432 severe macrocytic anemia (Bernstein et al., 1990; Waskow et al., 2004). The anemia likely

433 reflects the Kit requirement for HSPC genesis, maintenance and function (Bernstein et al.,

434 1990; Ding et al., 2012). Our analyses with primary fetal liver hematopoietic precursors

435 cultured under conditions that support erythroid precursor growth and differentiation

18 436 demonstrated that Exosc8 downregulation reduces Kit expression and signaling in

437 erythroid precursors. These findings would not have been predicted from prior work with W

438 mutant multipotent hematopoietic precursors in vivo, given the impact of Kit signaling on

439 multiple cell types.

440 Exosome complex functions have expanded considerably since the discovery of its

441 role in rRNA maturation (Mitchell et al., 1996) to include coding and non-coding RNA

442 degradation and processing (Kilchert et al., 2016; Schneider et al., 2012). The exosome

443 complex is enriched at actively transcribed genes (Andrulis et al., 2002; Lim et al., 2013),

444 regulates transcription start site usage (Jenks et al., 2008; Kuehner and Brow, 2008),

445 influences heterochromatin and post-transcriptional gene silencing (Eberle et al., 2015)

446 and regulates superenhancer activity (Pefanis et al., 2015). Gene promoters are

447 bidirectional in nature (Andersson et al., 2015), and the exosome complex rapidly

448 degrades PROMPTs (Lubas et al., 2011; Preker et al., 2011; Preker et al., 2008).

449 Depletion of exosome complex components stabilizes PROMPTs and additional cryptic

450 transcripts present at gene loci, including PSARs (promoter-associated small RNAs),

451 TSARs (terminator-associated small RNAs), PALRs (promoter-associated long RNAs),

452 TSSa RNAs (transcription start site-associated RNAs) and eRNAs (enhancer RNAs)

453 (Jensen et al., 2013; Lubas et al., 2015; Pefanis et al., 2015). The exosome complex is

454 also implicated in transcriptional regulation, and since regulatory RNAs can be integral

455 components of transcriptional mechanisms (Belostotsky, 2009; Jensen et al., 2013), this

456 can be explained, in part, through RNA-regulatory activity. While knowledge of molecular

457 mechanisms underlying exosome complex function are sophisticated, many questions

458 remain unanswered regarding its functions in distinct cellular contexts during development,

459 physiology and pathologies.

19 460 Genes encoding exosome complex components are linked to pathologies including

461 cancer (DIS3) (Chapman et al., 2011), immune disorders (EXOSC9 and EXOSC10)

462 (Allmang et al., 1999b) and congenital neurological disorders (EXOSC3 and EXOSC8)

463 (Boczonadi et al., 2014; Rudnik-Schoneborn et al., 2013; Wan et al., 2012). Despite these

464 human disease links, the apparent diversity of RNA targets and multiple functions of the

465 exosome complex, little is known about its role in cell fate decisions. Downregulating

466 EXOSC10, EXOSC9 or EXOSC7 expression caused differentiation of human epididymal

467 progenitor cells. Mechanistically, the exosome complex represses GRHL3, a key

468 transcription factor promoting epidermal differentiation (Mistry et al., 2012).

469 In summary, we have described the fundamental importance of the exosome

470 complex to orchestrate a critical developmental signaling transition that determines

471 whether erythroid precursors differentiate. Given the importance of ascribing biological

472 functions for regulatory RNAs, the machinery mediating their and exosome

473 complex functions to process diverse RNAs, it will be instructive to identify exosome

474 complex-regulated RNA ensembles mediating cell fate transitions. Furthermore, we

475 anticipate that the results described herein can be leveraged to improve strategies for the

476 industrial-level generation of erythroid cells to achieve therapeutic goals. Finally,

477 considering the common occurrence of Kit-activating mutations in (Lennartsson

478 and Ronnstrand, 2006), it will be of considerable interest to analyze the paradigm

479 described herein in physiological states in vivo, to determine if it can be extrapolated to

480 cancer, and to establish whether the exosome complex constitutes a promising therapeutic

481 target for Kit-driven pathologies.

482

483

20 484

485 MATERIALS AND METHODS

486 Generation of 3D exosome complex structures

487 Protein structure coordinate files for the human exosome complex (Liu et al., 2006) were

488 downloaded from the Research Collaboratory for Structural Bioinformatics Protein Data

489 Bank (www.RCSB.org, accession number 2NN6). Images were generated using PyMOL

490 (www.PyMOL.org).

491 Primary erythroid precursor cell isolation

492 Primary erythroid precursors were isolated from E14.5 mouse C57BL/6 fetal livers using

493 EasySep negative selection Mouse Hematopoietic Progenitor Cell Enrichment Kit

494 (StemCell technologies, Vancouver, Canada) as described (McIver et al., 2014).

495 Cell culture

496 G1E-ER-GATA-1 (RRID:CVCL_D047) cells were cultured in Iscove’s Modified Dulbecco’s

497 Medium (IMDM) (ThermoFisher, Waltham, MA) containing 15% FBS (Gemini, West

498 Sacramento, CA), 1% antibiotic/antimycotic (Corning, Tewksbury MA), 2 U/ml

499 erythropoietin, 120 nM monothioglycerol (Sigma, St, Louis, MO), 0.6% conditioned

500 medium from an SCF producing CHO cell line, and 1 μg/ml puromycin (Gemini) (Fujiwara

501 et al., 2009). G1E (RRID:CVCL_D046) cells were cultured without puromycin.

502 G1E cells were derived from GATA-1-null murine ES cells. ES cells were cultured under

503 conditions that promoted the development of definitive erythroid cells (Weiss et al., 1997).

504 G1E-ER-GATA-1 cells are G1E cells stably expressing GATA-1 fused to the ligand-binding

505 domain of human estrogen receptor (Gregory et al., 1999). G1E and G1E-ER-GATA-1

506 cells were a kind gift from Dr. Mitchell J. Weiss (St. Judes).

507

21 508 Fetal liver erythroid precursors cells were cultured in StemPro-34 (ThermoFisher) with 1x

509 nutrient supplement (ThermoFisher), 2 mM glutamax (ThermoFisher), 1% penicillin-

510 streptomycin (ThermoFisher), 100 μM monothioglycerol (Sigma), 1 μM dexamethasone

511 (Sigma), 0.5 U/ml of erythropoietin, and 1% conditioned medium from a kit ligand

512 producing CHO cell line. Cells were cultured in a humidified incubator at 37°C (5% carbon

513 dioxide).

514 shRNA-mediated knockdown.

515 The vectors expressing MiR-30 context luciferase, murine Exosc8 (Rrp43), Exosc9

516 (Rrp45) shRNAs were described (McIver et al., 2014).

517 MiR-30 context shExosc3 1 sequence:

518 TGCTGTTGACAGTGAGCGAACTGGCAGAGAGTTGACATATTAGTGAAGCCACAGAT

519 GTAATATGTCAACTCTCTGCCAGTCTGCCTACTGCCTCGGA.

520 MiR-30 context shExosc3 2 sequence:

521 TGCTGTTGACAGTGAGCGCAAGACCATTCAGCAGACGTTATAGTGAAGCCACAGAT

522 GTATAACGTCTGCTGAATGGTCTTATGCCTACTGCCTCGGA.

523 Bold sequences denote sense and antisense sequences.

524 Wild type Kit expression vector was a kind gift from Ruben Kapur. G1E-ER-GATA-1 cells

525 (5 x 105), and primary murine fetal liver erythroid precursors (2 x 105) were spinfected

526 (McIver et al., 2014).

527 Protein Co-Immunoprecipitation

528 48 h post-infection 20 x 106 G1E-ER-GATA1 cells were harvested and resuspended in 500

529 μl NP40 lysis buffer (50 mM Tris pH 8 (Sigma), 150 mM NaCl (Sigma), 5 mM DTT (Sigma),

530 0.5% NP40 (Sigma) supplemented with protease inhibitors (0.2mM PMSF (Sigma),

531 20μg/ml leupeptin (Roche, Basel, Switzerland)), 50 μg/ml RNase A (Sigma) and 2 μl/ml

22 532 DNase I (ThermoFisher). Cells were lysed on ice for 30min and insoluble cell debris were

533 removed by centrifugation. After taking an input sample (25 μl) the lysate was pre-cleared

534 with 20 μl protein-A Agarose (Sigma) and 5 μl rabbit pre-immune sera for 1 h at 4°C. After

535 pelleting the protein-A Agarose, the supernatant was incubated with 5 μg rabbit IgG or 5

536 μg rabbit anti-Exosc3 (Abcam, San Francisco CA, Ab156683) overnight at 4°C, before

537 addition of a 15 μl protein-A Agarose pellet for a further 2 h. The protein-A Agarose pellet

538 was washed once with NP40 lysis buffer and three times with TEG buffer (10 mM Tris pH

539 7.6, 50 mM NaCl, 4 mM EDTA, 5 mM DTT, 10% Glycerol). The protein-A Agarose was

540 resuspended in 50 μl of 2x SDS lysis buffer (25 mM Tris pH 6.8, 6% SDS, 4% β-

541 mercaptoethanol, 10% glycerol, 0.02% bromophenol blue), 25 μl of 2x SDS lysis buffer

542 was added to the input sample, and incubated at 100°C for 5 min. were resolved

543 on an 11% SDS-PAGE gel and Exosc2 (Abcam ab156698) was measured by semi-

544 quantitative Western blotting with ECL Plus (ThermoFisher).

545 Quantitative real-time RT-PCR

546 Total RNA was purified with Trizol (ThermoFisher). cDNA was prepared by annealing 1 μg

547 or 0.2 μg (sorted samples) of RNA with 250 ng of a 1:5 mixture of random hexamer and

548 oligo (dT) primers heated at 68°C for 10 min. This was followed by incubation with Murine

549 Moloney Leukemia Virus Reverse Transcriptase (ThermoFisher) with 10 mM DTT, RNasin

550 (Promega, Madison, Wi), and 0.5 mM dNTPs at 42°C for 1 h. The mixture was diluted to a

551 final volume of 100 μl and heat inactivated at 95°C for 5 min. cDNA was analyzed in

552 reactions (20 μl) containing 2 μl of cDNA, appropriate primers, and 10 μl of SYBR green

553 master mix (ThermoFisher). Product accumulation was monitored by SYBR green

554 fluorescence. A standard curve of serial dilutions of cDNA samples was used to determine

23 555 relative expression. mRNA levels were normalized to 18S rRNA. Primer sequences are

556 found in Supplementary File 1.

557 Flow cytometry and cell sorting

558 For quantitation of cell surface markers, 1 x 106 cells were stained in 100 μl PBS/10% FBS

559 with anti-mouse Ter119-APC (RRID:AB_469474) (1:100), CD71-PE (RRID:AB_465741)

560 (1:100) and Kit-PEcy7 (RRID:AB_469644) (1:100) (eBioscience, San Diego, CA), at 4°C

561 for 30 min in the dark. To quantitate apoptosis after CD71/Ter119 staining, cells were

562 washed in Annexin V Buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) then

563 stained with Annexin V-Pacific blue (ThermoFisher) (1:40) and DRAQ7 (Abcam) (1:100)

564 for 20 min in the dark at room temperature. To detect intracellular phosphorylated Akt,

565 erythroid precursor cells were expanded for 48 h and sorted into Ter119+ and Ter119-

566 populations using magnetic beads (StemCell technologies). Cells were serum-starved in

567 1% BSA in IMDM for 1 h at 37°C before stimulation with either 10 ng/ml SCF or 2 U/ml

568 Epo for 10 min and fixed in 2% paraformaldehyde for 10 min at 37°C. After

569 permeabilization overnight at -20°C in 95% methanol cells were incubated for 1 h in

570 HBSS/4% FBS at 4°C. Cells were stained with rabbit phospho-Akt or rabbit phospho-ERK

571 (1:200) for 30 min before incubation in goat anti rabbit-APC (1:200), Kit-PEcy7 (1:100) and

572 CD71-PE (1:100) for 30 min at room temperature. To analyze cell cycle of erythroid

573 precursor cells, fetal liver erythroid precursors were expanded for 24 or 72 h, and Ter119-

574 cells were isolated. Cells were fixed/permeabilized in 70% ethanol and stained with 5

575 μg/ml DAPI (Biolegend) overnight at -20°C. Cells were washed twice in PBS before

576 analysis. Samples were analyzed using a BD LSR II (BD Biosciences) or sorted into

577 distinct populations using a BD FACSAria II. DAPI or DRAQ7 (Abcam) were used for

24 578 apoptotic analyses, and for fixed cells, Zombie UV (Biolegend, San Diego, CA) staining

579 discriminated dead cells.

580 Colony Assay

581 R1 cells were FACS-sorted 24 h post-Exosc8-knockdown, and 5000 cells were plated in

582 duplicate in MethocultTM M3434 (StemCell Technologies) according to the manufacturer’s

583 instructions. CFU-E and BFU-E colonies were quantitated after culturing for 2 and 8 days,

584 respectively, at 37°C with 5% CO2

585 Total protein analysis

586 24 h post-Exosc8 knockdown, Ter119+ cells were depleted from the primary erythroid

587 precursor samples. Equal numbers of cells were boiled for 10 min in SDS lysis buffer.

588 Proteins were resolved by SDS-PAGE and incubated with rabbit anti-Kit (Cell Signaling,

589 Danvers, MA. D13A2 RRID:AB_1147633), rabbit anti-GATA-2 or rat anti-GATA-1 (Santa

590 Cruz, Dallas TX, sc-265 RRID:AB_627663).

591 Quantitative chromatin immunoprecipitation (ChIP) assay

592 Primary fetal liver erythroid precursor cells (2 x 106) were crosslinked with 1%

593 formaldehyde for 10 min. Lysates were immunoprecipitated with antibodies against

594 phospho-Ser5 Pol II (Covance, Princeton, NJ, H14 MMS-134R RRID:AB_10063994) or

595 GATA-1 (Grass et al., 2006) using rabbit pre-immune serum or control IgM as control. For

596 Exsoc9 ChIP, cells (8 x 106) were crosslinked, and lysates were immunoprecipitated with

597 anti-Exosc9 antibody (Novus Biologicals, Littleton, CO, NBP1-71702 RRID:AB_11026964)

598 using control IgG as control. DNA was quantitated by real-time PCR with SYBR green

599 fluorescence. Primers sequences used to assess protein occupancy are indicated in

600 Supplementary File 2.

601 Statistics

25 602 A Students T-test was used to compare experimental and control samples. When

603 comparing multiple groups, ANOVA was conducted to identify any significant variance

604 between samples, followed by a Tukey-Kramer test to identify statistical relationships

605 between each pair of samples within the experiment. All analysis was conducted using

606 JMP software (SAS Institute Inc.). Asterisks indicate significance relative to control, *

607 p<0.05, ** p<0.01 and *** p<0.001.

608

609 AUTHOR CONTRIBUTIONS

610 SCM, KRK and EHB designed experiments, analyzed data and wrote the manuscript.

611 SCM, KRK, and ED conducted experiments, PL conducted data mining and analysis. DY

612 conducted hematology analysis, and YK supplied reagents and edited the manuscript.

613

614 ACKNOWLEDGEMENTS

615 We thank R. Kapur for providing the wild type Kit expression vector and M.J. Weiss

616 for providing retroviral vectors and the cell culture protocol. This work was supported by

617 grants HL116365 and DK50107 to EHB and Cancer Center Support Grant P30 CA014520.

618 SCM was supported by an American Heart Association postdoctoral fellowship. The

619 authors have no conflict of interest to disclose.

620

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778 and are produced upstream of all three major types of mammalian promoters. Nucleic

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781 C.K., Schierup, M.H., and Jensen, T.H. (2008). RNA exosome depletion reveals

782 transcription upstream of active human promoters. Science 322, 1851-1854.

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797 and hDIS3L. EMBO J 29, 2342-2357.

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801 Wan, J., Yourshaw, M., Mamsa, H., Rudnik-Schoneborn, S., Menezes, M.P., Hong, J.E.,

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804 neuron degeneration. Nat Genet 44, 704-708.

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806 Rescue of lethal c-KitW/W mice by erythropoietin. Blood 104, 1688-1695.

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808 complex bound to poly(A) RNA. Nature 511, 435-439.

34 809 Weiss, M.J., Yu, C., and Orkin, S.H. (1997). Erythroid-cell-specific properties of

810 transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line. Mol

811 Cell Biol 17, 1642-1651.

812 Welch, J.J., Watts, J.A., Vakoc, C.R., Yao, Y., Wang, H., Hardison, R.C., Blobel, G.A.,

813 Chodosh, L.A., and Weiss, M.J. (2004). Global regulation of erythroid by

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815 Wessely, O., Deiner, E.M., Beug, H., and von Lindern, M. (1997). The glucocorticoid

816 receptor is a key regulator of the decision between self-renewal and differentiation in

817 erythroid progenitors. EMBO J 16, 267-280.

818 Wu, H., Klingmuller, U., Acurio, A., Hsiao, J.G., and Lodish, H.F. (1997). Functional

819 interaction of erythropoietin and stem cell factor receptors is essential for erythroid colony

820 formation. Proc Natl Acad Sci U S A 94, 1806-1810.

821 Wu, H., Klingmuller, U., Besmer, P., and Lodish, H.F. (1995a). Interaction of the

822 erythropoietin and stem-cell-factor receptors. Nature 377, 242-246.

823 Wu, H., Liu, X., Jaenisch, R., and Lodish, H.F. (1995b). Generation of committed erythroid

824 BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin

825 receptor. Cell 83, 59-67.

826 Zhu, H.H., Ji, K., Alderson, N., He, Z., Li, S., Liu, W., Zhang, D.E., Li, L., and Feng, G.S.

827 (2011). Kit-Shp2-Kit signaling acts to maintain a functional hematopoietic stem and

828 progenitor cell pool. Blood 117, 5350-5361.

829

830

35 831 FIGURES AND FIGURE LEGENDS 832

833 Figure 1. Exosc8 or Exosc9 downregulation disrupts protein-protein interactions

834 within the exosome complex.

835 (A) Crystal structure and model of the human exosome complex (Liu et al., 2006). Solid

836 line, direct interactions; Dashed line, indirect interactions. (B) Real-time RT-PCR analysis

837 of mRNA expression (mean +/- SE, 3 independent replicates) in G1E-ER-GATA-1 cells 48

838 h post-infection with either Exosc8 or Exosc9 shRNA expressing retrovirus. Values

839 normalized to 18S expression and relative to the control. (C) Left: representative image of

840 a semi-quantitative Western blot of Exosc2 co-immunoprecipitated with anti-Exosc3

841 antibody in G1E-ER-GATA-1 whole cell lysates prepared 48 h post-Exosc8 or Exosc9

842 knockdown. Right: densitometric analysis of band intensity relative to the input for each

843 knockdown condition (mean +/- SE, 3 independent replicates). Statistical analysis of

844 control and treatment conditions was conducted with the Student’s T-test. *p<0.05, **

845 p<0.01, ***p<0.001. Source data is available in Figure 1 – source data 1.

846

847 Figure 2. Exosome complex disruption renders primary erythroid cells

848 hypersensitive to limiting erythropoietin concentrations.

849 (A) Flow cytometric analysis with Annexin V and the cell-impermeable dye DRAQ7 to

850 quantitate apoptosis with control and Exosc8-knockdown primary erythroid cells expanded

851 for 48 h under Epo-limiting conditions. (B) Quantification of the percentage of primary

852 erythroid cells in live, late and early apoptotic populations (mean +/- SE, 4 biological

853 replicates). Statistical analysis of control and treatment conditions was conducted with the

854 Student’s T-test. *p<0.05, ** p<0.01, ***p<0.001. Source data is available in Figure 2 –

855 source data 1.

36 856

857 Figure 3. Erythropoietin is required for erythroid differentiation induced by

858 disrupting the exosome complex.

859 (A) Flow cytometric quantification of erythroid markers CD71 and Ter119 in live control

860 and Exosc8-knockdown erythroid precursor cells cultured for 48 h in Epo-limiting media.

861 Representative plots with R1–R5 gates denoted. (B) Quantitation of the percentage of live

862 cells in control and Exosc8-knockdown conditions from the R1-R4 and non erythroid gates

863 (mean +/- SE, 4 biological replicates). (C) Representative images of Wright-Giemsa-

864 stained, DRAQ7-negative erythroid precursor cells, infected with control or ShExosc8

865 retrovirus. Cells were cultured with or without Epo for 48 h (Scale bar, 10 μm). (D)

866 Representative images (left) and quantitation (right) of erythroid colony forming unit activity

867 with FACS-sorted R1 cells 24 h after Exosc8 knockdown (mean +/- SE, 6 biological

868 replicates) (Scale bar 200 μm). Statistical analysis of control and treatment conditions was

869 conducted with the Student’s T-test. *p<0.05, ** p<0.01, ***p<0.001. Source data is

870 available in Figure 3 – source data 1.

871

872 Figure 4. Exosome complex sustains proliferation signaling, while suppressing pro-

873 differentiation signaling.

874 (A) Experimental scheme: lineage-negative cells were isolated from E14.5 fetal livers, and

875 infected with luciferase or Exosc8 shRNAs. Cells were cultured for 48 h and sorted into

876 Ter119+ and Ter119- populations using beads. After 1 h of serum-starvation, cells were

877 stimulated for 10 min with 10 ng/ml SCF or 2 U/ml Epo and fixed/permeabilized before

878 staining for CD71 and p-Akt. (B) Top: p-Akt staining after stimulation with 10 ng/ml SCF in

879 control and Exosc8-knockdown cells (6 biological replicates). Bottom: Relative p-Akt MFI

37 880 after stimulation with 10 ng/ml SCF in control and Exosc8-knockdown cells. MFI expressed

881 relative to unstimulated Ter119-/CD71low control (mean +/- SE, 6 biological replicates). (C)

882 Top: p-Akt staining after stimulation with 2 U/ml EPO in control and Exosc8-knockdown

883 cells (6 biological replicates). Bottom: Relative p-Akt MFI after stimulation with 2 U/ml Epo

884 in control and Exosc8-knockdown cells. MFI expressed relative to unstimulated Ter119-

885 /CD71low control (mean +/- SE, 6 biological replicates). ANOVA identified any significant

886 variation within the experiment, and a Tukey-Kramer test identified the statistical

887 relationship between each pair of samples. *p<0.05, ** p<0.01, ***p<0.001. Source data is

888 available in Figure 4 – source data 1.

889

890 Figure 5: Exosome complex requirement for Kit expression.

891 (A) Left: surface Kit in R1-R3 populations 48 h post-Exosc8 knockdown. Representative

892 plots. Right: surface Kit MFI relative to control R1 (mean +/- SE, 5 biological replicates).

893 (B) Left: surface Kit in R1-R3 cells 48 h post-Exosc9 knockdown. Representative plots.

894 Right: surface Kit MFI relative to control R1 (mean +/- SE, 8 biological replicates). (C) Left:

895 real-time RT-PCR of Kit mRNA and primary transcripts in sorted R1-R3 populations 72 h

896 post-infection with shControl or shExosc8 normalized to 18S and relative to control R1

897 (mean +/- SE, 6 biological replicates). Middle: Kit Western blot with Ter119- cells 24 h

898 post-infection (mean +/- SE, 3 biological replicates). (D) Left: real-time RT-PCR of

899 erythroid mRNAs in sorted R1-R3 populations 72 h post-infection with shControl or

900 shExosc8 (mean +/- SE, 6 biological replicates). Middle: GATA-2 and GATA-1 Western

901 blot with Ter119- cells 24 h post-infection. Right: densitometric analysis normalized to

902 tubulin and relative to shControl (mean +/- SE, 3 biological replicates). (E) qRT-PCR of

903 Exosc8 and Kit mRNA and GATA-1/Exosc8-regulated cell cycle arrest genes in primary

38 904 erythroid precursor cells 24 h post-infection. Normalized to 18S and relative to the control

905 (mean +/- SE, 5 biological replicates). (F) Cell cycle analysis of control and Exosc8-

906 knockdown Ter119- cells 24 (top) and 72 h (bottom) post-infection (mean +/- SE, 6

907 biological replicates) (G) qRT-PCR analysis of Exosc8 and Kit mRNA in G1E cells 48 h

908 post-infection with shControl or shExosc8 retrovirus, normalized to 18S and expressed

909 relative to the control (mean +/- SE, 3 independent experiments) (H) Cell surface Kit

910 expression in infected (GFP+) and uninfected (GFP-) populations of G1E cells 48 h post-

911 infection with shExosc8 (mean +/- SE, 3 independent experiments). Statistical analysis of

912 control and treatment conditions was conducted with the Student’s T-test *p<0.05, **

913 p<0.01, ***p<0.001. Source data is available in Figure 5 – source data 1.

914

915 Figure 6: Exosome complex occupies the Kit locus and is required for active RNA

916 Polymerase II occupancy at Kit.

917 (A) qChIP of serine 5-phospho Pol II, and GATA-1 occupancy at Kit in control and Exosc8-

918 knockdown Ter119- erythroid precursor cells 24 h post-infection (mean +/- SE, 6

919 independent experiments). (B) qChIP of Exosc9 occupancy at Kit and promoters of other

920 exosome complex-regulated erythroid genes (Alas2, Hbb-b1 and Slc4a1) in erythroid

921 precursor cells after culturing for 48 h (mean +/- SE, 3 biological replicates). Statistical

922 analysis of control and treatment conditions was conducted with the Student’s T-test. * p <

923 0.05, ** p < 0.01, *** p < 0.001. Source data is available in Figure 6 – source data 1.

924

925 Figure 7: Functional link between Kit downregulation and erythroid differentiation

926 induced by disrupting the exosome complex.

39 927 (A) Erythroid maturation analyzed by flow cytometric quantitation of CD71 and Ter119

928 post-Exosc8 knockdown and/or Kit expression in primary erythroid precursor cells

929 expanded for 72 h. Representative flow cytometry plots, with the R1-R5 gates denoted. (B)

930 Left: relative Kit MFI post-Exosc8 knockdown and/or Kit overexpression (mean +/- SE, 4

931 biological replicates). Right: percentage of primary erythroid precursor cells in the R1-R4

932 gates (mean +/- SE, 4 biological replicates). (C) Left: relative Kit MFI 48 h post-Exosc8

933 knockdown in cells infected with increasing amounts of a Kit-expressing retrovirus. The

934 arrow depicts Kit downregulation resulting from knocking-down Exosc8. Right: percentage

935 of erythroid precursor cells in the R3 population 48 h post-infection with Exosc8 in cells

936 infected with increasing amounts of Kit-expressing retrovirus. The arrow depicts the

937 increased R3 population post-Exosc8 knockdown. ANOVA identified any significant

938 variation between experimental groups then a Tukey-Kramer test identified the statistical

939 relationship between each pair of samples, *p<0.05, ** p<0.01, ***p<0.001. Source data is

940 available in Figure 7 – source data 1

941

942 Figure 8: Exosome complex function to orchestrate developmental signaling

943 pathways that control proliferation versus differentiation. The master regulator of

944 erythropoiesis GATA-1 represses Kit transcription and upregulates EpoR transcription,

945 thus establishing the developmental signaling circuitry for erythroid maturation. GATA-1

946 represses genes encoding exosome complex subunits, which promotes erythroid

947 maturation. The exosome complex confers Kit expression and establishes competence for

948 SCF-induced Kit signaling. Disruption of this mechanism abrogates Kit signaling and

949 instigates Epo signaling, which favors erythroid precursor maturation versus self-renewal.

950

40 951 Figure 1 – figure supplement 1: The RNA binding exosome complex component

952 Exosc3 suppresses erythroid maturation.

953 (A) qRT-PCR analysis of Exosc3 mRNA in primary erythroid precursor cells 72 h post-

954 infection with shRNA-expressing retrovirus (mean +/- SE, 5 biological replicates). Values

955 are normalized to 18S expression and relative to the control. (B) Erythroid maturation

956 analyzed by flow cytometric quantitation CD71 and Ter119 staining 72 h post-Exosc3

957 knockdown in primary erythroid precursor cells. Representative flow cytometry plots, with

958 the R1-R5 gates denoted (5 biological replicates). (C) Percentage of primary erythroid

959 precursor cells in R1-R5 populations 72 h after Exosc3 knockdown (mean +/- SE, 5

960 biological replicates). Statistical analysis of control and treatment conditions was

961 conducted with the Student’s T-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

962

963 Figure 3 – figure supplement 1: Analysis of the exosome complex-mediated

964 erythroid maturation barricade using a distinct flow cytometric assay.

965 (A) Erythroid maturation of primary erythroid precursor cells 72 h post-infection with

966 shExosc8- or shExosc9-expressing retroviruses analyzed by flow cytometric quantification

967 of CD44 and side scatter (SSC). Representative flow cytometry plots with gates I to VI are

968 depicted. (B) Percentage of erythroid cells detected in gates I through VI (3 biological

969 replicates, mean +/- SE). (C) Representative images of Wright-Giemsa-stained erythroid

970 cells from the sorted, gated (I-IV) populations under control conditions (Scale bar, 10 μm).

971 Statistical analysis of control and treatment conditions was conducted with the Student’s T-

972 test. * p < 0.05, ** p < 0.01, *** p < 0.001.

973

41 974 Figure 4- figure supplement 1: Flow cytometric analysis of ERK phosphorylation

975 reveals Exosc8 requirement to confer Kit signaling and to suppress Epo signaling.

976 (A) Top: p-ERK staining after 10 min stimulation with 10 ng/ml SCF in control and Exosc8-

977 knockdown cells (5 biological replicates). Bottom: p-ERK MFI expressed relative to

978 unstimulated Ter119-/CD71low control (mean +/- SE, 5 biological replicates). (B) Top: p-

979 ERK staining after 10 min stimulation with 2 U/ml EPO in control and Exosc8-knockdown

980 cells (5 biological replicates). Bottom: p-ERK MFI expressed relative to the Ter119-/CD71

981 low control (mean +/- SE, 5 biological replicates). Initially ANOVA identified any significant

982 variation between experimental groups. A Tukey-Kramer test subsequently identified the

983 statistical relationship between each pair of samples. *p<0.05, ** p<0.01, ***p<0.001

984

985 Supplementary File 1: Primers used for analysis of mRNA expression by qRT-PCR

986

987 Supplementary File 2: Primers used for analysis of protein occupancy by qChIP

988

989 Figure 1 – Source Data 1

990 This Excel spreadsheet contains the values of each independent replicate for data

991 presented as histograms (mean +/- SE) in Figure 1. Sheet 1: Figure 1C mRNA expression

992 of Exosc8 and Exosc9 normalized to 18S. Sheet 2: Figure 1D densitometric analysis of

993 Exosc2 immunoblots (pull down/input) from an Exosc3 immunoprecipitation 48 h after

994 Exosc8 or Exosc9 knockdown.

995

996 Figure 2 – Source Data 1

42 997 This Excel spreadsheet contains the values of each biological replicate for data presented

998 as line graphs (mean +/- SE) in Figure 2. Sheet 1: Figure 2B percentage of cells from

999 each biological replicate found in the live, early apoptotic, late apoptotic and necrotic flow

1000 cytometry gates in control and Exosc8 knockdown cells after 48 h culture under Epo-

1001 limiting conditions.

1002

1003 Figure 3 – Source Data 1.

1004 This Excel spreadsheet contains the values for each biological replicate for data

1005 presented as either line graphs or histograms (mean +/- SE) in Figure 3. Sheet 1: Figure

1006 3B the percent live cells found in the R1, R2, R3, R4 and R5 flow cytometry gates in

1007 control and Exosc8 knockdown cells after 48 h culture in Epo-limiting conditions. Sheet 2:

1008 Figure 3D the CFU-E and BFU-E counts from colony assays performed after 24 h

1009 infection with shExosc8.

1010

1011 Figure 4 – Source Data 1

1012 This Excel spreadsheet contains the values for each biological replicate for data presented

1013 as histograms (mean +/- SE) in Figure 4. Sheet 1: Figure 4A and 4B p-Akt MFI after 10

1014 min stimulation with either SCF or Epo 48 h post-Exosc8 knockdown.

1015

1016 Figure 5 – Source Data 1

1017 This Excel spreadsheet contains the values for each biological replicate for data presented

1018 as either line graphs or histograms (mean +/- SE) in Figure 5. Sheet 1: Figure 5A Kit MFI

1019 in the R1, R2, R3, R4 and R5 population 48 h after Exosc8 knockdown. Sheet 2: Figure

1020 5B Kit MFI in the R1, R2, R3, R4 and R5 population 48h post-Exosc9 knockdown. Sheet

43 1021 3: Figure 5C Kit mRNA and primary transcript expression sorted R1-R3 populations 72 h

1022 post-infection with shControl or shExosc8 normalized to 18S and densitometry analysis of

1023 Kit protein in Ter119- cells 24 h post-knockdown. Sheet 4: Figure 5D mRNA expression of

1024 erythroid genes in sorted R1-R3 populations 72 h post-infection with shControl or

1025 shExosc8 and densitometry analysis of GATA1 and GATA2 protein Ter119- cells 24 h

1026 post-knockdown. Sheet 5: Figure 5E Expression of Exosc8, Kit and GATA-1/Exosc8-

1027 regulated cell cycle arrest genes in primary erythroid precursor cells 24 h post-infection,

1028 normalized to 18S. Sheet 5: Figure 5F Cell cycle analysis of control and Exosc8-

1029 knockdown Ter119- cells 24 and 72 h post-infection. Sheet 6: Figure 5G Exosc8 and Kit

1030 mRNA expression in G1E cells 48 h post-infection with shControl or shExosc8 retrovirus,

1031 normalized to 18S. Sheet 6 Figure 5H Kit MFI in infected (GFP+) and uninfected (GFP-)

1032 populations of G1E cells 48 h post-infection with shExosc8.

1033

1034 Figure 6 – Source Data 1

1035 This Excel spreadsheet contains the values for each biological replicate for data presented

1036 in histograms (mean +/- SE) in Figure 6. Sheet 1: Figure 6A qChIP of serine 5-phospho

1037 Pol II, and GATA-1 occupancy at Kit in control and Exosc8-knockdown Ter119- erythroid

1038 precursor cells 24 h post-infection. Sheet 2: Figure 6B qChIP of Exosc9 occupancy at Kit

1039 and promoters of other exosome complex-regulated erythroid genes (Alas2, Hbb-b1 and

1040 Slc4a1) in erythroid precursor cells after culturing for 48 h.

1041

1042 Figure 7 – Source Data 1

1043 This Excel spreadsheet contains the values for each biological replicate for data presented

1044 in line graphs (mean +/- SE) in figure 7. Sheet 1 2 and 3: Figure 7B Kit MFI and

44 1045 percentage of erythroid precursor cells in the R1, R2, R3 R4 and R5 populations 24, 48

1046 and 72 h post Exosc8 knockdown and/or Kit overexpression. Sheet 3: Figure 7C Kit MFI

1047 and percentage of erythroid precursor cells in the R1, R2, R3, R4 and R5 population 48 h

1048 post-Exosc8 knockdown in cells infected with increasing amounts of a Kit-expressing

1049 retrovirus.

45 Fig. 1

A Exosc4 Exosc2 Exosc3 Exosc9 Exosc7 9 4 Dis3 View 2 5 3 7 1

Exosc5 8 6

10 Exosc6 Exosc1 Exosc8

1.5 1.5 B Exosc8 Exosc9 C 140 100 70

-3 50 1.0 1.0 IgG

x 10 10 x 40 r 35 Exosc2 ** M 0.5 0.5 25 mRNA Levels mRNA

(RelativeUnits) *** ***

IP-Condition/Input *** 15 Exosc2Intensity Band 0 0 ShControl Sh Exosc8 Sh Exosc9 IgG IgG IgG Input Input Input Exosc3 Exosc3 Exosc3 Exosc3 IP IgG IP Exosc8Exosc9 Exosc8Exosc9 Exosc8 Exosc9 ShControlSh Sh ShControlSh Sh ShControl Sh Sh Fig. 2

Epo (U/ml) 0 0.001 0.005 0.05 0.5

A 5 10 Necrotic Late 3.98 25.3 3.23 22.5 6 13.2 6.57 11.1 4.12 22.8

10 4 DRAQ7

10 3

10 2 ShControl

0 Live Early 68 5.08 65.5 5.2 70.1 4.21 78.9 1.88 80.9 1.44

10 5 4.44 68.5 4.16 61.5 3.51 34.3 2.86 17 2.83 12.7

10 4

10 3 Exosc8

10 2 Sh

0 22.1 4.92 29.9 4.38 58.9 3.37 78.4 1.72 82.8 1.66 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 Annexin V

100 10 B Live Late Early Apoptosis Apoptosis 75 *** 7.5 *

50 * 5

25 *** 2.5 % Total Cells Total %

0 0 0 1 0 1 Epo (U/ml) 0 1 0.1 0.1 0.1 0.01 0.01 0.01 0.001 0.001 0.001 ShControl Sh Exosc8 Fig. 3

A Epo (U/ml) 0 0.001 0.005 0.05 0.5 10 5 R2 R3 45.5 43 41.3 47.8 44.8 48 44.2 49.3 44.1 43.5 CD71 10 4

10 3 R1 R4 11.5 0.505 10.9 0.344 10.2 0.337 6.59 0.358 6.1 0.153

2

ShControl 10

0 Other R5 0.407 4.21e-3 0.329 3.22e-3 0.33 3.3e-3 0.239 9.69e-4 0.216 1.97e-3

10 5 5.12 39 2.67 68.2 1.88 83.9 3.85 89 8.27 82.6

10 4

10 3

Exosc8 43.1 3.78 18.9 4.12 9 2.89 4.2 1.81 5.84 1.95

2

Sh 10

0 8.86 0.0526 6.11 0.0238 2.36 0.011 1.08 0.0183 1.19 0.0179 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 Ter119

B Erythroid Maturation 100 10 R1 R2 R3 ** R4 * Other ** ** 75 ** 7.5 * ** 50 ** 5.0 *** *** **

% Live/GFP % 25 * 2.5 *

Positive Cells Positive ** * *** ** ** ** ** * ** ** ** 0 0 0 0 1 1 0 0 0 1 1 1

Epo (U/ml) 0.1 0.1 0.1 0.1 0.1 0.01 0.01 0.01 0.01 0.01 0.001 0.001 0.001 0.001 0.001 ShControl Sh Exosc8

C ShControl Sh Exosc8 ShControl Sh Exosc8

0 U/ml Epo 0.5 U/ml Epo

D ShControl Sh Exosc8 BFU-E 120

90

60 BFU-E

Colonies/ 30 10,000Cells *** 0 ShControl Sh Exosc8 CFU-E 800

600

400 CFU-E

Colonies/ 200 10,000Cells

0 Exosc8 Sh ShControl Fig. 4

A Fetal Liver Ter119 Pos Lineage Negative Cells Infect Culture Serum Starve Stimulate (E14.5) Ter119 Neg B Erythroid Maturation SCF Dependance Vehicle SCF 100 Ter119 neg/ Ter119 neg/ Ter119 pos/ Ter119 pos/ CD71 low CD71 high CD71 high CD71 low 80

60

40 ShControl

Cell Number 20 (% of Maximum) 0 100 Ter119 neg/ Ter119 neg/ Ter119 pos/ Ter119 pos/ CD71 low CD71 high CD71 high CD71 low 80

60

40 Sh Exosc8

Cell Number 20 (% of Maximum)

0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 p-Akt Ter119 neg/CD71 low Ter119 neg/CD71 high Ter119 pos/CD71 high Ter119 pos/CD71 low 3 24 3 *** *** 2 16 2 * * * 1 8 1 p-Akt MFI 0 0 0 (Relative Units) SCF ShControl ShExosc8 ShControl ShExosc8 ShControl ShExosc8 ShControl ShExosc8

Erythroid Maturation Epo Dependance Vehicle Epo C 100 Ter119 neg/ Ter119 neg/ Ter119 pos/ Ter119 pos/ CD71 low CD71 high CD71 high CD71 low 80

60

40 ShControl

Cell Number 20 (% of Maximum) 0 100 Ter119 neg/ Ter119 neg/ Ter119 pos/ Ter119 pos/ CD71 low CD71 high CD71 high CD71 low 80

60

40 Sh Exosc8

Cell Number 20 (% of Maximum) 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 p-Akt Ter119 neg/CD71 low Ter119 neg/CD71 high Ter119 pos/CD71 high Ter119 pos/CD71 low 3 15 3 *** *

2 10 ** ** 2

1 5 1 p-Akt MFI 0 0 0 (Relative Units) Epo ShControl ShExosc8 ShControl ShExosc8 ShControl ShExosc8 ShControl ShExosc8 Fig. 5 4 4 A 100 R1 R2 R3 B 100 R1 R2 R3

80 3 80 3

60 60 2 2

40 40 Kit MFI Kit MFI 1 1 Cell Number Cell Number 20 20 * (Relative Units) (Relative Units) (% of Maximum) (% of Maximum) ** *** *** ** 0 0 2 3 4 5 2 3 4 5 2 3 4 5 0 2 3 4 5 2 3 4 5 2 3 4 5 0 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 R1 R2 R3 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 R1 R2 R3 Kit Kit ShControl ShControl ShControl ShExosc8 ShExosc8 ShControl ShExosc9 ShExosc9

C 2.0 2.0 2.0 Kit Kit 140 Kit Kit 3

- 1.5

1.5 1.5 0 100 1

70

x 1.0 1.0 1.0 r 50

M 0.5 0.5 0.5 α mRNA Level mRNA -Tubulin Band Intensity * ** * 50 (Relative Units) * (Relative Units) * (Relative Units) 0 0 Primary Transcript 0 R1 R2 R3 R1 R2 R3 ShControl ShExosc8 ShControl Sh Exosc8 ShControl Sh Exosc8 ShControl Sh Exosc8 ShControl Sh Exosc8

2.0 2.0 70 2.0 D Gata2 Vim GATA-2 50 GATA-2 1.5 1.5 1.5 40 35 1.0 1.0 1.0 25 **

3 0.5

0.5 - 0.5 0 50 α-Tubulin 1

0

x 70 0 0 2.0 GATA-1 r 2.4 2.0 50

M * Hdc SamD14 GATA-1 1.5 40 Band Intensity (Relative Units)

mRNA Level mRNA 1.8 1.5 35

(Relative Units) 1.0 ** 25 1.2 1.0 0.5 50 α-Tubulin 0.6 0.5 0

0 0 R1 R2 R3 R1 R2 R3 ShControl ShControl ShControl Sh Exosc8 Sh Exosc8 Sh Exosc8 ShControl ShControl ShExosc8 Sh Exosc8

shControl shExosc8 2 60 E Exosc8 Kit Cdkn1b Ddit3 F 24 h 24 h ** 1.5 40 *** 1

** 20 24 hours 0.5 **

0 0 2 60 Gadd54a Gas2l1 Trp53inp1 72 h 72 h

% Ter119 *** mRNA Level mRNA 1.5 Cell Number

Negative Cells *** (Relative Units) 40 ShControl Sh Exosc8 1

20 72 hours 0.5

0 0 DNA Content G1 S G2/M G1 ShControl S ShControl Sh Exosc8 ShControl Sh Exosc8 ShControl Sh Exosc8 G2/M ShExosc8

1.2 1.2 Exosc8 Kit 100 GFP GFP GFP pos GFP neg G H pos neg 0.9 80 0.9 ** 60 0.6 0.6 ***

40 Kit MFI 0.3 0.3 Cell Number mRNA Level mRNA *** (Relative Units)

(Relative Units) 20 (% of Maximum) 0 0 0 0 102 103 104 105 0 102 103 104 105 Kit ShControl ShExosc8 ShControl Sh Exosc8 ShControl ShControl ShControl Sh Exosc8 Sh Exosc8 Sh Exosc8 Fig. 6

Promoter 10 kb -114 +5 +58 3ʼUTR A Kit

0.03 0.09 0.03 0.24 * * IgM ** p-Pol II * 0.02 0.06 0.02 0.16 *

0.01 0.03 0.01 0.08 (Relative Units)

p-Pol II Occupancy 0 0 0 0 0.20 0.06 0.20 PI GATA-1 0.15 0.15 0.04 shControl shControl sh Exsoc8 sh Exsoc8 0.10 0.10 Rpb1 Krt5 0.02 Promoter Promoter 0.05 0.05 (Relative Units)

GATA-1 Occupancy GATA-1 0 0 0 shControl shControl shControl shControl shControl shControl shControl sh Exsoc8 sh Exsoc8 sh Exsoc8 sh Exsoc8 sh Exsoc8 sh Exsoc8 sh Exsoc8

-114 Promoter +0.2 +5 +57 +58 +80.8 3’UTR

B 0.025 *** IgG *** 0.020 ** Exosc9 *** * *** 0.015 ** 0.010 *

0.005 (Relative Units)

Exosc9 Occupancy 0 -1.1 +35 +58 +75 promoter promoter promoter promoter promoter +80.5 3’UTR +80.8 3’UTR

Kit Slc4a1 Hbb-b1 Alas2 MyoD Fig. 7

A ShControl Sh Exosc8 Empty Kit cDNA Empty Kit cDNA

10 5 57.5 30.6 67.4 22.2 43.2 39.9 58 27.7 CD71 10 4

10 3

24h 10.9 0.351 9.29 0.25 15.3 0.608 12.3 0.573

10 2

0 0.652 0.0145 0.854 6.75e-3 0.95 0.0201 1.39 6.29e-3 44 34.3 67.5 9.76 19.9 64.1 38.5 27.6 10 5

10 4

3

48h 10 18.3 2.1 20.2 0.312 8.82 4.84 26 1.48

10 2

0 1.38 0.0256 2.21 0 2.23 0.0534 6.37 0.0735 44.5 40.6 67.4 11.8 15.5 57.4 39.1 28.5 10 5

10 4

10 3 72h 11.1 2.84 17.7 0.995 5.57 18.6 21.8 5.66

10 2

0 0.891 0.0968 2.11 0.0314 1.97 0.965 4.52 0.418 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 Ter119

B R1 R2 R3 R4 10 28 80 80 28

7.5 21 60 60 21

5 14 40 40 14 Kit MFI Kit 2.5 7 20 20 7 % Live/GFP Live/GFP % Positive Cells Positive (RelativeUnits) 0 0 0 0 0 Time (h) 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72

ShControl/Empty ShControl/Kit Sh Exosc8 /Empty Sh Exosc8 /Kit

R3 C 5 50 4 40

3 30

2 20 Kit MFI Kit

% Live/GFP Live/GFP % 10

1 Cells Positive (RelativeUnits) 0 0 Kit vector (μl) 0 25 50 0 25 50

ShControl Sh Exosc8 Fig. 8

GATA-1

GATA-1 Epor

GATA-1 Kit

Exosome Complex

Proliferation Differentiation

Type 2 Coherent Feed-forward Loop Type 4 Coherent Feed-forward Loop Fig. S1

A Exosc3 B ShControl Sh Exosc3-1 Sh Exosc3-2 80 10 5 52.3 34.3 19.2 31.2 19.9 44.3

60 CD71 10 4 40 10 3 15.1 18.9 20 11.1 1.52 20.4 20.5

mRNA Levels mRNA *** *** 10 2 (RelativeUnits)

0 0

-1 -2 0.74 0.022 8.11 0.496 1.46 0.405 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 Ter119 Exosc3 Exosc3 ShControl Sh Sh

R1 R2 R3 R4 R5 C 20 80 20 **

15 60 15

10 40 10 *** *** Expansion 5 20 ** ** 5

*** ** 0 0

% GFP positive cells positive GFP % 0 -1 -1 -2 -2 -1 -2 -1 -2 -1 -2 Exosc3 Exosc3 Exosc3 Exosc3 Exosc3 Exosc3 Exosc3 Exosc3 Exosc3 Exosc3 ShControl ShControl ShControl ShControl ShControl Sh Sh Sh Sh Sh Sh Sh Sh Sh Sh Fig. S2

A ShControl Sh Exosc8 Sh Exosc9

10 5 CD44 38.2 4.03 11.3 10 4 17.6 4.51 2.11 I II V 10 3 2.62 12.9 III 3.59 13.7 2.47 16.4 VI IV

10 2 5.3 48.4 43.4 20.4 22.6 0 13.6

0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K FSC-A

B Erythroid Maturation 60 28 I II III IV V VI ** ** ** 45 *** 21

30 14 % GFP GFP % 15 ** 7 ** Positive Cells Positive ** ** *** 0 0 Exosc9 Exosc9 Exosc9 Exosc9 Exosc9 Exosc8 Exosc9 Exosc8 Exosc8 Exosc8 Exosc8 Exosc8 Sh Sh Sh ShControl ShControl ShControl Sh ShControl ShControl ShControl Sh Sh Sh Sh Sh Sh Sh Sh

C I II III

IV V VI Fig. S3

Erythroid Maturation A SCF Dependance Vehicle SCF 100 Ter119 neg/ Ter119 neg/ Ter119 pos/ Ter119 pos/ CD71 low CD71 high CD71 high CD71 low 80

60

40 ShControl

Cell Number Cell 20 (% of Maximum) of (% 0 100 Ter119 neg/ Ter119 neg/ Ter119 pos/ Ter119 pos/ CD71 low CD71 high CD71 high CD71 low 80

60 Exosc8 40 Sh

Cell Number Cell 20 (% of Maximum) of (% 0 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 p-ERK Ter119 neg/CD71 low Ter119 neg/CD71 high Ter119 pos/CD71 high Ter119 pos/CD71 low 4 *** 3 * ** 2

1 p-ERKMFI 0 (RelativeUnits) SCF ShControl Sh Exosc8 ShControl Sh Exosc8 ShControl Sh Exosc8 ShControl Sh Exosc8

Erythroid Maturation B Epo Dependance Vehicle Epo 100 Ter119 neg/ Ter119 neg/ Ter119 pos/ Ter119 pos/ CD71 low CD71 high CD71 high CD71 low 80

60

40 ShControl

Cell Number Cell 20 (% of Maximum) of (% 0 100 Ter119 neg/ Ter119 neg/ Ter119 pos/ Ter119 pos/ CD71 low CD71 high CD71 high CD71 low 80

60

40 Exosc8 Sh

Cell Number Cell 20 (% of Maximum) of (% 0 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 p-ERK Ter119 neg/CD71 low Ter119 neg/CD71 high Ter119 pos/CD71 high Ter119 pos/CD71 low 4 16 4

3 * 12 3

*** *** ** 2 8 ** 2 1 4 1 p-ERKMFI 0 0 0 (RelativeUnits) Epo ShControl Sh Exosc8 ShControl Sh Exosc8 ShControl Sh Exosc8 ShControl Sh Exosc8