The Super Elongation Complex Drives Neural Stem Cell Fate Commitment

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1 Article The Super Elongation Complex Drives Neural Stem Cell Fate Commitment Graphical Abstract Authors Kun Liu, Dan Shen, Jingwen Shen,..., Chouin Wong, Weidong Feng, Yan Song Correspondence In Brief Liu et al. implicate the super elongation complex (SEC), best known for transcription elongation checkpoint control, in driving Drosophila neural stem cell (NSC) fate commitment. SEC is highly expressed in NSCs, where it interacts directly with the Notch signaling pathway in a self-reinforcing feedback loop for timely stem cell fate lock-in. Highlights d Super elongation complex (SEC) subunits are highly expressed in neural stem cells d d d SEC forms a positive feedback loop with Notch signaling in promoting NSC self-renewal Overactivation of SEC induces neural progenitor dedifferentiation and tumorigenesis SEC acts as an intrinsic amplifier driving neural stem cell fate commitment Liu et al., 2017, Developmental Cell 40, March 27, 2017 ª 2017 Elsevier Inc.

2 Developmental Cell Article The Super Elongation Complex Drives Neural Stem Cell Fate Commitment Kun Liu, 1,2 Dan Shen, 1 Jingwen Shen, 1 Shihong M. Gao, 1 Bo Li, 1 Chouin Wong, 1 Weidong Feng, 1 and Yan Song 1,2,3, * 1 Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences 2 Peking-Tsinghua Center for Life Sciences Peking University, Beijing , China 3 Lead Contact *Correspondence: yan.song@pku.edu.cn SUMMARY Asymmetric stem cell division establishes an initial difference between a stem cell and its differentiating sibling, critical for maintaining homeostasis and preventing carcinogenesis. Yet the mechanisms that consolidate and lock in such initial fate bias remain obscure. Here, we use Drosophila neuroblasts to demonstrate that the super elongation complex (SEC) acts as an intrinsic amplifier to drive cell fate commitment. SEC is highly expressed in neuroblasts, where it promotes self-renewal by physically associating with Notch transcription activation complex and enhancing HES (hairy and E(spl)) transcription. HES in turn upregulates SEC activity, forming an unexpected self-reinforcing feedback loop with SEC. SEC inactivation leads to neuroblast loss, whereas its forced activation results in neural progenitor dedifferentiation and tumorigenesis. Our studies unveil an SEC-mediated intracellular amplifier mechanism in ensuring robustness and precision in stem cell fate commitment and provide mechanistic explanation for the highly frequent association of SEC overactivation with human cancers. INTRODUCTION Both normal development and tissue homeostasis rely on the remarkable capacity of stem cells to divide asymmetrically, simultaneously generating one identical stem cell and one differentiating progeny (Clarke and Fuller, 2006; Lin, 2008). Extensive studies have unveiled how extrinsic niche signals and intrinsic cell polarity cues ensure proper orientation of mitotic spindle and, hence, asymmetric division of stem cells (Inaba and Yamashita, 2012; Knoblich, 2010; Morrison and Kimble, 2006; Williams and Fuchs, 2013). However, it remains unclear whether the initial fate bias, established by unequal exposure to niche signals or differential partitioning of cell fate determinants, can be immediately and automatically consolidated and stabilized into distinct and irreversible cell fate outcomes. In fact, in vivo timelapse imaging of the developing zebrafish hindbrain using the Notch activity reporter showed that, immediately after the asymmetric division of a radial glia progenitor, Notch activity is not noticeably biased in the paired daughter cells (Dong et al., 2012). Instead, the differential Notch activity in the pair of daughter cells only gradually increases afterward, over a time span of 3 8 hr (Dong et al., 2012), indicating the existence of a progressive and tightly regulated transition phase between the initial cell fate decision and the ultimate cell fate commitment. Stem cells and progenitors, especially the fast-cycling ones, face the daunting challenges of ensuring timely, precise, and robust cell fate determination in every cell cycle and are likely to achieve so through rapid amplification of the initial small fate bias upon their asymmetric division. In electronics, a device called an amplifier magnifies a small input signal to a large output signal until it reaches a desired level. Conceivably, a similar amplifier mechanism could be employed in the stem cells or progenitors to accelerate the transition phase and drive cell fate commitment. Dysregulation of such an amplifier could cause an imbalance between self-renewal and differentiation, resulting in impaired tissue homeostasis. However, the regulatory modules governing the transition phase from stem cell fate decision to fate commitment, especially the identity and control of this putative amplifier, remain largely unexplored. Drosophila type II neural stem cells (NSCs), known as neuroblasts (NBs), provide an excellent model system for studying stem cell fate commitment (Brand and Livesey, 2011; Chang et al., 2012; Homem and Knoblich, 2012; Sousa-Nunes et al., 2010). Firstly, distinct from type I NB lineages, type II NB lineages contain transit-amplifying cells called intermediate neural progenitors (INPs), similar to mammalian NSC lineages in both functional and molecular criteria, yet with much simpler anatomy and lineage composition (Brand and Livesey, 2011; Homem et al., 2015). Each type II NB undergoes stereotypic, self-renewing divisions to produce immature INPs, which, upon maturation, undergo a few rounds of asymmetric, self-renewing divisions to give rise to ganglion mother cells (GMCs) that subsequently generate post-mitotic neurons or glia (Figures 1A and 1B) (Bello et al., 2008; Boone and Doe, 2008; Bowman et al., 2008). Secondly, the identity of each cell type in the NB lineages can be unambiguously determined by a combination of cell fate makers as well as by their geological positions within the lineages (Figures 1A and 1B) (Brand and Livesey, 2011; Homem and Knoblich, 2012; Sousa-Nunes et al., 2010). Thirdly, the molecular mechanisms underlying initial NB versus INP fate decision are well understood. Unidirectional Notch signaling is both necessary and sufficient to promote type II NB self-renewal (Bowman et al., Developmental Cell 40, , March 27, 2017 ª 2017 Elsevier Inc. 537

3 Figure 1. SEC Is Highly Expressed in NBs and Is Essential for NB Self-Renewal (A and B) Diagrams depicting the lineage composition (A) and hierarchy (B) of type II NBs. (C) Schematic of the SEC. Note that EAF and EAR are not shown in this diagram. (D F) High accumulation of daff-lacz (D), daff- GFP (E), or endogenous daff (F) in type II NBs, colocalizing with NB marker Deadpan (Dpn). In this and subsequent micrographs, NB lineages are outlined by white dashed lines, whereas NBs, immature INPs, and mature INPs are marked with brackets, white arrowheads, and cyan arrowheads, respectively. Membrane-bound GFP, F-actin, or Dlg outline the cell cortex. (G) Expression pattern of dell-myc or NLS-RFP transgenes in type II NB lineages. Note that NSL- RFP but not dell-myc were detected in mature INPs. (H) Endogenous dell is highly expressed in a type II NB, quickly downregulated in INPs, and not detectable in GMCs or neurons (nuclear Prospero [Pros]: GMC/neuron marker). (I J) shmirna-mediated knockdown of dell led to severe and selective type II NB loss (I). Individual type II NB lineages are encircled by white dashed lines. In this and subsequent micrographs, yellow dotted line marks the boundary between the optic lobe (left) and the central brain (right) areas. Closeup images are displayed in (J). (K and L) Quantification of total type II or type I NB number in brain lobe of indicated genotypes. WT, wild-type. **p < (n = 10 19). NS, not significant. In this and subsequent quantifications, data are presented as mean ± SD. Scale bars, 10 mm (D H and J) and 25 mm (I). See also Figure S ; Song and Lu, 2011, 2012; Wang et al., 2006; Weng et al., 2010). At each division, type II NBs asymmetrically segregate differentiation-promoting determinants, such as Notch antagonist Numb, into immature INPs. As a consequence, Notch pathway effector HES (hairy and E(spl)) genes, such as E(spl)mg, are highly expressed in NBs but not in immature INPs (Almeida and Bray, 2005; Song and Lu, 2011; Zacharioudaki et al., 2012). HES genes, encoding basic helix-loop-helix (bhlh) transcription factors, are crucial for promoting NB self-renewal (Zacharioudaki et al., 2012). Importantly, numb mutant immature INPs fail to complete maturation but instead revert fate back into NBs and result in tumorigenesis (Bowman et al., 2008; Wang et al., 2006), indicating that the asymmetric segregation of Numb protein is critical for establishing the initial fate bias between a type II NB and its sibling INP. However, whether such initial bias is sufficient to confer differential Notch activity and achieve definitive fate commitment is currently unclear. Lastly, type II NBs undergo fast cell divisions, dividing every 2 hr (Cabernard and Doe, 2013; Homem et al., 2013), placing them under huge pressure to timely yet precisely achieve differential fate outcomes. Therefore, within type II NB lineages, a regulatory module that drives robust cell fate commitment is likely to exist, plausibly with high activity. Overactivation of Notch signaling leads to immature INP dedifferentiation and tumorigenesis (Bowman et al., 2008; Song and Lu, 2011, 2012; Zacharioudaki et al., 2012), providing a sensitized background for identifying factors pivotal for NB or INP fate commitment. In such a genetic background we carried out a genome-wide RNAi-based screen for genes whose downregulation specifically suppresses the supernumerary NB phenotype induced by Notch overactivation (K.L., B.L., D.S., and Y.S., unpublished data), and identified subunits of the super elongation complex (SEC). The SEC is composed of the elongation factor ELL (eleven-nineteen lysine-rich leukemia) 1/2/3, the flexible scaffolding protein AFF (AF4/FMR2 family) 1/2/3/4, the ELL-associated factor EAF1/2, eleven-nineteen leukemia (ENL)/AF9, as well as the Pol II elongation factor P-TEFb consisting of cyclin T (CycT) and cyclin-dependent kinase 9 (CDK9) (Figure 1C) (Lin et al., 2010; Luo et al., 2012; Smith et al., 2011). Our screen identified all subunits of SEC except EAF and ENL/AF9, suggesting that SEC interplays with Notch signaling in promoting NB selfrenewal. The SEC subunits were originally identified as frequent 538 Developmental Cell 40, , March 27, 2017

4 Figure 2. SEC Promotes NB Self-Renewal by Stimulating dhes Transcription (A) Single frames of time-lapse movies of a wild-type (WT) or a CycT-RNAi dividing type II NB expressing both E(spl)mg-GFP and CD8-RFP. Yellow and blue brackets indicate NB and progenitor respectively; time in hr:min. NE: nuclear envelope. (B) Quantification of relative E(spl)mg-GFP fluorescence intensity (FI) in type II NBs and immature INPs in WT or CycT-RNAi NB lineages over time. (legend continued on next page) Developmental Cell 40, , March 27,

5 translocation partners of MLL (mixed-lineage leukemia) in inducing leukemogenesis, and play key roles in c-myc-dependent carcinogenesis and HIV viral DNA transcription (Luo et al., 2012; Smith et al., 2011). Previous studies demonstrated that SEC executes its functions by inducing rapid gene transcription, mainly through phosphorylating RNA polymerase II (Pol II) C-terminal domain and releasing it from promoter-proximal pausing (Fuda et al., 2009; Levine et al., 2014; Lin et al., 2011). Here we show that the SEC is specifically expressed in Drosophila NBs, where it acts as an amplifier to drive type II NB fate commitment. SEC exerts its function by physically associating with Notch transcription activation complex to stimulate dhes (Drosophila HES) transcription. dhes in turn promotes SEC expression/activity. Thus, driven by a self-reinforcing feedback loop between SEC and Notch signaling, an initial small bias of Notch activity between an NB and its sibling INP is rapidly amplified and consolidated into robust and irreversible fate commitment. RESULTS SEC Subunits Are Highly Expressed in Drosophila Neural Stem Cells To assess the expression pattern of SEC subunits in NB lineages, we employed a daff-lacz enhancer trap, in which the lacz expression levels reflect the promoter activity of Drosophila AFF (daff). Unexpectedly, daff-lacz highly and specifically accumulated in NBs and became quickly downregulated in differentiating progeny cells (Figure 1D). We next examined the expression pattern of daff protein using a bacterial artificial chromosome (BAC) transgene that expresses a daff-gfp fusion protein under control of the daff endogenous promoter and regulatory sequences. daff-gfp largely recapitulated the expression pattern of daff-lacz (Figure 1E). To unambiguously verify the NB-specific expression of the endogenous daff protein, we further generated its specific antibody (Figures S1A S1C). Interestingly, antibody staining also revealed high expression of the endogenous daff protein in type II NBs (Figure 1F). Thus, we conclude that daff is predominantly expressed in NBs and that such a distribution pattern was established, at least partly, at the transcriptional level. To elucidate the expression pattern of Drosophila ELL (dell) in NB lineages, we expressed a dell-myc transgene in all cells within NB lineages by insc-gal4. dell-myc, but not a control NLS-RFP transgene, displayed specific accumulation in NBs and immature progenitors (Figure 1G). Since the dell-myc fusion did not carry any transcriptional regulatory sequence of dell, such a unique expression pattern of dell was likely established by a post-translational mechanism. We next generated a specific antibody against the dell protein (Figures S1D S1F). Interestingly, the endogenous dell expression was also predominantly found in NBs and strongly colocalized with daff- GFP in type II NB lineages (Figures 1H and S1G). Considering that daff is a scaffolding subunit, whereas dell is a stoichiometrically limiting factor of SEC (Chou et al., 2013; He et al., 2010; Hill and Sundquist, 2013; Schulze-Gahmen et al., 2013), our results strongly suggest that the activity of SEC is mainly restricted to NBs. Importantly, both dell and daff were symmetrically partitioned into daughter cells of dividing NBs in cytokinesis (Figure S1H), indicating that the biased accumulation of SEC subunits in NBs were established after NB asymmetric division. SEC Is Essential for Maintaining NB Cell Fate The specific expression of SEC subunits in NBs prompted us to investigate whether SEC plays a critical role in maintaining NB self-renewal. Knockdown of dell in type II NB lineages using short hairpin micrornas (shmirnas) (Ni et al., 2011), by PntP1-Gal4, led to severe NB loss (4.12 ± 0.45 type II NBs in each dell-ir brain lobe versus 8 in each wild-type brain lobe; n = 10 19; Figures 1I 1K). The remaining type II NBs had few progeny (Figures 1I and 1J), suggesting that the proliferative capacity of both NBs and mature INPs were severely compromised upon dell loss. Importantly, such type II NB loss phenotype can be completely reverted by coexpressing an shmirna-resistant dell transgene but not by the coexpression of apoptosis inhibitor p35 (Figures 1I 1K), indicating that the phenotype was not caused by off-target effects of the shmirna or apoptotic cell death, but more likely NB premature differentiation. Knockdown of daff in NB lineages by insc-gal4 largely phenocopied the dell-rnai phenotype (Figure 1K). In contrast, downregulation of either daff or dell, by ase-gal4, exhibited mild effects on the maintenance and proliferative ability of type I NBs that are relatively insensitive to Notch downregulation (Figures 1L and S1I S1L). Therefore, SEC is essential for maintaining type II NB stem cell fate. dhes Genes Are the Main Targets of SEC in NBs To test whether SEC maintains type II NB self-renewal through promoting Notch signaling, we performed in vivo Notch reporter assays utilizing E(spl)mg-GFP, a faithful reporter for Notch activity (Almeida and Bray, 2005; Song and Lu, 2011; Zacharioudaki et al., 2012). In vivo time-lapse imaging of dividing type II NBs in intact larval brains revealed that Notch activity was uniformly distributed in two daughter cells shortly after NB asymmetric division. Upon degradation of maternally inherited E(spl)mg-GFP protein, Notch activity started to increase in NBs and decrease in progenitors, progressively establishing its asymmetric distribution over a time span of about 1 hr (Figures 2A and 2B; Movie S1). Importantly, such differential Notch activity between (C) Expression of E(spl)mg-GFP and Dpn in type II NB lineages of indicated genotypes. Type II NBs and immature INPs are marked with white brackets and white arrowheads, respectively. (D and E) Quantification of relative E(spl)mg-GFP fluorescence intensity (D) in type II NBs or the ratio of E(spl)mg-GFP FI in NB/immature INP (E) of indicated genotypes. **p < (n = 12 15). (F) Asymmetric cortical distribution of apical marker atypical PKC (apkc) and basal maker Miranda (Mira) in metaphase NBs of indicated genotypes. (G) Quantification of relative Dpn fluorescence intensity in type II NBs of indicated genotypes. **p < (n = 10 15). (H) Larval brain lobes of indicated genotypes were stained for Dpn and Pros. (I) Quantification of total NB number per brain lobe of indicated genotypes. **p < (n = 12 18). Scale bar, 10 mm (A, C, and F); 50 mm (H). See also Figure S2; Movies S1 and S Developmental Cell 40, , March 27, 2017

6 Figure 3. SEC Physically Associates with Notch Transcription Complex (A C) Schematic drawings of daff, dcsl and dell domains and truncated constructs of daff (A) and dcsl (B). (D F) Coimmunoprecipitation (coip) of full-length (FL) or truncated FLAG-tagged daff and HA-tagged dcsl in 293T cells. Note that dcsl interacts tightly with daff-3 and weakly with daff-4 (F), yet fails to interact with daff-1 or daff-2 (E) in coip assays. In these and subsequent panels, GFP served as a negative control and input represents 4% of total in coip experiments. (G) dcsl directly interacts with daff-3 in GST pull-down assays. Purified recombinant MBP-dCSL was used as prey and was pulled down by GST-dAFF-3 but not GST alone. CB, Coomassie blue staining. (H) The reciprocal coip of FL or truncated HA-dCSL and FLAG-dAFF in 293T cells. (I) CoIP of dell-flag and HA-dCSL. HA-dAFF was coexpressed to stabilize dell-flag. (J and K) CoIP of NICD-V5 and HA-dAFF in the presence or absence of FLAG-dCSL (J), and an illustration of putative interactions among SEC-Notch transcription super complex (K). (legend continued on next page) Developmental Cell 40, , March 27,

7 an NB and its sibling progenitor could be much less effectively established upon CycT knockdown (Figures 2A and 2B; Movie S2). As a consequence, the expression of E(spl)mg-GFP was evidently reduced upon downregulation of each SEC subunit in NB lineages (Figures 2C, 2D, S2A, and S2B). Furthermore, NB-specific overexpression of a kinase-dead form of CDK9 (CDK9-KD) drastically attenuated E(spl)mg-GFP levels (Figures 2C and 2D), indicating that the kinase activity of CDK9 is essential for SEC to promote Notch signaling. Conversely, overexpression of individual SEC subunits, except CDK9, led to a prominent upregulation of E(spl)mg-GFP in both NBs and mature progenitors (Figures 2C and 2D). Significantly, the ratio of E(spl)mg-GFP expression between an NB and its adjacent progenitor, directly reflecting the differential Notch activity, prominently reduced upon SEC inactivation (Figure 2E). Upon attenuation of SEC activity, the mitotic NBs displayed normal apical-basal cortical polarity (Figure 2F), arguing against the possibility that SEC promotes Notch activity through regulating NB asymmetric cell division. Furthermore, in response to changes in SEC activity, the alteration in the expression of enhancer reporter for E(spl)mg, mg-phstinger, resembles that of E(spl)mg-GFP (Figure S2C), demonstrating that SEC is both necessary and sufficient to promote E(spl)mg transcription. Taken together, our studies strongly suggest that SEC promotes differential Notch activity after NB asymmetric cell division, likely at the amplification step, by inducing E(spl)mg transcription. Similar to E(spl)mg, the expression of an enhancer reporter for E(spl)m8, another dhes family gene, is also positively regulated by SEC (Figure S2D). By contrast, the expression of other selfrenewal factors, including bhlh family transcription factor Deadpan (Dpn) and Ets transcription factor PntP1 (San-Juan and Baonza, 2011; Zacharioudaki et al., 2012; Zhu et al., 2012), remain unchanged within NBs and mature INPs upon alteration in SEC function (Figures 2C, 2G, and S2D), indicating that SEC specifically regulates dhes transcription in NBs. Taken together, our results support an attractive model whereby, upon SEC inactivation, the differential dhes expression between type II NBs and their progenitor siblings is too small to confer a robust stem cell fate commitment, resulting in type II NBs with an identity crisis and eventual cell fate loss. To investigate how SEC impinges on the Notch pathway to promote dhes expression, we investigated its epistatic relationship with dcsl (Drosophila homolog of CSL, also named Su(H)). Overexpression of dcsl-vp16 (Furriols and Bray, 2000), a fusion between dcsl and the VP16 transcriptional activation domain, in NB lineages was sufficient to drive immature INP dedifferentiation and tumorigenesis (Figures S2E and S2F), bypassing Notch pathway upstream events. Attenuation of SEC activity by expressing CDK9-KD or dell-rnai effectively suppressed such supernumerary NB phenotype (Figures S2E and S2F), placing SEC in parallel or downstream of dcsl in regulating dhes transcription. If dhes genes are key functional targets of SEC in promoting self-renewal, then overexpression of dhes gene should rescue the reduction in self-renewal capacity resulting from SEC inactivation. Indeed, the potent suppression effects of CDK9-KD on Notch overactivation-induced excessive NB self-renewal were largely abolished upon coexpression of E(spl)mg (Figures 2H and 2I). Thus, dhes genes are major functional targets of SEC in NBs. SEC Physically Associates with Notch Transcription Activation Complex Given that SEC is a transcription regulatory complex, it is likely that SEC cooperates with Notch transcription activation complex in inducing dhes transcription. We therefore assessed whether SEC (Figures 3A 3C) promotes dhes transcription by physically associating with the CSL-NICD-MAM ternary transcription activation complex (Kopan and Ilagan, 2009). Indeed, dcsl was specifically coimmunoprecipitated with the full length, N terminus, or C terminus of daff (Figure 3D) from HEK293T cell extracts. Further domain-mapping analysis revealed that the daff-3 fragment (amino acids 808 1,211) exhibited a strong binding activity to dcsl (Figures 3E and 3F). We next performed in vitro glutathione S-transferase (GST) pull-down assay using purified recombinant proteins and found that dcsl was specifically pulled down by GST-dAFF-3 but not GST alone (Figure 3G). We thus conclude that daff directly interacts with dcsl. Consistent with this notion, reciprocal coimmunoprecipitation (coip) assay showed that the full length, but neither the N nor C terminus, of dcsl interacted with daff (Figures 3H, S3A, and S3B). In addition, coip experiments further demonstrated physical interaction between dell and dcsl (Figure 3I). Intriguingly, whereas daff strongly associated with dcsl, the interaction between daff and NICD (Notch intracellular domain) was barely detectable (Figure 3J), prompting us to investigate the possibility that dcsl acted as an adaptor linking daff to NICD (Figure 3K). Indeed, upon dcsl coexpression, strong interaction between daff and NICD was detected (Figures 3J and 3K). Furthermore, V5-dCSL expressed in NB lineages was specifically coimmunoprecipitated with FLAG-dAFF from fly larval brain extracts (Figure 3L) and vice versa (Figure S3C), validating the in vivo protein-protein interaction. CoIP experiments further revealed physical interactions among human CSL, AFF4, and ELL2 proteins (Figure 3M), indicating that the formation of SEC-Notch transcription activation super complex is evolutionarily conserved. Finally, our DamID-qPCR analysis showed that daff preferentially bound to the promoter regions of dhes genes (Figure 3N). Together, our data clearly indicate that SEC is recruited by the Notch transcription activation complex to the promoter-proximal regions of Notch target genes to promote Notch signaling. We next investigated whether SEC promotes dhes gene transcription by releasing paused Pol II from their promoter-proximal (L) CoIP of FLAG-dAFF and V5-dCSL in Drosophila larval brains. FLAG-dAFF and V5-dCSL were specifically expressed in NB lineages by insc-gal4, and V5-dCSL was coimmunoprecipitated with FLAG-dAFF from larval brain extracts. (M) CoIP of Myc-tagged human ELL2 or FLAG-tagged human AFF4 and HA-tagged human CSL in 293T cells. (N) DamID-qPCR analysis of daff occupancy at the promoter-proximal regions of indicated genes in central brain NB lineages. Data are presented as log 2 (Dam-dAFF/Dam). **p < ; NS, not significant. See also Figure S Developmental Cell 40, , March 27, 2017

8 Figure 4. Notch Signaling and dhes Promote dell and daff Expression (A and C) Expression pattern of dell (A) or daff (C) in wild-type (WT) control, ada mutant, dcsl mutant, or brat mutant type II NB MARCM clones marked by CD8-GFP. In these and subsequent panels, NB-like dedifferentiating progenitors are indicated by yellow arrowheads. (B and D) Quantification of relative dell (B) or daff (D) FI in control immature INPs versus dedifferentiating INPs of indicated genotypes, or in WT versus dcsl mutant type II NBs. **p < 0.001; NS, not significant (n = 15 20). (legend continued on next page) Developmental Cell 40, , March 27,

9 sites. Surprisingly, our Dam-Pol II occupancy analysis revealed paused Pol II at the 5 0 end of hsp70 but not E(spl)mg or E(spl) m8 loci (Figures S3D and S3E), strongly suggesting that these two dhes genes are non-pausing genes. Consistent with this notion, whereas the expression levels of pausing genes have been found to increase upon loss of the NELF (negative elongation factor) complex activity (Kwak and Lis, 2013; Zhang et al., 2007), depletion of various subunits of NELF led to no change in dhes gene transcription (Figures S3F and S3G). Therefore, SEC is likely to promote the transcription of dhes genes in the absence of paused Pol II. dell and daff Expression Is Promoted by Notch Signaling If the main task of SEC within NB lineages is to promote self-renewing fate by upregulating dhes transcription, how is the expression and activity of SEC restricted to NBs in the first place? We reasoned that such an expression pattern of SEC can be achieved via NB-specific upregulation by self-renewal factors, such as Notch, or via progenitor-specific inhibition by differentiation-promoting factors, such as Brain tumor (Brat) or Prospero (Pros) (Betschinger et al., 2006; Bowman et al., 2008; Choksi et al., 2006; Lee et al., 2006), or both. To distinguish these possibilities, we assessed the expression levels of daff and dell upon alteration of various cell fate determinants. Compared with wild-type immature INPs, the expression levels of daff and dell are significantly increased in dedifferentiating progenitors induced by loss of the Notch antagonist a-adaptin (ada) (Figures 4A 4D) (Song and Lu, 2012) or overexpression of Notch (Figures 4B and 4D 4F), implying that Notch signaling enhances daff/dell expression. Conversely, the high expression of dell and daff in type II NBs were largely diminished by loss of dcsl (Figures 4A 4D), indicating that Notch signaling is both necessary and sufficient to promote SEC expression. By contrast, although loss of brat or pros drove immature progenitor or GMC dedifferentiation and severe brain tumor phenotypes, the expression levels of daff or dell in reverting progenitors or GMCs remained largely unaltered (Figures 4A 4D and S4A). Taken together, our results demonstrate that it is Notch activation, but not cell fate changes (progenitor or GMC dedifferentiation) or brain tumor formation per se, that upregulates dell/daff expression in NB lineages. We next assessed whether Notch signaling promotes daff/dell expression directly or through its target dhes. Overexpression of E(spl)mg results in supernumerary NB phenotypes, coinciding with elevated expression of daff and dell in reverting progenitors (Figures 4B and 4D 4F). If SEC subunits are functional targets of dhes, we reason that attenuation of SEC might abolish the brain tumor-inducing effect of dhes. Indeed, dell knockdown or CDK9 inactivation potently rescued the brain tumor phenotype induced by E(spl)mg overexpression, whereas Notch knockdown failed to do so (Figures 4G, 4H, and S4B). Therefore, our results revealed a Notch-dHES-SEC regulatory module in promoting SEC activity. More importantly, our results reveal that dhes, as a major target of SEC, forms a positive feedback loop with SEC to further boost the transcriptional response. Given that dhes mainly acts as a transcription repressor (Kageyama et al., 2007), we explored a scenario whereby dhes inhibits the expression of transcription repressor Earmuff (Erm), which in turn inhibits SEC expression, based on previous observations: (1) Erm is specifically expressed in immature progenitors to prevent INP fate reversion and tumorigenesis (Janssens et al., 2014; Weng et al., 2010); (2) dhes overexpression-induced brain tumor phenotype can be potently rescued by Erm overexpression (Janssens et al., 2014); and (3) bioinformatics analysis identified the DNA-binding motif of the Erm/Brahma (Brm) complex at the daff enhancer region (Koe et al., 2014). Indeed, erm transcription, as reflected by its enhancer reporter erm-9d10-lacz, was ectopically activated in NBs upon loss of dhes, and was abolished in immature INPs upon dhes overactivation (Figures S4C and S4D), in accordance with a recent study utilizing a different erm transcription reporter (Li et al., 2016). Furthermore, multiple putative HES-binding sites are present in the promoter region of erm (Figures S4E) (Giagtzoglou et al., 2003; Kageyama et al., 2007; Kobayashi and Kageyama, 2014). Therefore, despite the lack of biochemical evidence, dhes is likely to inhibit erm transcription in a direct manner. On the other hand, we found that endogenous dell or daff protein or a daff transcription reporter containing the putative Erm/Brm-binding motif were ectopically expressed in dedifferentiating INPs upon Erm/Brm inactivation (Figures 4I, 4J, and S4F). Consistently, SEC inactivity potently suppressed brain tumor phenotypes induced by loss of Erm/Brm activity (Figures 4K and 4L). Taken together, we conclude that dhes promotes SEC expression in NBs through a dhes-erm/brm-sec double-negative regulatory mechanism. In summary, our results presented so far suggest a model whereby the unequal partitioning of Notch antagonist Numb into immature INPs initiates a small bias in the dhes levels between NBs and their sibling INPs; a slightly higher abundance of dhes in NBs enhances SEC activity that in turn cell-autonomously drives even higher levels of dhes. Thus, this self-reinforcing positive feedback loop between SEC and dhes quickly amplifies the initial small bias in Notch activity above a certain threshold, resulting in a timely and definitive stem cell fate commitment. (E and F) Close-up images of the expression pattern of dell (E) or daff (F) in type II NB lineages of indicated genotypes. Note that the cell size of dedifferentiating INPs (yellow arrowheads; Dpn + ) is smaller than that of primary type II NBs (white brackets) and larger than that of wild-type immature INPs (white arrowheads). (G) Supernumerary NB phenotype induced by E(spl)mg overexpression was potently suppressed by CDK9 inactivation but not by Notch depletion. (H) Quantification of total NB number per brain lobe of indicated genotypes. **p < 0.001; NS, not significant (n = 10 18). (I and J) The expression of dell protein (I) or daff-stinger reporter (J) was barely detectable in WT INPs (white arrowheads) but was significantly elevated in dedifferentiating INPs (yellow arrowheads) induced by simultaneous depletion of Erm and Brm complex subunit Snr1. Note that daff-stinger was also expressed in glial cells (open arrowheads). (K and L) Supernumerary NB phenotype induced by snr1 depletion was potently suppressed by dell-rnai (K). Quantification of type II NB number per brain lobe of indicated genotypes is shown in (L). **p < (n = 10 15). Scale bars, 10 mm (A, C, E, F, I, and J) and 50 mm (G and K). See also Figure S Developmental Cell 40, , March 27, 2017

10 SEC Overactivation Drives Progenitor Dedifferentiation If the model presented above is correct, one important prediction is that forced activation of SEC within immature INP may be sufficient to upregulate dhes expression, initiate and sustain the amplification loop, and lead to cell fate reversion of INPs back to NB-like cells and brain tumor formation. Indeed, while overexpression of either dell or daff transgene alone failed to alter cell fate, simultaneous overexpression of both transgenes in all type II NB lineages by PntP1-GAL4 caused formation of supernumerary highly proliferative Dpn +, Mira +, Ase NB-like cells (Figures 5A, 5B, S5A, and S5B). These observations indicate that daff synergized with dell to promote self-renewing cell fate. In contrast, overexpression of both SEC subunits in all type I NB lineages by ase-gal4 had no effect (Figures 5C and 5D), strongly suggesting that the SEC overactivationinduced ectopic NBs were originated from INP dedifferentiation. Furthermore, the cell polarity of ectopic NBs induced by dell and daff coexpression remained unaltered (Figure 5E), arguing against the possibility that these ectopic NBs result from NB symmetric divisions. Thus, our observations indicate that the reverting neural progenitors are the origin of brain tumor induced by SEC overactivation. We next investigated whether tumorigenesis induced by SEC overactivation is due to dhes upregulation. In control type II NB lineages, E(spl)mg-GFP is highly expressed in NBs, lowly expressed in the mature INPs, and undetectable in immature INPs (Figures 2C and 5F). The expression levels and pattern of this Notch reporter remained unchanged 48 hr after temperature shift (Figure 5G). By sharp contrast, in type II NB lineages concomitantly overexpressing dell and daff for 48 hr, newly born Dpn immature INPs adjacent to NBs (pink arrowheads) showed a dramatically elevated E(spl)mg-GFP expression of a 100-fold increase (Figures 5F and 5G). Interestingly, in comparison, overexpression of Notch in this genetic setting only led to a 30-fold increase in E(spl)mg expression (Figures S5C and S5D). While being pushed away from the primary NBs by newly born cells, the immature INPs coexpressing dell and daff (yellow arrowheads) started to express NB marker Dpn but not progenitor marker Ase (Figure 5F), indicating that a surge in dhes expression rapidly reverted their cell fate back to an NB-like state. Consistent with this notion, removing one copy of dhes gene cluster almost completely prevented SEC overactivationinduced tumorigenesis, whereas increasing one copy of the E(spl)mg genomic sequence evidently enhanced the tumor phenotype (Figures 5H and 5I). Therefore, SEC overactivationinduced brain tumors were driven by dhes overexpression and originated from reverting immature INPs. dell and daff Promote Each Other s Expression The synergistic effect of dell and daff in escalating E(spl)mg-GFP expression and inducing brain tumors prompted us to examine whether these two factors regulate each other. daff or dell protein levels in NB lineages were evidently reduced upon dell or daff knockdown, respectively (Figures S5E S5G). Furthermore, cotransfection of increasing amounts of dell-myc led to increased FLAG-dAFF protein levels, and vice versa (Figures S5H and S5I). Thus, dell and daff are both necessary and sufficient to promote each other s expression, explaining their synergistic effects. SEC Functionally Interplays with Notch Signaling to Promote Self-Renewal We next investigated whether SEC functionally interplays with Notch signaling to drive progenitor dedifferentiation. Either knockdown of the Notch receptor or overexpression of a dominant negative form of the Delta ligand completely suppressed the supernumerary NB phenotype induced by daff and dell coexpression (Figures 6A, 6B, and S6A), confirming that SEC promotes self-renewing cell fate in a Notch signaling-dependent manner. Strongly supporting our model whereby SEC is recruited mainly by the docking component of the Notch transcription activation complex CSL to enhance dhes transcription and promote self-renewal, increasing the expression levels of dcsl dramatically enhanced the supernumerary NB phenotype induced by dell and daff coexpression (Figures 6A and 6B) and caused formation of highly proliferative and invasive tumors upon transplantation (Figures 6C, 6D, and S6B). Importantly, overexpression of dcsl alone by insc-gal4 neither upregulated the abundance of E(spl)mg-GFP in NB lineages nor caused tumorigenesis (Figures 6A, 6B, and S6C). Furthermore, whereas overexpression of dcsl alone in Dpn Ase progenitors by erm-gal4(ii) showed no phenotypes (Figure 6E), coexpression of dcsl together with dell and daff in Dpn Ase progenitors, but not in Dpn Ase + progenitors by erm-gal4(iii), resulted in immature INP dedifferentiation and tumorigenesis (Figure 6E), further confirming that Dpn Ase reverting immature progenitors are the cellular origin of SEC overactivation-induced brain tumors. Not surprisingly, coexpression of CDK9-KD largely abolished the brain tumor-inducing capacity of SEC (Figures 6B and S6A), indicating that the catalytic activity of P-TEFb is indispensable for SEC in promoting self-renewal. Taken together, our data indicate that SEC, by forming a reinforcing positive feedback loop with Notch signaling, acts as an intrinsic amplifier to magnify and consolidate initial small fate bias into distinct and definitive fate outcomes, ensuring precise and robust stem cell fate commitment (Figure 7A). To further test this model, we assessed whether SEC is capable of compensating the even smaller fate differences between daughter cells due to defective asymmetric cell division and resolving such cell fate ambiguity. Type II NB loss resulting from defective asymmetric NB division in apkc-rnai background was effectively rescued by a mild overactivation of SEC (Figures 7B 7D), further substantiating our model. DISCUSSION SEC as an Intrinsic Amplifier to Ensure Neural Stem Cell Fate Commitment Is the establishment of an initial fate bias at the end of stem cell asymmetric division truly the end, or just the beginning of the end? Our findings revealed that a progressive and tightly controlled transition phase exists between the initial fate decision and the final definitive fate commitment. Our results identified the evolutionarily conserved SEC as a crucial intrinsic amplifier, accelerating this previously overlooked fate transition phase and ensuring NSC fate commitment in Drosophila type II NB lineages. Inactivation of SEC prevents the self-reinforcing feedback loop between SEC and Notch signaling from running, resulting in NBs with ambiguous stem cell identity and ultimate Developmental Cell 40, , March 27,

11 Figure 5. SEC Overactivation Drives INP Dedifferentiation (A) Flip-out clonal analysis of type II NB lineages in control or PntP1-Gal4>dELL;dAFF backgrounds. WT, wild-type. (B) Quantification data showing that, while control type II NB lineages contained one and only one type II NB, type II NB lineages simultaneously overexpressing dell and daff contained on average 5.4 type II NBs (R10 mm) at 72 hr ACI (after clone induction). **p < (n = 18 20). (C and D) Overexpression of dell and daff in type I NB lineages by ase-gal4 did not cause brain tumor formation (C). Quantification of type I NB number in brain lobes of indicated genotypes is shown in (D). NS, not significant. (E) As in control type II NBs, NBs overexpressing both dell and daff showed normal cortical distribution of apkc and Mira (n = 40). (F) Time-course analysis of E(spl)mg-GFP expression in PntP1-Gal4>dELL;dAFF;Gal80ts type II NB lineages. Note the drastic increase of E(spl)mg-GFP expression in newly born INPs (pink arrowhead) at 48 hr after temperature shift (ATS; from 22 C to 29 C). Also note that some immature INPs residing one cell diameter away from the primary NBs (yellow arrowheads) became E(spl)mg-GFP+, Dpn+, Ase NB-like cells. White arrowheads indicate E(spl)mg-GFP, Dpn immature INPs. (G) Quantification of relative E(spl)mg-GFP expression levels in newly born INPs of control background or in newly born INPs (pink arrowheads), NBs or newly born INP/NB of PntP1-Gal4>dELL;dAFF background at indicated time. ***p < ; NS, not significant (n = 8 12). Note that E(spl)mg-GFP levels in newly born INPs became comparable with their levels in NBs directly before these INPs dedifferentiated into NB-like cells. (H) Larval brains of indicated genotypes were stained for Dpn and Pros. (I) Quantification of total type II NB number per brain lobe of indicated genotypes. **p < (n = 12 17). Scale bars, 10 mm (A, E, and F) and 50 mm (C and H). See also Figure S Developmental Cell 40, , March 27, 2017

12 Figure 6. SEC Synergizes with Notch Signaling in Causing Transplantable Tumors (A) Larval brain lobes of indicated genotypes were stained for Dpn and Pros. (B) Quantification of total type II NB number per brain lobe of indicated genotypes. WT, wild-type. **p < 0.001; NS, not significant (n = 11 15). (C) Upon transplantation into the abdomens of adult host files, GFP + tumor pieces from larval brains coexpressing dell;daff or dell;daff;dcsl grew into large tumor mass (yellow brackets) and metastasized to distal organs such as the eye (yellow arrowheads). In contrast, GFP + tissue from wild-type or dcslexpressing larval brains caused neither tumorous growth (white brackets) nor metastasis (white arrowheads) in the host flies. (D) GFP + tumor tissues from the transplanted hosts were isolated and stained for NB markers Mira and Dpn, or cell proliferation marker phosphor-histone H3 (ph3). Note that most of the extracted GFP + tumor cells were Mira +, Dpn + NBs and highly proliferative. (E) Coexpression of dell, daff, and dcsl by erm-gal4 (II) but not erm-gal4 (III) caused formation of supernumerary Dpn +, Ase ectopic NBs (yellow arrowhead). Scale bars, 50 mm (A) and 10 mm (D and E). See also Figure S6. fate loss (Figure 7A). Conversely, ectopic overactivation of SEC initiates and sustains this positive feedback loop within progenitors, driving dedifferentiation and tumorigenesis (Figure 7A). It is interesting to note that, as one of the most active P-TEFb-containing complexes in controlling rapid transcriptional induction in response to dynamic developmental or environmental cues (Lin et al., 2011; Luo et al., 2012), SEC is particularly suitable for being an amplifier in driving timely cell fate commitment. Since fast-cycling stem cells are under huge pressure to achieve robust fate determination in every cell cycle, it is not surprising that they employ SEC as a regulatory component to induce immediate activation of master fate-specifying genes that in turn form a self-amplifying loop with SEC to rapidly magnify the initial fate bias and ensure prompt fate commitment. Such an intracellular amplifier mechanism revealed by our studies might complement the well-established intercellular lateral inhibition mechanism and represent a general, cell-autonomous paradigm to ensure robustness and precision in binary cell fate commitment. Lateral inhibition is a widely used mechanism underlying cell fate diversification (Artavanis-Tsakonas et al., 1999; Losick and Desplan, 2008), whereby unidirectional Notch signaling utilizes intercellular feedback loops to amplify an initial small difference between adjacent daughter cells, and eventually confers distinct cell fates. Lateral inhibition relies on intercellular interactions between adjacent cells (Artavanis-Tsakonas et al., 1999; Losick and Desplan, 2008). Here, we propose a model whereby an intracellular amplifier mechanism may also diversify cell fates. The intracellular amplifier and intercellular lateral inhibition mechanisms, both acting through feedback loops, are not mutually exclusive. Instead, they are complementary to each other and can be used concomitantly or sequentially to achieve differential fate outcomes in a timely, precise, and robust manner. An amplifier design often employs negative feedback to prevent excessive amplification. Here the dhes-erm/brm-sec double-negative regulatory mechanism that we revealed in NBs might also operate in neural progenitors, where the Erm/Brm complex could serve as a crucial brake to prevent the Notch- SEC-Notch self-reinforcing positive feedback loop from starting. Notch signaling plays a conserved role during vertebrate embryonic neurogenesis in maintaining the undifferentiated status of NSCs (Pierfelice et al., 2011). Intriguingly, expression of HES-1, a primary target of Notch pathway in mammalian neural development, oscillates every 2 hr (Shimojo et al., 2008). It has been proposed that oscillations in HES-1 expression drive fluctuations in gene expression, resulting in differential expressions between neighboring cells, which needs be further amplified to confer distinct cell fates (Pierfelice et al., 2011). How such an amplification step is triggered and modulated remains elusive. Given that SEC is highly conserved in mammals, it is interesting to speculate that a similar amplifier mechanism is employed to Developmental Cell 40, , March 27,

13 Figure 7. SEC Acts as an Amplifier to Drive Neural Stem Cell Commitment (A) A schematic model depicting SEC-mediated stem cell fate commitment. SEC (purple) normally acts as an intrinsic amplifier in magnifying and consolidating initial small fate bias between a stem cell (green) and its sibling progenitor (light gray). Upon SEC inactivation, a stem cell loses its self-renewing fate. Conversely, upon SEC overactivation, a progenitor reverts its fate back into a stem cell-like state and initiates tumorigenesis. (B D) SEC resolves cell fate ambiguity resulting from defective asymmetric NB division. (B) WT or apkc-rnai telophase NBs were stained for apkc and Mira. Note that apkc knockdown led to uniform cortical distribution of Mira and reduced NB cell sizes. (C) Larval brain lobes of various genotypes as indicated were stained for Dpn and Ase. Type II NB (Dpn + Ase ; arrowheads) loss phenotype caused by apkc depletion was effectively rescued by weak coexpression of dell and daff. Note that 103UAS-CD8-GFP (CD8-GFP) was used as control for ruling out the titration effect of Gal4 protein. Also note that a combination of UAS-dELL and UAS-dAFF transgenes of very weak expression levels were utilized here to ensure that their coexpression alone did not alter type II NB number per brain lobe. Quantification of type II NB number per brain lobe of indicated genotypes in shown in (D). WT, wild-type. **p < 0.001; NS, not significant (n = 15 30). Scale bars, 10 mm (B) and 25 mm (C). ensure mammalian NSC fate commitment. Whether SEC interplays with Notch signaling to drive cell fate commitment in other stem cell lineages (Bertet et al., 2014; Koch et al., 2013) also warrants future investigation. SEC Function in Development and Tumorigenesis Despite extensive studies elucidating how SEC regulates transcription elongation, the in vivo function of SEC in normal development and physiology remains enigmatic. Our results indicate that SEC is highly expressed in Drosophila NSCs, where it is recruited by the Notch transcription activation complex to stimulate the transcription of dhes genes and promote self-renewing fate. Interestingly, the dhes genes in fly larval brain NB lineages are non-pausing genes (Southall et al., 2013)(Figures S3D S3G), raising the possibility that SEC promotes the transcriptional acti- vation of dhes in the absence of paused Pol II. Consistent with this view, recent studies have demonstrated that the rapid transcriptional induction of some nonpausing genes, such as Cyp26a1 in human embryonic stem cells and a subset of pre-cellular genes in early Drosophila embryos, depends on SEC activity and Pol II occupancy (Dahlberg et al., 2015; Levine et al., 2014; Lin et al., 2011). Our findings that SEC physically and genetically interplays with the dcsl-nicd- MAM transcription activation complex to activate dhes transcription thus provide a unique physiological context for elucidating the detailed molecular mechanisms underlying transcriptional induction of non-pausing genes by SEC. The upstream signals and molecular mechanisms controlling SEC activity in normal development or physiology are just unfolding (Lin et al., 2011). It has been previously shown that the activity of SEC could be regulated by modulating the kinase activity of CDK9, the catalytic subunit of SEC (He et al., 2010; Lin et al., 2011). Our results unveil a new and unexpected mechanism underlying the control of SEC: the Notch-HES axis spatially restricts SEC activity within NSCs by cell-autonomously promoting the protein abundance of daff and dell, two regulatory subunits of SEC. Consistently, overactivation of Notch signaling led to dedifferentiation of immature INPs, in which the expression levels of daff/dell and, hence, the activity of SEC evidently increase. 548 Developmental Cell 40, , March 27, 2017