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provided by Caltech Authors DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 1207

Development 140, 1207-1219 (2013) doi:10.1242/dev.088559 © 2013. Published by The Company of Biologists Ltd Architecture of a lymphomyeloid developmental switch controlled by PU.1, Notch and Gata3 Marissa Morales Del Real and Ellen V. Rothenberg*

SUMMARY Hematopoiesis is a classic system with which to study developmental potentials and to investigate regulatory networks that control choices among alternate lineages. T-cell progenitors seeding the thymus retain several lineage potentials. The PU.1 is involved in the decision to become a T cell or a myeloid cell, and the developmental outcome of expressing PU.1 is dependent on exposure to Notch signaling. PU.1-expressing T-cell progenitors without Notch signaling often adopt a myeloid program, whereas those exposed to Notch signals remain in a T-lineage pathway. Here, we show that Notch signaling does not alter PU.1 transcriptional activity by degradation/alteration of PU.1 . Instead, Notch signaling protects against the downregulation of T-cell factors so that a T-cell transcriptional network is maintained. Using an early T-cell line, we describe two branches of this network. The first involves inhibition of E- by PU.1 and the resulting inhibition of Notch signaling target . Effects of E- protein inhibition can be reversed by exposure to Notch signaling. The second network is dependent on the ability of PU.1 to inhibit important T-cell transcription factor genes such as Myb, Tcf7 and Gata3 in the absence of Notch signaling. We show that maintenance of Gata3 protein levels by Myb and Notch signaling is linked to the ability to retain T-cell identity in response to PU.1.

KEY WORDS: Gene regulatory network, Lineage decision, Myb, Quantitative analysis, T-cell development, Sfpi1, Mouse

INTRODUCTION on early T-cell progenitors, with cells partitioning between those T-cell development depends on the correct expression of an that maintain a T-cell gene expression pattern and those that shift intricate transcription factor network and on signaling from the towards a myeloid pattern (Dionne et al., 2005; Franco et al., 2006). environment. T cells develop from multipotent progenitors that This suggests competition between two self-reinforcing network migrate from the bone marrow to the thymus, where they become states. However, the actual gene network underlying this choice has dependent upon Notch signaling for their development and been obscure. survival (Yang et al., 2010). At the early CD4– CD8– double Here, we explore the mechanisms that mediate the regulatory negative (DN) stages, pro-T cells retain lineage plasticity until the competition between PU.1 and Notch signals, using primary mouse DN2b stage where they become committed pre-T cells. The ETS fetal thymocytes and a clonal pro-T-cell line system to dissect the family transcription factor PU.1 (Sfpi1 – Mouse Genome regulatory impacts of PU.1 and Notch signaling. We show that Informatics) is important during early T-cell development (Back Notch signaling does not inactivate PU.1 protein but re-channels its et al., 2005; Nutt et al., 2005), and is highly expressed initially but transcriptional effects. However, PU.1 and Notch signaling are repressed during commitment (Fig. 1A). This pattern must be involved in a mutually inhibitory network, as PU.1 can repress maintained for development to succeed. In early T-cell stages, Notch targets. Our results further reveal two branches of the T-cell PU.1 drives expression of cytokine receptors such as Il7r and Flt3, gene network that collaborate against the PU.1-mediated diversion: and of genes that are important for cell communication one involving basic helix-loop-helix E proteins in a tight positive- (Turkistany and DeKoter, 2011). However, it is also required for feedback loop with Notch; and a separate branch for Gata3 and the the development and function of other cell types, including Gata3-activating factor Myb. We show that PU.1 undermines Gata3 hematopoietic stem cells (Iwasaki et al., 2005), multipotent expression, foreshadowing diversion in individual cells. The two T- progenitors (Wontakal et al., 2011), myeloid cells (Ghani et al., cell lineage protective pathways converge as Myb and Notch 2011) and B cells (Houston et al., 2007). Forced overexpression of signaling each enable Gata3 expression to be maintained in the face PU.1 can divert early T cells to a myeloid lineage (Anderson et of high levels of PU.1. al., 2002; Dionne et al., 2005; Lefebvre et al., 2005; Laiosa et al., 2006b). However, in the context of T-cell development the MATERIALS AND METHODS progenitors are normally protected from diversion, even while Mice expressing high levels of PU.1, by their exposure to Notch C57BL/6-Bcl2tg mice [B6.Cg-Tg(BCL2)25Wehi/J] were housed under specific pathogen-free conditions, bred and cared for by Caltech Animal signaling from the environment (Franco et al., 2006; Laiosa et al., Facility staff. Embryonic day (E) 14.5 or 15.5 fetal thymocytes were used. 2006b) (Fig. 1A). All animal work followed protocols approved by the Institutional Animal Tracking the effects on several dozen genes has shown that the Care and Use Committee. interaction between PU.1 and Notch can have dichotomous effects Cell culture Scid.adh.2C2 cells were cultured in RPMI1640 with 10% fetal bovine Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, serum (Sigma-Aldrich), sodium pyruvate, non-essential amino acids, USA. penicillin/streptomycin/glutamine (Gibco/Life Technologies/Invitrogen) *Author for correspondence ([email protected]) and 50 μM β-mercaptoethanol. Cells were incubated at 5% CO2 and 37°C. For Notch signaling inhibition, InSolution γ-Secretase Inhibitor X (EMD

Accepted 21 December 2012 Millipore) were added at 0.5 μM. DEVELOPMENT 1208 RESEARCH ARTICLE Development 140 (6)

A Myeloid Notch Notch Signaling Signaling Committed-T

MPP Survival PU.1+ PU.1+ PU.1-

(DN1, DN2) (DN3) Thymus

B C Infection with Cells cultured with ab Bcl2tg Fetal PU.1 GFP and IL7 and Flt3L in Empty Vector PU.1 Empty Vector PU.1 Thymocytes Empty vector GFP differing Notch 100 100 104 104 for 4 hours conditions for 6 3 29 3 14 3 17 17 3 days. 10 10 102 102 OP9-

Flow cytometry 1 10 101 Control 20 20 Analysis (GFP+)

1caM 0 0 0 0 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Notch Signaling: 104 104 Empty Vector PU.1 0.9 26 100 100 % of Max 103 15 0.1 103 21 18

4 Day 1 Day 2 Day 3 104 10 102 102 OP9- 3 103 0.1 10 4 DL1 101 101 2 102 10

a 0 0 1 1 10 20 20 +++ 10 10 10 0.2 20 100 101 102 103 104 100 101 102 103 104 0 0 0 0 10 10 0 1 2 3 4 100 101 102 103 104 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

4 104 10 PU.1 c 3 103 2 10 43 Mac1

2 b 102 10 97 98 --- +1ca 100 92

1 101 10 80 74 PU1 Low 13 80 0 0 1yhT 10 10 0 1 2 3 4 60 53 0 1 2 3 4 10 10 10 10 10

10 10 10 10 10 M

PU1 Inter. 4 104 10 40 32

%

3 103 0.1 10 7 20 PU1 High

2 c + -- 102 10 Notch 1 1 ++ 10 + 10 Signaling - - - 0.3 24 0 100 10 0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 Low PU.1 Int. PU.1 High PU.1

4 10 104

3 10 0.8 103 18

2 d - ++ 10 102

1 10 101 6 58 0 10 100 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 * GFP+ Cells analyzed Mac1

Fig. 1. Continuous Notch signaling is not required to protect fetal thymocytes from diversion. Notch signaling can protect cells with high PU.1 protein levels. (A) PU.1 and Notch signaling interactions during early T-cell development. (B) E15.5 fetal thymocytes transduced with PU.1 and empty vector were cultured in different Notch signaling conditions (a-d) for 3 days with Il7 and Flt3 ligand. The transduced cells were analyzed for the expression of the T-cell marker Thy1 and the myeloid marker Mac1. (C) E15.5 thymocytes were transduced with PU.1 or an empty vector (a-c). The percentage of Mac1+ cells in samples expressing high, intermediate and low levels of PU.1 protein were obtained using flow cytometry.

Fetal thymocytes were cultured on OP9-Delta-like1 (OP9-DL1) or OP9- RNA extraction and quantitative real-time RT-PCR control stroma in α-MEM with 20% fetal bovine serum, cDNA was prepared from total RNA using RNeasy extraction kits (Qiagen) penicillin/streptomycine/glutamine, 50 μM β-mercaptoethanol, 5 ng/ml Il7, and reverse transcribed using random primers and SuperscriptIII 5 ng/ml Flt3 ligand (cytokines from Peprotech). (Invitrogen). Specific gene expression in cDNA samples was measured by qRT-PCR Cell staining, flow cytometry and sorting (ABI Prism 7900HT) using SyberGreenER mix (Invitrogen). Results were FITC, PE, APC, APCe780, Pacific Blue and PerCPCy5.5-conjugated calculated (ΔCt method) and normalized to Actinb levels. For actual values antibodies from eBioscience or Cell Signaling (against CD25, CD44, CD45, see supplementary material Table S1A,B (Fig. 2) and Table S2A-E (Figs 3- Mac1/CD11b, CD11c, Thy1, NGFR and human CD8) were used for cell 7). Primers used for qRT-PCR were described previously (David-Fung et al., surface staining. Fc receptors were first blocked with 2.4G2. PU.1 2009; Li et al., 2010; Yui et al., 2010), or are listed in supplementary intracellular staining using the BD cytofix/cytoperm kit (Becton Dickinson material Table S3. Immunocytometry Systems) was carried out using PU.1 (9G7) rabbit mAb- AlexaFluor 647 and rabbit mAb IgG (#2985, Cell Signaling) as an isotype Heatmap generation control. Gata3 intracellular staining using Foxp3 Staining Buffer Kit Heatmaps were generated using a Matlab (MathWorks) script written by Dr (eBioscience) was carried out with mouse anti-Gata3-AlexaFluor 647 and Hao Yuan Kueh (California Institute of Technology, Pasadena, CA, USA). mouse IgG1 κ as an isotype control (BD Biosciences Pharmingen). Data Briefly, values are log10-transformed averages of expression levels presented are representative of multiple independent experiments with n=2 determined by qRT-PCR from 2-4 independent experiments: n=2 or 3 (Fig. 1C; Fig. 7C,D) and n=3-5 (Fig. 1B; Fig. 3A,B; Fig. 4A; Fig. 5A; Fig. (Fig. 2C,D), n=4 (Fig. 3C; Fig. 4B), n=2 (Fig. 5B; Fig. 6D; Fig. 7E). Levels 6B,C; Fig. 7B). for each gene in different samples are presented relative to the level in the Cells were sorted using BDIS FACS Aria IIu or iCyt Mission Technology control sample (empty vector transduced=1.0). The color scale ranges from Reflections cell sorters and analyzed using FACSCalibur (BDIS) or ~10−2 to 102 times this reference value, as indicated. Ordering of genes was MACSQuant (Miltenyi Biotec) analyzers and FlowJo software (Tree Star). by hierarchical clustering (median method, Matlab). DEVELOPMENT Myeloid versus T-cell fate choice RESEARCH ARTICLE 1209

Cloning/subcloning include some cells with natural myeloid potential, but few were Gfi1-, Id2-, Bambi- and Cebpa-coding sequences were purchased from Thy1+, i.e. derived from T-lineage precursors (Fig. 1Bb: 13% total Genscript and subcloned into retroviral vectors: LZRS or MIGR1 with a Mac1+ versus 2% Thy1+ Mac1+). Fetal thymocytes transduced with GFP marker, or derivatives with an NGFR marker (New England Biolabs PU.1 generated far more Mac1+ cells than thymocytes transduced reagents). For retroviral packaging, Phoenix-Eco cells were transfected with with empty vector under all conditions (Fig. 1B): if cultured in the long-term puromycin selection for LZRS-based vectors, whereas 293T cells absence of Notch signals, most became Mac1+, over 50% derived were transiently co-transfected with pCL-Eco plasmid for MIGR1-based + vectors. Tcf7 in a retroviral vector with a Vex reporter and ICN1 and from Thy1 cells. As expected, the samples cultured in the presence dnMAML in MIGR1 were kind gifts from Avinash Bhandoola and Warren of Notch signaling on OP9-DLl cells throughout the 3-day culture + + Pear, respectively (University of Pennsylvania, Philadelphia, USA). contained a far smaller percentage of Thy1 Mac1 diverted cells as + Gata3shRNA in the Banshee retroviral vector was made by Gabriela well as fewer Mac1 cells overall than those cultured on OP9- Hernandez-Hoyos (California Institute of Technology, Pasadena, CA, control. Notch signals restored for the last 2 days after an initial day USA). of deprivation also reduced diversion, as these samples mimicked the conditions we had used previously (Franco et al., 2006). Retroviral infection Non-tissue culture treated plates (Corning) were incubated with Retronectin Notably, however, samples that were initially cultured on OP9-DL1 (Takara) at 40-50 ng/ml overnight at 4°C. Retronectin was removed and for only 1 day and then shifted to OP9-control were also protected, viral supernatant added and spun at 2000 g for 2 hours at 32°C. Unbound almost as strongly as in continuous presence of DL1 (Fig. 1Bc). virus was removed and cells added in their preferred medium at 1×106 Thus, Notch signaling through the onset of PU.1 overexpression cells/ml, then incubated for 4 hours or overnight. could establish a regulatory state making Thy1+ fetal thymocytes relatively resistant to diversion. Western blots Cell extracts in Laemmli sample buffer were boiled for SDS-PAGE. Proteins were transferred to PVDF Immobilin (Millipore) and blots were Pro-T cells with high levels of PU.1 protein are blocked with 5% milk in TBS-T (Tris-buffered saline, 0.5% Tween-20), able to resist Mac1 upregulation in the presence incubated with SP1 (sc-59) or PU.1 (sc-352) antibody (Santa Cruz of Notch signaling Biotechnology, 1:1000 dilution) and then with secondary antibody (1:2000). A possible mechanism for protection of pro-T cells from PU.1- Samples were then incubated with substrate (SuperSignal, Pierce) for film mediated diversion could be to inactivate PU.1 protein. PU.1 detection. phosphorylation can affect its DNA binding (Seshire et al., 2011) and transactivation domain engagement (Hamdorf et al., 2011). Notch RESULTS signaling can regulate protein phosphorylation (Vo et al., 2011) and Notch signaling protects against diversion at trigger protein degradation by promoting ubiquitylation (Lim et al., early and late time points after PU.1 2011). To test directly whether Notch-Delta signaling resulted in overexpression changes in PU.1 protein levels, fetal thymocytes were infected with In the early T-cell stages when PU.1 is active, it provides cells with PU.1 or empty vector and cultured on either OP9-DL1 or OP9-control access to developmental alternatives and is therefore a risk to T- for 2 days, and then stained for both intracellular PU.1 and cell- lineage fidelity. We have shown previously that thymocytes can be surface Mac1. The intermediate and high levels of PU.1 protein in protected from PU.1-mediated lineage diversion if they receive transduced cells matched the levels of endogenous PU.1 in those Notch signals (Franco et al., 2006), as they would in the normal control thymocytes that revealed natural myeloid potential when thymus in vivo. However, the mechanism through which Notch Notch signals were removed (Fig. 1Ca, empty vector, OP9-control). signaling counteracts the activity of PU.1 has been obscure. The distributions of PU.1 protein in PU.1-transduced cells were To investigate the critical time interval in which Notch signaling not globally altered by the presence or absence of Notch signals. affects thymocyte responses to PU.1, we forced fetal thymocytes to However, the response to a given level of intracellular PU.1 express PU.1 by retroviral transduction and exposed them to differing depended strongly on Notch signaling, as cells made all-or-none Notch signaling conditions for 3 days, using switch cultures based on choices between remaining Mac1– and diverting to high Mac1+ co-culture with OP9-DL1 or OP9-control stromal cells. BCL2- states (Fig. 1Ca,c). High, intermediate and low levels of intracellular transgenic (Bcl2tg) thymocytes were used to enhance recovery of PU.1 protein all drove over 90% of cells to become Mac1+ in the cells after regulatory perturbation (supplementary material Fig. S1) absence of Notch ligand (Fig. 1Cb). In the presence of Notch ligand, (Franco et al., 2006; Taghon et al., 2007). OP9-control stroma Mac1 could still be induced at the highest levels of PU.1 protein, supports B cell, natural killer cell and myeloid development, but when and Notch signaling did not affect the levels of Mac1 expressed transfected to express the Notch ligand Delta-like1 (DL1), OP9-DL1 (Fig. 1Cc). However, the high, intermediate and low PU.1 level cells cells support T-cell development (Schmitt and Zúñiga-Pflücker, each generated substantially lower Mac1+ percentages in the 2002). Thus, thymocytes were infected with empty vector or with presence of Notch signaling. Importantly, cells that now resisted PU.1-expressing retrovirus during a 4-hour incubation, cultured with Mac1 upregulation (Fig. 1Cb,c) expressed the same levels of PU.1 OP9-DL1 or OP9-control stroma for 1 day, and then either returned protein that promoted Mac1 expression when Notch signals were to the same Notch signaling condition or switched to the opposite absent. Thus, Notch signaling can sharply raise the dose-dependent condition for 2 more days (Fig. 1B). Thy1, which is itself Notch threshold for PU.1 to induce expression of Mac1, without affecting insensitive, was used to identify cells that had entered the T-cell accumulation of PU.1 protein itself. pathway (Taghon et al., 2007), as Mac1 (CD11b; encoded by Itgam) marked entrance to the myeloid pathway (Dionne et al., 2005). As PU.1 protein is intact in the presence of Notch these markers are normally mutually exclusive, activation of Mac1 on signaling Thy1+ cells identifies T-lineage cells beginning myeloid diversion. Similarly, when PU.1 was introduced into a pro-T-cell-like cell line, Later, these become Mac1+ Thy1–. Scid.adh.2C2, western blotting measurements showed that Notch On OP9-control stroma in the absence of Notch signals, the signaling affected the PU.1 dose threshold for the cells to divert to a + empty vector-transduced thymocyte population was revealed to Mac1 state (supplementary material Fig. S2). However, qualitative DEVELOPMENT 1210 RESEARCH ARTICLE Development 140 (6)

PU.1 electrophoretic mobility patterns were the same in diverted and sorting for RNA analysis, separately isolating transduced DN2 and diversion-resistant cells, whether Notch signaling was active or DN3 cells (Fig. 2; supplementary material Table S1A,B). inhibited, offering no evidence for differential phosphorylation or The genes analyzed showed four different patterns of response ubiquitylation. This suggests that PU.1 itself remains biochemically to PU.1 and Notch-DL1 interaction, as illustrated by representative competent in the presence of Notch signaling. bar graphs of expression for individual genes (Fig. 2A,B) and summary heatmaps of PU.1 effects on DN2 and DN3 cell gene Notch signaling effects on initial changes in gene expression (Fig. 2C,D). The normal developmental expression expression in fetal thymocyte responses to high- patterns of key genes are also shown (Fig. 2E). One group of genes level PU.1 was upregulated efficiently by PU.1 overexpression, whether Notch Because even transient exposure to Notch signaling could protect signaling was present or absent (e.g. Fig. 2A,C,D). This group PU.1-overexpressing thymocytes from diversion, Notch signaling included the stem and progenitor cell-associated genes Lyl1, Bcl11a might alter the earliest responses to PU.1. Previous studies had and Hhex, and the myeloid gene Fes. The effectiveness of PU.1 was shown that Notch signals protect important T-cell genes from partly constrained by the natural limits of the expression of these repression 40-48 hours after PU.1 transduction (Franco et al., 2006). genes from DN2 to DN3 (Zhang et al., 2012) (Fig. 2E): e.g. effects Those analyses were potentially skewed toward diversion, however, on Hhex and Lmo2 were seen in DN2 cells but not significantly in because the cells were initially deprived of Notch signals during the DN3 cells (supplementary material Table S1C). Only select genes, >16-hour transduction. In addition, survival effects could obscure e.g. Lmo2 (Fig. 2A) and Mac1 (Itgam), were inhibited from gene-specific regulation, for thymocytes naturally increase Notch- responding to PU.1 by Notch-DL1 interaction. Thus, PU.1 can dependence as they progress from DN2 to DN3, when many T-cell indeed act positively on many target genes, even in the presence of genes are induced (Yui et al., 2010). Therefore, we infected fetal Notch signaling. thymocytes with PU.1 or empty vector for only 4 hours, then Genes specifically expressed in the T lineage showed three cultured them with or without Notch signaling for 16 hours before patterns of response (Fig. 2B-D). Some were downregulated by

A Bcl11a DN2 Hhex DN2 CD Fig. 2. Gene expression profile of 0.01 0.1 DN2 DN3 fetal thymocytes in response to 0.01 0.001 high-levels of PU.1 in short-term 0.001 PU.1EV PU.1EV Notch Notch cultures. E15.5 fetal thymocytes 0.0001 0.0001 Signaling++ -- Signaling ++-- Notch 2 were infected with PU.1-GFP or ++--Notch Signaling ++-- Dtx1 Dtx1 2 EV PU.1 Signaling EV PU.1 empty vector-GFP and transferred to Tcf7 Fes Lmo2 DN2 Zfpm1 1.5 Bcl11a OP9-DL1 or OP9-control cells 0.01 1.5 + Gfi1 Itgam overnight. DN2 and DN3 GFP cells 0.001 Myb 1 Pou6f1 were sorted and gene changes were 1 Hes1 Bambi detected using qRT-PCR. (A) Genes 0.0001 HEBalt Meis1 Notch 0.5 upregulated with PU.1. (B) Genes ++-- Nrarp 0.5 Signaling EV PU.1 Lyl1 downregulated in DN2 and DN3 cells Ets1 HEBalt Gata3 0 with PU.1. Data are mean±s.d. B Hes1 0 DN2 DN3 Runx1 Nrarp (C,D) Heatmaps of gene expression Itgam Nrarp Nrarp –0.5 Runx1 –0.5 obtained by qRT-PCR in DN2 and 0.1 0.1 Hhex Gata3 DN3 fetal thymocytes expressing Bcl11a 0.01 0.01 Ets1 Fes –1 –1 PU.1 for 16 hours in the presence or Tcf7 absence of Notch signaling. (E) Early 0.001 0.001 Bambi Myb Lmo2 –1.5 –1.5 T-cell regulatory gene expression 0.0001 0.0001 Lef1 Notch ++--Notch ++-- Zfp710 Zfpm1 patterns. Signaling Signaling Meis1 EV PU.1 EV PU.1 –2 Gfi1 –2 Tcf7 Tcf7 1 1 E 0.1 0.1 NOTCH SIGNALS Nrarp, Hes1/5, Dtx1 DN1 0.01 0.01

0.001 0.001 myeloid DN2 DN3 Later stages of (non-T) T-cell development 0.0001 0.0001 Notch ++--Notch ++ CD25+ CD25+ Signaling Signaling -- EV PU.1 EV PU.1 Myb, Gfi1, Fog1 (Zfpm1) Gata3, Tcf7 Myb Myb Cd3e, Cd3g, Bcl11b 0.1 0.1 Rag1, Ptcra 0.01 0.01 PU.1, Lyl1, Hhex, Bcl11a 0.001 0.001 Fes, Csf1r, Cebpa/b, Itgam

0.0001 0.0001 Notch ++Notch ++-- Signaling --Signaling EV PU.1 EV PU.1

Gata3 Gata3 0.1 0.1

0.01 0.01

0.001 0.001

0.0001 0.0001 Notch ++--Notch ++ Signaling Signaling -- EV PU.1 EV PU.1 DEVELOPMENT Myeloid versus T-cell fate choice RESEARCH ARTICLE 1211

PU.1 whether or not Notch signaling was present. These included then cultured them for 2 more days with or without GSI and assessed Ets1 and the crucial T-cell regulatory gene Tcf7, a gene that is whether they remained Mac1 negative (Fig. 3B). Some cells in the initially induced by Notch (Germar et al., 2011; Weber et al., 2011) vehicle control samples did upregulate Mac1 after 2 days, but the cells but is not acutely dependent on Notch signaling for its maintenance. cultured in GSI generated a much higher percentage of Mac1+ cells Another pattern was defined by Notch target genes [e.g. Deltex1, (Fig. 3B). Thus, Scid.adh.2C2 cells expressing levels of PU.1 that are Hes1, HEBalt (Tcf12) and Nrarp], which depended on Notch signals barely adequate for diversion can be efficiently diverted when even in control cells: e.g. Deltex1. A third group consisted of genes endogenous Notch signaling is blocked. that were downregulated by PU.1, but much more severely if Notch signaling was absent. These included genes important for T-cell Diversion depends on PU.1-mediated inhibition of development, such as Myb, Fog1 (Zfpm1) and Gfi1. However, in Notch signaling in Scid.adh.2C2 cells general, the Notch target genes were also PU.1 inhibited, and Although inhibition of Notch signaling facilitated diversion, the additively affected by Notch deprivation and PU.1 (Fig. 2B-D: final molecular phenotype of the diverted cells was the same with Nrarp, HEBalt and Hes1). or without Notch inhibition, and the features of this response Though we anticipated Notch to influence PU.1 effects, these largely matched those of fetal thymocytes. Fig. 3C and Table 1 results suggest that PU.1 in early T cells also antagonizes responses (values in supplementary material Table S2A; Fig. S4A) to Notch. Thus, PU.1+ cells may demand higher-intensity Notch summarizes gene expression patterns in cells that were transduced signaling to maintain expression of directly and indirectly Notch- with PU.1 or empty vector and cultured for 2 days with GSI or regulated genes. control vehicle, then sorted to separate Mac1+ diverted cells from cells remaining Mac1–. A set of Notch-dependent target genes was A clonal early T-cell line can be used to study detectably inhibited by GSI, both in the absence of PU.1 and in Notch signaling protection against diversion of PU.1-transduced cells (Table 1). In addition, PU.1 turned on one pro-T cells set of genes that was neither dependent on Notch signaling nor on The lasting protective effects of Notch signaling in early pro-T cells Notch inhibition (Table 1). These were activated in Mac1+ and and its impact on early responses to PU.1 overexpression imply that Mac1– PU.1-expressing cells alike, showing that PU.1 is active in these early affected genes may be involved in deciding between all these cellular contexts. However, the induction of Mac1 by protection and diversion in cells with high PU.1 expression. Testing PU.1 heralded a global gene expression shift. Macrophage- these genes for epistatic or synergistic effects by co-transfection associated genes such as Csf1r and Mac1 (Itgam) were would be difficult in fetal thymocytes. Therefore, we used a upregulated by PU.1 selectively in the cells becoming Mac1+ previously described early T-cell line (Dionne et al., 2005) devoid (Table 1). As in fetal thymocytes, PU.1 also inhibited T-cell genes of intrinsic myeloid potential, which is much more permissive for (Table 1). Unlike activation, repression primarily occurred in co-transduction experiments. Mac1+ cells, not in cells remaining Mac1– (Table 1), implying that Scid.adh.2C2 cells, DN3-like cells that do not express these genes are repressed only when the regulatory threshold for endogenous PU.1, have previously been used to demonstrate the diversion has been crossed. Notably, cells becoming Mac1+ in all-or-none diversion response of early T-cells after PU.1 response to PU.1 alone maximally downregulated the Notch target overexpression (Dionne et al., 2005). Scid.adh.2C2 cells were genes, with or without GSI (Table 1). Thus, forced PU.1 cloned from a cell line, Scid.adh, which is derived from a expression can initiate a mechanism that leads to severe Notch spontaneous pro-T cell tumor (Carleton et al., 1999), and show pathway inhibition in Scid.adh.2C2 cells, and this event is tightly spontaneous, ligand-independent Notch pathway activation. We correlated with diversion. tested whether the Scid.adh.2C2 response to PU.1 was also subject to Notch-dependent protection. Notch signaling in these cells was Dissection of PU.1-dependent gene expression inhibited by γ-secretase inhibitor (GSI), as shown by the effects in the presence and absence of Notch downregulation of the Notch-dependent marker, CD25 (Fig. 3A, signaling ‘empty vector’, 0.5 μM GSI). Cells survived well with or without To dissect the mechanism of Notch pathway interaction with PU.1, Notch signaling. Scid.adh.2C2 cells transduced with PU.1 we used Scid.adh.2C2 cells for co-transduction experiments to upregulated Mac1 in a fraction of the population, and the percentage combine PU.1 with constitutively active Notch1 (ICN1) or the of cells becoming Mac1+ increased with the addition of GSI dominant-negative inhibitor of Notch-dependent transcription, (Fig. 3A). Interestingly, another Scid.adh subclone that was unable dnMAML (Maillard et al., 2004). Doubly transduced cells were to divert in response to PU.1 alone (6D4) (Dionne et al., 2005) also sorted based on their co-expression of both viral vectors after 2 days. showed strong diversion when Notch signaling was inhibited When ICN1 was co-expressed with PU.1, most of the cells (supplementary material Fig. S3). Thus, Notch signaling limits the remained CD25+ and did not upregulate Mac1. This protection response to PU.1 in these Scid.adh-derived clonal cell lines as in depended on Notch-dependent transcription, as the addition of primary thymocytes. dnMAML with PU.1 not only extinguished CD25 expression but Although many PU.1-overexpressing Scid.adh.2C2 cells also caused most of the cells to upregulate Mac1 (Fig. 4A). upregulated Mac1, a population of Mac1-CD25+ cells still remained. However, PU.1 could still induce gene expression changes in CD25 is encoded by a Notch target gene, Il2ra (Maillard et al., 2006), Scid.adh.2C2 cells, including expression of the dendritic-cell and expression levels of other Notch target genes correlate with CD25 marker CD11c, even in the presence of ICN1 (Fig. 4A). levels (M.M.D.R., unpublished). Individual Scid.adh.2C2 cells that The ability to manipulate Notch signaling independently of PU.1, remain Mac1 negative might simply express insufficient PU.1 to while maintaining viability, enabled us to ask how much of the divert, or they might resist because of higher Notch signaling, ‘PU.1’ effect on T-cell gene expression depended on its Notch suggested by their high CD25 expression. To distinguish these inhibition effects (Fig. 4B). Doubly transduced cells (Fig. 4A) were possibilities, we transduced Scid.adh.2C2 cells with PU.1 for 2 days, sorted for RNA analysis (fewer than 12% of PU.1+ ICN1+ cells were + – + + +

sorted the apparently diversion-resistant PU.1 Mac1 CD25 cells, Mac1 ; over 70% of PU.1+ dnMAML+ cells were Mac1 ). As DEVELOPMENT 1212 RESEARCH ARTICLE Development 140 (6)

A BCEV PU.1 Infection with PU.1 Infection with PU.1 Mac1- Mac1+ or empty vector and culture w/o GSI GSI -+ - + -+ Scid.adh.2c2 cells Scid.adh.2c2 cells Rag1 2 Days Hes5 Ptcra Culture +/-GSI 2 Days Notch1 Lef1 2.5 PU1+ cells (+Vehicle) Dtx1 Flow cytometry analysis Nrarp 105 Myb 2

4 10 Tcf7

1

ca 3 Sort Mac1- 10 Ets1

M CD25 high cells Flt3 102 GFP SSC

Mac1 1.5 7AAD 0 Aiolos

FSC FSC FSC 2 3 4 CD25 5 0 10 10 10 10 Trib2

250K 5 5 HEBalt 10 105 10 0.29 CD25 1 200K 4 36 4 Zfpm1 10 104 10 150K Hes1 3 3 Culture 2 Days 10 99 103 10 EV + 100K +/- GSI Il7Ra 2 2 102 Vehicle 50K 10 10 0 E2A 0.5 73 0 0 0 2 3 4 5 Bcl11b 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 010 10 10 10

250K 5 5 5 Gfi1 10 10 10 0.22 200K 4 40 4 Csf1r 10 104 10 CD25 High 0 150K 4 Lyl1 3 3 10 10 99 103 10 EV + 100K 15 22 Bcl11a 2 2 103 10 102 10 0.5µM GSI 50K Bambi 66 0 0 0 2 0 2 3 4 5 10 Vehicle Itgam −0.5 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 010 10 10 10

250K 5 5 1 Zeb2 10 105 10 18 10 200K Egr2 4 4 59 4 10 10 0 3 60 10 10 0 1 2 3 4 150K 10 10 10 10 10 Runx3

3 3 1c −1 10 98 103 10 PU.1 + 100K 104 Pou6f1

2 a 102 2 10 50K 10 Vehicle 40 31 Id2 3 69 0 0 0 M 10 0 2 3 4 5 Id3 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 010 10 10 10 102 0.5µM GSI −1.5 250K 5 5 Gata3 10 105 10 39 101 Psen2 200K 4 59 4 10 104 10 HEBcan 150K 0 14 15 3 3 10 100 3 10 0 1 2 3 4 10 10 PU.1 + 10 10 10 10 10 100K Maml1 −2 2 2 102 50K 10 10 0.5µM GSI CD25 Ski 57 0 0 0 0 2 3 4 5 Smad3 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 010 10 10 10 Nkap Tgfbr1 −2.5 Helios Spen Ikaros Runx1

Fig. 3. Scid.adh.2C2 cells can be used to study PU.1 and Notch signaling interactions. (A) Scid.adh.2C2 cells expressing PU.1 or empty vector were cultured with or without GSI for 48 hours. Mac1 and CD25 expression levels were measured using flow cytometry. (B) PU.1+Mac1– Scid.adh.2C2 cells were cultured in the presence or absence of GSI for 2 days. Mac1 and CD25 expression levels were measured using flow cytometry. (C) Heatmap of gene expression in Scid.adh.2c2 cells expressing PU.1 or empty vector with or without GSI for 2 days and sorted according to Mac1 expression. expected, PU.1 with dnMAML mimicked the full range of the these genes through a mechanism that depends on something diverted phenotype. However, separate regulatory components were besides Notch inhibition. Second, expression of some PU.1- distinguished with dnMAML alone, and when PU.1 expression was dependent genes were actually enhanced by ICN1, implying distinct combined with ICN1 (Fig. 4B; supplementary material Table S2B; gene-specific rules for interaction (supplementary material Table Fig. S4B). Forced expression of ICN1 could protect classic Notch S2B, group 4). Third, importantly, T-cell regulatory genes, including target genes, even in the presence of PU.1 (supplementary material Myb, Tcf7 and, to a lesser extent, Gata3 were only downregulated Table S2B, ‘response group’ 5), and these genes could be by PU.1 when combined with loss of Notch signaling upregulated by ICN1 alone (supplementary material Table S2B, (supplementary material Table S2B, group 7): they were minimally group 2), implying an additive effect. However, three additional affected by ICN1, dnMAML or PU.1 alone. dnMAML alone was relationships emerged. highly effective at blocking Notch target gene expression First, ICN1 could not protect all T-cell genes from PU.1 (supplementary material Table S2B, group 1), and yet had (supplementary material Table S2B, group 6). Thus, PU.1 represses absolutely no effect on Myb, Tcf7, Gata3 or Fog1. However, in a

Table 1. Gene responses to PU.1 forced expression and Notch signaling Response group Genes (1) Notch dependent Rag1, Hes5, Ptcra, Notch1, Lef1, Dtx1, Nrarp (2) PU.1 activated Lyl1, Bcl11a, Bambi, Itgam, Zeb2, Egr2, Runx3, Pou6f1, Id2 (3) PU.1 activated only (more) in Mac1+ diverted cells Csf1r, Egr2, Id2 (Bambi, Itgam) (4) Downregulated by PU.1 Ets1*, Aiolos*, Flt3* (5) Downregulated by PU.1 only (more) in diverted cells Myb, Tcf7, Trib2, HEBalt, Zfpm1, Hes1, Il7ra, Tcfe2a, Bcl11b, Gfi1, Gata3, HEBcan, Psen2,(Ets1), (Flt3) (6) Downregulated in diverted cells and in Notch-inhibited cells Rag1, Hes5, Ptcra, Notch1, Lef1, Dtx1, Nrarp Summary of results in Fig. 3C. Genes in parentheses show a greater change in these conditions. *Slight but reproducible effects. DEVELOPMENT Myeloid versus T-cell fate choice RESEARCH ARTICLE 1213

ABFig. 4. PU.1-dependent gene expression effects in controlled Notch signaling Infection with combinations of viral vectors conditions using Scid.adh.2C2 cells. Scid.adh.2c2 cells (A) Experimental set-up and flow cytometric Culture for 1 + dnMAML analysis of CD25, Mac1 and CD11c expression. 2 Days EV + EV EV + ICN EV + dnMAML PU.1 + EV PU.1 + ICN PU. (B) Heatmap of gene expression in sorted Scid.adh.2C2 cells expressing PU.1 with Analyze cells expressing Sfpi1 both vectors for the Bambi dnMAML, ICN1 or empty vector obtained from Vector 1 Vector expression of Mac1, CD11c Csf1r 2.5 qRT-PCR. Vector 2 and CD25 Bcl11a Lyl1 SpiB Dtx1 2

Mac1 Nrarp Mac1 CD11c Hes5 CD25 CD25 CD11c Zfpm1 3 3 3 1.5 10 10 10 Rag1

2 2 10 25 74 10 32 67 102 99 Ptcra

101 101 1 HEBalt 10 EV + EV Il2Ra 1 0 0 10 10 100 Lef1

-1 -1 10 10 10-1 -1 0 1 2 3 -1 0 1 2 3 10 10 10 10 10 10 10 10 10 10 10-1 100 101 102 103 Notch3

3 3 10 10 103 Notch1 0.5 2 2 10 10 102 Gfi1b 99 3 97 99 Tgfbr1 1 1 1 10 10 10 EV + ICN Runx3 0 0 10 10 100 Zeb2 0

-1 -1 10 10 10-1 Pou6f1 -1 0 1 2 3 -1 0 1 2 3 10 10 10 10 10 10 10 10 10 10 10-1 100 101 102 103 Id2 3 3 3 10 10 10 Id3 2 2 10 10 102 −0.5 99 99 99 Egr3

1 1 10 10 101 EV + dnMAML Gata3

100 100 0 Myb 10 HEBcan −1 -1 -1 10 10 10-1 -1 0 1 2 3 -1 0 1 2 3 10 10 10 10 10 10 10 10 10 10 10-1 100 101 102 103 Aiolos 3 3 10 10 103 43 5 42 11 14 36 Tcf7 2 2 10 10 102 Trib2 14 39 20 27 35 15 −1.5 1 1 10 10 101 Egr2 PU1 + EV Limd1 0 0 10 10 100 Hes1

-1 -1 10 10 10-1 -1 0 1 2 3 -1 0 1 2 3 10 10 10 10 10 10 10 10 10 10 10-1 100 101 102 103 Il7Ra −2

3 3 3 Runx1 10 10 10 3 6 8 26 2 9 Smad3 2 2 10 3 87 10 3 63 102 66 23 Ikaros

1 1 10 10 101 −2.5 PU1 + ICN E2A 0 0 10 10 100 Psen1 Spen -1 -1 10 10 10-1 -1 0 1 2 3 -1 0 1 2 3 10 10 10 10 10 10 10 10 10 10 10-1 100 101 102 103 Gfi1

3 3 10 10 103 Bcl11b 68 0.6 42 0.4 39 33 2 2 2 10 31 1 10 57 0.6 10 22 7

1 1 10 10 101 PU1 + dnMAML

0 0 10 10 100

-1 -1 10 10 10-1 -1 0 1 2 3 -1 0 1 2 3 10 10 10 10 10 10 10 10 10 10 10-1 100 101 102 103

CD11c+ Mac1– intermediate stage leading to diversion, Myb, Tcf7 (encoded by Tcf7), Myb, Gfi1 or Gata3, but it might also be basic and Gata3 also remained less affected (supplementary material Fig. helix-loop-helix E protein (E2A, HEB, TCF12) activity, which S5). Thus, to complete diversion (Fig. 3C), PU.1 must shut off these reportedly controls both T-cell differentiation genes such as Rag1 genes by another mechanism, beyond antagonism of Notch, even and other T-cell regulatory genes (Ikawa et al., 2006; Schwartz et al., though Notch signaling maintains the inputs that protect their 2006). Indeed, PU.1 could neutralize E proteins: in Mac1+ diverted expression. cells, the E protein antagonist Id2 is upregulated, and this upregulation is blocked by Notch signaling. Although this response Id2 co-infection with PU.1 increases diversion to is weak on its own, PU.1 overexpression also reduces expression Mac1+ cells via inhibition of the Notch pathway in of the E proteins E2A, HEB (canonical) and HEBalt in Scid.adh.2C2 cells Scid.adh.2C2 cells and fetal thymocytes alike (Franco et al., 2006). The data thus far indicate that diversion to a Mac1+ state is linked To test whether E protein activity could set the threshold for with PU.1-dependent repression of at least two distinct groups of diversion in response to PU.1, we co-expressed Id2 with PU.1 in T-cell genes. Of these, Notch-dependent target genes such as those Scid.adh.2C2 cells. In fact, Id2 and PU.1 together reproducibly inhibited by dnMAML (supplementary material Table S2B, group increased the percentage of cells becoming Mac1+ when compared 1) represent one component, but others such as Myb, Gata3, Gfi1 with PU.1 alone (Fig. 5A). This distinguished Id2 from two other and Tcf7 represent a separate, possibly rate-limiting, component. regulators that we tested as alternative candidates for collaborators These genes encode among the most important transcription factors with PU.1. Both the well-known myeloid factor C/EBPα and the known for T-cell development (Rothenberg et al., 2008) and may PU.1-induced factor BAMBI failed to increase the percentage of themselves play a role in maintaining T-cell identity. PU.1-transduced Scid.adh.2C2 cells becoming Mac1+, although We reasoned that extinction of the T-cell program must occur C/EBPα did reduce CD25 expression (supplementary material Fig. only when the T-cell gene(s) that resist(s) diversion was finally S6A and data not shown). Id2 overexpression alone also decreased

turned off or neutralized. This resistance factor might be TCF1 CD25 levels, although it did not upregulate Mac1. This suggested DEVELOPMENT 1214 RESEARCH ARTICLE Development 140 (6)

Fig. 5. E-protein inhibition is a mechanism for

EV reducing Notch signaling, but does not account

A B for all PU.1-mediated effects. (A) Scid.adh.2c2 cells EV + EV + ID2 PU.1 + EV PU.1 + ID2 expressing PU.1 and Id2, or an empty vector and

EV + EV EV + PU1 Bambi PU.1, Id2 and ICN were cultured for 2 days and then 3 10 103 Id2 0.07 0.3 31 2.7 Spen analyzed for their expression of Mac1 and CD25. 2 Mac1 10 102 Meis1 (B) Heatmap of Scid.adh.2C2 gene expression in Tgfbr1 1 1 sorted cells co-expressing Id2 and PU.1 obtained by CD25 10 10 Gata3 Csf1r qRT-PCR. 100 0 2.5 10 Lyl1 24 76 -1 -1 23 43 10 10 Itgam -1 0 1 2 3 10 10 10 10 10 10-1 100 101 102 103 Bcl11a 2 Pou6f1 ID2 + EV ID2 + PU1 Runx3 1.5 3 103 10 Rag1 0.15 0.09 54 1.7 Zeb2 2 102 10 Hes5 1 Ptcra 1 101 10 Notch3 0.5 0 0 HEBAlt 10 10 Il2ra 11 81 19 -1 33 10-1 10 Nrarp 0 -1 0 1 2 3 10-1 100 101 102 103 10 10 10 10 10 Notch1 Dtx1 −0.5 ID2 + PU1 (+ ICN)* Egr3

103 Tcf7 33 6.9 Aiolos −1 102 Zfpm1 Psen2 1 −1.5 10 Ikaros Smad3 0 10 Il7R −2 22 38 E2A 10-1 10-1 100 101 102 103 Hes1 Ets1 −2.5 Gfi 1 Myb Egr1 Trib2 HEBcan Bcl11b Runx1 Id3 that the Id2 effect might involve inhibition of Notch signaling. E Myb protects against PU.1-driven diversion proteins have been shown to be rate-limiting positive regulators of Myb and Tcf7 were consistently downregulated in response to PU.1 Notch1 (Yashiro-Ohtani et al., 2009), as well as positive contributors during diversion, and were prominent candidates as diversion to the expression of some Notch target genes (Ikawa et al., 2006) ‘barriers’ because the cells do not turn on Mac1 until these two such as Ptcra. genes are downregulated (Fig. 3C; supplementary material Fig. S3). Gene expression analysis confirmed that Notch target genes are Myb is already expressed strongly during the first stage of T-cell downregulated maximally in samples with Id2 alone, as well as in development (DN1), increasing slightly in the DN2 and DN3 stages samples co-expressing PU.1 and Id2 (Fig. 5B; supplementary (Tydell et al., 2007) (Fig. 2E). To test whether forced expression of material Table S2C, group 1). If Id2 overexpression affects the same Myb could block the ability of PU.1 to upregulate Mac1, we infected pathway as Notch inhibition, then forced Notch signaling in PU.1 Scid.adh.2C2 cells with retroviral Myb for 24 hours, then and Id2 co-expressing samples might be epistatic to Id2. In a triple- superinfected them with PU.1 and cultured the cells for an transduction experiment, Scid.adh.2C2 cells were infected with additional 48 hours (Fig. 6A). Despite increasing Myb less than PU.1, Id2 and ICN1. Cell surface staining of these cells after 2 days threefold over the level normally expressed in Scid.adh.2C2 cells, showed that the effect of Id2 to enhance diversion to Mac1+ cells co-transduction of Myb with PU.1 modestly but reproducibly was indeed canceled out when Notch signaling was enforced by the decreased the percentage of Mac1+ cells (Fig. 6B,C). addition of ICN1 (Fig. 5A, bottom). Thus, E protein antagonism Gene expression analysis (Fig. 6D; supplementary material Table does play a role in diversion, and induction of Id2 and repression of S2D) showed that Myb did not inhibit PU.1 from upregulating E2A and HEB probably provide one part of the mechanism through targets such as Bcl11a. Importantly, protection by Myb did not seem which PU.1 inhibits Notch activation in a positive feedback to to be mediated primarily through Notch signaling either, as Myb promote a myeloid fate. did not prevent PU.1 repression of Notch target genes However, Id2 alone had minimal effect on Gfi1, Myb or Tcf7 (supplementary material Table S2D3). However, Gfi1, Tcf7, Gata3 expression (supplementary material Table S2C, groups 4 and 5). and HEBalt were expressed at higher levels in cells with Myb and Furthermore, as reported in earlier E2A knockdown studies (Xu and PU.1 compared with those with PU.1 alone (supplementary material Kee, 2007), we detected an upregulation of Gata3 with Id2 alone Table S2D, groups 2 and 4). This group of protected genes was (supplementary material Table S2C, group 2), an effect reversed tested in turn for protection against PU.1-mediated diversion, but when PU.1 was present and quite different from the phenotype of they did not perform as well as Myb. TCF1 (Tcf7) was a high diverted cells. Therefore, the mechanism through which these T- priority candidate; however, the percentage of cells co-expressing lineage regulatory target genes are inhibited by PU.1 to complete TCF1 and PU.1 that were Mac1+ was the same as the percentage of diversion is not simply by blocking E protein activity, any more cells expressing PU.1 and an empty vector (supplementary material

than it is simply by blocking Notch activity. Fig. S6B), and Tcf7-shRNA did not increase diversion (not shown). DEVELOPMENT Myeloid versus T-cell fate choice RESEARCH ARTICLE 1215

A D E

V 100 Infection with an empty vector, Myb, + E + Myb Gata3shRNA, or Tcf7 80 PU1 high Scid.adh.2c2 cells Culture for EV + EV EV+ Myb PU.1 PU.1 60 2.5 PU1 low 24 hrs Bambi EV Control

% of Max 40 Infection with an empty vector or PU.1 Isotype Control Bcl11a 1.5 Culture for Csf1r 20 48 hrs Id2 0.5 0 3 Lyl1 10-1 100 101 102 10 Itgam 0 Analyze cells expressing –0.5 GATA3 both vectors for the Gata3

Vector 1 Vector expression of Mac1, Rag1 –1.5 Vector 2 CD11c, and CD25 Hes5 F 100 Egr3 B –2.5 80 Runx3

Mac1 Mac1 Mac1+ CD11c Pou6f1 1. 5 60 CD25 CD25 CD11c Mac1- Trib2 5 5 40 105 10 10 EV Control

0.21 0.61 0.03 0.15 Notch3 % of Max 4 4 4 10 10 10 99 27 72 27 73 Empty vector + Psen2 20 Isotype Control 3 3 103 10 10 HEBcan 1 2 Empty vector 2 102 10 10 0 0 0 0 Ikaros 3 2 3 4 5 1 2 3 4 1 2 3 4 010 10 10 10 -1 0 1 2 10 10 10 10 10 10 10 10 HEBalt 10 10 10 10 10

5 5 105 0.06 0.28 10 0.03 0.37 10 Gfi1 4 4 4 10 10 20 80 10 19 81 99 Tcf7 0. 5 GATA3 3 Myb + 3 3 10 10 10 Smad3 2 2 2 10 Empty Vector 10 10 0 0 0 Ptcra G

1 2 3 4 2 3 4 5 101 102 103 104 10 10 10 10 010 10 10 10 Nrarp 100

5 5 105 10 0 10 44 9 21 15 26 27 Aiolos 4 4 4 Myb + PU1 10 80 10 5.2 42 10 28 36 39 8.9 Empty vector + Zfpm1 3 3 103 10 10 PU1 + EV PU.1 Dtx1 60 2 2 102 10 10 0 0 0 Myb Myb + EV

2 3 4 5 1 2 3 4 1 2 3 4 010 10 10 10 % of Max 10 10 10 10 10 10 10 10 –0. 5 40 Il2Ra EV Control 5 5 5 10 10 27 12 10 15 18 16 22 Notch1 4 20 4 4 10 10 10 Isotype Control 6 55 16 50 51 12 Myb + Lef1 3 3 3 10 10 10 PU.1 Runx1 0 2 2 102 10 3 10 –1 -1 0 1 2 0 0 0 Id3 10 10 10 10 10 2 3 4 5 1 2 3 4 1 2 3 4 010 10 10 10 10 10 10 10 10 10 10 10 Hes1 C E2A GATA3 Spen * –1. 5 1.2 p-value=0.003 Il7R n=3 1 Bcl11b

0.8 0.6 0.4 0.2 0 -0.2 Fold Change in % Mac1+ EV Myb EV Myb EV PU.1 Fig. 6. Co-expression of Myb and PU.1 in Scid.adh.2C2 cells reduced the percentage of Mac1+ cells. This is mediated in part by the protection of Gata3. (A) Experimental set-up. (B,C) Mac1, CD25 and CD11c flow cytometric analysis of Scid.adh.2C2 cells expressing PU.1 and Myb for 2 days. Data are mean±s.d. Asterisk indicates P<0.05. (D) Heatmap of gene expression analysis of Scid.adh.2C2 cells. (E) Gata3 intracellular staining of Scid.adh.2C2 cells expressing PU.1. (F) Gata3 intracellular staining of Mac1+ and Mac1– PU.1-expressing Scid.adh.2C2 cells. (G) Gata3 protein levels in Scid.adh.2C2 cells expressing a combination of PU.1, Myb and empty vector.

Co-expression of Gfi1 or HEBalt with PU.1 also did not block Mac1+ cells (Fig. 3C). It was lower priority to test for control of induction of Mac1 (supplementary material Fig. S6C,D). In fact, pro-T-cell lineage fidelity only because the magnitudes of PU.1 and Gfi1 actually exacerbated the diversion response in the Notch effects on Gata3 RNA were small. To test for Gata3 effects Scid.adh.2C2 cells, and in fetal thymocytes, when co-expressed more sensitively at the single-cell level, we performed intracellular with PU.1 (supplementary material Fig. S6C and data not shown). staining of the Gata3 protein in Scid.adh.2C2 cells with and without Thus, although incomplete, the protective effect of Myb against overexpressed PU.1 (Fig. 6E). In fact, PU.1 overexpression diversion was specific, implicating Myb and Notch signaling as markedly downregulated Gata3 protein levels in one subset of the separate control points for resistance to diversion. transduced cells, even though it slightly upregulated Gata3 protein, relative to controls, in another subset. This split had the same all-or- A specific effect of PU.1 on Gata3 protein: Myb none quality as the diversion response itself. Gata3 downregulation protects Gata3 protein levels was seen at a much greater frequency in cells expressing high levels The gene that was most affected by Myb overexpression, one that of PU.1 (Fig. 6E), in which Gata3 levels were 5- to 10-fold reduced. Myb rendered most resistant to PU.1, was Gata3. Indeed, Myb- Those cells that downregulated Gata3 protein were also the ones transduced cells expressed higher levels of Gata3 RNA than that upregulated Mac1 (Fig. 6F). controls with or without PU.1, raising the issue of whether Gata3 Myb may positively regulate Gata3 in later T-cell development could help to resist diversion. Gata3 is essential and rate limiting (Maurice et al., 2007; Gimferrer et al., 2011). To test whether Myb

for T-lineage development and is specifically downregulated in could also maintain Gata3 despite PU.1 overexpression, we compared DEVELOPMENT 1216 RESEARCH ARTICLE Development 140 (6)

Gata3 protein levels in Scid.adh.2C2 cells co-expressing PU.1 and compared with unperturbed cells. This Gata3 reduction made PU.1- Myb with Gata3 in cells co-expressing PU.1 and an empty vector expressing cells more susceptible to diversion. As shown in Fig. 7B, (Fig. 6G). Cells co-expressing PU.1 with an empty vector showed the fraction of cells remaining Mac1– CD11c– was halved, while lowered Gata3 protein levels, but Gata3 was rescued to normal levels increased percentages of cells acquired these myeloid markers. in cells co-expressing PU.1 and Myb (Fig. 6G). Guaranteed Reduced Gata3 protein by itself had little effect on gene expression of Myb thus seems to protect Scid.adh.2C2 cells from the expression in the Scid.adh.2C2 cells, but the combination of PU.1 PU.1-driven mechanism that downregulates Gata3 protein. expression and Gata3 knockdown had a powerful effect on gene expression (Fig. 7E; supplementary material Table S2E). Gata3 Gata3 as a gatekeeper: Gata3 knockdown in PU.1- knockdown did not generally cause further upregulation of genes expressing cells enhances Mac1 upregulation induced by PU.1, and it did not exacerbate PU.1-mediated To investigate whether Gata3 downregulation was simply a marker repression of several Notch targets (supplementary material Table or actually caused differences in the ability of PU.1 to divert the S2E, group 3). However, we found that the multiple T-cell genes cells, we used shRNA to reduce Gata3 expression in PU.1- downregulated by PU.1 were further downregulated in cells with transduced cells and measured the impact on diversion. PU.1 and lowered Gata3 (supplementary material Table S2E, group Scid.adh.2C2 cells were first infected with a construct expressing a 4). Notch1 and Notch3 themselves were affected. Loss of Gata3 thus short hairpin RNA against Gata3 (Hernández-Hoyos and Alberola- sensitizes cells to the effects of PU.1, with a potency comparable to Ila, 2005); then after 24 hours the cells were infected with a PU.1- Notch inhibition. expressing vector and cultured for 48 hours more (Fig. 7) before analysis. The Gata3-shRNA alone knocked down Gata3 protein to Notch and Gata3 pathway interlinkage: Notch levels that were comparable with the lowest Gata3 protein levels in signaling makes Gata3 resistant to PU.1 PU.1-expressing cells (Fig. 7A). Unexpectedly, the Scid.adh.2C2 These results imply that Gata3 downregulation can complement the cells co-expressing Gata3 shRNA together with PU.1 had even inhibition of Notch responses by PU.1 and make cells susceptible to lower levels of Gata3 protein, some with 20-fold reduction diversion. However, our earlier results indicate that direct

AB E 100

80 Mac1 CD11c Mac1 CD25 CD25 CD11c A3shRNA

60 5 5 10 105 44 9 10 21 15 26 27

1 + EV 4 104 39 4 10 10 5 42 28 36 9 V + GAT % of Max 40 Empty vector 3 EV + EV E PU. PU.1 + GATA3shRNA 103 10 103 + PU.1 2.5 2 102 10 20 102 0 0 0 Egr3

2 3 4 5 101 102 103 104 010 10 10 10 101 102 103 104 Bcl11a 0 3 5 0 -1 0 1 2 5 5 10 10 10 10 10 10 10 1 3 10 0.1 0.5 3 0.1 Itgam

4 4 4 10 10 18 78 10 19 81 96 0.5 Bambi Gata3shRNA 3 GATA3 3 103 10 10 + EV Csf1r −2. 5 2 2 2 10 10 10 0 0 0 Lyl1 2 3 4 5 1 2 3 4 Gata3 shRNA + PU.1 101 102 103 104 10 10 10 10 010 10 10 10 Gata3 shRNA Pou6f1 5 5 5 10 PU.1 10 51 16 10 36 28 18 48 Id2 4 4 4 10 19 10 5 28 10 20 17 15 EV Control Gata3shRNA Runx3 3 3 3 10 1.5 10 10 + PU.1 2 2 2 10 Gata3 Isotype Control 10 10

0 0 0

2 3 4 5 1 2 3 4 Notch3 101 102 103 104 10 10 10 10 010 10 10 10 Trib2 1 Il7r C D HEBcan 100 100 Hes1 0.5 80 80 Id1 PU.1 + dnMAML 48 hrs Id3 60 60 PU.1 + dnMAML 24 hrs Bcl11b EV Control

% of Max 40 % of Max 40 Spen 0 Isotype Control Aiolos 20 20 Notch1 0 0 E2A -1 0 1 2 3 -1 0 1 2 3 −0.5 10 10 10 10 10 10 10 10 10 10 Lef1 GATA3 GATA3 Myb 100 Rag1 PU.1 + dnMAML Hes5 −1 80 PU.1 + ICN Tcf7 PU1 + dnMAML 48 hrs EV + PU.1 60 Zfpm1 EV Control PU1 + dnMAML 24 hrs Ptcra −1.5 Isotype Control 40 EV + EV % of Max Nrarp 20 Dtx1

0 HEBalt -1 0 1 2 3 Gfi1 10 10 10 10 10 Mac1

Fig. 7. Inhibition of Gata3 protein enhances PU.1-driven Mac1 upregulation in Scid.adh.2C2 cells. Notch signaling blocks PU.1-driven Gata3 protein inhibition. (A) Gata3 intracellular staining of samples expressing PU.1 and Gata3 shRNA after 3 days. (B) Experimental set-up as in Fig. 6A. Cells were analyzed for their expression of CD25, Mac1 and CD11c. (C) Gata3 protein levels in cells expressing PU.1 and ICN1, dnMAML or empty vector for 2 days. (D) Gata3 protein levels in Scid.adh.2C2 cells co-expressing PU.1 and dnMAML for 24 and 48 hours (left panel). Mac1 expression in the same cells at 24 and 48 hours (right panel). (E) Heatmap of gene expression in Scid.adh.2C2 cells expressing a combination of Gata3 shRNA, PU.1 and empty vectors

for 3 days. DEVELOPMENT Myeloid versus T-cell fate choice RESEARCH ARTICLE 1217 manipulations of Notch signaling were also sufficient to regulate Csf1r, whereas another involves expression of stem-cell or PU.1-driven diversion, despite little detectable effect of Notch progenitor-cell genes such as Bcl11a, Lyl1 and possibly also Bambi. inhibition on Gata3 RNA. In fact, dnMAML alone could slightly Environmental Notch signaling blocks activation of Itgam and elevate Gata3 RNA (Fig. 4B). To revisit whether there is any Csf1r, but not PU.1-dependent activation generally. Concomitantly, convergence between these two regulatory mechanisms for there are two network branches through which PU.1 can negatively protecting T-cell identity, we tested whether manipulations of Notch regulate the T-cell differentiation program. Extinction of T-lineage signaling in the context of PU.1 activity might have clearer effects regulatory gene expression is most tightly correlated with a switch on Gata3 protein. to myeloid fate. One branch involves the ability of PU.1 to attenuate By themselves, transduction with dnMAML or ICN1 had transcriptional responses to Notch signaling: PU.1 raises the virtually no effect on Gata3 protein levels in Scid.adh.2C2 cells threshold of Notch signaling needed for expression of Notch target (supplementary material Fig. S7). However, when PU.1 genes. This occurs in part through inhibition of an E protein – transduction was combined with dnMAML or ICN1, the effect on Notch-positive feedback circuit. In parallel, however, we show that Gata3 was dramatic (Fig. 7C). Cells co-expressing PU.1 and ICN1 PU.1 also antagonizes expression of a second set of T-cell regulatory uniformly expressed Gata3 at the highest level. By contrast, cells co- genes, including Myb and its activation target Gata3. These seem expressing PU.1 and dnMAML shifted almost completely to the crucial for sustaining Gfi1, Zfpm1 (Fog1) and Tcf7 expression in the low level of Gata3, normally seen only in cells with the highest presence of PU.1. All these genes can also be protected against PU.1 expression of PU.1. Thus, Notch signaling affects not only Notch by Notch signals, but are not otherwise Notch dependent, implying target gene expression but also the mechanism for Gata3 that the protective effect of Notch on this gene set is indirect, e.g. via stabilization, with later impact on Gata3 targets. maintenance of Gata3. Myeloid-lymphoid lineage choice is thus a Kinetically, the impact of Notch inhibition on Gata3 levels bifurcation between opposing feed-forward network circuits, one could precede appearance of the diverted phenotype (Fig. 7D). dominated by PU.1, and the other by Notch signals that protect both Mac1 expression is not evident on PU.1-transduced Scid.adh.2C2 Notch-E protein targets and Myb-Gata3 targets. cells until 48 hours (Fig. 7D, right panel). However, the Our results suggest that the balance may be tipped from resistance combination of PU.1 and dnMAML began to downregulate Gata3 to diversion by initial weakening of either protective mechanism in protein in the whole population of transduced Scid.adh.2C2 cells PU.1-expressing cells. Reduction either of Gata3 or of Notch by 24 hours, falling lower by 48 hours. Thus, any decrease in signaling can sensitize the cells to diversion, and Notch signaling Notch signaling undermined the resistance of Gata3 in the cells not only protects Gata3 but also protects its positive regulator, Myb, to inhibition by PU.1, precipitating the positive-feedback cascade from inhibition by PU.1. However, it is notable that when PU.1 and that eventually silences genes dependent on Gata3 and Notch Notch signals ‘balance’, T-cell regulatory gene expression can be signaling alike. maintained, along with expression of specific progenitor-associated PU.1 target genes. This is exactly the situation in early T-cell DISCUSSION precursors before lineage commitment (Fig. 1A). Our results with T-lineage specification of blood-cell precursors is promoted by Bcl11a, Lyl1 and possibly also Hhex and Bambi regulation, all of Notch interaction, with Delta expressed in the thymic micro- which are naturally expressed in early thymocytes, thus open the environment. However, throughout multiple cell cycles in this way for PU.1 to play a stage-specific positive role for early T cells. environment, the differentiating precursors continue to express Our results are drawn from both primary fetal thymocytes and a transcription factors such as PU.1 that are associated with DN3-like clonal cell line, and the relationships are similar if not multipotentiality. Their access to other fates is revealed if removed completely identical. Scid.adh.2C2 cells do not perfectly match the from the thymus. How does the thymus predictably manage to gene expression states of the primary cells, and as magnitudes of impose a T-cell fate on virtually all these cells, despite their intrinsic specific gene expression responses to PU.1 change with normal delay of commitment? Our results reveal the architecture of a developmental progression, they also differ between the cell line regulatory gene network switch circuit through which and the primary cells. These probably reflect differences in basal environmental Notch signaling interacts with PU.1 to determine T- Notch transduction machinery, E protein activity and Gata3 cell, myeloid or progenitor-cell status (Fig. 8). expression between these cell types (data not shown). Tcf7 is less Two branches of this network are positively regulated by PU.1. protected by Notch signaling in the primary cells than in One involves upregulation of myeloid genes such as Itgam and Scid.adh.2C2 cells, whereas genes such as Ptcra are more protected. However, these are not the PU.1 repression targets that appear to set the threshold against diversion. Instead, the key components of High the network core architecture shown in Fig. 8 are consistent with Other results in both types of cells. PU.1 Myb Genes PU.1 opposition to Gata3 recalls the PU.1:GATA1 opposition that underlies erythroid/myeloid fate determination, which is based in Gata3 part on protein-protein interaction (reviewed by Cantor and Orkin, 2002; Laiosa et al., 2006a). Here, Gata3 appears to be important for Itgam E Proteins Zfpm1 maintenance against PU.1, like GATA1 in erythroid Csf1r development. ChIP-seq analysis shows that Zfpm1, Gfi1, Myb and T-Cell Pathway T-Cell Myeloid Pathway Notch Tcf7 are all linked with Gata3 binding sites in early T cells (Zhang Signaling et al., 2012) (Bambi, Bcl11a, Itgam and Id2 are not), suggesting that high PU.1 may primarily inhibit these T-cell genes by blocking Fig. 8. Interactions between PU.1, Notch signaling and regulatory positive Gata3 inputs. However, Gata3-PU.1 antagonism itself is genes that partially define a lymphomyeloid switch during early T- more conditional. Although PU.1 reduces Gata3 protein when

cell development. Summary of discussion. See text for details. Notch signaling is inhibited, PU.1 slightly upregulates Gata3 when DEVELOPMENT 1218 RESEARCH ARTICLE Development 140 (6)

Notch signals are active. PU.1 binds multiple sites around Gata3 in Franco, C. B., Scripture-Adams, D. D., Proekt, I., Taghon, T., Weiss, A. H., Yui, early T cells (Zhang et al., 2012), potentially contributing to both M. A., Adams, S. L., Diamond, R. A. and Rothenberg, E. V. (2006). Notch/Delta signaling constrains reengineering of pro-T cells by PU.1. Proc. effects. PU.1 can be repressed by high-level Gata3 (Taghon et al., Natl. Acad. Sci. USA 103, 11993-11998. 2007), but the genomic sites through which GATA1 silences PU.1 Germar, K., Dose, M., Konstantinou, T., Zhang, J., Wang, H., Lobry, C., expression (Chou et al., 2009) are not bound by Gata3 in early T Arnett, K. L., Blacklow, S. C., Aifantis, I., Aster, J. C. et al. (2011). T-cell factor cells (Zhang et al., 2012). Furthermore, although reduced Gata3 1 is a gatekeeper for T-cell specification in response to Notch signaling. Proc. Natl. Acad. Sci. USA 108, 20060-20065. makes cells more diversion sensitive, increased Gata3 cannot bypass Ghani, S., Riemke, P., Schönheit, J., Lenze, D., Stumm, J., Hoogenkamp, M., the need for Notch signaling to make cells diversion-resistant (data Lagendijk, A., Heinz, S., Bonifer, C., Bakkers, J. et al. (2011). Macrophage not shown). This suggests that PU.1-Gata3 relationships are development from HSCs requires PU.1-coordinated microRNA expression. Blood 118, 2275-2284. probably asymmetric. Gimferrer, I., Hu, T., Simmons, A., Wang, C., Souabni, A., Busslinger, M., The relationship between PU.1 and Notch signaling provides a Bender, T. P., Hernandez-Hoyos, G. and Alberola-Ila, J. (2011). Regulation discrete, micro-environmental threshold setter for lymphoid of GATA-3 expression during CD4 lineage differentiation. J. Immunol. 186, 3892-3898. precursor fate determination. In normal thymocytes though not in Hamdorf, M., Berger, A., Schüle, S., Reinhardt, J. and Flory, E. (2011). PKCδ- Scid.adh.2C2 cells, the signals actually received depend on induced PU.1 phosphorylation promotes hematopoietic stem cell environmental density of Notch ligands. Within the pro-T cells, differentiation to dendritic cells. Stem Cells 29, 297-306. Hernández-Hoyos, G. and Alberola-Ila, J. (2005). Analysis of T-cell signaling not only requires E proteins to maintain Notch1 development by using short interfering RNA to knock down protein expression but also a positive-feedback loop with E protein activity, expression. Methods Enzymol. 392, 199-217. as expression of both Id2 and Id3 E protein antagonists increases Houston, I. B., Kamath, M. B., Schweitzer, B. L., Chlon, T. M. and DeKoter, R. when Notch signaling is reduced. The molecular mechanism P. (2007). Reduction in PU.1 activity results in a block to B-cell development, abnormal myeloid proliferation, and neonatal lethality. Exp. Hematol. 35, 1056- through which PU.1 inhibits Notch-dependent transcription still 1068. requires more investigation. However, our results show that the Ikawa, T., Kawamoto, H., Goldrath, A. W. and Murre, C. (2006). E proteins and expression of PU.1 in the earliest T-cell precursors itself becomes a Notch signaling cooperate to promote T cell lineage specification and commitment. J. Exp. Med. 203, 1329-1342. sensor that determines what level of Notch signal from the Iwasaki, H., Somoza, C., Shigematsu, H., Duprez, E. A., Iwasaki-Arai, J., environment will suffice to promote entry and forward progression Mizuno, S., Arinobu, Y., Geary, K., Zhang, P., Dayaram, T. et al. (2005). along the T-cell pathway. Distinctive and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their differentiation. Blood 106, 1590-1600. Laiosa, C. V., Stadtfeld, M. and Graf, T. (2006a). Determinants of lymphoid- Acknowledgements myeloid lineage diversification. Annu. Rev. Immunol. 24, 705-738. We thank Avinash Bhandoola, Warren Pear and Gabriela Hernandez-Hoyos for Laiosa, C. V., Stadtfeld, M., Xie, H., de Andres-Aguayo, L. and Graf, T. (2006b). constructs; the entire Rothenberg lab for help and valuable discussions; Robert Reprogramming of committed T cell progenitors to macrophages and Butler for technical expertise; Va Si for pilot Gata3 staining experiments; dendritic cells by C/EBP α and PU.1 transcription factors. Immunity 25, 731-744. Rochelle Diamond, Diana Perez and Josh Verceles for cell sorting; and Scott Lefebvre, J. M., Haks, M. C., Carleton, M. O., Rhodes, M., Sinnathamby, G., Washburn for mouse care. Simon, M. C., Eisenlohr, L. C., Garrett-Sinha, L. A. and Wiest, D. L. (2005). Enforced expression of Spi-B reverses T lineage commitment and blocks β- Funding selection. J. Immunol. 174, 6184-6194. The work was supported by the National Institutes of Health (NIH) [CA90233 Li, L., Leid, M. and Rothenberg, E. V. (2010). An early T cell lineage and CA90233-08S1], by the Garfinkle Memorial Laboratory Fund, by the Al commitment checkpoint dependent on the transcription factor Bcl11b. Sherman Foundation, by a NIH predoctoral training grant [T32GM07616 to Science 329, 89-93. M.M.D.R.] and by the Albert Billings Ruddock Biology Professorship (E.V.R.). Lim, S. O., Kim, H. S., Quan, X., Ahn, S. M., Kim, H., Hsieh, D., Seong, J. K. and Deposited in PMC for release after 12 months. Jung, G. (2011). Notch1 binds and induces degradation of Snail in hepatocellular carcinoma. BMC Biol. 9, 83. Competing interests statement Maillard, I., Weng, A. P., Carpenter, A. C., Rodriguez, C. G., Sai, H., Xu, L., Allman, D., Aster, J. C. and Pear, W. S. (2004). Mastermind critically regulates The authors declare no competing financial interests. Notch-mediated lymphoid cell fate decisions. Blood 104, 1696-1702. 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