expression patterns define novel roles for E47 in cell cycle progression, cytokine-mediated signaling, and T lineage development

Ruth Schwartz*†, Isaac Engel*†‡, Mohammad Fallahi-Sichani§, Howard T. Petrie§, and Cornelis Murre*¶

*Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0377; and §Scripps͞Florida Research Institute, 5353 Parkside Drive, RF-1, Jupiter, FL 33458

Communicated by Michael S. Levine, University of California, Berkeley, CA, May 5, 2006 (received for review April 7, 2006) In maturing T lineage cells, the helix–loop–helix E47 has analyze the global patterns of in E2A-deficient been shown to enforce a critical proliferation and developmental lymphomas in the absence and presence of E47. Cluster analysis checkpoint commonly referred to as ␤ selection. To examine how identified groups of that were regulated by E47 and shared E47 regulates cellular expansion and developmental progression, biochemical or physiological properties. These included genes we have used an E2A-deficient lymphoma cell line and DNA involved in cell survival, cell cycle progression, stress response, lipid microarray analysis to identify immediate E47 target genes. Hier- metabolism, ␣␤, and natural killer T (NKT) cell function. Based on archical cluster analysis of gene expression patterns revealed that these observations, we suggest that E47 functions in molecular E47 coordinately regulates the expression of genes involved in cell pathways to coordinate cell survival, cell growth, and developmen- survival, cell cycle progression, lipid metabolism, stress response, tal maturation. We identified cyclin-dependent kinase 6 (Cdk6) as and lymphoid maturation. These include Plc␥2, Cdk6, CD25, Tox, an E47 target gene, providing a potential mechanism underlying a Gadd45a, Gadd45b, Gfi1, Gfi1b, Socs1, Socs3, Id2, Eto2, and Xbp1. role for E47 in cell cycle progression. Socs1 and Socs3 were also We propose a regulatory network linking Janus kinase (JAK)͞signal regulated by E47, and we propose that E47 functions in a cytokine transducer and activator of transcription (STAT)-mediated signal- -mediated regulatory network to regulate Socs gene ex- ing, E47, and suppressor of cytokine signaling (SOCS) in a pression. Interestingly, the regulation of two E47 targets, Hes1 and common pathway. Finally, we suggest that the aberrant activation Gfi1, reflects a conserved regulatory network used in Drosophila of Cdk6 in E47-deficient T lineage cells contributes to the devel- melanogaster sensory organ development. opment of lymphoid malignancy. Results ͉ ͉ ͉ E2A Gfi SOCS Xbp1 Global Gene Expression Patterns in E2A-Deficient Lymphomas in Response to E47 Activity. Our previous observations have indicated ymphocyte development is regulated, in part, by a distinct class a critical role for the E2A proteins in cell survival, growth, and early Lof helix–loop–helix (HLH) proteins, named E proteins (1). Four thymocyte development (1). However, the mechanisms underlying E proteins, E12, E47, E2-2, and HEB, are expressed in developing E47-mediated control of proliferation and differentiation remain to lymphocytes. E12 and E47 are encoded by one gene, designated as be elucidated. As a first approach to this question, we have used E2A, and arise through differential splicing in an exon encoding the oligonucleotide microarrays to identify genes regulated by E47 in HLH domain. E2A-deficient mice exhibit a complete block in B cell E2A-deficient lymphoma cell lines. For this study, we used the 1F9 development at the onset of lineage commitment (2–4). cell line, originally isolated from E2A-deficient thymomas (15). To development is partially blocked in E2A-deficient mice before the identify E47 target genes, 1F9 cells were transduced with a retro- onset of T cell receptor (TCR) ␤ V(D)J gene rearrangement (5–7). virus carrying an E47͞estrogen receptor (E47ER) hybrid protein. E proteins, and E2A proteins in particular, play critical roles in Control cells were transduced with virus carrying the basic HLH regulating developmental progression at the pre-TCR and TCR region but lacking the N-terminal transactivation domain. Both checkpoints (8, 9). Signals emanating from the pre-TCR and TCR retroviral constructs also directed the expression of human CD25 complex act to suppress E protein DNA-binding activity, induce the (hCD25) to allow rapid isolation of transduced cells (16). One day expression of the E protein inhibitor Id3, and lower the abundance after infection, cells were incubated with 4-hydroxytamoxifen (4- of E47 protein levels (10). Lowering the dose of E47 has been OHT) for a period of 6 hours, to activate the E47ER fusion protein. demonstrated to release the block in differentiation and prolifer- hCD25-positive cells were purified by using magnetic beads, and ation observed in thymocytes with defects in pre-TCR expression RNA was isolated and used to generate probes for hybridization to and pre-TCR-mediated signaling (8, 9, 11). The E proteins also murine oligonucleotide microarrays. Approximately 225 genes were function at the TCR checkpoint, because an E2A deficiency has identified whose transcript levels were modulated by the enforced been shown to accentuate positive selection (12). In contrast, expression of E47 (data not shown). The expression pattern asso- positive selection in Id3-ablated mice is blocked, albeit partially ciated with E47 expression appeared to reflect the role of E47 in cell (13). Taken together, these data suggest that the E2A proteins act cycle regulation and T lineage developmental maturation, as dem- as gatekeepers at the pre-TCR and TCR checkpoints, and passage onstrated by the induction of pT␣, Hes1, Xbp1, Eto2, Gadd45a, through these checkpoints requires pre-TCR or TCR-mediated modulation of E2A activity. E proteins also function as tumor suppressors. E2A-deficient Conflict of interest statement: No conflicts declared. mice rapidly develop thymic lymphoma (5, 14). Reintroduction of Abbreviations: DP, double positive; DN, double negative; HLH, helix–loop–helix; Cdk6, E2A activity into cell lines adapted from lymphomas that developed cyclin-dependent kinase 6; TCR, T cell receptor; NKT, natural killer T; T-ALL, T-acute in E2A-ablated mice caused rapid (15). In contrast, lymphoblastoid leukemia. enforced expression of E47 in conjunction with Bcl-2 induced cell †R.S. and I.E. contributed equally to this work. cycle arrest, suggesting a direct role for E2A in the suppression of ‡Present address: Division of Developmental Immunology, La Jolla Institute for Allergy and cell growth (11). As a first approach to determine the mechanism Immunology, 10355 Science Center Drive, San Diego, CA 92122. by which E47 inhibits cellular proliferation and promotes develop- ¶To whom correspondence should be addressed. E-mail: [email protected]. mental progression, we have used oligonucleotide microarrays to © 2006 by The National Academy of Sciences of the USA

9976–9981 ͉ PNAS ͉ June 27, 2006 ͉ vol. 103 ͉ no. 26 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603728103 Downloaded by guest on September 26, 2021 Fig. 1. E47 target genes expressed in an E2A-deficient lymphoma cell line. The 1.F9 cell line was transduced with retrovirus encoding E47 or a truncated form of E47 encoding the basic HLH domain. RNA was isolated from transduced cells and analyzed for target gene expression by using microarray analysis. Selected

activated and repressed target genes by the overexpression of E47 are depicted on the top (A) and bottom (B), respectively. Values indicated on the vertical axis IMMUNOLOGY show the fold induction or repression of E47 target transcript levels.

Gadd45b, Ets2, Mef2b, Gfi1, Gfi1b, Id2, Socs1, Socs3, and Ror␥,as Cdk6 transcription upon enforced expression of E47 was particu- well as repression of Cdk6, Pou2af1, Icos, Gata3, Ror␣, Foxo1, larly striking. Specifically, Cdk6 transcript levels were reduced by a Lmo4, CD25, and Tox (Fig. 1). To verify the observations obtained factor of 2-fold, 6 hours after tamoxifen treatment (Table 1). This from the microarray analysis, we have used quantitative PCR result was further corroborated by quantitative PCR analysis (Fig. analysis to quantitatively determine transcript levels of CD25, 4). Because Cdk6 activity acts to phosphorylate Rb to regulate cell Socs3, Gadd45a, GAdd45b, and Ror␥ (Fig. 4, which is published as cycle progression, we examined the phosphorylation status of Rb supporting information on the PNAS web site, and results not using Western blotting and Rb-specific antibodies. One day after shown). The data point to a role for E47 in contributing to the induction, Rb phosphorylation significantly declined, consistent transcriptional program that controls lymphoid development by with cell cycle arrest of E2A-deficient lymphomas (R.S. and I.E., acting upstream of transcriptional regulators known to play key unpublished observations). E47 also induced the expression of p21, roles in various proliferation and developmental pathways, includ- Rb, and , albeit modestly (Table 1). The growth arrest and ing Hes1, Ets2, Gata3, Pou2af1, Foxo1, Xbp1, Ror␥, Gfi1, Gfi1b, DNA damage inducible genes, Gadd45a and Gadd45b, were also and Tox. coordinately induced by E47 expression (Table 1 and data not shown). Taken together, these data indicate that a subset of genes E47 Regulates the Expression of the Cell Cycle Regulator Cdk6. To link involved in cell cycle progression are regulated by E47 in E2A- genes that act together in a physiological pathway, hierarchical deficient lymphoma cells, most notably Gadd45 members and clustering analysis of gene expression patterns was performed on Cdk6. group samples according to their shared biochemical and functional pathways. Interestingly, this analysis revealed a large cluster of E47 Modulates the Expression of Genes Involved in Cell Survival, Lipid genes previously shown to play critical roles in cell cycle progression Metabolism, and T Lineage Maturation. E47 targeted the expression and cell survival. Among these were Cdk6, p21, retinoblastoma of genes involved in cell survival, including Bid, Bim, caspase-3, and (Rb), cyclin E1, cyclin E2, and E2F4 (Table 1). The repression of caspase-6 (Table 1). Hierarchical clustering of E47 target signatures

Schwartz et al. PNAS ͉ June 27, 2006 ͉ vol. 103 ͉ no. 26 ͉ 9977 Downloaded by guest on September 26, 2021 Table 1. Cluster of E47 targets potentially regulating cell growth Table 3. Cluster of E47 targets regulating genes involved in lipid in E2A-deficient lymphomas metabolism Identified Fold Identified Fold target Function modulation target Function modulation

Gadd45a Growth arrest and DNA-damage-inducible 45 ␣ 2.4 Cyp11a Cytochrome P450 14.7 Gadd45b Growth arrest and DNA-damage-inducible 45 ␤ 2.2 Plc␥2 Phospholipase C␥2 2.8 Ccne2 Cyclin E2 2.1 Dgke Diacylglycerol kinase ␧ 2.0 Cdkn1A Cyclin-dependent kinase inhibitor 1A (p21) 2.0 Cerk Ceramide kinase 1.9 Ccne1 Cyclin E1 1.8 Psap Prosaposin 1.6 Cdc45L Cell division cycle homolog (S. cerevisiae like) 1.5 Pafah1B3 Platelet-activating factor acetylhydrolase 1B 1.5 Rb Retinoblastoma gene 1.5 Gpam Glycerol-3-phosphate acyltransferase 1.5 E2F4 1.5 SC4mol Sterol-C4-methyl oxidase-like Ϫ1.4 Jund1 Jun protooncogene related gene D1 1.3 Cpt1␤ Carnitine palmitoyltransferase 1 Ϫ1.4 c- Myelocytomatosis oncogene Ϫ1.4 Gm2a Gm2 ganglioside activator protein Ϫ1.8 Axin2 Axin2 Ϫ3.7 Gpld1 Glycosylphosphatidylinositol phospholipase D1 Ϫ11.8 Casp3 Caspase 3 Ϫ1.3 Sult2B1 Sulfotransferase 2B Ϫ41.9 Cdk6 Cyclin-dependent kinase 6 Ϫ2.6 Bid BH3-interacting domain death agonist 1.7 Bim Bcl2-interacting mediator of cell death 1.5 Casp 6 Caspase 6 Ϫ1.4 double-negative thymocyte DN2 and DN3 compartments but is Perp-pending apoptosis effector related to PMP22 Ϫ2.0 rapidly down-regulated during the maturation of murine thymo- cytes from the DN3 to DP stages, such that the vast majority of DP cells do not express detectable levels of CD25. The CD25 levels in 6also revealed groups of genes that act during various stages in T E47-deficient DN2 and DN3 cells are similar to that of wild-type lineage development. These include CD25, Rag1, Rag2, Socs1, thymocytes, indicating that E47 activity is not essential to induce Socs3, CD3␧, and CD8b (Table 2). Several transcriptional regula- CD25 expression in the DN compartment. However, the data described above raise the possibility that E47 activity is required to tors that function to promote lymphoid development at various ␤ stages, including Gata3, Foxo1, Ror␥, Gfi1, Ets2, and Xbp1, were repress CD25 transcription upon selection. To investigate this also among the immediate targets of E47 (Table 2). Interestingly, possibility, we monitored the expression of CD25 in E2A-deficient we found that clustering analysis showed gene expression signatures thymocytes using flow cytometry. Interestingly, we observed that a that grouped samples according to their shared roles in NKT cell large fraction of E2A-deficient ISP and DP thymocytes expressed development, including CD1d, AP3D, AP3B1, cathepsin, and low but significant levels of CD25 (Fig. 2A). These data suggest that prosaposin (Table 2). Furthermore, this analysis revealed that E47 E47 functions in both the ISP and DP cell stages to suppress CD25 induced the expression of a subset of genes involved in lipid expression. biosynthesis, notably Plc␥2 (Table 3). Collectively, this analysis To examine whether E47 regulated CD25 gene expression in DP elucidated biochemical and functional pathways in which E47 acts thymocytes, we prepared RNA from E47-null, heterozygous, and to control cell cycle progression, cell survival, lipid biosynthesis, and wild-type DP cells that were purified by electronic sorting. We then T lineage maturation. used these RNA samples to generate cDNA and measured CD25 transcript levels using quantitative PCR. Strikingly, CD25 tran- Ϸ Ϫ͞Ϫ E2A Proteins Are Required for the Complete Inhibition of CD25 scripts were found to be present at 8-fold higher levels in E47 Expression in Immature Single-Positive (ISP) and Double-Positive (DP) DP thymocytes than in either the wild-type or heterozygous litter- Thymocytes. The data described above indicate that CD25 is re- mate samples (data not shown). These data suggest that E2A pressed by E47. CD25 is expressed at relatively high levels in the proteins repress CD25 in DP thymocytes largely through pretrans- lational mechanisms. Thus passage through the ␤ selection check- point modifies E2A activity, leading to changes in the subset of Table 2. Cluster of E47 targets potentially regulating genes regulated by E47. hematopoiesis and immune response These observations suggested that complete repression of CD25 Identified Fold in E2A-deficient thymocytes might be initiated during maturation target Function modulation within the DP stage. To test this hypothesis, we used flow cytometry-based strategies to analyze CD25 expression within both Rag1 Recombination-activating gene 1 1.9 relatively immature and mature subsets of DP thymocytes from Rag2 Recombination-activating gene 2 1.7 E47-null and heterozygous littermates. To define immature DP CD8b CD8 antigen 1.7 CD3␧ CD3⑀ polypeptide Ϫ1.6 thymocytes, we injected mice with BrdU and performed CD25 Interleukin 2 receptor ␣ Ϫ2.7 extraction. We then stained these BrdU-pulsed thymocytes with Ror␥ RAR-related orphan receptor ␥ 4.3 antibodies against CD4, CD8, CD25, and BrdU. Thymocyte pro- Socs3 Suppressor of cytokine signaling 3 3.9 liferation largely ceases shortly after progression to the DP stage, Socs1 Suppressor of cytokine signaling 1 3.5 thus a short pulse of BrdU will be incorporated only by the most Xbp1 X-box-binding protein 1 3.1 immature DP thymocytes (17). We observed that nearly all BrdUϩ Ets2 E26 avian leukemia oncogene 2 3.0 DP thymocytes from E47Ϫ͞Ϫ mice expressed relatively high levels Mef2b Myocyte enhancer factor 2B 2.7 of CD25 (Fig. 2B). To identify relatively mature populations of DP Gfi1 Growth factor independent 1 2.0 Gfi1b Growth factor independent 1b 2.7 cells, we stained thymocytes using antibodies against CD4, CD8, Gata3 GATA-binding protein 3 Ϫ2.0 and CD25 together with either anti-TCR␤ or CD69. Thymocytes Foxo1 Forkhead box O1 Ϫ2.9 up-regulate both TCR and CD69 expression just before exit from Ctsl Cathepsin L 2.2 the DP stage, thus the most mature fractions of the DP population Psap Prosaposin 1.6 can be identified by increased levels of either TCR or CD69. We AP3D Adaptor-related protein complex 3, ␦ subunit 1.2 observed that either TCR moderate͞high or CD69ϩ DP thymo- ␤ AP3B1 Adaptor-related protein complex 3, 1 subunit 1.2 cytes from E47-null mice expressed relatively low levels of CD25 CD1D CD1D1 antigen Ϫ1.5 (Fig. 2B and data not shown). These data demonstrate that

9978 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603728103 Schwartz et al. Downloaded by guest on September 26, 2021 Fig. 2. Aberrant expression of CD25 levels in E47Ϫ͞Ϫ thymocytes. (A) Thymocytes from E47Ϫ͞Ϫ and ϩ͞Ϫ littermates were stained with antibodies against CD8a, CD4, TCR␤, and CD25 and analyzed by flow cytometry. Shown are the CD25 staining histograms for CD8highCD4low TCR␤-immature single-positive thymocytes (Upper Left), CD8highCD4high DP (Upper Right), CD4highCD8low CD4SP (Lower Left), and CD8highCD4lowTCRhigh mature CD8SP (Lower Right). Black tracings: E47Ϫ͞Ϫ. Gray tracings: E47ϩ͞Ϫ. Solid tracings: anti-CD25 staining. Dotted tracings: isotype control staining. (B) Thymocytes from BrdU pulse-labeled E47Ϫ͞Ϫ and E47ϩ͞Ϫ littermates were stained for CD8a, CD4, CD25, and either BrdU or CD69. Histograms depict CD25 fluorescence in CD8high CD4high BrdUϩ (BrdUϩ DPs, Left), total DPs (Center), and CD8highCD4highCD69high (CD69highDPs, Right). Black solid tracings: E47Ϫ͞Ϫ. Gray dotted tracings: E47ϩ͞Ϫ.

maturation events occurring within the DP stage are necessary for We have shown that E47 is necessary to prevent the develop- the down-regulation of CD25 in E2A-deficient thymocytes. mental progression of DN3 thymocytes in the absence of pre-TCR signaling, and that signaling from the TCR and pre-TCR complexes The Expression of a Substantial Proportion of E47 Target Genes Is acts to down-regulate E47-binding activity (8, 10, 11). These data Modulated During the DN3 to DN4 Transition. The data described led us to conclude that TCR signals function largely to inhibit E47 above indicate that E47 is required to repress CD25 expression activity, which in turn allows for thymocyte developmental pro- beyond the pre-TCR checkpoint. As a first approach to identify gression. However, we now show that E47 activity is required to additional E47 target genes that are regulated during ␤ selection, we repress CD25 expression upon transition from the DN to DP cell have compared the programs of gene expression regulated by E47 stages. In addition, we also observed that L-selectin is down- in the 1F9 cell line with that of genes whose expression patterns are regulated by E47 in 1F9 cells as well as during ␤ selection. IMMUNOLOGY modulated during ␤ selection (18). We observed 13 genes whose Furthermore, Ror␥ transcripts, which are induced by E47 in 1F9 mRNA levels were significantly elevated in DN4 cells, whereas the cells, are also up-regulated after passage of the ␤ selection check- transcript levels encoded by 74 genes were lower in DN4 cells point. These observations necessitate that we revise our model of (H.T.P., unpublished observations). Interestingly, Ϸ10% of the the effects of pre-TCR signaling on E2A protein activity (Fig. 3A). genes modulated in expression levels during the DN3 to DN4 cell Although it is clear that pre-TCR signaling acts to relieve the stages were also regulated by E47 in E2A-deficient lymphomas (Fig. inhibitory effects of E2A proteins on much of the differentiation 5, which is published as supporting information on the PNAS web program induced by ␤ selection, it is now also apparent that signals site). In addition to CD25, this common set of target genes included arising as a consequence of pre-TCR expression elicit novel E Gpr56, Dhrs3, Ptpre, Socs3, Hes1, Xbp1, L-selectin, and Ror␥. protein activities. Taken together, these data indicate that the expression of a We observed that E47 regulates a significant number of tran- significant fraction of E47 target genes in E2A-deficient lympho- scripts encoding factors involved in cell cycle progression. These mas is modulated during ␤ selection. include p21 and Cdk6. A role for E47 in modulating p21 expression has been described (19). However, lymphomagenesis in p21- Discussion deficient mice is rare and in fact appears to be promoted rather than Previous observations have indicated a critical role for E47 in being suppressed by p21 in ataxia telangiectasia mutated-deficient lymphocyte survival, growth, and developmental progression. Ad- mice (20). Thus, it appears unlikely that E47 acts to prevent the ditionally, E47 has been demonstrated to function as a tumor development of lymphoma through the induction of p21 expression. suppressor, because E47-deficient mice rapidly develop T cell A more attractive candidate is Cdk6. Cdk6 mRNA levels decreased lymphoma. These observations brought into question how E2A 2-fold within a 6-hour period. Furthermore, we have observed that proteins regulate such a diverse set of cellular activities. The data cells in which E47 activity has been enforced for 24 hours expressed presented here identify previously unrecognized E47 target genes increased levels of hypophosphorylated Rb, an effect consistent that may provide insight into how E47 acts to regulate cell cycle with a reduction in Cdk6 activity (data not shown). These data progression, cytokine-mediated signaling, lipid metabolism, stress provide a possible mechanism for how E47 expression promotes cell response, survival, and T lineage maturation. cycle arrest (11).

Schwartz et al. PNAS ͉ June 27, 2006 ͉ vol. 103 ͉ no. 26 ͉ 9979 Downloaded by guest on September 26, 2021 Fig. 3. Model diagrams indicating regulation and function of E47 activity. (A) Model diagram depicting the effects of pre-TCR signaling on E2A function. Signals from the pre-TCR complex abrogate the inhibitory effect of E2A proteins on the proliferation and differentiation of DN3 thymocytes to the DP stage. However, pre-TCR signaling also induces a function of E2A proteins in the down-regulation of CD25 expression. The asterisk indicates novel E-protein activities in response to pre-TCR signaling. Genes regulated by E47 in E2A-deficient lymphomas and during ␤ selection are indicated. (B) Model diagram showing a regulatory network involving cytokine receptor-mediated signaling, E47, and SOCS activities in a common pathway. (C) Similarities of regulatory networks in D. melanogaster sensory organ development and T lineage cells.

These results bring into question whether the regulation of Cdk6 Thymocyte development in transgenic mice overexpressing Tox transcription by E47 plays a role in the development of human shows significant similarities with that observed for E2A-deficient T-acute lymphoblastoid leukemia (T-ALL). The most common mice, raising the possibility that TCR-mediated signaling, E2A, and defect with human T-ALL is the activation of the TAL͞SCL1 and Tox are linked in a linear pathway (30). TAL͞SCL2 (TAL1 and TAL2, respectively) genes (21, 22). The Our microarray analysis showed that the transcriptional regulator TAL gene products are members of the HLH family and readily Xbp1 is regulated by E47. Xbp1 functions to promote the devel- form heterodimers with E2A. They become aberrantly activated in opment of activated B lineage cells into antibody-secreting cells, T lineage cells upon somatic genomic rearrangement. We have regulates the unfolded protein response, and promotes phospho- previously suggested that the TAL protooncogenes act to promote lipid biosynthesis (31, 32). Additionally, clustered analysis showed human T-ALL by suppressing the ability of E2A to induce down- that E47 also modulates the expression of a significant subset of stream target gene expression (5). Mouse models using E protein- genes involved in lipid metabolism and cell growth. It is conceivable deficient mice and transgenic mice expressing TAL1 in the thymo- that during maturation of mature B lineage cells into plasma cells, cyte compartment have provided further support for this hypothesis E47 activity is modulated to induce the expression of Xbp1 and and show interestingly common patterns of gene expression with genes involved in lipid biogenesis to promote development of the E47 target genes, including ROR␥, RAG2, and CD3␧ (23–25). Our plasma cell secretory apparatus. Likewise, it would be of interest to data also imply that TAL proteins may act to prevent the repression determine the role of Xbp1 during ␤ selection. of Cdk6 by E47 as well, consistent with the presence of high-affinity Several genes that are coordinately, albeit modestly, regulated by E2A͞Tal1-binding sites that are present in the Cdk6 promoter E47 are involved in NKT cell function. Among these are CD1d, region. Thus TAL proteins may promote T-ALL by interfering with AP3D, AP3B1, prosaposin, and cathepsin L. AP3D and AP3B otherwise repressive effects of E2A proteins on Cdk6 expression, interact with the cytoplasmic tail of the CD1 proteins and function and it will be important to examine the potential role of Cdk6 in the to deliver CD1B to late endosomes and lysosomes (33). Prosaposins development of human T-ALL. act as endosomal lipid transfer proteins to edit Cd1b-bound lipid Two modulators of E protein activity, Id2 and Eto2, were antigens (34). The lysosomal cysteine protease cathepsin L is activated by E47 expression. Id2 functions by interfering with the essential to promote NKT cell development (35). The coordinate DNA-binding activity of E47, whereas Eto2 acts to repress target regulation of genes involved in NKT cell development and lipid gene expression upon interacting with the E47 N-terminal trans- metabolism is intriguing, and a significant role for E proteins in activation domain (26, 27). The activation of Id2 by E47 is remi- NKT cell development cannot be excluded. niscent of the feedback mechanism regulating I␬B expression by Interestingly, the expression of Socs1 and Soc3 was substantially NF-␬B, and it will be interesting to examine how E47, Id2, and Eto2 induced by E47 expression. SOCS proteins are feedback suppres- act in a regulatory network to control cell growth and cell fate (28). sors of the Janus kinase (JAK) and signal transducer and activator The high mobility group box-containing transcription factor Tox, of transcription (STAT) signaling module. Cytokine signaling in- is also negatively regulated by E47 activity. Tox expression is duces the expression of SOCS proteins that act in turn to antagonize transiently activated during both ␤ and positive selection (29). further signaling. In vivo experiments have indicated that SOCS1

9980 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603728103 Schwartz et al. Downloaded by guest on September 26, 2021 and SOCS3 act in response to IL-7, IL-6, IL-4, IL-12, IL-15, and opmental progression. They also suggest novel and unexpected granulocyte colony-stimulating factor (36). Our data indicate that functions for E47, including the control of lipid biosynthesis, E47 regulates the expression of SOCS proteins, and it will be cytokine-mediated signaling, and stress response. particularly interesting to examine whether JAK͞STAT signaling directly or indirectly activates E47 expression, which in turn may act Methods to induce SOCS gene expression (Fig. 3B). E2A proteins have been Retroviral Infection and Purification of Transduced Cells. Retroviral demonstrated to play a critical role in the IL-7-dependent prolif- supernatant preparation and spin infection into the E2AϪ͞Ϫ eration of pro-B lineage cells (37). Furthermore, modulation of lymphoma line 1.F9 were performed as described (15). For prep- IL-7 signaling has been shown to be a required step in B lineage aration of 4-hydroxy tamoxifen-treated RNA samples, cells were commitment (38). It is conceivable that E47 acts in prepro-B and spin-infected with E47-ER-Tac or basic HLH-ER-Tac virus and pro-B cells to regulate SOCS expression to modulate IL-7R␣- cultured for 20 hours in Iscove’s modified Dulbecco’s media mediated signaling. Likewise, E proteins may promote the survival without phenol red plus 5% FBS. The retrovirally transduced cells of IL-7-dependent developing double-negative thymocytes by mod- were then cultured for an additional 6 hours with or without 1 ␮M ulating Socs gene expression (Fig. 3B). 4-hydroxytamoxifen before harvest and isolation of transduced The induction of Gfi1, Gfi1b, and Hes1 expression by E47 is cells by magnetic selection of human CD25 (Tac antigen), as intriguing, because a similar regulatory network regulates Drosoph- described (16). ila sensory organ development (Fig. 3C). In D. melanogaster, sensory organ development is regulated in part by the proneural RNA Purification and Microarray Analysis. Isolated RNA samples proteins, achaete and scute, that form heterodimers with daugh- were converted into double-stranded cDNA. The resulting cDNA terless, a protein closely related to E47, to regulate Enhancer of samples were then used to synthesize biotinylated cRNA, which in Split transcription (39). Similarly, our data demonstrate that E47 turn was hybridized to CODELINK mouse oligonucleotide arrays acts to induce Hes1 expression in T lineage cells. Sensory organ (Amersham Pharmacia). After hybridization and washing, the development also requires the activity of a zinc-finger-containing arrays were incubated with streptavidin-coupled fluorophore, protein named Senseless. The expression of Senseless is regulated washed, scanned, and analyzed using CODELINK analysis software. by the proneural genes that act as heterodimers with daughterless Expression levels from scanned images were determined by using (40). Notably, here we demonstrate that two mammalian homo- CORGON software (42, 43). Genes with P values for presence of Յ0.1 logues of Senseless, Gfi1 and Gfi1b, are regulated by E47 in T were considered for further analysis. Expression levels from all lineage cells. A role for E47 in modulating the expression of Gfi1 experimental conditions were normalized simultaneously by using was not unexpected, because Gfi1-null mutant mice show similar the multi-loess technique, as described (43). We calculated the defects in thymocyte development as described for E47-deficient absolute and relative changes of the expression levels for every gene mice (12, 41). Taken together, these data indicate that an ancient and sorted the genes based on their interest statistics. The interest regulatory network underlying Drosophila sensory organ develop- statistic design was based on the software package FOCUS (44). ment has been conserved in mammalian cells. It will be interesting to determine how this network acts in developing T cells to We thank Jessica Novak for technical assistance and Carol Katayama for modulate developmental progression and cellular expansion. help with oligonucleotide design for quantitative PCR. This work was Collectively, these data provide a conceptual framework in which supported by National Institutes of Health grants to C.M. and H.T.P. and E47 acts to regulate cell cycle regulation, cell survival, and devel- a Lymphoma Research Foundation fellowship to I.E.

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