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Protein Stability and Transcription Factor Complex Assembly Determined by the SCL-LMO2 Interaction

2007; Elsevier BV; Volume: 282; Issue: 46 Linguagem: Inglês

10.1074/jbc.m703939200

ISSN

1083-351X

Autores

Éric Lécuyer, Simon Larivière, Marie‐Claude Sincennes, André Haman, Rachid Lahlil, Margarita Todorova, Mathieu Tremblay, Brian C. Wilkes, Trang Hoang,

Tópico(s)

Viral Infectious Diseases and Gene Expression in Insects

Resumo

Gene expression programs are established by networks of interacting transcription factors. The basic helix-loop-helix factor SCL and the LIM-only protein LMO2 are components of transcription factor complexes that are essential for hematopoiesis. Here we show that LMO2 and SCL are predominant interaction partners in hematopoietic cells and that this interaction occurs through a conserved interface residing in the loop and helix 2 of SCL. This interaction nucleates the assembly of SCL complexes on DNA and is required for target gene induction and for the stimulation of erythroid and megakaryocytic differentiation. We also demonstrate that SCL determines LMO2 protein levels in hematopoietic cells and reveal that interaction with SCL prevents LMO2 degradation by the proteasome. We propose that the SCL-LMO2 interaction couples protein stabilization with higher order protein complex assembly, thus providing a powerful means of modulating the stoichiometry and spatiotemporal activity of SCL complexes. This interaction likely provides a rate-limiting step in the transcriptional control of hematopoiesis and leukemia, and similar mechanisms may operate to control the assembly of diverse protein modules. Gene expression programs are established by networks of interacting transcription factors. The basic helix-loop-helix factor SCL and the LIM-only protein LMO2 are components of transcription factor complexes that are essential for hematopoiesis. Here we show that LMO2 and SCL are predominant interaction partners in hematopoietic cells and that this interaction occurs through a conserved interface residing in the loop and helix 2 of SCL. This interaction nucleates the assembly of SCL complexes on DNA and is required for target gene induction and for the stimulation of erythroid and megakaryocytic differentiation. We also demonstrate that SCL determines LMO2 protein levels in hematopoietic cells and reveal that interaction with SCL prevents LMO2 degradation by the proteasome. We propose that the SCL-LMO2 interaction couples protein stabilization with higher order protein complex assembly, thus providing a powerful means of modulating the stoichiometry and spatiotemporal activity of SCL complexes. This interaction likely provides a rate-limiting step in the transcriptional control of hematopoiesis and leukemia, and similar mechanisms may operate to control the assembly of diverse protein modules. Tissue-specific programs of gene expression are largely controlled by networks of interacting transcription factors that provide flexibility and specificity in gene regulation (1Tjian R. Maniatis T. Cell. 1994; 77: 5-8Abstract Full Text PDF PubMed Scopus (954) Google Scholar, 2Grosschedl R. Curr. Opin. Cell Biol. 1995; 7: 362-370Crossref PubMed Scopus (150) Google Scholar). This theme finds particular relevance in the context of the hematopoietic system, where the generation of diverse cellular lineages is controlled by combinations of hematopoietic-specific and broadly expressed transcription factors (3Shivdasani R.A. Orkin S.H. Blood. 1996; 87: 4025-4039Crossref PubMed Google Scholar, 4Sieweke M.H. Graf T. Curr. Opin. Genet. Dev. 1998; 8: 545-551Crossref PubMed Scopus (145) Google Scholar, 5Swiers G. Patient R. Loose M. Dev. Biol. 2006; 294: 525-540Crossref PubMed Scopus (128) Google Scholar). Regulation by networking is exemplified by the SCL/TAL-1 (hereafter referred to as SCL) transcription factor, which is essential for the establishment of the hematopoietic system and plays important functions at several branch points in the hematopoietic hierarchy (6Lecuyer E. Hoang T. Exp. Hematol. 2004; 32: 11-24Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). SCL is a member of a subfamily of tissue-specific bHLH transcription factors that includes two other hematopoietic factors, TAL2 and LYL-1 (7Begley C.G. Green A.R. Blood. 1999; 93: 2760-2770Crossref PubMed Google Scholar), and two neurogenic factors, NSCL1/nHLH-1 and NSCL2 (8Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1373) Google Scholar). Transcription regulation by SCL requires its integration within multifactorial complexes (SCL complexes) containing the ubiquitously expressed bHLH factors encoded by the E2A gene (E47 and E12), LMO 5The abbreviations used are: BFU-E, burst-forming unit erythroid; bHLH, basic helix-loop-helix; CFU-GM/-Meg, colony-forming unit granulocyte-macrophage/megakaryocyte; EMSA, electrophoretic mobility shift assay; Epor, erythropoietin receptor gene; F-LMO2, FLAG-LMO2; GFP, green fluorescence protein; GPA, glycophorin A; IB, immunoblotting; IP, immunoprecipitation; IRES, internal ribosome entry site; Ldb1, LIM domain-binding protein-1; LIM, Lin-Isl-Mec; LMO, LIM-only; MSCV, murine stem cell virus; NSCL1, neuronal SCL-1; SCL/TAL1, stem cell leukemia/T-cell acute leukemia-1; IF, immunofluorescence; PBS, phosphate-buffered saline; HA, hemagglutinin; RT, reverse transcription; GST, glutathione S-transferase. 5The abbreviations used are: BFU-E, burst-forming unit erythroid; bHLH, basic helix-loop-helix; CFU-GM/-Meg, colony-forming unit granulocyte-macrophage/megakaryocyte; EMSA, electrophoretic mobility shift assay; Epor, erythropoietin receptor gene; F-LMO2, FLAG-LMO2; GFP, green fluorescence protein; GPA, glycophorin A; IB, immunoblotting; IP, immunoprecipitation; IRES, internal ribosome entry site; Ldb1, LIM domain-binding protein-1; LIM, Lin-Isl-Mec; LMO, LIM-only; MSCV, murine stem cell virus; NSCL1, neuronal SCL-1; SCL/TAL1, stem cell leukemia/T-cell acute leukemia-1; IF, immunofluorescence; PBS, phosphate-buffered saline; HA, hemagglutinin; RT, reverse transcription; GST, glutathione S-transferase. proteins, the LIM domain-binding protein Ldb1, and hematopoietic GATA family members (9Visvader J.E. Mao X. Fujiwara Y. Hahm K. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13707-13712Crossref PubMed Scopus (137) Google Scholar, 10Wadman I.A. Osada H. Grutz G.G. Agulnick A.D. Westphal H. Forster A. Rabbitts T.H. EMBO J. 1997; 16: 3145-3157Crossref PubMed Scopus (724) Google Scholar, 11Grutz G.G. Bucher K. Lavenir I. Larson T. Larson R. Rabbitts T.H. EMBO J. 1998; 17: 4594-4605Crossref PubMed Scopus (97) Google Scholar). These complexes govern the switch between a proliferative state in erythroid progenitors and a commitment to terminal differentiation (12Goardon N. Lambert J.A. Rodriguez P. Nissaire P. Herblot S. Thibault P. Dumenil D. Strouboulis J. Romeo P.H. Hoang T. EMBO J. 2006; 25: 357-366Crossref PubMed Scopus (115) Google Scholar), and different variants of these complexes activate transcription of target genes such as c-kit, glycophorin A (GPA), and protein 4.2 in progenitor and erythroid cells (13Lecuyer E. Herblot S. Saint-Denis M. Martin R. Begley C.G. Porcher C. Orkin S.H. Hoang T. Blood. 2002; 100: 2430-2440Crossref PubMed Scopus (138) Google Scholar, 14Xu Z. Huang S. Chang L.S. Agulnick A.D. Brandt S.J. Mol. Cell. Biol. 2003; 23: 7585-7599Crossref PubMed Scopus (119) Google Scholar, 15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). These observations are consistent with the view that subtle variations in the composition of transcription factor complexes modulate choices in cell fate. Interestingly, SCL complexes exhibit an all-or-nothing behavior in transcription activation requiring the simultaneous presence of each partner for robust target gene activation (13Lecuyer E. Herblot S. Saint-Denis M. Martin R. Begley C.G. Porcher C. Orkin S.H. Hoang T. Blood. 2002; 100: 2430-2440Crossref PubMed Scopus (138) Google Scholar, 15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). These complexes have, therefore, been proposed to form multi-input motifs within the hematopoietic regulatory hierarchy (5Swiers G. Patient R. Loose M. Dev. Biol. 2006; 294: 525-540Crossref PubMed Scopus (128) Google Scholar), a network feature that ensures specificity and flexibility in gene regulation. Despite these findings, the mechanisms governing the assembly of SCL-containing complexes on target gene regulatory elements remain ill-defined. It has recently been shown that the SCL-LMO2 interaction is essential for hematopoietic cell fate specification (16Mead P.E. Deconinck A.E. Huber T.L. Orkin S.H. Zon L.I. Development. 2001; 128: 2301-2308Crossref PubMed Google Scholar, 17Schlaeger T.M. Schuh A. Flitter S. Fisher A. Mikkola H. Orkin S.H. Vyas P. Porcher C. Mol. Cell. Biol. 2004; 24: 7491-7502Crossref PubMed Scopus (34) Google Scholar, 18Patterson L.J. Gering M. Eckfeldt C.E. Green A.R. Verfaillie C.M. Ekker S.C. Patient R. Blood. 2007; 109: 2389-2398Crossref PubMed Scopus (115) Google Scholar), consistent with the phenotypic similarity of SCL-/- and LMO2-/- embryos, which exhibit early lethality due to a complete absence of blood cells (19Warren A.J. Colledge W.H. Carlton M.B. Evans M.J. Smith A.J. Rabbitts T.H. Cell. 1994; 78: 45-57Abstract Full Text PDF PubMed Scopus (538) Google Scholar, 20Robb L. Lyons I. Li R. Hartley L. Kontgen F. Harvey R.P. Metcalf D. Begley C.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7075-7079Crossref PubMed Scopus (483) Google Scholar, 21Shivdasani R.A. Mayer E.L. Orkin S.H. Nature. 1995; 373: 432-434Crossref PubMed Scopus (778) Google Scholar, 22Porcher C. Swat W. Rockwell K. Fujiwara Y. Alt F.W. Orkin S.H. Cell. 1996; 86: 47-57Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar, 23Robb L. Elwood N.J. Elefanty A.G. Kontgen F. Li R. Barnett L.D. Begley C.G. EMBO J. 1996; 15: 4123-4129Crossref PubMed Scopus (285) Google Scholar, 24Yamada Y. Warren A.J. Dobson C. Forster A. Pannell R. Rabbitts T.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3890-3895Crossref PubMed Scopus (263) Google Scholar). In addition to their important function during normal hematopoiesis, the SCL and LMO2 genes are the most frequent targets of chromosomal rearrangements in pediatric T-cell acute lymphoblastic leukemia (7Begley C.G. Green A.R. Blood. 1999; 93: 2760-2770Crossref PubMed Google Scholar, 25Rabbitts T.H. Genes Dev. 1998; 12: 2651-2657Crossref PubMed Scopus (144) Google Scholar). Furthermore, retroviral integration within the LMO2 gene, leading to a severe lymphoproliferative disorder, has been noted after gene therapy in one clinical trial (26Hacein-Bey-Abina S. Von Kalle C. Schmidt M. McCormack M.P. Wulffraat N. Leboulch P. Lim A. Osborne C.S. Pawliuk R. Morillon E. Sorensen R. Forster A. Fraser P. Cohen J.I. de Saint Basile G. Alexander I. Wintergerst U. Frebourg T. Aurias A. Stoppa-Lyonnet D. Romana S. Radford-Weiss I. Gross F. Valensi F. Delabesse E. Macintyre E. Sigaux F. Soulier J. Leiva L.E. Wissler M. Prinz C. Rabbitts T.H. Le Deist F. Fischer A. Cavazzana-Calvo M. Science. 2003; 302: 415-419Crossref PubMed Scopus (2978) Google Scholar). Finally, SCL genetically interacts with LMO1/2 to induce aggressive T-cell tumors in transgenic mice (7Begley C.G. Green A.R. Blood. 1999; 93: 2760-2770Crossref PubMed Google Scholar), underscoring the importance of the SCL-LMO2 interaction in vivo. Despite this extensive body of evidence conveying the biological importance of the SCL-LMO2 interaction, it is not clear why this interaction is important at the molecular level. In addition, because LIM domain proteins, including LMO2, are known to be targeted for proteasomal degradation (27Ostendorff H.P. Peirano R.I. Peters M.A. Schluter A. Bossenz M. Scheffner M. Bach I. Nature. 2002; 416: 99-103Crossref PubMed Scopus (138) Google Scholar, 28Xu Z. Meng X. Cai Y. Liang H. Nagarajan L. Brandt S.J. Genes Dev. 2007; 21: 942-955Crossref PubMed Scopus (68) Google Scholar), this raises the possibility that LMO2 degradation may represent a rate-limiting step in the assembly of SCL complexes. In the present study we show that SCL prevents the degradation of LMO2 via direct protein-protein interaction, allowing for the nucleation of multifactorial complexes with proper SCL/LMO2 stoichiometry. Plasmids and Mutagenesis—A detailed description of plasmid construction methodology is provided as supplemental material. Cell Culture, Infections, Transfections, Immunofluorescence (IF), and Protein Extraction—NIH 3T3, 293, ts20, and TF-1 cells were cultured as described previously (15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar, 29Krosl G. He G. Lefrancois M. Charron F. Romeo P.H. Jolicoeur P. Kirsch I.R. Nemer M. Hoang T. J. Exp. Med. 1998; 188: 439-450Crossref PubMed Scopus (61) Google Scholar, 30Coulombe P. Rodier G. Bonneil E. Thibault P. Meloche S. Mol. Cell. Biol. 2004; 24: 6140-6150Crossref PubMed Scopus (111) Google Scholar). Primary fetal liver cells were isolated from E12.5 Epor-/- and SCLlacZ/WT embryos, sorted for TER119 expression, and cultured as described before (15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). Fetal liver cell infections were performed by overnight co-culture on GP+E packaging cells stably expressing the viruses encoding SCL and SCL-M13. After infection, the cells were cultured in the presence of interleukin-3 (50 ng/ml) and steel factor (5 ng/ml) for an additional 24 h before performing further analyses. Infections of TF-1 cells with control retroviruses (MSCV-neo) or viruses encoding SCL and SCL-M13 were performed as detailed previously (15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). Transient transactivation assays were performed as previously described (13Lecuyer E. Herblot S. Saint-Denis M. Martin R. Begley C.G. Porcher C. Orkin S.H. Hoang T. Blood. 2002; 100: 2430-2440Crossref PubMed Scopus (138) Google Scholar, 15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). Expression vector doses used in specific experiments are indicated in figure legends. In all samples the amount of total DNA was kept constant at 4.5 μg with pGem4, and 100 ng of the cytomegalovirus-β-galactosidase vector was included to normalize luciferase values. For IF analysis, TF-1 or transfected 293 cells were harvested, washed in PBS, and fixed in 500 μl of Bouin'ns fixative for 15 min at room temperature. Cells were then washed several times in PBS by successive centrifugations and spun onto glass slides. In a humidified chamber at room temperature, the samples were blocked with PBS plus 15% normal goat serum for 15 min, washed in PBS, and incubated for 2 h with primary mouse anti-SCL and goat anti-LMO2 or anti-Ldb1 antibodies (see description below). After washing the samples 5× for 10 min in PBS, they were incubated for 2 h with secondary donkey anti-mouse and anti-goat antibodies coupled to fluorescein isothiocyanate and Cy3, respectively. All antibodies were used at a 1/100 dilution in PBS. Cells were then counter-stained with Hoechst for 1 min, washed, and resuspended in Vectashield (Vector Laboratories Inc, Burlington, ON, Canada) before analysis on an LSM 510 laser scanning confocal system (Carl Zeiss Inc., Maple Grove, MN). TF-1 and 293 cell nuclear extracts were prepared as documented previously (15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). Protein extracts from primary fetal liver from wild type and SCLlacZ/WT embryos were prepared as detailed before (31Calkhoven C.F. Muller C. Martin R. Krosl G. Pietsch H. Hoang T. Leutz A. Genes Dev. 2003; 17: 959-964Crossref PubMed Scopus (56) Google Scholar). For 293 cell extracts used in immunoprecipitations (IPs) and electrophoretic mobility shift assays (EMSA), 4.2×106 cells were seeded in 100-mm plates and transfected with combinations of expression vectors (5 μg of each) as described in the figure legends. The total amount of DNA was kept at 25-30 μg using pGem4. For studies of LMO2 protein expression/stability in 293 cells, 7×105 cells/well were seeded in 6-well plates and transfected with 1 μg of each expression vector indicated in figure legends. The total amount of DNA was kept at 5 μg with pGem4. Proteasomal inhibitor (MG132, lactacystin) or cycloheximide treatments were carried out as described in figure legends from 4 h to overnight. After treatment, the cells were washed twice with cold PBS and instantaneously frozen on liquid nitrogen. Total cell protein extracts were then prepared by lysing the cells for 20 min at 4 °C with 400 μl of radioimmunoprecipitation assay buffer (10 mm Tris-HCl (pH 8.0), 140 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.1% SDS, 0.1% deoxycholate) supplemented with protease inhibitors. EMSA, Pulldown Assays, IBs, and IPs—For EMSA, each binding reaction was performed using 10 μg of TF-1 or 293 cell nuclear extract in 20 mm HEPES (pH 7.5), 50 mm KCl, 1 mm dithiothreitol, 1 mm EDTA, 5% glycerol, 10 μg bovine serum albumin, 0.5 μg of dI-dC, and the GPA-84 probe (10,000 cpm) in a final volume of 20 μl. After 15 min at room temperature, protein-DNA complexes were resolved at 150 V on a 4% PAGE gel in 0.5× Tris-buffered EDTA at 4 °C for 4 h. Pulldown experiments were performed as previously described (13Lecuyer E. Herblot S. Saint-Denis M. Martin R. Begley C.G. Porcher C. Orkin S.H. Hoang T. Blood. 2002; 100: 2430-2440Crossref PubMed Scopus (138) Google Scholar, 15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). For IPs, 293 (10-60 μg) or TF-1 (500 μg) cell nuclear extracts were incubated overnight at 4 °C with 3 μg of antibody in 1 ml of IP buffer (20 mm Tris-HCl (pH 8.0), 137 mm NaCl, 1% Nonidet P-40, 10% glycerol, 1 mm EDTA). Protein complexes were precipitated by adding appropriately conjugated Pansorbin cells (Calbiochem) for 30-120 min at 4 °C, washed 3 times with 1 ml of IP buffer, and subjected to SDS-PAGE. After transfer on PVDF membranes, proteins were visualized by immunoblotting (IB) using ECL plus (GE Healthcare). The following antibodies were used for IP, IB, and IF analysis. The mouse anti-E2A (YAE), rat anti-GATA-1 (N6), rabbit anti-GFP (FL), goat anti-LMO2 (N-16), and goat anti-Ldb1/CLIM-2 (N-18) were all from Santa Cruz Biotechnology Inc (Santa-Cruz, CA). The BTL-73 and 2TL-136 mouse anti-SCL antisera were provided by Dr. D. Mathieu (Institut de Génétique Moléculaire, Montpellier, France). The mouse anti-HA, anti-FLAG, and anti-P21 antibodies were obtained from Covance (Richmond, VA), Stratagene (La Jolla, CA), and BD Biosciences (Mississauga, ON, Canada), respectively. RT-PCR Analysis—Total RNA was extracted from TF-1 or 293 cells as previously described (15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). To eliminate contaminant DNA molecules, 500 ng of each sample was then subjected to digestion with EcoRI and DNase I in REact3 buffer (Invitrogen). After the nucleases were heat-inactivated for 10 min at 65 °C, the samples were subjected to reverse transcription using the Superscript first strand cDNA synthesis system (Invitrogen). The quantification of GPA, β-major, LMO2, and S14 and S16 mRNAs was performed by real time PCR on a MX3000 apparatus (Stratagene, La Jolla, CA) using QuantiTect SYBR green PCR kit (Qiagen, Mississauga, ON, Canada) according to the manufacturer'ns instructions and under the following conditions: 95 °C for 15 min followed by 50 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s. Oligonucleotides used for RT-PCR analysis were described previously (15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). Structural Modeling—The structure of the E47 homodimer-DNA complex (32Ellenberger T. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Crossref PubMed Scopus (353) Google Scholar) was obtained from the Brookhaven protein data base and was subjected to 1000 steps of conjugate gradient minimization. One of the E47 moieties was transformed into human SCL using homology modeling techniques. After the structure of the DNA bound SCL/E47 heterodimer was obtained, the side chains of the mutated amino acids were packed using SYBYL (Tripos, St. Louis, MO). In this procedure the backbone dihedral angles are held fixed, whereas the side chains of the individual amino acids are rotated one at a time until a sterically acceptable conformation was obtained. This structure was again minimized, at which time no major conformational changes were observed. SCL and LMO2 Are Predominant and Specific Interaction Partners—The SCL and LMO2 interaction is essential for hematopoiesis. To determine what proportion of LMO2 is associated with SCL in hematopoietic cells, we performed quantitative IPs of nuclear extracts of CD34+ TF-1 cells with an antibody against SCL or control immunoglobulins. As shown in Fig. 1A, all of the SCL protein was precipitated with an anti-SCL antibody (lanes 2 and 4), whereas the protein remained in the supernatant with a control antiserum (lanes 1 and 3). Under these conditions, most of LMO2 co-precipitated with SCL, and only 20% of LMO2 was found in the supernatant (lane 2). Furthermore, by performing successive SCL and Ldb1 IPs, we found that the majority LMO2 that did not come down with the SCL antibody was co-precipitated with anti-Ldb1 antisera (data not shown). In contrast, all of LMO2 was recovered in the supernatant of control IPs (lane 1). Therefore, we conclude that the majority (∼90%) of LMO2 is associated with SCL and Ldb1 in TF-1 cell extracts. In agreement with these findings, all three of these factors were found to exhibit overlapping nucleoplasmic localization patterns, as determined by IF analysis in TF-1 cells (Fig. 1B). Together, our observations concur with the view that SCL, LMO2, and Ldb1 are predominant interaction partners in hematopoietic cells. SCL has recently been shown to exhibit exquisite specificity in comparison to other bHLH factors in inducing hematopoietic cell fate commitment in embryonic stem cells (17Schlaeger T.M. Schuh A. Flitter S. Fisher A. Mikkola H. Orkin S.H. Vyas P. Porcher C. Mol. Cell. Biol. 2004; 24: 7491-7502Crossref PubMed Scopus (34) Google Scholar). SCL shares 64% identity within the bHLH domain with the closely related neurogenic factor NSCL1, and both proteins heterodimerize with the ubiquitous bHLH factor E47. Furthermore, LMO2, which is 59% identical to LMO1 within the LIM domains, interacts with SCL but not E47. To assess the specificity of the SCL-LMO2 interaction, we compared the binding properties of SCL and NSCL1 in pulldown assays. As expected, both SCL and NSCL1 interact with GST-E47 (Fig. 1C). Interaction with LMO proteins, however, revealed a striking difference. Both of the main isoforms of SCL (p42 and p22) associate equally well with LMO1 and LMO2. In contrast, NSCL1 was only weakly retained on GST-LMO1 columns and was not retained on GST-LMO2 columns. These results indicate that interaction with LMO2 is specific to SCL. To ascertain whether this interaction specificity is also observed in transfected cells, we next performed IPs using nuclear extracts from 293 cells expressing SCL or HA-NSCL1, in combination with FLAG-LMO2 (F-LMO2), E47, and Ldb1. Increasing amounts of nuclear extracts were subjected to IP using anti-SCL, anti-FLAG, or anti-HA antibodies. In SCL-containing extracts, IP with both the anti-SCL and anti-FLAG antibodies efficiently precipitated SCL, F-LMO2, and E47 (Fig. 1D, lanes 1-7), showing that these factors associate in transfected cells. With HA-NSCL1 extracts, however, the anti-HA antibody brought down HA-NSCL1 and E47 but was unable to co-precipitate F-LMO2 (lanes 9-11). In agreement, the anti-FLAG antibody precipitated F-LMO2 but not HA-NSCL1 or E47 (lanes 12-14). Finally, co-expressed GFP protein, included as a negative control, was not precipitated with any of the antibodies. Because LMO2 and E47 only co-precipitate in the presence of SCL, we conclude that SCL acts as a bridging factor between its two partners. More importantly, whereas both SCL and NSCL1 are able to associate with E47, interaction with LMO2 is restricted to SCL. Specificity of SCL in Hematopoietic Target Gene Activation and Assembly of Transcription Factor Complexes—To determine whether SCL also demonstrates specificity at the molecular level during hematopoietic target gene activation, we next performed transactivation assays of the c-kit and GPA promoters, two well characterized targets of SCL-containing complexes (13Lecuyer E. Herblot S. Saint-Denis M. Martin R. Begley C.G. Porcher C. Orkin S.H. Hoang T. Blood. 2002; 100: 2430-2440Crossref PubMed Scopus (138) Google Scholar, 15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). This approach previously revealed an essential requirement for each partner of the SCL complex in transcriptional regulation (13Lecuyer E. Herblot S. Saint-Denis M. Martin R. Begley C.G. Porcher C. Orkin S.H. Hoang T. Blood. 2002; 100: 2430-2440Crossref PubMed Scopus (138) Google Scholar, 15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). As suspected, although SCL-containing complexes efficiently activate the c-kit and GPA promoters, neither the neurogenic bHLH factor NSCL1 nor the myogenic factor MyoD is able to substitute for SCL function (Fig. 2A, left panels) despite being efficiently expressed in transfected cells (Fig. 2A, right panel). In contrast to SCL, other partners of the complex are functionally redundant with members of their respective families (supplemental Fig. S1).FIGURE 2Non-redundant function of SCL in hematopoietic gene activation and complex formation on DNA. A, SCL is non-redundant with other bHLH factors in c-kit and GPA promoter activation. The kit-1146-Luc or GPA-84-Luc reporter constructs (1500 ng) were transfected into NIH 3T3 cells with expression vectors for E47 (150 ng), LMO2 (750 ng), Ldb1 (750 ng), and GATA-1 or -2 (150 ng) and the indicated amounts of SCL, NSCL1, and MyoD vectors (50-450 ng). Transactivation assays with kit-1146-Luc were conducted using GATA-1-containing complexes (upper graph), whereas GPA-84-Luc was assayed with complexes containing GATA-2 (lower graph). Results are expressed as fold activation relative to the reporter vector transfected alone, represent the average ± S.D. of triplicate determinations, and are representative of (n) independent experiments. Right panel, nuclear extracts of TF-1 cells (10 μg) or NIH 3T3 cells (10 μg) expressing SCL or HA-NSCL1 were analyzed by IB with the indicated antibodies. Expression of GFP in transfected cells was monitored as a control for equal loading. Arrowheads indicate specific bands. B, SCL is specifically required to assemble a high molecular weight complex on the GPA promoter sequences. EMSAs were performed using the GPA-84 probe (arrow), which covers positions -84 to -30 of the human GPA promoter (15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar), and nuclear extracts (10 μg) of TF-1 cells (lane 2) or 293 cells transfected with the indicated expression vectors (lanes 3-6). Note the low mobility complex containing SCL and its partners (arrowhead) (15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar), which is not formed in the presence of NSCL1. Asterisks indicate nonspecific complexes. Proteins expressed in the 293 cell extracts (10 μg) used for EMSA were revealed by IB with the indicated antibodies (lanes 7-10).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess whether SCL is specifically required for the assembly of SCL complexes on DNA, we next performed EMSA using a probe derived from the GPA promoter (GPA-84), to which the SCL complex was previously shown to bind with high affinity (15Lahlil R. Lecuyer E. Herblot S. Hoang T. Mol. Cell. Biol. 2004; 24: 1439-1452Crossref PubMed Scopus (128) Google Scholar). As shown in Fig. 2B, SCL and its partners, expressed in 293 cells, form a low mobility complex (arrowhead) on the GPA-84 probe (lane 5), which is comparable with that seen with endogenous proteins from hematopoietic TF-1 cells (lane 2) and is distinct from nonspecific complexes (asterisks) observed with untransfected 293 cell extracts (lane 3). This low mobility complex is supershifted by antibodies against SCL and its partners (data not shown; Ref. 13Lecuyer E. Herblot S. Saint-Denis M. Martin R. Begley C.G. Porcher C. Orkin S.H. Hoang T. Blood. 2002; 100: 2430-2440Crossref PubMed Scopus (138) Google Scholar) and is not seen with 293 extracts lacking SCL but expressing the other partners of the complex (lane 4). Interestingly, NSCL1 was unable to substitute for SCL in nucleating the assembly of this complex on DNA (lane 6), in agreement with transactivation experiments. The expression of SCL and its partners as well as NSCL1 in 293 cell extracts was confirmed by IB (lanes 7-11). We conclude that SCL confers specificity in hematopoietic target gene activation and in the assembly SCL complexes on DNA. SCL Interacts with LMO2 via Multiple Residues in the Loop and Helix 2—Schlaeger et al. (17Schlaeger T.M. Schuh A. Flitter S. Fisher A. Mikkola H. Orkin S.H. Vyas P. Porcher C. Mol. Cell. Biol. 2004; 24: 7491-7502Crossref PubMed Scopus (34) Google Scholar) previously identified residues of the HLH domain that are critical for interaction with LMO2 and for the rescue of primitive

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