Title: Transcriptional Activation by MEIS1A in Response to Protein Kinase A Signaling Requires the Transducers of Regulated CREB Family of CREB Co-activators
Abstract: The transcription factor encoded by the murine ecotropic integration site 1 gene (MEIS1) is a partner of HOX and PBX proteins. It has been implicated in embryonic patterning and leukemia, and causally linked to restless legs syndrome. The MEIS1A C terminus harbors a transcriptional activation domain that is stimulated by protein kinase A (PKA) in a manner dependent on the co-activator of cAMP response element-binding protein (CREB), CREB-binding protein (CBP). We explored the involvement of another mediator of PKA-inducible transcription, namely the CREB co-activators transducers of regulated CREB activity (TORCs). Overexpression of TORC1 or TORC2 bypassed PKA for activation by MEIS1A. Co-immunoprecipitation experiments demonstrated a physical interaction between MEIS1 and TORC2 that is dependent on the MEIS1A C terminus, whereas chromatin immunoprecipitation revealed PKA-inducible recruitment of MEIS1, PBX1, and TORC2 on the MEIS1 target genes Hoxb2 and Meis1. The MEIS1 interaction domain on TORC1 was mapped to the N-terminal coiled-coil region, and TORC1 mutants lacking this domain attenuated the response to PKA on a natural MEIS1A target enhancer. Thus, TORCs physically cooperate with MEIS1 to achieve PKA-inducible transactivation through the MEIS1A C terminus, suggesting a concerted action in developmental and oncogenic processes. The transcription factor encoded by the murine ecotropic integration site 1 gene (MEIS1) is a partner of HOX and PBX proteins. It has been implicated in embryonic patterning and leukemia, and causally linked to restless legs syndrome. The MEIS1A C terminus harbors a transcriptional activation domain that is stimulated by protein kinase A (PKA) in a manner dependent on the co-activator of cAMP response element-binding protein (CREB), CREB-binding protein (CBP). We explored the involvement of another mediator of PKA-inducible transcription, namely the CREB co-activators transducers of regulated CREB activity (TORCs). Overexpression of TORC1 or TORC2 bypassed PKA for activation by MEIS1A. Co-immunoprecipitation experiments demonstrated a physical interaction between MEIS1 and TORC2 that is dependent on the MEIS1A C terminus, whereas chromatin immunoprecipitation revealed PKA-inducible recruitment of MEIS1, PBX1, and TORC2 on the MEIS1 target genes Hoxb2 and Meis1. The MEIS1 interaction domain on TORC1 was mapped to the N-terminal coiled-coil region, and TORC1 mutants lacking this domain attenuated the response to PKA on a natural MEIS1A target enhancer. Thus, TORCs physically cooperate with MEIS1 to achieve PKA-inducible transactivation through the MEIS1A C terminus, suggesting a concerted action in developmental and oncogenic processes. The homeodomain is a DNA-binding structure shared by numerous transcription factors throughout eukaryotes, and is most commonly 60 amino acids in length (1.Gehring W.J. Qian Y.Q. Billeter M. Furukubo-Tokunaga K. Schier A.F. Resendez-Perez D. Affolter M. Otting G. Wüthrich K. Cell. 1994; 78: 211-223Abstract Full Text PDF PubMed Scopus (706) Google Scholar). The three-amino acid loop extension class of homeoproteins is so named for an extra 3 residues in the loop between helices 1 and 2 in the typical homeodomain (2.Bertolino E. Reimund B. Wildt-Perinic D. Clerc R.G. J. Biol. Chem. 1995; 270: 31178-31188Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Members of the three-amino acid loop extension class in mammals include the MEIS, PREP, and PBX families, which participate in relatively complex interactions between themselves and with the products of another group of homeodomain-containing proteins, the HOX family (3.Featherstone M. Lufkin T. Murine Homeobox Gene Control of Embryonic Patterning and Organogenesis. Elsevier, Amsterdam2003: 1-42Google Scholar). PBX proteins form cooperative DNA-binding heterodimers with MEIS, PREP, or HOX proteins, and coordinate the formation of higher order heterotrimeric complexes of PBX, HOX and MEIS, or PREP (3.Featherstone M. Lufkin T. Murine Homeobox Gene Control of Embryonic Patterning and Organogenesis. Elsevier, Amsterdam2003: 1-42Google Scholar, 4.Moens C.B. Selleri L. Dev. Biol. 2006; 291: 193-206Crossref PubMed Scopus (396) Google Scholar). DNA-binding PBX homodimers have also been noted, further extending the possible permutations for these partners (5.Calvo K.R. Knoepfler P. McGrath S. Kamps M.P. Oncogene. 1999; 18: 8033-8043Crossref PubMed Scopus (38) Google Scholar). Targets of PBX·MEIS heterodimers include the bovine CYP17 gene (6.Bischof L.J. Kagawa N. Moskow J.J. Takahashi Y. Iwamatsu A. Buchberg A.M. Waterman M.R. J. Biol. 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Montminy M. Mol. Cell. 2003; 12: 413-423Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 39.Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates 3rd, J.R. Takemori H. Okamoto M. Montminy M. Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). To construct FLAG-TORC1, TORC1 coding sequence was PCR-amplified from template pCMV-SPORT6-TORC1 purchased from Open Biosystems (catalogue number MHS1010–7507865; accession number BC028050), and cloned into BamHI and XhoI sites of pcDNA3.1(+) that had already been inserted with a FLAG tag. FLAG-TORC1-(47–634), FLAG-TORC1-(47–290), and FLAG-TORC1-(148–290) were subcloned as EcoRI-NotI fragments. FLAG-TORC1-(1–431), FLAG-TORC1-(1–518), Flag-TORC1-(1–493), and FLAG-TORC1-(1–627) were generated by removal of ClaI-XhoI, BsrGI-XhoI, SfiI-XhoI, and BspEI-XhoI fragments, respectively, and ligated following a blunt ending treatment by T4 DNA polymerase (Fermentas). Anti-MEIS NT is an affinity purified rabbit polyclonal antibody raised in-house against amino acid residues 1–34 of MEIS1 (8.Huang H. Rastegar M. Bodner C. Goh S.L. Rambaldi I. Featherstone M. J. Biol. Chem. 2005; 280: 10119-10127Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 44.Huang H. Paliouras M. Rambaldi I. Lasko P. Featherstone M. Mol. Cell. Biol. 2003; 23: 3636-3645Crossref PubMed Scopus (35) Google Scholar). The anti-MEIS1/2/3 mouse monoclonal antibody was purchased from Upstate Biotechnology (catalogue number 05-779). Goat anti-rabbit IgG conjugated to alkaline phosphatase (catalogue number sc-2007) was used as a nonspecific antibody control in chromatin immunoprecipitation (ChIP) assays, the anti-PBX1 (catalogue number sc-889) and anti-GAL4 (catalogue number sc-577) rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology. We purchased the anti-FLAG M2 affinity agarose (catalogue number A2220) as well as the anti-β-tubulin (catalogue number T4026) and anti-FLAG (catalogue number F3165) mouse monoclonal antibodies from Sigma. The rabbit polyclonal anti-TORC2 antibody was raised against amino acid residues 454–607 of murine TORC2 (39.Screaton R.A. Conkright M.D. Katoh Y. Best J.L. Canettieri G. Jeffries S. Guzman E. Niessen S. Yates 3rd, J.R. Takemori H. Okamoto M. Montminy M. Cell. 2004; 119: 61-74Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). The secondary antibodies used in this study were horseradish peroxidase-conjugated goat anti-mouse IgG, F(ab′)2 fragment specific (catalogue number 115-035-072, Jackson ImmunoResearch Laboratories) and mouse anti-rabbit IgG, light chain specific (catalogue number 211-032-171, Jackson ImmunoResearch Laboratories). HEK293 and P19 mouse embryonal carcinoma cells were cultured in Dulbecco's modified Eagle's medium and α minimal essential medium, respectively, supplemented with 10% fetal bovine serum, l-glutamine, and penicillin/streptomycin. To differentiate P19 cells, cells were aggregated in 100-mm diameter bacterial Petri dishes at a density of 105 cells/ml and treated with 0.3 μm retinoic acid (catalogue number R2625, Sigma) for 48 h. HEK293 cells were seeded at 75 to 90% confluence in 60-mm diameter tissue culture dishes for immunoprecipitation and in 12-well plates for luciferase assay. The cells were allowed to attach overnight and then transfected by Lipofectamine 2000 reagent (Invitrogen, catalogue number 11668-019). MG132 (Merck, catalogue number 474790) was used at 10 μm for 5 h. HEK293T cells were seeded at 1.0 × 105 cells/ml in 40 mm-diameter tissue culture dishes lined with ethanol- and acid-washed coverslips and allowed to attach overnight. Medium was changed 6 h after transfection. 24 h after transfection cells were fixed with 4% paraformaldehyde (Sigma) for 20 min at room temperature and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 min. Cells were blocked with 10% goat serum (Hyclone) in 0.1% Triton/PBS for 10 min and incubated with primary antibody. The antibody used was goat polyclonal anti-TORC2 (sc-46274) (Santa Cruz Biotechnology, Inc.). Secondary antibody used was anti-goat 488 (Invitrogen). Slides were mounted with Vectorshield 4′,6-diamidino-2-phenylindole (Vector Laboratories) before microscopy. For Fig. 1, 10 ng of TORC1, 5 ng of TORC2, and 15 ng of PKAα expression plasmids were transfected into HEK293 cells. For Fig. 2, 800 ng of total DNA consisted of 200 ng of the pMLHoxb1ARE luciferase reporter and 100 ng of each expression plasmid. A lacZ reporter was co-transfected to normalize transfection efficiency. For Fig. 3A, the amounts of pENTR T2i shRNA (TORC2 shRNA) are given within the panel. For Fig. 3B, 25,000 cells per well were plated in 48-well plates and transfected using Lipofectamine 2000 with the following vectors: 40 ng of pML5xUAS, 40 ng of pGAL-DBD or pGAL-MEIS1A(335–390), 40 ng of pRSV-PBS or pRSV-PKA, 40 ng of pENTR U6 (control shRNA) or pENTR T2i shRNA (TORC2 shRNA), and 100 ng of pRSV-β-galactosidase. At 48 h post-transfection, cell lysates were prepared using 100 μl per well of lysis buffer (1% Triton X-100, 1 mm dithiothreitol, 92.8 mm K2HPO4, pH 7.8, 9.2 mm KH2PO4, pH 7.8) and centrifuged for 5 min at 4 °C. A 20-μl aliquot of the supernatant was added to 12.5 μl of assay buffer (20 mm ATP, 40 mm MgCl2, 0.4 m Tris-Cl, pH 7.8). The mixture was immediately quantified for luciferase activity using a Lumat LB 9507 luminometer (EG&G Berthold) that dispensed 100 μl per reaction of luciferin solution, which contains 1 mm d-(−)-luciferin (catalogue number 11626353001, Roche) and 0.1 m Tris-Cl, pH 7.8.FIGURE 2TORCs mediate transcriptional activation through the MEIS1A C terminus at a natural MEIS1 target enhancer. HEK293 cells were transiently transfected with the pMLHoxb1ARE luciferase reporter and expression plasmids as indicated. Luciferase activities in the absence or presence of the PKA catalytic domain (control or PKA) were measured at 48 h post-transfection.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Knockdown of TORCs prevents PKA-mediated activation of the MEIS1A C terminus. A, upper panel, effect of TORC2 shRNA or non-silencing control (CTRL) shRNA on GAL-MEIS1A-(335–390) luciferase transcription augmented by TORC2. The indicated plasmids were co-transfected with the pML5xUAS reporter in HEK293 cells. Lower panel, knockdown of FLAG-TORC2 protein levels in TORC2 or control shRNA-treated cells was verified by immunoprecipitation with M2 beads followed by Western blot (WB) analysis with an anti-FLAG antibody. Cell extracts were probed for tubulin, confirming equivalent protein concentrations in each sample. FLAG-TORC2(Wobble) served as an RNA interference-resistant control. B, the role of endogenous TORC2 on transcriptional activation through the MEIS1A C terminus. Cells were transfected with the pML5xUAS reporter and expression vectors for either the GAL DBD or GAL-MEIS1A-(335–390), along with a PKA expression vector or empty plasmid. Transcriptional activation by PKA through the MEIS1A C terminus was abrogated by coexpression with the TORC2-specific shRNA but not the control shRNA. The experiment was conducted in triplicate. Error bars are S.D., and p signifies the results of the Student's t test applied to values for PKA-induced activity in the presence of control shRNA versus TORC2 shRNA. RLU, relative luciferase units.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cells were washed twice in ice-cold PBS 48 h post-transfection and harvested in 500 μl of Buffer B (150 mm KCl, 0.1% Nonidet P-40, 20 mm Tris-Cl, pH 8.0, 5 mm MgCl2, 10% (w/v) glycerol) added with protease inhibitor mixture (catalogue number 11873580001, Roche). Following two freeze-thaw cycles, cells were spun down at 4 °C for 10 min. The supernatant was incubated with the appropriate primary antibody from 5 h to overnight at 4 °C, followed by 3 h incubation at 4 °C with 30 μl of a 50% slurry of Protein A-agarose (catalogue number 16-156, Upstate Biotechnology), unless the primary antibody was in the form of anti-FLAG M2 affinity agarose. The precipitates were washed three times, each with 500 μl of Buffer B. Precipitates of Protein A-agarose were eluted with 1× SDS sample buffer and boiling. Elution from anti-FLAG M2 affinity agarose was done by adding a 7.5-μg FLAG peptide (catalogue number F3290, Sigma) for 1 h at 4 °C. Protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to 0.45-μm nitrocellulose membrane. The membranes were blocked with 5% nonfat milk powder in 0.1% Tween 20 in PBS (PBS-T) for 1 h at room temperature to reduce nonspecific background, followed by primary antibody incubation for 3 h at room temperature or overnight at 4 °C. The membranes were then washed four times, 10 min each with PBS-T, and incubated with secondary antibody conjugated with horseradish peroxidase for 45 min at room temperature. Subsequent to three 10-min PBS-T washes, bound antibodies were detected with a chemiluminescent kit (catalogue number KP-54-61-00, Mandel). ChIP assays were performed according to the protocol from Upstate Biotechnology with minor changes as reported previously (8.Huang H. Rastegar M. Bodner C. Goh S.L. Rambaldi I. Featherstone M. J. Biol. Chem. 2005; 280: 10119-10127Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 45.Rastegar M. Kobrossy L. Kovacs E.N. Rambaldi I. Featherstone M. Mol. Cell. Biol. 2004; 24: 8090-8103Crossref PubMed Scopus (57) Google Scholar). P19 cells induced to differentiate down the neural pathway by aggregation in the presence of retinoic acid (see above) were treated with 20 μm forskolin for 2 h, cross-linked with 1% formaldehyde for 10 min at 37 °C, collected, and washed twice with ice-cold PBS containing protease inhibitor mixture. A 200-μl aliquot of SDS lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-Cl, pH 8.0, protease inhibitor mixture) was added to each 1 × 106 cells and incubated on ice for 10 min. The 200-μl lysates were sonicated at 4 °C with 10 sets of 10-s pulses at 30% amplitude of a Betatec Sonics Vibra Cell sonicator to an average DNA length of 200 bp and then centrifuged for 10 min at 4 °C. Each 100-μl sonicated cell supernatant was diluted 10-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tris-Cl, pH 8.0, 167 mm NaCl, protease inhibitor mixture) and pre-cleared with 40 μl of a 50% slurry of salmon sperm DNA/Protein A-agarose (catalogue number 16-157, Upstate Biotechnology) for 30 min at 4 °C with rotation. After an overnight incubation with anti-MEIS NT, anti-PBX1, anti-TORC2, or anti-rabbit IgG antibodies, 30 μl of salmon sperm DNA/Protein A slurry was added for 1 h at 4 °C, along with a no antibody control. To remove nonspecific DNA from the protein A-antibody-histone complex, we performed extensive washes with 500 μl of each buffer in the following sequences: once with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-Cl, pH 8.0, 150 mm NaCl), once with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-Cl, pH 8.0, 500 mm NaCl), once with lithium chloride buffer (0.25 m LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mm EDTA, 10 mm Tris-Cl, pH 8.0), and twice with TE buffer (1 mm EDTA, 10 mm Tris-Cl, pH 8.0). Each wash was done by first pipetting up and down for 10 times, and then an 8–10-min incubation on a rotating platform at 4 °C. Subsequently, the histone complex was eluted from the antibody by incubating twice with 125 μl of elution buffer (1% SDS, 0.1 m NaHCO3) for 15 min at room temperature. Cross-links were reversed at 65 °C for 4 h in the presence of 0.2 m NaCl. DNA was phenol-chloroform-extracted, ethanol precipitated, and resuspended in 40 μl of distilled water (catalogue number 15230-147, Invitrogen). Five percent (by volume) of the immunoprecipitated DNA was served as template in quantitative real-time PCR by a SYBR Green JumpStart Taq ReadyMix kit (catalogue number S1816, Sigma) with a Roche LightCycler. The sequences of ChIP primers used in this study were as follows: for Hoxb1 ARE, 5′-CTCTGGTCCCTTCTTTCC and 5′-GGCCAGAGTTTGGCAGTC; for Hoxb2 r4 enhancer, 5′-AGGCCTTTTTAAGGGATATGC and 5′-AGGCCTCAAAGCTGAAAATGA; for Meis1 promoter, 5′-TTAGGACTGATTCAAGGAAAGC and 5′-GCCCCTCAGACCCAACTAC; and for gapdh, 5′-AACGACCCCTTCATTGAC and 5′-TCCACGACATACTCAGCAC. The primers for the murine Meis1 gene flank a consensus PBX·MEIS binding site having the sequence 5′-TGATTGACAG-3′. To study the me