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.

FIGURE 1.

CREB co-activators TORC1 and TORC2 mediate the MEIS1A C terminus transcriptional activity. A, HEK293 cells were transfected with expression vectors for GAL-DBD, GAL-MEIS1A-(335–390), or GAL-MEIS1A-(GQWHYM) plus the pML5xUAS luciferase reporter. The effect of the PKA catalytic domain and/or TORC1 on luciferase activity is shown in the upper panel, whereas that of the PKA catalytic domain and/or TORC2 is shown in the lower panel. RLU, relative luciferase units. β-gal, β-galactosidase. B, TORC2 subcellular localization was quantitated in non-transfected cells and cells overexpressing TORC2. Top panels, green signal corresponds to TORC2. Middle panels, 4′,6-diamidino-2-phenylindole-stained nuclei (blue). Bottom panels, overlay. N, exclusively nuclear signal; N>C, nuclear signal stronger than cytoplasmic; N=C, signals in the nucleus and cytoplasm approximately equal; N<C, nuclear signal weaker than cytoplasmic; C, exclusively cytoplasmic signal. Over-exp, images of cells overexpressing TORC2. 100 cells each were scored for the distribution of endogenous versus overexpressed TORC2. The exposure of images showing endogenous TORC2 was increased in Adobe Photoshop to visualize clearly the relatively weak signal.

FIGURE 2.

TORCs 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.

FIGURE 3.

Knockdown 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.

FIGURE 4.

MEIS1A interacts with TORC1 and TORC2. A, co-immunoprecipitation assay of FLAG-tagged TORC1 and untagged MEIS1A in transfected HEK293 cells. Anti-MEIS NT and anti-FLAG Western blot (WB) analyses were performed on FLAG-TORC1 immunoprecipitates (IP) prepared with anti-FLAG M2 affinity agarose. 10% input levels of MEIS1A and FLAG-TORC1 are indicated. B, upper panel, Western blot analysis of transfected and endogenous MEIS1A detected in immunoprecipitates of endogenous TORC2 from HEK293 cells. The experiment was performed in triplicate and the results of each assay are shown. Lower panel, anti-TORC2 Western blot analysis showing immunoprecipitated TORC2 by the anti-TORC2 antibody but not the control anti-GAL4 antibody. 10% input levels of MEIS1A and TORC2 are shown. C, a MEIS1A mutant lacking the C terminus fails to co-immunoprecipitate with TORC1. HEK293T cells were co-transfected with a FLAG-tagged TORC1 expression vector and a vector encoding either wild-type MEIS1A or a mutant lacking the TORC-responsive C terminus (MEIS1A-(Δ334–390)). On the second day following transfection, cells were treated with the proteasome inhibitor MG132 and cell lysates prepared 5 h later. Immunoprecipitation of TORC1 was performed with an anti-FLAG antibody, and the presence of MEIS1 proteins in the immunoprecipitates was subsequently assessed by Western blotting.

FIGURE 5.

A MEIS1 interaction domain maps to a coiled-coil region at the TORC1 N terminus. Upper panel, co-immunoprecipitation between untagged MEIS1A and full-length FLAG-tagged TORC1 (Flag-TORC1) or its deletion derivatives in transfected HEK293 cells. MEIS1A proteins co-precipitated with FLAG-TORC1 derivatives prepared using anti-FLAG M2 affinity agarose were revealed by Western blot (WB) analysis with anti-MEIS NT antibody. The bottom two panels show inputs of MEIS1A and FLAG-TORC1 derivatives, respectively. Lower panel, schematic diagram of TORC1 constructs and their MEIS1A binding activities. WB, Western blot; IP, immunoprecipitation. The plus and minus signs below Binding correlate with the extent of binding to MEIS1A by the various TORC1 mutants.

FIGURE 6.

MEIS1, PBX1, and TORC2 are recruited to endogenous MEIS1 targets. The results of ChIP assays in untreated (−FSK) or forskolin-treated (+FSK) mouse P19 cells are presented. Values obtained by LightCycler quantification were normalized against the corresponding input and nonspecific IgG antibody, and expressed as relative occupancy. The data are presented as the mean ± S.E. of three experiments. A, the Meis1 promoter, which harbors a consensus PBX-MEIS binding site. B, the Hoxb2 r4 enhancer. C, the Hoxb1 ARE. D, glyceraldehyde-3-phosphate dehydrogenase (gapdh) served as an internal control.