FHL2 is coexpressed with Hand1 in the developing heart. (A and B) FHL2 mRNA expression in E9.5 wild-type embryos as detected by whole-mount in situ hybridization. (C and D) Sagittal sections through E9.5 wild-type hearts following whole-mount in situ hybridization for FHL2 and counterstaining with eosin. Abbreviations: aa, arch arteries; ao, dorsal aorta; at, atrium; ba, branchial arch; baII, second branchial arch artery; baIII, third branchial arch artery; fd, foregut diverticulum; lv, left ventricle; my, myocardium; ot, outflow tract; v, ventricle.

Hand1 and FHL2 interact in vitro and in vivo in embryonic cardiomyocytes. (A) In vitro pull-down assay showing Hand1 interaction with FHL2. SDS-PAGE of ³⁵S-labeled FHL2 were incubated with 0.5 μg of GST-Hand1 or GST-bHLH, respectively. FHL2 was pulled down by GST-Hand1 and GST-bHLH but not by GST alone. (B) Immunoprecipitation of MYC-FHL2 by full-length FLAG-Hand1 demonstrating that Hand1 and FHL2 can interact in vivo. Transient transfections of full-length MYC-FHL2 together with full-length FLAG-Hand1 constructs (lanes 7 and 8) were performed in NIH 3T3 cells as described in Materials and Methods. Immunoprecipitation (IP) was performed with α-FLAG M2 antibody and immunoblotting with α-MYC 9E10.2. Lanes 1 and 2 represent untransfected and lanes 3 to 6 represent single transfection controls (lanes 3 and 4, FLAG-Hand1; lanes 5 and 6, MYC-FHL2). Note the single band in lane 8, which represents MYC-FHL2 following immunoprecipitation by α-FLAG compared to the blank control lanes 2, 4, and 6. (C) Immunoprecipitation of endogenous FHL2 and Hand1 in P19-derived cardiomyocytes, demonstrating that the two proteins interact in the heart. Cardiomyocytes were derived from aggregated P19 cells differentiated in 0.8% DMSO as described in Materials and Methods. IP was performed on lysates with a monoclonal α-FHL2 antibody and immunoblotting with α-Hand1. Lane 1 represents 5% of the lysate input. Lane 2, CO-IP lane; lane 3, a no-primary-antibody negative control. Note the single band in lane 2, representing Hand1 following IP by α-FHL2 compared to the blank control lane 3. Expression of FHL2 in the starting lysate and CO-IP lanes was confirmed by immunoblotting with a polyclonal α-FHL2 antibody.

Mammalian two-hybrid analyses of Hand1 and FHL2 interactions in NIH 3T3 cells. NIH 3T3 cells were cotransfected with GAL4-DBD, GAL4-Hand1, and the CMV_min(Gal)5-luc reporter gene along with MVP16 as a positive control, Hand1, E12, or FHL2 or Hand1 plus FHL2 or E12 plus FHL2. MVP16 dramatically activated luciferase expression to confirm that the system is working (lane 2). Hand1, E12, or FHL2 in isolation had no effect on luciferase expression (lanes 4 to 6). GAL4-Hand1 activated luciferase expression by approximately threefold (lane 7) compared to reporter alone (lane 1). Coexpression of FHL2 had no significant effect on this level of activation (lane 8). Hand1 in combination with GAL4-Hand1 (lane 9) inhibited the activation observed with GAL4-Hand1 alone (lane 7). FHL2 had no effect on the Hand1-induced inhibition (lane 10). Coexpression of Gal4-Hand1 with E12 significantly activated luciferase expression (approximately sixfold; lane 11). FHL2 significantly inhibited GAL4-Hand1/E12-induced activation (lane 12), suggesting that FHL2 can functionally interact with Hand1-E protein heterodimers in vitro. Error bars reflect the standard errors of the means from a minimum of three independent transfections. **, P < 0.05.

Subcellular colocalization of endogenous Hand1 and FHL2 in H9c2 cells. H9c2 cells were cultured on coverslips, fixed with 4% paraformaldehyde, and immunostained with polyclonal α-Hand1 (A) and α-FHL2 (B) antibodies followed by fluorescein isothiocyanate-conjugated anti-goat (A) and tetramethyl rhodamine isocyanate-conjugated anti-rabbit (B) antibodies, respectively. (C to E) Nuclei were visualized following staining with bis-benzamide (Hoechst 33342 [D]). Merged images of immunostained H9c2 cells demonstrated nuclear colocalization of Hand1 and FHL2 (C). Secondary antibody-alone negative controls are shown in panels D and E.

Hand1 and FHL2 are spatially associated in intact living cells. FRET analysis of Hand1-YFP and FHL2-CFP fusion proteins in cotransfected NIH 3T3 cells. (A) N-terminal fluorescent fusion proteins were constructed as shown (numbers refer to size of each cassette, in base pairs) with Hand1-YFP as the donor and FHL2-CFP as the acceptor. (B) Images of the nuclear localization of the FHL2-CFP acceptor and Hand1-YFP donor were merged to confirm areas of overlap, and then three regions were selected prior to FRET analysis. The red region represents overlap of donor and acceptor in which the acceptor was not bleached. Blue represents a region of overlap where the acceptor was bleached. Green represents a mutually exclusive region not bleached by way of a background control. (C) Acceptor bleach FRET analysis was carried out whereby the three selected regions (red, blue, and green) were excited by the donor YFP channel (excitation, 500/20 nm), following in the case of the blue region appropriate prebleaching of the acceptor. Emission was recorded using the CFP channel (emission, 470/30 nm) as a measure of fluorescence intensity over time. A distinct increase in CFP intensity was observed for the blue overlap region, postbleaching of the acceptor and following donor YFP excitation, indicating efficient FRET and a direct physical association of the two fusion proteins. The data presented in panel C represent the averages of 10 independent FRET measurements.

FHL2 inhibits Hand1/E-protein heterodimer-induced transcriptional activation but has no effect on Hand1 homodimer activity. (A) Mock Hand1 target gene reporter, consisting of six concatemerized Thing1 boxes upstream of an α-CA minimal promoter driving luciferase, was cotransfected into NIH 3T3 cells with either Hand1, E12, or Hand1 plus E12 in the presence or absence of FHL2. Hand1 activated the system approximately two- threefold above reporter alone (lanes 1 and 3). FHL2 (150 ng) had no significant effect on Hand1 homodimer activation of the reporter (lanes 3 and 4). Cotransfection with E12 alone had no effect on reporter activation, and this remained unaltered in the presence of FHL2 (lanes 5 and 6). Cotransfection of Hand1 with E12 resulted in a sevenfold activation above that of reporter alone (lane 7). FHL2 significantly repressed Hand1/E12 coactivation of the mock target gene. Titration experiments with an increasing amount of FHL2 (50 to 150 ng) revealed a dose-dependent effect of FHL2 on the level of repression (lanes 8 to 10). (B) Increasing the dose of FHL2 (200 and 400 ng) had no effect on Hand1-induced activation of the mock target reporter (lanes 2 to 5). These findings suggest that the interaction of FHL2 with Hand1 impairs Hand1-E12 heterodimer function but has no effect on Hand1 homodimer activity. (C) Characterization of the FHL2 LIM domains involved in inhibition of Hand1-E12 transcriptional activity. Full-length FHL2 is required for efficient inhibition of Hand1-E12 activity (lanes 1 and 2). The effects of different FHL2 deletion constructs were analyzed on the Hand1-E12-activated mock target reporter. The N-terminal FHL2 (1/2-2) and the 2-3 constructs retained some inhibitory activity (lanes 3 and 5), suggesting that LIM domain 2 may play a prominent role. Single LIM domains in isolation had no effect on Hand1-E12 activity (lanes 6 to 9). Error bars reflect the standard errors of the means from a minimum of three independent transfections. **, P < 0.05; *, P < 0.1.

FHL2 inhibition of Hand1-E12-induced transcription is not mediated by an effect on heterodimerization per se. Cotransfection of NIH 3T3 cells with the mock target gene and a Hand1∼Hand1-tethered homodimer resulted in a sixfold activation of the reporter gene which was not effected by increasing amounts (200 to 400 ng) of FHL2 (lanes 1 to 5). Coexpression of reporter with a Hand1∼ITF2-forced heterodimer induced reporter gene activation by approximately sixfold (lane 6). FHL2 was still able to significantly repress Hand1∼ITF2-induced activation in a dose-dependent manner (lanes 7 to 9), suggesting a mechanism of FHL2 function distinct from disruption of Hand1-E protein heterodimerization. Error bars reflect the standard errors of the means from a minimum of three independent transfections. **, P < 0.05.

FHL2 does not appear to affect Hand1-E12 DNA binding. EMSAs with IVT Hand1, E12, and FHL2 and proteins from cotransfected NIH 3T3 cell lysates were incubated with ³²P-labeled Thing1 box and scrambled control oligonucleotide probes, and the generated protein-DNA complexes were resolved by native PAGE and autoradiography. (A) IVT FHL2 alone does not bind to the Thing1 box sequence (lane 2). Hand1 and E12 in combination bind the optimal Thing1 box such that a shifted band representing a Hand1-E12 heterodimer complex bound to DNA is observed (21, 46), and this can be competitively blocked by an excess of cold Thing1 probe (lanes 5 and 6). Preincubation of Hand1/E12 with IVT FHL2 actually resulted in a slight enhancement in the bound Hand1-E12 complex (lane 7). (B) The enhanced Hand1-E12 DNA binding brought about by the addition of FHL2 was confirmed using cell lysates from NIH 3T3 cells cotransfected with all three proteins (lanes 1 and 3). Arrowheads indicate specific DNA binding complexes. ns, nonspecific bands.