Volume 16, Issue 6, Pages 867-880 (December 2004) The SRF Target Gene Fhl2 Antagonizes RhoA/MAL-Dependent Activation of SRF  Ulrike Philippar, Gerhard Schratt, Christoph Dieterich, Judith M. Müller, Petra Galgóczy, Felix B. Engel, Mark T. Keating, Frank Gertler, Roland Schüle, Martin Vingron, Alfred Nordheim  Molecular Cell  Volume 16, Issue 6, Pages 867-880 (December 2004) DOI: 10.1016/j.molcel.2004.11.039

Figure 1 Genes Inducible by SRF-VP16 Display Different Serum Induction Profiles (A) Serum induction profiles of genes in E14 Srf+/+ ES cells were clustered based on the self-organizing map (SOM) algorithm. Four different clusters were obtained: a, weak; b, moderate and transient; c, no; and d, strong and sustained serum induction. (B) Individual serum induction profiles of genes grouped into clusters a–d according to Figure 1A. In addition to E14 Srf+/+ ES cells, the induction kinetics for 100 Srf−/− and 100-2 Srf−/−rescue ES cells are shown. Degree of induction is reflected by relative color coding: blue, low expression; red, high expression. The numbers to the right of the induction profiles represent the fold activation for serum-treated E14 Srf+/+ ES cells at the 60 min time point. Molecular Cell 2004 16, 867-880DOI: (10.1016/j.molcel.2004.11.039)

Figure 2 Validation of SRF-Dependent Activation of Selected Genes Identified by Microarray Profiling (A–I) Quantitative RT-PCR was performed with mRNA from 100 Srf−/− ES cells, transiently transfected with SRF-VP16 or SRF-ΔM-VP16, using specific primers for GAPDH (A), Egr-1 (B), SM22α (C), SMA (D), tuftelin (E), CTGF (F), KRT-17 (G), ET-1 (H), and Fhl2 (I). Values represent transcript levels relative to the endogenous housekeeping gene HPRT and are the mean of three independent experiments. Asterisk denotes statistically significant induction (p < 0.05, Student's t test). (J) ChIP analysis of SRF binding to promoters of SRF target genes in vivo. Chromatin of day 8 differentiated 100 Srf−/− and E14 Srf+/+ ES cells was immunoprecipitated using a polyclonal anti-SRF antibody (lanes 3 and 6). Bound DNA fragments were amplified by PCR using primers specific for the CArG-containing promoter regions of the indicated genes. Incubation without antibody was used as control (lanes 2 and 5). 1% of the isolated genomic DNA served as input (lanes 1 and 4). Molecular Cell 2004 16, 867-880DOI: (10.1016/j.molcel.2004.11.039)

Figure 3 Expression of FHL2 Is Regulated by SRF in a RhoA- and Differentiation-Dependent Manner (A) 81 Srf−/− and 100 Srf−/− ES cells were transiently transfected with SRF-VP16 or SRF-ΔM-VP16, and protein extracts were prepared 72 hr after transfection. Western blotting was performed using polyclonal anti-FHL2 or anti-α-actinin antibody. (B) NIH3T3 cells were transfected with luciferase reporter constructs driven by the human FHL2 promoter with an intact or mutated CArG sequence (500 ng), in the presence or absence of RhoAV14 (100 ng). Values are given as fold activation over vector-transfected samples and represent the mean of four independent experiments. (C) 100 Srf−/− and E14 Srf+/+ ES cells were differentiated by LIF removal under monolayer conditions for up to 8 days. Protein lysates were prepared on the indicated days and subjected to Western Blotting using polyclonal anti-FHL2 or anti-α-actinin antibody. FHL2 protein is indicated by the arrow. (D) 99 Srf−/+ ES cells were differentiated in the presence of retinoic acid for up to 8 days and mRNA expression was analyzed by quantitative RT-PCR. Values represent the mean of two independent experiments ± SD. Molecular Cell 2004 16, 867-880DOI: (10.1016/j.molcel.2004.11.039)

Figure 4 SRF and FHL2 Interact In Vitro and In Vivo (A) GST pull-down assays were performed with the indicated GST fusion proteins and in vitro-translated 35S-labeled SRF. 1% of 35S-labeled SRF was loaded as input. Coomassie staining shows comparable expression of the GST fusion proteins used. (B) Mapping of the SRF interaction domain of FHL2. GST pull-down assays were performed as in (A). (C) SRF Core is sufficient to interact with FHL2. GST pull-down assay was performed with GST-FHL2 and in vitro translated 35S-labeled SRF Core (aa 133–265). 10% of 35S-labeled SRF Core was loaded as input. (D) Immunoprecipitation of 293T cells transfected with HA-SRF and Flag-FHL2 in the absence or presence of RhoAV14 was performed using M2 anti-Flag or an IgG control antibody. Immunoprecipitated SRF or FHL2 protein was detected by Western blotting with anti-SRF or anti-Flag M2 antibody, respectively. Input represents 4% of the starting protein lysate. (E) Immunoprecipitation of rat embryonic heart extracts was performed using anti-SRF or an IgG control antibody. Immunoprecipitated SRF or FHL2 protein was detected by Western blotting with anti-SRF or anti-FHL2 antibody. (F) SRF and FHL2 bind to the promoters of SM genes. Differentiated E14 Srf+/+ ES cells were transfected with HA-SRF, Flag-FHL2, and RhoAV14 as indicated. ChIP assay was performed using an anti-SRF (lanes 3 and 7) or anti-Flag antibody (lanes 4 and 8). No antibody incubation served as control (lanes 2 and 6). 1% of the isolated genomic DNA served as input (lanes 1 and 5). Molecular Cell 2004 16, 867-880DOI: (10.1016/j.molcel.2004.11.039)

Figure 5 FHL2 Represses SRF-Dependent SM Promoters (A–E) 293T cells were transiently transfected with the indicated luciferase promoter constructs, increasing amounts of FHL2 expression plasmid (100 ng, 500 ng, 1 μg, 1.4 μg), and 100 ng RhoAV14 as indicated. Values represent fold activation compared to vector control-transfected cells and are the mean of three independent experiments. Asterisk denotes statistically significant repression (p < 0.05, Student's t test). (F) FHL2-mediated repression is dependent on nuclear localization. 293T cells were transfected with SM22α-Luc and the FHL2 or C7A mutant expression plasmid in the presence or absence of RhoAV14. Values represent percent activation relative to RhoAV14-transfected cells and are the mean of three independent experiments. (G) FHL2-mediated repression is dependent on interaction with SRF. 293T cells were transfected with SM22α-Luc and the indicated Flag-FHL2 expression plasmid in the presence or absence of RhoAV14. Values represent percent activation relative to RhoAV14 transfected cells and are the mean of three independent experiments. Asterisk denotes statistically significant repression (p < 0.05, Student's t test). Molecular Cell 2004 16, 867-880DOI: (10.1016/j.molcel.2004.11.039)

Figure 6 FHL2 Antagonizes MAL/RhoA-Mediated Activation of SM Promoters and Competes with MAL for SRF Binding (A–E) 293T cells were transiently transfected with the indicated luciferase promoter constructs, 1.4 μg FHL2 expression plasmid, 100 ng RhoAV14, and 25 ng MAL as indicated. Values represent fold activation compared to vector control-transfected cells and are the mean of three independent experiments. Asterisk denotes statistically significant repression (p < 0.05, Student's t test). (F and G) Undifferentiated 100 Srf−/− ES cells were transiently transfected with SRF expression plasmid (100 ng). Cotransfection with MAL (20 ng), FHL2 (1.4 μg), and RhoAV14 (100 ng) expression plasmids was performed as indicated. mRNA was prepared 48 hr after transfection and subjected to quantitative RT-PCR analysis. Values were normalized to the average activation by MAL as deduced from four independent experiments. Asterisk denotes statistically significant repression (p < 0.05, Student's t test). (H) EMSA was performed using a 32P-labeled CArG box oligonucleotide, 293T cell extracts expressing MALΔN, and in vitro-translated FHL2. DNA complexes of SRF and SRF/MALΔN are indicated. Molecular Cell 2004 16, 867-880DOI: (10.1016/j.molcel.2004.11.039)