MIM/BEG4, a Sonic hedgehog-responsive gene that potentiates Gli-dependent transcription - PubMed (original) (raw)

MIM/BEG4, a Sonic hedgehog-responsive gene that potentiates Gli-dependent transcription

Christopher A Callahan et al. Genes Dev. 2004.

Abstract

Sonic hedgehog (Shh) signaling plays a critical role during development and carcinogenesis. While Gli family members govern the transcriptional output of Shh signaling, little is known how Gli-mediated transcriptional activity is regulated. Here we identify the actin-binding protein Missing in Metastasis (MIM) as a new Shh-responsive gene. Together, Gli1 and MIM recapitulate Shh-mediated epidermal proliferation and invasion in regenerated human skin. MIM is part of a Gli/Suppressor of Fused complex and potentiates Gli-dependent transcription using domains distinct from those used for monomeric actin binding. These data define MIM as both a Shh-responsive gene and a new member of the pathway that modulates Gli responses during growth and tumorigenesis.

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Figures

Figure 1.

Figure 1.

MIM is a Shh-responsive gene. (A) Strategy to isolate novel Shh-responsive genes. (B,C) In situ hybridizations with anti-sense probes demonstrate that similarly to Gli1 (C), MIM (B) transcripts accumulate in human BCC tumor epithelium (transcript staining is red with toluidine blue counterstain). (D) MIM anti-sense probes show that MIM transcripts also accumulate in the outer root sheath of human anagen hair follicles (red staining highlighted with arrowhead). No staining is seen with control MIM sense probes (lack of red staining highlighted by arrowhead in E). (F) MIM transcripts are not detected within the interfollicular epithelium in human scalp. (G) An affinity-purified, polyclonal, anti-MIM antibody shows MIM protein expression within the epithelium of human BCCs (brown signal highlighted by arrowhead). (H) Tumor epithelium (arrowhead) is only highlighted by the Mayer's hematoxylin counterstain when treated with control preimmune serum from the same animal. MIM immunoreactivity is also seen within the ectopic epithelial skin proliferations formed in embryonic K14-Shh transgenic mice (arrowhead in I) and the BCC-like skin tumors arising in K5-Gli2 transgenic mice (arrowhead in J). (K) No MIM protein is detected in K5-Gli2 transgenic skin tumors stained with preimmune serum. Bars: B_–_F, 50 μm; G,H, 15 μm; I, 100 μm; J,K, 50 μm. (L) Cultured ptch1 mutant MEFs demonstrate constitutive pathway activation, and accumulate high levels of MIM transcripts. In contrast, in cells in which Shh target genes are suppressed, such as ptch1+/– MEFs or _ptch1_-null MEFs rescued by retroviral gene transfer with the wild-type ptch1 gene, there are dramatically lower levels of MIM RNA. Similarly to known Shh targets (Taipale et al. 2000), treatment of ptch1 mutant MEFs for 72 h with 8 μM cyclopamine, an inhibitor of smoothened (Chen et al. 2002), or for 24 h with 40 μM forskolin, a negative regulator of Ci/Gli (Wang et al. 1999), reduces MIM accumulation. Reactivating Shh target genes in cultured _ptch1_–/–; ptch1+ cells by infecting with a Shh retrovirus (_ptch_–/–; ptch1+; shh+) increases MIM transcripts.

Figure 2.

Figure 2.

MIM synergizes with Gli to recapitulate Shh effects in skin. Representative sections of EGFP (A), Shh (B), Gli1 (C), MIM (D), and Gli1 + MIM (E) regenerated human skin stained with anti-Ki67 antibody, a marker of cell proliferation. Keratinocyte proliferative activity is measured by the number of keratinocytes with brown nuclear staining. (F) Quantification of data in which each graft condition is plotted on the _X_-axis, and the _Y_-axis represents the average number of Ki-67 positive keratinocytes per 100 μm of graft tissue. Digits placed over error bars show the number of grafts examined per condition. p values are calculated versus EGFP control grafts. (G_–_J) H&E-stained sections showing the deeply invasive ingrowths identified in both Shh (H) and Gli1 + MIM (I,J) skin. (K) The number and depth of the invasive ingrowths identified in each graft condition. At top, the histogram lists the number of skin grafts and total graft distance (in millimeters) examined per graft condition. Below, each histogram bar represents one invasive epithelial ingrowth measuring at least 250 μm in depth (e.g., two deep ingrowths were seen in Shh grafts). The height of each bar shows that ingrowth's maximum depth of invasion (_Y_-axis). Bars: A_–_E,G,H,J, 100 μm; I, 25 μm.

Figure 3.

Figure 3.

MIM potentiates Gli-dependent transcription. Polyclonal K17 antibody (A) but not secondary antibody alone (B) reacts with tumor epithelium. Coincident expression of Gli1 (C) and K17 (D) in a skin mosaic demonstrates that Gli activates endogenous K17. Bars: A,B, 100 μm; C,D, 25 μm. (E) Luciferase activities in lysates from primary keratinocytes showing Gli1 (white bars) and Gli2 (gray bars) activate the K17 promoter, and MIM potentiates Gli-dependent transcription in a dosage-sensitive manner. (F) Luciferase activity showing MIM does not potentiate NF-κB transcriptional activity in keratinocytes. (G) Luciferase activity with K17 promoter deletions identifies a 41-bp region required for both Gli responsiveness and MIM potentiation in keratinocytes. (H) MIM potentiates Gli-mediated transcription from multimerized Gli response elements in keratinocytes. Luciferase activities were measured from reporter constructs carrying eight directly repeated copies of wild-type Gli response sequences from the mouse K17 promoter (8xK17BS-luc) (Supplementary Fig. S2), the mouse FOXA2 promoter (16-2K17p-luc), or a mutated element from the mouse FOXA2 promoter (16-3K17p-luc) (Sasaki et al. 1997). (I) Luciferase activities showing that MIM does not potentiate Gli-mediated transcription from multimerized Gli response elements when cotransfected with a Gli2 DNA-binding mutant (Gli2-ZFD) or a Gli2 transactivation mutant (Gli2-TADD). (E_–_I) Error bars, ±SEM.

Figure 4.

Figure 4.

MIM associates with Gli and Sufu. (A, top row) A MIM column retains HA-Gli2 and endogenous Sufu as seen by retention in eluate fractions. (Middle row) Coexpressed GFP is not retained. (Lower row) GST fusion proteins. Neither GST nor mutant MIMΔN399 retains Gli2 or Sufu. Lines separate portions of the blot probed with different antibodies. The input represents 1% of total 293 cell lysate loaded on column. (B) MIM column also retains HA-Gli1. The input represents 1% of total 293 cell lysate loaded on column. (C) Immunoprecipitated MIM-myc pellets HA-Gli1 in 293 cell lysates. (i) Input; (b) beads. Immunoprecipitated Gli1-V5 pellets MIM-myc in 293 cell lysates. Immunoprecipitated Sufu-myc also pellets HA-Gli1 in 293 cell lysates. The input in each case represents 0.5% of total input. (D, top row) The MIM column retains HASufu as seen by retention in eluate fractions. (Middle row) Coexpressed GFP is not retained. (Lower lane) GST fusion proteins. Neither GST nor MIMΔN399 retains Sufu. The input represents 1% of total eluate loaded. (E) Immunoprecipitated Sufu-myc pellets MIM-GFP and MIM-myc pellets HA-Sufu when coexpressed in 293 cell lysates. (i) Input; (b) beads. The input represents 0.5% of lysate. No binding of GFP to Sufu-myc was detected (data not shown). (F) In vitro translated Gli1 and Sufu bind to GST-MIM. (Top row) The indicated GST beads were incubated with 35S-labeled protein, and the spun pellet was examined by autoradiography. Note no increase in binding of Sufu or Gli to MIM when both are present. The input represents 50% of reaction used. (G) Luciferase activity of BEG4 mutants missing the monomeric actin-binding domain (MIMN538), the F-actin bundling domain (MIMΔN159), or the N-terminal domain required for association with the Gli complex (MIMΔN399). Error bars, ±SEM.

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