Journal: Journal of Biological Chemistry

Article Title: GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells

doi: 10.1074/jbc.m707117200

Figure Lengend Snippet: FIGURE 1. FST expression is preferentially induced by GLI2 in human keratinocytes. A, qRT-PCR analysis of FST mRNA levels in HaCaT keratinocytes expressing either GLI1 (GLI1-HaCaT) or GLI2act (GLI2act-HaCaT) under tetracycline control for the time indicated. As a control for GLI activity the known target gene PTCH is shown (inset). B, FST and PTCH mRNA levels in human N/TERT-1 keratinocytes retrovirally transduced with GLI1 or GLI2act measured by qRT-PCR. The -fold change refers to the mRNA ratios for induced to uninduced cells (A) and cells infected with a GLI1/2 to control EGFP expressing virus (B). C, Western blot analysis of FST protein levels in GLI2act-HaCaT cells. Samples were taken from tetracycline-treated and untreated GLI2act-HaCaT cells as indicated. The upper panel shows protein expression of GLI2act transgene. FST protein was detected using a specific antibody recognizing all FST isoforms (lower panel). For control HaCaT cells were transiently transfected with an expression plasmid expressing human FST isoform 344 (p4TO-FST344) or empty expression vector (p4TO). *, unspecific signal.

Article Snippet: A mouse monoclonal antibody directed against human FST (R&D Systems, dilution, 1:100) was used.

Techniques: Expressing, Quantitative RT-PCR, Control, Activity Assay, Transduction, Infection, Virus, Western Blot, Transfection, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells

doi: 10.1074/jbc.m707117200

Figure Lengend Snippet: FIGURE3.TheFSTpromotercontainsthreeputativeGLIbindingsites.A,overviewofthelocalizationandsequenceofthreeputativeGLIbindingsitesinthe human FST promoter. Numbers indicate distance from the transcription start site (according to RefSeq NM_013409). B, cross-species alignment of the region surrounding the putative GLI binding sites shows high conservation between the indicated species. Putative GLI binding sites are highlighted in gray boxes, in the first line, the consensus GLI binding site is indicated, and diverging bases are shown in lowercase. Arrows indicate the orientation of the putative binding sites. C, EMSA analysis shows specific binding of recombinant GLI2 zinc finger domain protein to all three putative GLI binding sites in vitro. Specificity of binding was tested by competing with increasing amounts of unlabeled oligonucleotide corresponding to either the wild type binding sequence (bs) or mutated binding site (bsm) sequences or unspecific competitor dI-dC. D, chromatin immunoprecipitation shows specific binding of GLI2 to the FST promoter in vivo. Chromatin isolated from induced GLI2act- or GLI1-HaCaT cells was precipitated with either specific (GLI2 and GLI1) or unspecific (normal IgG) antibodies. A 127-bp fragment (asterisk in A) spanning bs2 and bs3 of the FST promoter was amplified only from the specific precipitate, a 148-bp fragment from the PTCH promoter was used as positive control. No amplification was detected from the unspecific precipitates and for a 284-bp fragment from the promoter of the human acidic ribosomal protein P0 (RPLP0) (negative control). A representative of three independent experiments is shown.

Article Snippet: A mouse monoclonal antibody directed against human FST (R&D Systems, dilution, 1:100) was used.

Techniques: Binding Assay, Recombinant, In Vitro, Sequencing, Chromatin Immunoprecipitation, In Vivo, Isolation, Amplification, Positive Control, Negative Control

Journal: Journal of Biological Chemistry

Article Title: GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells

doi: 10.1074/jbc.m707117200

Figure Lengend Snippet: FIGURE 4. The human FST promoter is predominantly activated by GLI2. A, luciferase reporter assay with a cloned 3088-bp fragment of the human FST promoter (FSTprom, schematic drawing). HaCaT cells were co-transfected with FSTprom reporter construct and GLI expression constructs as indicated. Only GLI2act expression resulted in strong activation of the FST promoter. B, activity of the expression constructs used in A was tested on a reporter construct (PTCHprom)containinga1313-bpfragmentofthehumanPTCHpromoter.C,activationoftheFSTpromoterisdependentontwoGLIbindingsites.Aschematic view of constructs with mutated bs1, bs2, and bs3 is shown. D, luciferase reporter constructs were either co-transfected with GLI2act expression construct or emptyvectorascontrol(pc,rightgraph).Mutationofbs1orbs3oracombinationofbothabolishedGLI2act-dependentactivationoftheFSTpromoter,whereas mutation of bs2 showed no influence on activation.

Article Snippet: A mouse monoclonal antibody directed against human FST (R&D Systems, dilution, 1:100) was used.

Techniques: Luciferase, Reporter Assay, Clone Assay, Transfection, Construct, Expressing, Activation Assay, Activity Assay, Mutagenesis