1. Volume 2, Issue 1, Pages 109-120 (July 1998)
Smad2 and Smad3 Positively and Negatively Regulate TGFβ-Dependent Transcription through the Forkhead DNA-Binding Protein FAST2 
Etienne Labbé, Cristoforo Silvestri, Pamela A. Hoodless, Jeffrey L. Wrana, Liliana Attisano 
Molecular Cell 
Volume 2, Issue 1, Pages (July 1998)
DOI: /S (00)

2. Figure 1 Analysis of FAST2 Amino Acid Sequence and Expression Pattern
(A) Comparison of the sequence of mouse FAST2 with Xenopus FAST1. Conserved residues (shaded boxes), the forkhead domain (solid underline), the Smad-interacting domain (dashed underline), and the location of degenerate primers used to amplify Fast2 (arrows) are indicated.
(B) A schematic representation of the conservation (shown in percent identity) between different regions of FAST2 and FAST1 is shown.
(C) Temporal expression of FAST2 during mouse development. RNA isolated from embryonic carcinoma cells and from mouse embryos (E6.5–12.5) was subjected to RT–PCR using primers to Fast2 and Hprt (as control). PCR products were visualized by autoradiography.
Molecular Cell 1998 2, DOI: ( /S (00) )

3. Figure 2 FAST2, Smad2, and Smad4 Mediate Induction of the gsc Promoter
(A) HepG2 cells were transiently transfected with gsc-Lux and the indicated DNA and luciferase activity in lysates from cells incubated in the presence (closed bars) or absence (open bars) of TGFβ determined. Luciferase activity was normalized to β-galactosidase activity and is expressed as the mean ± SD of triplicates from a representative experiment.
(B) HepG2 cells were transiently transfected with wild-type (open bars) or constitutively active (closed bars) versions of the activin (ActRIB), TGFβ (TβRI), or BMP (ALK3, ALK6) type I receptors, with or without FAST2 and Smads, as indicated. Luciferase activity was determined as in (A).
(C) Interaction of FAST2 with Smads. COS-1 cells were transiently transfected with various combinations of myc/FAST2 and Flag-tagged Smads (F-Smad), wild-type (wt), or activated (act) versions of activin (ActRIB) or BMP (ALK6) receptors, or with TGFβ type I and type II receptors, as indicated. Cells were labeled with [35S]methionine, incubated with (lane 5) or without TGFβ (lanes 1–4), and lysates immunoprecipitated with anti-Flag antibodies. Coprecipitating FAST2 was visualized by SDS–PAGE and autoradiography. Constant expression of FAST2 was confirmed by immunoprecipitation with anti-myc antibodies (bottom panel).
Molecular Cell 1998 2, DOI: ( /S (00) )

4. Figure 3
Electrophoretic Mobility Shift Assays with FAST2, Smad2, and Smad4
Nuclear extracts from transiently transfected COS-1 cells were incubated with a 32P-labeled 111 bp gsc promoter probe and protein/DNA complexes visualized by autoradiography.
(A and B) COS-1 cells were transiently transfected with various combinations of myc/FAST2, Flag/Smad2, Smad4/HA, and wild-type (wt) or constitutively active activin (ActRIB*; act) or TGFβ (TβRI*) type I receptors.
(C) Cells were transfected with constitutively active activin type I receptors and various combinations of myc/FAST2, Flag/Smad4, and HA/Smad2 as indicated. For supershifting assays (B and C) anti-Flag (F), anti-myc (M), or anti-HA (H) antibodies were added. The migration of complexes containing FAST2, the TGFβ/activin response factor (TRF), and the supershifted complexes (SS) are indicated.
Molecular Cell 1998 2, DOI: ( /S (00) )

5. Figure 4 FAST2 Binds to the TGFβ/Activin Response Element (TARE)
(A) DNaseI footprint analysis. Increasing amounts of bacterially expressed FAST2 (wedge) or control GST protein (−) were incubated with a wild-type (WT) or mutant (M) 32P-labeled gsc promoter fragment and was subjected to DNaseI digestion followed by denaturing electrophoresis and autoradiography. The FAST2-protected region (lanes 2–4) and the corresponding nucleotide sequence (determined from a Maxam-Gilbert G+A sequencing reaction carried out in parallel) is indicated. The location of the two point mutants, which prevent FAST2 binding (lanes 5–7), are shown.
(B) The FAST2-binding region of the mouse goosecoid promoter is compared to related regions in the Xenopus and human goosecoid and the Xenopus Mix.2 gene. A predicted consensus binding site is shown. The FAST2-binding region in the mouse gsc promoter, as determined by DNaseI footprinting and deletional analysis is indicated (shaded box). Point mutations that are known to disrupt activin-dependent signaling are indicated (arrows).
(C) A schematic representation of the 3′ end deletion constructs and the corresponding nucleotide sequence is shown (top panel). The construct nomenclature (left side), FAST2 DNaseI-protected region (uppercase letters), the nonprotected nucleotides (lowercase), and the introduced point mutations (asterisks) are indicated. HepG2 cells were transiently transfected with the indicated 3′ end deletion constructs, in the presence (+) or absence (−) of FAST2. Luciferase activity from cells incubated with (closed bars) or without (open bars) TGFβ is plotted as in Figure 2.
(D) Electrophoretic mobility shift assays of FAST2. Bacterially expressed FAST2 or GST control protein were subjected to gel shift assays using 32P-labeled probes corresponding to the indicated gsc promoter fragments. Protein/DNA-binding complexes were visualized by autoradiography.
Molecular Cell 1998 2, DOI: ( /S (00) )

6. Figure 5
Smad4 DNA Binding Is Required for Formation of Transcriptional Activation Complexes and Efficient Induction of the gsc Promoter
(A) A schematic representation of the 5′ end deletion constructs and the corresponding construct name is shown (left panel). HepG2 cells were transiently transfected with the indicated (right panel) 5′ end deletion constructs of the gsc promoter, in the presence (+) or absence (−) of FAST2. Luciferase activity from cells incubated with (closed bars) or without (open bars) TGFβ is plotted as in Figure 2.
(B) Electrophoretic mobility shift assays of FAST2 with various 5′ end–deleted versions of the gsc promoter. Bacterially expressed FAST2 or GST control protein were subjected to gel shift assays using 32P-labeled probes corresponding to the indicated gsc promoter fragments (left panel, lanes 1–4). For examination of TRF formation, nuclear extracts were prepared from COS-1 transfected with constitutively active ActRIB, together with FAST2 or Smads, as indicated (right panel, lanes 5–16).
(C) DNaseI footprint analysis of Smad4. Bacterially expressed GST control (lanes 1 and 3), FAST2 (lane 2), full-length Smad4 (FL; lane 4), Smad4, MH1 domain (lane 5), and FAST2 with full-length Smad4 (lane 6) were incubated with a 32P-labeled gsc promoter fragment and were subjected to DNaseI digestion. The FAST2 and Smad4 protected regions are shown. The nucleotide sequence, determined from a Maxam-Gibert G+A sequencing reaction (lane 7), is indicated.
Molecular Cell 1998 2, DOI: ( /S (00) )

7. Figure 7
The Smad3 MH1 Domain Mediates DNA Binding and Blocks TGFβ-Dependent Induction of gsc-Lux
(A) Electrophoretic mobility shift assays with Smad1, -2, -3, and -4. Bacterially expressed GST control (lanes 1 and 6), full-length (lanes 2–5), or MH1 domain versions (lanes 7–10) of Smad1 (S1), Smad2 (S2), Smad3 (S3), and Smad4 (S4) were subjected to gel shift assays using the 111 bp gsc promoter fragment. The migration of complexes containing Smads is indicated.
(B) DNaseI footprint analysis of Smad2, -3, and -4. Varying concentrations of bacterially expressed MH1 domains of Smad4 (lanes 2–3), Smad3 (lanes 4–5), Smad2 (lanes 6–7), and the GST control (lanes 1 and 8) were incubated with a 32P-labeled gsc promoter fragment and were subjected to DNaseI digestion. The Smad3 and Smad4 protected regions are shown (left).
(C) A schematic representation of wild-type and chimeric versions of Smad2 and Smad3 are shown (top panel). HepG2 cells were transiently transfected with FAST2, Smad4, and increasing concentrations of wild-type and chimeric versions of Smad2 and Smad3. Luciferase activity is plotted as in Figure 2.
(D) A model for positive and negative transcriptional regulation by Smad2 and Smad3. TGFβ/activin signaling induces formation of a DNA-binding complex containing FAST2, Smad4, and either Smad2 or Smad3. Since Smad2 cannot directly bind to DNA, Smad4 binds to its target sequence and promotes efficient induction of the promoter. Smad3 competes with Smad4 for the same target sequence; thus, we postulate that inhibition of Smad4 DNA contact causes a conformation change in the complex such that transcriptional activation of the gsc sequence cannot occur.
Molecular Cell 1998 2, DOI: ( /S (00) )

8. Figure 6 Smad3 Negatively Regulates the gsc Promoter
(A and B) HepG2 cells were transiently transfected with the indicated constructs and luciferase activity from lysates of cells incubated in the presence (closed bars) or absence (open bars) of TGFβ determined. For (B), cells were transfected with 0.1 μg of Smad2 and Smad4 and varying amounts of Smad3 (0.3 ng to 0.17 μg).
(C) Electrophoretic mobility shift assays with FAST2, Smad3, and Smad4. COS-1 cells were transiently transfected with combinations of myc/FAST2, Flag/Smad3 (F-Smad3), and Smad4/HA. Nuclear extracts were incubated with a 32P-labeled 111 bp gsc probe. For supershifting assays, anti-Flag (F), anti-myc (M), or anti-HA (H) antibodies were added with the gsc probe. The migration of complexes containing FAST2, the TGFβ/activin response factor (TRF), and the supershifted complexes (SS) are indicated.
Molecular Cell 1998 2, DOI: ( /S (00) )