Cloning and characterization of SNIP1. (A) Putative genomic structure of human SNIP1 gene is shown. Black boxes represent untranslated regions and the white boxes represent translated regions. The exon numbers are noted inside their respective white boxes with the sizes of introns written at the bottom. Numbers at the edges of the boxes denote the amino acid number from the human protein sequence. (B) Schematic diagram of human SNIP1 protein is shown with two conserved domains noted in shaded boxes, which are a bipartite NLS and a FHA. Numbers at the edges of the boxes denote the amino acid number from the deduced protein sequence. The sequence of the human SNIP1 protein is shown with the arrow indicating the starting methionine and an asterisk star representing the stop codon. The NLS is enclosed in a box and the FHA domain is underlined. (C) Commercially available Northern blot was purchased from Clontech and hybridized with the carboxy-terminal fragment of SNIP1. The sizes of three transcripts are indicated. (D) COS-1 cells were transfected with HA-tagged SNIP1 constructs as indicated. The specificity of an antibody against SNIP1 was monitored by Western blotting of the lysates. HA–SNIP1 and HA–SNIP1-C are indicated by arrows. (NS) Nonspecific band. (E) Western blot of cell lysates from various cell lysates. A single band of ∼50 kD was detected. (F) Western blot of lysates from various adult mouse tissues.

SNIP1 interacts with Smad proteins in vivo and in vitro. (A) Truncated SNIP1 cloned from the yeast two-hybrid assay was cloned into the prey vector pJG4-5 and was transformed into yeast. The truncated SNIP1 transformants were then transformed again with the indicated LexA–Smad fusion constructs in the bait construct pEG202. Four separate colonies from each group of transformants were streaked. (Blue) A positive interaction was detected for the transformants of SNIP1/Smad1 and SNIP1/Smad2. (B) NMuMg cell lysates treated with or without 5 ng/ml TGF-β or 50 ng/ml BMP2 for 1 hr was subjected to immunoprecipitation with α-SNIP1 and blotted with antibodies against indicated Smads. No IP lane was immunoprecipitated with rabbit IgG. The presence of SNIP1 and Smads in these cells were monitored by direct immunoblotting using antibodies against SNIP1 and Smads. (C) In vitro product of Smad4 made using reticulocyte lysate was incubated with various GST–SNIP1 deletion constructs. (Arrow) Smad4 product. (D) GST–SNIP1 (25%) constructs used in the reactions were subjected to SDS-PAGE and stained.

Deletion mapping of the interacting regions of SNIP1 and Smad4. (A) Schematic diagram of the SNIP1 protein with the GST fusion protein of SNIP1 deletion constructs used in these experiments shown below (left). Numbers indicate the starting amino acid number of the deletion constructs. Smad4 and its deletion constructs used to produce in vitro transcribed/translated products used in the experiment (right). (B) Smad4 deletion constructs from the above diagram were used to make protein using reticulocyte lysate. The Smad4 deletion proteins were used in in vitro-binding assay using various bacterially expressed GST fusion of SNIP1 deletions constructs as indicated. After extensive washing, the beads were subjected to SDS-PAGE and autoradiography. (C) Twenty-five percent of the reticulocyte lysates used in the reaction was subjected to SDS-PAGE and autoradiography to monitor for expression of Smad4 constructs (top) GST fusion proteins (25%) were also subjected to standard SDS-PAGE and stained as per experimental protocol to control for equal loading of proteins in the experiments (bottom). (D) NMuMg cell lysates transfected with the indicated deletion constructs Smad4 were treated with or without 5 ng/ml TGF-β for 1 hr. These lysates were subjected to immunoprecipitation with α-SNIP1 and blotted with α-Flag and α-HA. No IP lane was immunoprecipitated with rabbit IgG. The presence of SNIP1 in these cells was monitored by direct immunoblotting using antibodies against SNIP1. These lysates were also used for direct immunoblotting with antibodies against Flag and HA to determine the expression of transfected Smad4 deletion constructs. HC and LC denote heavy and light chain of mouse IgG, respectively. (NS) Nonspecific band.

SNIP1 localizes to the nucleus constitutively and is involved in transcriptional inhibition. (A) HA–SNIP1, HA–SNIP1-N, and HA-SNIP1-C expression vectors were transiently cotransfected into NMuMg cells. After an overnight serum starvation, cells were analyzed by indirect immunofluorescence using α-HA monoclonal antibody. (Top) FITC staining of the cells to localize transfected SNIP1 and its deletion constructs. (Bottom) DAPI staining for the nuclei. (B) Cellular localization was determined by an independent observer, blinded as to the construct transfected, counting 200 cells per well to determine the percentage of each SNIP1 construct present in each of the sub-cellular compartments. Cytoplasmic (open bars) represents fluorescent signal present only in the cytoplasm of the cell, weak nuclear (hatched bars) represent signal in both the cytoplasm and nucleus, and nuclear (solid bars) represent signal present only in the nucleus. Results are presented from a single experiment, representative of three independent experiments. (C) NMuMg cells were cotransfected with 500 ng of 3TP–Lux, or SBE4–Luc with indicated amounts of HA–SNIP1 and its deletions. TGF-β was either not added (open bars) or added (solid bars) at a final concentration of 5 ng/ml, 24 hr before lysis, and analyzed for luciferase activity. Results are expressed as means (±s.d.) of triplicate assays, normalized for transfection efficiency using β-galactosidase activity.

Injection of SNIP1 into Xenopus embryos leads to a suppression of Gsc expression and anterior truncations. A total of 1 ng of SNIP1-FL (C,D,H), or SNIP1-N (E,F,I) or 2 ng of SNIP1-C (B,G) RNA were injected into the two dorsal blastomeres of a 4-cell embryo. Embryos were allowed to develop until stage 10.5 and fixed for whole mount in situ analysis for Xgsc expression, or embryos were cultured until stage 33, fixed, and photographed. SNIP1-FL or SNIP1-N-injected embryos exhibited a suppression of Xgsc expression as compared with the uninjected control (A) or to the SNIP1-C injected embryos. At stage 33, SNIP1-C injected embryos were phenotypically normal, whereas SNIP1-FL and SNIP1-N-injected embryos often showed loss of anterior structures.

Endogenous SNIP1 interacts with endogenous CBP/p300 through its amino terminus. NMuMg cell lysates treated with or without TGF-β were subjected to immunoprecipitation using α-SNIP1 and blotted with α-p300 (A) or α-CBP (B). The expression of these proteins in the cells was monitored by direct immunoblotting as shown at bottom. (C) Schematic diagram of p300 and location of the deletion constructs used to make an in vitro-transcribed/translated product. (D) p300 deletion constructs from the above diagram were used to make protein using reticulocyte lysate. The p300 deletion proteins were used in in vitro-binding assay using various bacterially expressed GST fusion of SNIP1 deletions constructs as indicated. After extensive washing, the beads were subjected to SDS-PAGE and autoradiography. (E) A total of 25% of the reticulocyte lysates used in the reaction was subjected to standard SDS-PAGE and autoradiography to detect the amounts used in each reactions (top). A total of 25% of GST fusion proteins used in the experiments was subjected to standard SDS-PAGE and stained as per experimental protocol to control for equal loading of GST proteins in the experiments (bottom). (F) NMuMg cells were cotransfected with pG5E1B with (+) or without (−) 0.5 μg of HA–SNIP1-FL, as indicated along with Gal4 fusion proteins of p65, p53, and VP16. Results are expressed as means (±s.d) of triplicate assays, normalized for transfection efficiency using β-Galactosidase activity.

SNIP1 is able to inhibit the interaction of Smad4 with p300. (A) NMuMg cells were transfected with HA-tagged SNIP1 constructs and indicated Flag-tagged full-length Smad2 and Myc-tagged Smad4 expression constructs. The activation of the system was achieved by treating the cells with (+) or without (−) 5ng/ml TGF-β for 1 hr before lysis. Interaction between p300 and Smad4 was analyzed by immunoblotting the α-p300 immunoprecipitates with α-Myc antibody. The amount of immunoprecipitated p300 was monitored by immunoblotting 25% of p300 immunoprecipitate with α-p300 antibody. The expression of the transfected constructs was monitored by immunoblotting with antibodies against HA for SNIP1 or Myc and Flag for Smads. The control lane was immunoprecipitated with normal rabbit IgG. (B) COS-1 cells were transfected with HA-tagged SNIP1 constructs and increasing amounts of Myc-tagged Smad4 expression construct. The activation of the system was achieved by treating the cells with (+) or without (−) 5ng/ml TGF-β for 1 hr before lysis. Interaction between p300 and SNIP1 was analyzed by immunoblotting the α-p300 immunoprecipitates with α-HA antibody. The amount of immunoprecipitated p300 was monitored by immunoblotting 25% of p300 immunoprecipitate with α-p300 antibody. The expression of the transfected constructs was monitored by immunoblotting with antibodies against HA for SNIP1 or Myc Smad4. The control lane was immunoprecipitated with normal rabbit IgG.