Verschueren K et al. (1999), SIP1, a novel zinc finger/homeodomain repressor...

XB-ART-12719

J Biol Chem 1999 Jul 16;27429:20489-98.

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SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5'-CACCT sequences in candidate target genes.

Verschueren K , Remacle JE , Collart C , Kraft H , Baker BS , Tylzanowski P , Nelles L , Wuytens G , Su MT , Bodmer R , Smith JC , Huylebroeck D .

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Activation of transforming growth factor beta receptors causes the phosphorylation and nuclear translocation of Smad proteins, which then participate in the regulation of expression of target genes. We describe a novel Smad-interacting protein, SIP1, which was identified using the yeast two-hybrid system. Although SIP1 interacts with the MH2 domain of receptor-regulated Smads in yeast and in vitro, its interaction with full-length Smads in mammalian cells requires receptor-mediated Smad activation. SIP1 is a new member of the deltaEF1/Zfh-1 family of two-handed zinc finger/homeodomain proteins. Like deltaEF1, SIP1 binds to 5'-CACCT sequences in different promoters, including the Xenopus brachyury promoter. Overexpression of either full-length SIP1 or its C-terminal zinc finger cluster, which bind to the Xbra2 promoter in vitro, prevented expression of the endogenous Xbra gene in early Xenopus embryos. Therefore, SIP1, like deltaEF1, is likely to be a transcriptional repressor, which may be involved in the regulation of at least one immediate response gene for activin-dependent signal transduction pathways. The identification of this Smad-interacting protein opens new routes to investigate the mechanisms by which transforming growth factor beta members exert their effects on expression of target genes in responsive cells and in the vertebrate embryo.

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Species referenced: Xenopus
Genes referenced: cd40 gemin2 mmut myc myod1 nherf2 otx2 pigy smad1 smad2 tbxt zeb1

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	FIG. 1. Association of SIP1TH1 with different XSmad and mouse Smad baits (MH2 domains or full-length) in yeast twohybrid assays, as illustrated by staining for LacZ (blue colonies indicate positive LacZ staining, i.e. interaction of prey with bait). Four representative colonies are shown for each interaction analyzed. Numbering of deleted or substituted amino acids is according to their position in the full-length protein.
	FIG. 2. A, schematic representation of the domain structure of SIP1 protein and its similarities with mouse dEF1. The putative zinc fingers are shown (dark gray boxes for C2H2-type zinc fingers and light gray boxes for C3H type) together with the homeodomain-like sequence (HD). The oval indicates the domain essential for interaction of SIP1with the Smad MH2 domain (SBD). SIP1 cDNAs used in this study are indicated (SIP1 full-length shown in red, SIP1TH1 and SIP1CZF shown in yellow). The sequence of SIP1 is available from GenBankTM under accession number AF033116. B, amino acid sequence comparison between mouse SIP1 and mouse dEF1. Gray boxes reflect identical or biochemically similar amino acids. The position of the C2H2 type zinc fingers in SIP1 is indicated by a bold overline, other zinc fingers in SIP1 are indicated by thin double overlines.
	FIG. 3. Mapping of the Smad-binding domain in SIP1. Schematic representation of deletion constructs to map the segment required for interaction of SIP1 with the XSmad1 MH2 domain. The original Smad-binding domain (spanning amino acids 315â507; shown in brown) was used as a starting point for the detailed mapping. The right column summarizes interaction data obtained in yeast, as assayed by ability to grow on plates lacking histidine and in the presence of 5 mM 3-amino-triazole and by staining of b-galactosidase. The Smadbinding domain (SBD, shown in green) is a 51-amino acids-long peptide defined as essential for the interaction; its amino acid sequence (aa seq) is shown in the lower part of the figure.
	FIG. 4. In vitro association of SIP1 with the MH2 domains of different Smad proteins. Myc-tagged full-size SIP1 protein was expressed in COS1 cells. Upper panel, equal amounts of the same cell extract were used in lanes 1â6. The 145-kDa SIP1 protein (indicated by the arrow) was efficiently pulled down from this cell lysate using the different GST-Smad fusion proteins (lanes 1 and 4â6; visualization is by Western blotting using anti-Myc antibody) but not by an unrelated GST-fusion protein (GST-CD40; lane 3) and GST (lane 2). GST fusions included the MH2 domains of XSmad1(G418S) (lane 4), mouse Smad5 (lane 5), mouse Smad2 (lane 6), and wild type XSmad1 (lane 1), respectively. Lane 7 provides a negative control with proteins pulled-down by GST-XSmad1 from a cell lysate of mock-transfected cells. Lower panel, estimate of the amount of GST-fusion proteins used in the pull-down experiments by Ponceau S staining of the used blot. Lower amounts of GST-Smad fusion proteins were used in lanes 1 and 4â7.
	FIG. 5. A, interaction of SIP1 with the MH2 domain of XSmad1 in mammalian cells. An expression construct encoding a fusion between GST and the MH2 domain of XSmad1 was transfected in COS1 cells together with expression constructs for Myc-tagged SIP1. As shown by immunoblotting of pulled-down material from cell extracts, SIP1 specifically interacted with the GST-XSmad1 fusion protein (middle panel, lane 1), whereas deletion of the SBD51 disrupted the interaction (middle panel, lane 2). Comparable affinity purification of the GST-fusion protein and equal expression of SIP1 were confirmed by immunoblotting of the pulled down material using polyclonal anti-GST antibody (upper panel) and of total cell extracts using monoclonal anti-myc antibody (lower panel), respectively. B, ligand-dependent interaction of SIP1 with full-length Smads in mammalian cells. Lanes 1â5, lanes 6â11, and lanes 11â17 contain data from three independent experiments. HEK293T cells were transiently transfected with various combinations of expression constructs encoding Myc-tagged SIP1, Flag-tagged Smads, and type I receptors, as indicated. Cell lysates were immunoprecipitated with anti-Flag antibodies, and the precipitated proteins were visualized by SDS-polyacrylamide gel electrophoresis and immunoblotting using anti-Myc (upper panel) or anti-Flag (middle panel) antibodies. The middle panel shows the comparable immunoprecipitations of Flag-tagged Smads in each experiment, whereas the lower panel shows immunoblotting of total cell extracts using anti-Myc antibody, to confirm comparable expression of SIP1. *, indicates the heavy chain of the anti-Flag antibody used in the immunoprecipitations; ca, constitutively active; wt, wild type; DSBD indicates SIP1 in which the 51-amino acids-long SBD was deleted.
	FIG. 6. Interaction of the DNA-binding domain of SIP1 (SIP1CZF) with target sites of different promoters by gel retardation analysis. A, the fusion protein GST-SIP1CZF (10 ng) was incubated with the indicated 32P-labeled double-stranded oligonucleotides. No binding was observed with negative control GST-PLAG1DBD (Control; the Brabinding site was used here). B, interaction of SIP1CZF with E2 box sequences. The experiment was carried out as described in panel A, but MyoD and MyoD-Mut labeled probes were used. Competition experiments were carried out with the oligonucleotides listed above the lanes. In the negative control lanes (Control), both MyoD and MyoD-Mut probes were incubated with GST-PLAG1DBD, and no competitor was added. No, no competitor added.
	FIG. 7. SIP1 binds to the Xbra2 promoter. Fifty pg of 32P-labeled Xbra SIP1 probes (WT or D) were incubated with extracts from COS1 cells transfected with expression constructs for Myc-tagged SIP1CZF (lanes 1â2) or full-length SIP1 (SIP1FS, lanes 3â4). Lane 5 shows binding of endogenous proteins in cell extracts from mock-transfected cells. Specific SIP1 complexes are indicated (*) as well as endogenous complexes (l). Xbra-WT probe contains the sequence 59-ATCCAGGCCACCTAAAATATAGAATGATAAAGTGACCAGGTGTCAGTTCT, and Xbra-D contains 59-ATCCAGGCCACCTAAAATATAGAATGATAAAGTGACCAGATGTCAGTTCT. SIP1-binding sites are in bold, and the substituted nucleotide is underlined.
	FIG. 8. SIP1 polypeptides repress endogenous Xbra gene expression in early Xenopus embryos. Xenopus embryos at the 2- to 4-cell stage were left uninjected (A) or were injected with 1 ng of RNA encoding SIP1CZF (B), XOtx2(K3E) (C), or full-length SIP1 (D). Both SIP polypeptides caused repression of Xbra expression. In panels B and D, expression is down-regulated in half the embryo because one of two cells of the early embryo was injected. In a typical experiment, 14 of 16 embryos injected with RNA encoding SIP1CZF displayed down-regulation of Xbra expression, as did, in a separate experiment, 18 of 19 embryos injected with full-length SIP1. Slight down-regulation of Xbra expression was seen in only 1 of 13 embryos injected with RNA encoding Otx2(K3R).