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First published online 2 February 2005
doi: 10.1242/dev.01664

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Sumoylation of LIN-1 promotes transcriptional repression and inhibition of vulval cell fates

Department of Molecular Biology and Pharmacology Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (22K): [in a new window]   Fig. 1. UBC-9 binds two consensus sumoylation motifs of LIN-1. (A) Schematic of LIN-1: ETS DNA-binding domain (black) and consensus sumoylation motifs (above); the D domain (D) and the FQFP motif (F) are docking sites for ERK. The positions of the e1275 and n1790 mutations and amino acid numbers are shown below. (B) The interaction of GAL4AD:UBC-9(1-166) with the indicated LexA DNA-binding domain (LA):LIN-1 fusion protein was monitored using the two-hybrid system. Bars represent the average LexA-dependent ß-galactosidase activity from at least five independent yeast transformants and lines indicate the standard deviation. The values were normalized by setting the interaction with LA:LIN-1(1-252) equal to 100 RLU.

View larger version (12K): [in a new window]   Fig. 5. SUMO is sufficient to increase binding of MEP-1 to LIN-1. The association of MEP-1 with the indicated LA fusion proteins was monitored using the yeast two-hybrid system. Bars represent the average LexA-dependent ß-galactosidase activity from three independent yeast transformants grown to logarithmic phase in selective media, and lines indicate the standard deviation. The values were normalized by setting the interaction of each protein with LA:LIN-1(1-64) to 100 RLU. The LA fusion proteins were expressed at similar levels as determined by western blotting (data not shown).

View larger version (18K): [in a new window]   Fig. 3. Sumoylation promotes transcriptional repression by LIN-1. (A) The promoter region of the L8G5 reporter plasmid: eight LexA-binding sites (white boxes), five GAL4-binding sites (black circles), an E1A promoter (arrow) and a luciferase-coding region. (B) 293 HEK cells were transiently transfected with: (1) the L8G5 reporter plasmid; (2) an expression plasmid that encodes the GAL4 DNA-binding domain (G4) alone or fused to the indicated fragments of LIN-1 and/or C. elegans SMO-1; (3) an expression plasmid that encodes the LexA:VP16 fusion protein (+ or -); and (4) a reporter plasmid that encodes ß-galactosidase to measure transfection efficiency. Bars indicate luciferase activity divided by ß-galactosidase activity. Values are the average and standard deviation of three to four independent transfections conducted in parallel. Values were normalized by setting the value for G4 alone equal to 100 RLU. Western blotting demonstrated that the LexA:VP16 fusion protein was expressed at equivalent levels independent of the co-expressed G4 fusion protein, and that each G4:LIN-1 fusion protein was expressed, although the levels could not be estimated because of a crossreactive protein of similar size (data not shown).

View larger version (29K): [in a new window]   Fig. 2. LIN-1 is covalently-modified by SUMO-1. (A) Extracts from yeast expressing LA:LIN-1(1-252) alone (-) or with His- and FLAG-tagged SUMO1/Smt3 (HF-SUMO) (+) were subjected to metal affinity chromatography. Bound proteins were separated by SDS-PAGE and immunoblotted (IB). An anti-FLAG antibody detected all proteins modified by HF-SUMO; an anti-LA antibody detected LA:LIN-1(1-252) complexes. The arrows indicate high molecular weight forms of LA:LIN-1(1-252) that appear to be covalently modified by one or multiple HF-SUMO moieties (14 kDa), and may also contain endogenous SUMO1/Smt3 (11 kDa). Bands present in lanes 3 and 4 are a cross-reactive endogenous yeast protein that was present in strains lacking LA:LIN-1 (data not shown) and 60 kDa unmodified LA:LIN-1(1-252) that bound the affinity matrix in a Ni2+-independent manner (data not shown). Molecular weight markers (in kDa) are indicated. (B) Extracts from Sf9 cells that were not infected (Mock) or infected with viruses that express GST:LIN-1 alone (-) or with His- and FLAG-tagged C. elegans SMO-1 (HF-SUMO) (+) were subjected to glutathione sepharose affinity chromatography to purify the GST fusion proteins. Bound proteins were separated by SDS-PAGE and immunoblotted. An anti-GST antibody detected all LIN-1 species; an anti-FLAG antibody detected LIN-1 that was covalently modified by HF-SUMO. Arrows indicate sumoylated isoforms of LIN-1 (lanes 3 and 7) that were absent in extracts containing mutant GST:LIN-1(1-64; 9-16A) (lanes 4 and 8). (C) Extracts from Sf9 cells were analyzed as in B. Arrows indicate sumoylated isoforms of LIN-1 (lane 5) that were absent in extracts containing mutant GST:LIN-1(1-64; K10A) (lane 6).

View larger version (26K): [in a new window]   Fig. 4. The sumoylation motifs of LIN-1 are necessary for the interaction with MEP-1. (A) Schematic of MEP-1 with zinc-finger motifs (black) and glutamine-rich region (gray) (Belfiore et al., 2002). (B) The interactions between LA:LIN-1 fusion proteins and MEP-1(155-859) fused to the GAL4 activation domain (GAL4AD) were measured qualitatively using the yeast two-hybrid system. A (+) indicates robust activation of a LexA-dependent lacZ reporter gene. (C) The interactions between wild-type LA:LIN-1(1-64) or the indicated mutant and MEP-1 were measured quantitatively. Bars represent the average LexA-dependent ß-galactosidase activity and lines indicate the standard deviation of at least six independent yeast transformants. The signal with LA:LIN-1(1-64) was set to 100 relative light units (RLU); the signals with mutant proteins are proportional. (D) To monitor expression of LA:LIN-1 proteins, we analyzed protein extracts from transformed yeast by western blotting using an anti-LA antibody. Lanes 1-14 correspond to LA fusion proteins listed as 1-14 in C. (E) The interaction of MEP-1 with the indicated LA:LIN-1 fusion protein was measured quantitatively. Bars represent the average of six independent yeast transformants and lines indicate the standard deviation. The signal with LA:LIN-1(158-180) was set to 100 RLU and signals with mutant proteins are proportional.

View larger version (136K): [in a new window]   Fig. 6. mep-1 inhibits vulval cell fates of P3.p, P4.p and P8.p. A wild-type hermaphrodite at the `Christmas tree' stage of vulval development treated with extensive exposure to mep-1 RNAi. A bracket indicates the vulval invagination formed by P5.p-P7.p; arrows indicate ectopic invaginations formed by the descendants of P3.p and P4.p.

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