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Six3-mediated auto repression and eye development requires its interaction with members of the Groucho-related family of co-repressors

¹ Department of Genetics, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794, USA
² Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
³ Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

View larger version (31K): [in a new window]   Fig. 1. Six3 and members of the Groucho family of corepressors. (A) The pc97-Six3 and pc86-Grg5 constructs used in the yeast transformation assay. Gal4-DB, Gal4 DNA-binding domain; SD, Six domain; HD, homeodomain; Gal4-AD, Gal4 activation domain; Q, glutamine-rich domain; GP, glycine and proline-rich domain. (B) Comparison of murine Grg family members with Drosophila Groucho protein. Mouse Grg5 lacks the CcN domain (potential phosphorylation sites for casein kinase II and cdc2), SP domain (serine- and proline-rich) and WD40 repeat domain (40 amino acid repeats separated by tryptophan and aspartic acid), but it contains the Q and GP domains. The Q domain of mouse Grg5 shares as much as 64% amino acid identity with Drosophila Groucho.

View larger version (45K): [in a new window]   Fig. 2. Binding of Six proteins to Grg proteins in vitro and in vivo. (A) Six family proteins bind to Grg5 in vitro. Coomassie staining of a gel containing GST (0.2 µg) and GST-Grg5 fusion proteins (0.2 µg) used in the GST pull-down experiments is shown on the top row. The input lane shows 10% of the total protein used in the GST pull-down experiments. Full-length Six3 migrated as a 37 kDa protein. The faster migrating bands may have been shorter forms of Six3 that originated from internal translation start sites in the Six3 transcript. (B) Six family proteins interact in vitro with mouse Grg4 and with Drosophila Groucho. Input lane shows 10% of the total [35S]methionine-labeled proteins used in the GST pull-down experiments. Specifically, mouse Six3 and Six6 protein bound to GST-Grg4 fusion protein (GST-Grg4 lane) but not to GST alone (GST lane). Drosophila Optix bound only weakly to GST-Grg4 fusion protein, whereas Six3F88E mutant protein did not. Fly Groucho strongly bound to mouse Six3 (lower panel). (C) Mouse Six3 bound to Grg4 in mammalian cells. NIH3T3 cells were transfected with either Six3 expression vector alone (lane 1) or Six3 expression vector and Flag-tagged Grg4 (lane 2). Co-immunoprecipitation (IP) was performed with anti-Flag antibody, and precipitated Six3 was detected with rabbit anti-mouse Six3 antibody (lane 2). Input lane shows 10% of the total protein used in the IP experiment. Six3 and Flag-Grg4 proteins in the crude cell lysate underwent western blot analysis. (D) Fly Groucho immunoprecipitated with mouse Six3. NIH3T3 cells were transfected with either Flag-tagged Groucho expression construct alone (lane 1) or Flag-tagged Groucho expression construct and Six3 expression vector (lane 2). Immunoprecipitation was carried out with anti-Six3 antibody. The precipitated Flag-tagged Groucho protein underwent western blot analysis with anti-Flag antibody. The input lane shows 10% of the total cell lysate used in the IP experiment. Flag-Groucho and Six3 in the crude cell lysate was subjected to western blot analysis.

View larger version (27K): [in a new window]   Fig. 3. Mapping of the interaction domains of Six3 and Grg5 using a yeast two-hybrid assay. (A) Alignment of eh1-like motif identified in mouse Six3 and Six6 with the corresponding eh1 motifs present in the Drosophila, C. elegans and mouse engrailed protein. (B) Mapping of the interaction domains by using a yeast two-hybrid assay. The strength of the interaction between each pair of proteins was reflected by the growth rate of the transformants on both uracil-selective and histidine-selective plates. The N terminus and SD (Six31-183) of Six3 bound to Grg5 similarly to the full-length Six3. Removal of amino acids 1-183 (Six3184-333) abolished the interaction with Grg5. Binding to Grg5 was restored when the construct Six31-120 was used. Six373-120 also interacted strongly with Grg5; however, Six3121-183 did not interact with Grg5. Construct Six3F88E, including a point mutation at position 88 of the eh1-like motif of Six3 (phenylalanine was replaced by glutamic acid), abolished the interaction with Grg5. The fragment containing the Q domain and four amino acids of the SP domain of Grg5 (Grg51-134) interacted with Six3, whereas the fragment containing the C terminus of Grg5 (Grg5135-197) did not.

View larger version (55K): [in a new window]   Fig. 4. Expression of Grg5 in the developing mouse forebrain and eye tissue. (A) Digoxigenin-labeled Grg5 antisense probe revealed expression of this gene in the ventral forebrain (VB), optic vesicle (OV) and surface ectoderm (SE) of E9.5 mouse embryos (left). A similar expression is also seen at E10.5 (middle) but is now more evident in the developing optic vesicle and invaginating surface ectoderm. At E11.5 (right) expression is seen in the retina (R) and lens (L). (B) Confocal images showing the colocalization of Grg5 and Six3 in the nuclei of transfected NIH3T3 cells. NIH3T3 cells were transfected with a Flag-tagged Grg5 and a CMV-based Six3 expression construct. Immunostaining was performed using mouse anti-Flag antibody and a rabbit anti-Six3 antibody; DNA was stained with TOTO-3.

View larger version (45K): [in a new window]   Fig. 5. Identification of the DNA sequence motif bound by Six3. (A) The sequence of the 19 oligonucleotides containing a core ATTA (bold) motif selected by the GST-Six3 fusion protein are aligned for comparison. The oligonucleotide at the top was used in the electrophoretic mobility shift assay (EMSA) experiments depicted below. The DNA sequence identified by Kawakami et al. (Kawakami et al., 1996b) as recognized by Six2, Six4 and Six5 is shown at the bottom. (B) Six3 bound specifically to the identified oligonucleotides in an EMSA. Double-stranded DNA of the first oligonucleotide represented in A was end-labeled with 32P and used as a probe. Lane 1, 32P-labeled probe alone; lane 2, GST and 32P-labeled probe; lane 3, when the GST-Six3 fusion protein was combined with the 32P-labeled probe, specific retardation was observed (bottom arrow); lane 4, a super-shift (top arrow) of the GST-Six3-probe complex was seen when using an anti-Six3 antibody; lane 5, the binding complex was competed when adding 100 times more of the nonradioactive probes than of the radioactive ones; lane 6, competition of the binding complex with 300 times more nonradioactive probes than radioactive probes; lanes 7 and 8, no competition of the binding complex was observed when using either 100 or 300 times more nonradioactive mutated probes, in which the core motif ATTA was mutated into AGCA.

View larger version (41K): [in a new window]   Fig. 6. Six3 binds to its own promoter. (A) The Six3 promoter region (GenBank Accession Number, AD487887). The putative Six3 recognition sequences identified in that genomic fragment are labeled I, II and III. (B) Bacterially expressed Six3 protein (full length) and 32P-labeled DNA probes I, II and III were subjected to an electrophoretic mobility shift assay (EMSA). The upper band containing the Six3-bound DNA fragment is indicated (arrow). The binding specificity was determined by the ability of the complex to be competed by nonradioactive wild-type oligonucleotides (lanes 3, 7 and 11), but not by nonradioactive mutated oligonucleotides (lanes 4, 8 and 12). The lower band seen in lanes 2 and 4 (arrowhead) probably represents truncated form of Six3 protein bound to the probe. This band was also efficiently competed by the cold wild-type probe but not by the mutated form. (C) The sense strand sequences of the oligonucleotide probes I, II and III used in the EMSA are represented. The core sequence motif ATTA of probes I, II and III was mutated into AGCA in the mutated oligonucleotide probes mut1, mut2 and mut3.

View larger version (24K): [in a new window]   Fig. 7. Grg5 and Grg4 mediate Six3 autorepression. (A) The expression vector Gal4-DB has no effect on the Gal4 UAS-TK-CAT reporter gene, but co-transfection of Gal4-DB-Grg5 fusion gene expression plasmid (Gal4 DB-Grg5) resulted in about 80% repression of the basal activity of the CAT reporter gene. Similar repression activity was observed when using a construct containing the Gal4-DB fused to the Grg5 Q domain (Gal4 DB-Grg5Q). (B) Co-transfection of a CMV-based Six3 expression plasmid (Six3), together with the Six3pro-luc reporter gene into NIH3T3 cells led to about 60% repression of the activity of the luciferase reporter, whereas transfections using the CMV expression vector alone showed no repression of the activity of the reporter gene. Co-transfection of the CMV-based Grg5 (Grg5) and Six3 expression plasmids increased the Six3-mediated transcriptional repression to about 80%, while the use of the Grg5 expression plasmid alone had no effect on the activity of the reporter gene. No repression by Six3 was observed when co-transfecting the Six3 expression plasmid together with the Six3pro-luc reporter gene in which the identified Six3 DNA recognition motifs I, II and III were removed. (C) Co-transfection of Gal4-DNA-binding domain (BD)-Six31-183 fusion gene expression plasmid (Gal4-DB-Six3N) with the Gal4 UAS-TK-luciferase reporter plasmid (Gal4 UAS-TK-luc) resulted in about 50% repression of the reporter activity in NIH3T3 cells. The Gal4-DNA binding domain and Six3184-333 fusion gene expression construct (Gal4-DB-Six3C) had a similar repression effect on the reporter gene activity; however, only the plasmid Gal4-DB-Six3N containing the identified Grg-interacting domain was responsive to co-transfected Grg5 and repressed the activity of the reporter gene. The Gal4-DB-Six3C that did not include the Grg-interacting domain was not responsive to Grg5. (D) Grg4 enhances Six3-mediated autorepression in NIH3T3 cells. Co-transfection of Grg4 and Six3 expression constructs together with the Six3pro-luc reporter increased the repression activity of Six3. Unlike wild-type Six3, the construct containing the mutated Six3F88E in which interaction with Grg proteins was abolished, failed to repress Six3 promoter activity, and did not respond to Grg4.

View larger version (97K): [in a new window]   Fig. 8. Six3 expression in the developing mouse retina. (A-C) Six3 is expressed in the murine embryonic retina. Immunostaining of a cryosection of E14.5 retina revealed Six3 nuclear staining in the inner neuroblastic layer (inbl), where newly postmitotic cells are differentiating (arrow), and in the outer neuroblastic layer (onbl), containing mitotic progenitor cells (open arrowhead). The expression in onbl cells is generally lower compared with that in inbl cells at this stage. A similar pattern of expression was detected at E17.5 (D-F), P0 (G-I) and P6 (J-L). Immunostaining of a 3-week-old mouse retina (M-O) revealed nuclear staining in the inner nuclear layer (INL) and ganglion cell layer (GCL) (M, arrows) and a punctuated pattern in the photoreceptors found in the outer nuclear layer (ONL) (O, arrows). PE, pigmented epithelium; H, horizontal cell; Am, amacrine cell. Scale bars: 25 µm (low magnification) and 10 µm (high magnification) in A-H,J-O; 10 µm in I.

View larger version (62K): [in a new window]   Fig. 9. Overexpression of Six3 and Six3F88E in the postnatal retina. (A) Postnatal day 0 (P0) retinal progenitor cells were infected with replication incompetent retroviral vectors carrying one of two different forms of the Six3 cDNA upstream of an internal ribosome entry site (IRES) and a human placental alkaline phosphatase reporter gene. LIA-ESix3 contains the full-length mouse Six3 cDNA. LIA-ESixF88E contains the full-length Six3 cDNA with the single amino acid substitution (F to E) at position 88. Each retroviral stock (0.5 µl) (LIA-E, LIA-ESix3 and LIA-ESixF88E) was injected into the eyes of newborn rats. Three weeks later, the retinae were harvested, stained for alkaline phosphatase expression and sectioned. Clones of cells derived from individual retinal progenitor cells were scored for cell number and cell composition. (B) Normal morphology of photoreceptor cells in LIA-ESixF88E-infected cells. (C) When the Six3 protein was overexpressed in the developing retinal progenitor cells, nearly 50% of the clones (see Table 1) exhibited an altered photoreceptor phenotype. For simplicity, we have designated this ‘Clone Type A’. Processes were found in the outer nuclear layer similar to rod photoreceptor processes but the outer segments were absent (arrow) and the termini normally associated with rod photoreceptors were malformed (open arrowhead). The cell bodies in these clones tend to lie at the outer nuclear layer/inner nuclear layer boundary. OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 25 µm.

View larger version (74K): [in a new window]   Fig. 10. Overexpression of Six3, but not of Six3F88E disrupts lens morphogenesis. Stage 10 chicken embryos were electroporated in ovo with the expression vectors indicated, together with a GFP expression vector around the head ectoderm. Twenty-four hours later, embryos were photographed for GFP fluorescence (second row), then fixed and hybridized for -crystallin mRNA (top row) and sectioned along the planes indicated in the top row (EP, third row). The shape of the lens tissue is demarcated by a white line for clarity. In the third row, the non-electroporated control side (left) of the same embryo is shown for reference, which usually bears lower hybridization signals because the side faced the bottom of the tubes during the hybridization process. A and P indicate anterior and posterior sides, respectively. (A) Control embryo electroporated with the insert-less expression vector. (B) After electroporation with Six3, -crystallin-expressing domains and -crystallin-negative domains were seen within the lens tissue (indicated by the line in the inset). Sections of the same embryo showed that the invagination of the lens placode was inhibited and confirmed the segregation of -crystallin-expressing and non-expressing domains within the placode. (C) Co-electroporation of Six3 and Grg5 caused essentially the same effect as Six3 alone. (D) Electroporation with Six3F88E showed no effect.

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