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, 19 (12), 8581-90

Identification of CtBP1 and CtBP2 as Corepressors of Zinc Finger-Homeodomain Factor deltaEF1

Affiliations

Identification of CtBP1 and CtBP2 as Corepressors of Zinc Finger-Homeodomain Factor deltaEF1

T Furusawa et al. Mol Cell Biol.

Abstract

deltaEF1, a representative of the zinc finger-homeodomain protein family, is a transcriptional repressor which binds E2-box (CACCTG) and related sequences and counteracts the activators through transrepression mechanisms. It has been shown that the N-proximal region of the protein is involved in the transrepression. Here we demonstrate that deltaEF1 has a second mechanism of transrepression recruiting CtBP1 or CtBP2 as its corepressor. A two-hybrid screen of mouse cDNAs with various portions of deltaEF1 identified these proteins, which bind to deltaEF1 in a manner dependent on the PLDLSL sequence located in the short medial (MS) portion of deltaEF1. CtBP1 is the mouse orthologue of human CtBP, known as the C-terminal binding protein of adenovirus E1A, while CtBP2 is the second homologue. Fusion of mouse CtBP1 or CtBP2 to Gal4DBD (Gal4 DNA binding domain) made them Gal4 binding site-dependent transcriptional repressors in transfected 10T1/2 cells, indicating their involvement in a transcriptional repression mechanism. When the MS portion of deltaEF1 was used to Gal4DBD and used to transfect cells, a strong transrepression activity was generated, but this activity was totally dependent on the PLDLSL sequence which served as the site for interaction with endogenous CtBP proteins, indicating that CtBP1 and -2 can act as corepressors. Exogenous CtBP1/2 significantly enhanced transcriptional repression by deltaEF1, and this enhancement was lost if the PLDLSL sequence was altered, demonstrating that CtBP1 and -2 act as corepressors of deltaEF1. In the mouse, CtBP1 is expressed from embryo to adult, but CtBP2 is mainly expressed during embryogenesis. In developing embryos, CtBP1 and CtBP2 are expressed broadly with different tissue preferences. Remarkably, their high expression occurs in subsets of deltaEF1-expressing tissues, e.g., cephalic and dorsal root ganglia, spinal cord, posterior-distal halves of the limb bud mesenchyme, and perichondrium of forming digits, supporting the conclusion that CtBP1 and -2 play crucial roles in the repressor action of deltaEF1 in these tissues.

Figures

FIG. 1
FIG. 1
Identification of CtBP1 and CtBP2 as interacting factors with δEF1 and their primary structures. (A) Interaction of CtBP1 and CtBP2 with the middle portion of δEF1 in yeast cells. (Left) Portions of δEF1 protein fused to Gal4DBD. Amino acid residue numbers of the termini are indicated. N-fin, HD, and C-fin indicate N-proximal zinc finger cluster, homeodomain, and C-proximal zinc finger clusters, respectively. (Middle) Growth of yeast cells cotransformed with plasmids expressing Gal4AD-CtBP and Gal4DBD-δEF1 on plates containing 5 or 15 mM 3-AT. At 5 mM 3-AT, cells carrying Gal4DBD fused to MS-δEF1 or ML-δEF1 grew well, while those with full-length δEF1 (Full-δEF1) showed attenuated growth. At 15 mM 3-AT, growth of cells with Gal4DBD–Full-δEF1 was totally inhibited, and that of cells with Gal4DBD–ML-δEF1 was reduced. The results indicate that CtBP interacts with the MS portions of δEF1, but the transcriptional activation levels attained by bait-prey interaction are variable using MS, ML, and Full portions of δEF1 in the order MS > ML > Full. This was confirmed by measurement of β-galactosidase (β-gal) activity in a liquid culture of each yeast colony (right). (B) Alignment of amino acid sequences of mouse and human CtBP1, human CtBP2, and dCtBP. Identical amino acid residues are highlighted; similar residues are shaded. The first methionine codon of the mouse CtBP1 open reading frame which satisfies Kozak’s consensus was designated the initiation codon. In the mouse CtBP2 cDNA sequence, a stop codon immediately precedes the coding sequence. The human CtBP2 sequence is from the EST (expressed sequence tag) database. Nucleotide accession numbers for human CtBP1, human CtBP2, and dCtBP cDNAs are g1063638, g2909777, and g2950374, respectively.
FIG. 2
FIG. 2
Binding of CtBP proteins is dependent on the PLDLSL sequence of δEF1. (A) Conservation of the CtBP binding motif PLDLSL among mouse δEF1 and adenovirus (Ad) E1A proteins. (B) β-Galactosidase (β-gal) activities generated by interaction between δEF1 (MS) and CtBP1/2 in the yeast two-hybrid assay. Yeast cells cotransformed with Gal4AD-CtBP1/CtBP2 and Gal4DBD-MS (normal or mutant ASDLSL, PLASSL, or PLDLAS were grown in liquid culture, and β-galactosidase activity was measured. (C) Binding of CtBP1 and CtBP2 to full-length δEF1 in vitro. GST, GST-CtBP1, or GST-CtBP2 bound to glutathione beads was mixed with in vitro-translated and N-terminally Xpress-tagged δEF1 (Input). The bound proteins were analyzed by Western blotting using an anti-Xpress antibody. The arrow indicates the position of Xpress-δEF1 on the blot. (D) Interaction of full-length or truncated forms of CtBP proteins with δEF1 in yeast cells. Full-length CtBP1/2 or fragments thereof (left) were fused with Gal4AD, and interaction with Gal4DBD-MS was assessed by growth of yeast cells on plates containing 10 mM 3-AT.
FIG. 3
FIG. 3
CtBP proteins exhibit transrepression when bound to DNA by fusion with Gal4DBD or by interaction with MS-Gal4DBD. (A) Structure of the luciferase reporter plasmid 4xGAL-TK-Luc. (B) Effects of gal4DBD-CtBP fusion proteins indicated at the left. The effect on transcription of the 4xGAL-TK-Luc reporter gene was assessed by expression of luciferase in transfected 10T1/2 cells (right panel; average of three transfections). CtBP1 or CtBP2 can actively repress the reporter activity only when fused to Gal4DBD; Gal4DBD or CtBP1/2 alone had no appreciable effect. (C) Effects of MS-gal4DBD fusion proteins and of exogenous CtBP1/2 on expression of 4xGAL-TK-Luc in 10T1/2 cells.
FIG. 4
FIG. 4
CtBP proteins act as corepressors of δEF1. (A) Structure of the reporter plasmid (MCK4R-δ51-Luc) carrying four copies of the E2-box element (R) of the mouse MCK enhancer and basal promoter sequence (−51 to +57) of the chicken δ1-crystallin gene (15). (B) Effect of CtBP1 on transcriptional repression by δEF1. 10T1/2 cells were transfected with the reporter plasmid (0.3 μg) and with effector plasmids for expression of MyoD (0.75 μg), δEF1 or its mutant PLASSL (0.25 μg), and CtBP1. Relative luciferase expression levels averaged over three transfection experiments are indicated. (C) Western blot analysis of the wild-type and mutant (PLASSL) forms of δEF1 expressed in COS-7 cells. An equivalent amount of nuclear extract from the transfected cells was analyzed by Western blotting using anti-δEF1 antibodies. In vitro-translated δEF1 protein was included as the size marker. Exogenous δEF1 of mouse origin, both wild-type and PLASSL mutant forms, produced the same band intensities. The bands of exogenous δEF1 (mouse) were positioned slightly lower than that of endogenous simian δEF1 (open arrowhead), presumably reflecting the lack of exon 3 in rodent δEF1 (30).
FIG. 5
FIG. 5
Northern blot analysis of the expression of CtBP1 and CtBP2 of the mouse in comparison with δEF1. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) message is used to control the RNA loaded on the filter. CtBP1 has a transcript of 2.4 kb and is expressed from embryo to adult stages and widely among adult organs. CtBP2 transcript has two sizes, 2.8 kb (major) and 5.6 kb (minor), and expression in the embryo is much stronger than in the adult tissues. 10T1/2 cells express both CtBP1 and CtBP2 strongly, probably reflecting their origin of 13.5-day mouse embryo (25). δEF1 expression represented by the 5.6-kb transcript occurs among the various developmental stages, adult organs, and 10T1/2 cells.
FIG. 6
FIG. 6
Whole-mount in situ hybridization analysis of CtBP1 and CtBP2 expression in mouse embryos. (A and B) Side views of 10.5-day embryos hybridized with CtBP1 antisense probe (A) and CtBP2 antisense probe (B). (C and D) Dorsal views of the same embryos. The insets show embryos hybridized with the corresponding sense probes. Major expression sites: CG, cephalic ganglia; DRG, dorsal root ganglia; PL, posterior-distal portion of the limb bud mesenchyme; SC, spinal cord; H, heart. The heart lacks expression of CtBP1/2. (E and F) Forelimbs and trunks of 12.5-day embryos hybridized with the CtBP1 (E) or CtBP2 (F) probe. Some of the perichondria are marked by arrowheads. PMG, primordia of mammary glands.

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