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Structurally related Arabidopsis ANGUSTIFOLIA is functionally distinct from the transcriptional corepressor CtBP

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Abstract

ANGUSTIFOLIA (AN) controls leaf morphology in the plant Arabidopsis thaliana. Previous studies on sequence similarity demonstrated that the closest proteins to AN are members of animal C-terminal-binding proteins (CtBPs) found in nematodes, arthropods, and vertebrates. Drosophila CtBP (dCtBP) functions as a transcriptional corepressor for deoxyribonucleic acid (DNA)-binding repressors containing the short amino acid motif, PXDLS, to regulate tissue specification and segmentation during early embryogenesis. It has previously been shown that AN was thought to repress transcription similar to the function of CtBPs; however, AN lacks some of the structural features that are conserved in animal CtBPs. In this paper, we examined whether AN is functionally related to dCtBP. Firstly, we re-examined sequence similarity among AN and various CtBPs from several representative species in the plant and animal kingdoms. Secondly, yeast two-hybrid assays demonstrated that AN failed to interact with an authentic CtBP-interacting factor, adenovirus E1A oncoprotein bearing the PXDLS motif. Thirdly, AN tethered to DNA was unable to repress the expression of reporter genes in transgenic Drosophila embryos. Fourthly, overexpression assays suggested that dCtBP and AN function differently in Drosophila tissues. Finally, AN failed to rescue the zygotic lethality caused by dCtBP loss-of-function. These data, taken together, suggest that AN is functionally distinct from dCtBP. Likely, ancestral CtBPs acquired corepressor function (capability of both repression and binding to repressors containing the PXDLS motif) after the animal–plant divergence but before the protostome–deuterostome split. We therefore propose to categorize AN as a subfamily member within the CtBP/BARS/RIBEYE/AN superfamily.

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Acknowledgments

We thank Anna Di Gregorio and Monn M. Myat for critical reading of the manuscript and Susan Lee for technical help. We also thank Eric Lai, Makoto Sato, Richard H. Goodman, Wilhelm Grussem, M. Sekine, and the Bloomington stock center for providing fly strains and plasmids. H.A. is supported by postdoctoral fellowships from the Uehara Memorial Foundation and the Japan Society for the Promotion of Science (JSPS). This work was supported by grants from the Charles A. Frueauff Foundation and from the Speaker’s Fund for Biomedical Research awarded by the City of New York to Y.N. and by a Grant-in-Aid from JSPS and grants from the Toray Science Foundation and the Sumitomo Foundation to H.T.

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Author notes

  1. Kiu-Hyung Cho & Gyung-Tae Kim

    Present address: Division of Molecular Biotechnology, Don-A University, Busan, 604-714, South Korea

  2. Dereje Negeri

    Present address: Signalling pathways, cell biology and cancer, Max-Delbrueck Center for Molecular Medicine (MDC) Berlin-Buch, Robert-Roessle Str. 10, 13125, Berlin, Germany

Authors and Affiliations

  1. Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, Box 60, New York, NY, 10021, USA

    Mark D. Stern, Hitoshi Aihara, Giorgio A. Roccaro, Elizabeth Guevara, Huan Huan Sun, Dereje Negeri & Yutaka Nibu

  2. National Institute for Basic Biology, National Institutes of Natural Sciences, Myodaiji-cho, Okazaki, Aichi, 444-8585, Japan

    Kiu-Hyung Cho, Gyung-Tae Kim & Hirokazu Tsukaya

  3. Graduate School of Science, University of Tokyo, Science Build no. 2, 7-3-1 Hongo, Tokyo, 113-0033, Japan

    Gorou Horiguchi & Hirokazu Tsukaya

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Fig. S1

Amino acid alignment of Ce-CtBP-S and Ce-CtBP-L. Amino acid sequences of Ce-CtBP-L (accession number NP_508983) and Ce-CtBP-S (accession number T34290) were compared (GIF 72.6 KB)

High-resolution image file (TIFF 27.8 MB)

Fig. S2

Amino acid alignment of Sp-CtBP-S and Sp-CtBP-L. Amino acid sequences of Se-CtBP-L (accession number XP_001176951) and Sp-CtBP-S (accession number XP_780717) were compared. Sp-CtBP-S seems to be a truncation of the THAP domain, as this shorter isoform starts from a methionine residue located at position 105 aa in reference to Sp-CtBP-L (GIF 50.7 KB)

High-resolution image file (TIFF 20.4 MB)

Fig. S3

Comparison of N-terminal regions between Ce-CtBP-L and Sp-CtBP-L. The N terminus of Ce-CtBP-L contains the THAP domain (1–89 aa), which may bind to DNA. This domain is well conserved in the N terminus of Sp-CtBP-L (GIF 10.8 KB)

High-resolution image file (TIFF 6.74 MB)

Fig. S4

dCtBP binds to the PXDLS motifs. GST pull-down assays as well as mutations of the PXDLS motifs of Knirps and Snail by alanine substitution were previously described (Nibu et al. 1998a,b). A full-length wild-type Knirps protein labeled with 35S-methionine was incubated with the GST-dCtBP 383aa fusion protein or with GST alone. Bound complexes were loaded onto a polyacrylamide gel. For comparison, 10% of the total amount of 35S-labeled Knirps used in the binding reaction (lane 10% input). Knirps does not bind GST (lane GST on the left side of the panel indicated as wild-type) but interacts with GST-dCtBP (lane GST/dCtBP 383). Four alanine substitutions in the PXDLS motif (conversion from PMDLS to AAAAS) of Knirps in the full-length context diminish binding to GST-dCtBP, consistent with previous results obtained with a truncated form of Knirps (Keller et al. 2000; Nibu et al. 1998b). The 35S-labeled full-length wild-type Snail proteins interact with GST-dCtBP but not with GST (left side of the panel indicated as wild-type). A mutant form of Snail lacking both its PXDLS motifs (in which the normal motif, PQDLS and PEDLS, were converted to AQAAA and AEAAA, respectively; AQAAA AEAAA) interacts with GST-dCtBP more weakly than a form of Snail mutated mutations only in the second motif (Nibu et al. 1998b) (GIF 26.4 KB)

High-resolution image file (TIFF 5.66 MB)

Fig. S5

AN fails to interact with the retinoblastoma (Rb) tumor suppressor in yeast two-hybrid assays. A DB-Rb plasmid (the GAL4-DNA-binding domain fused to Rb in pAS2-1) and an AD-D3 plasmid (Cyclin D3 fused to the GAL4-activation-domain in pACT2) were kindly provided by Drs. W. Grussem and M. Sekine, respectively. The method described in Fig. 2 was employed. Yeast colonies carrying both AD-AN and BD-Rb plasmids did not grow in the selective medium (SD-Trp-Leu-His, containing 25 mM 3-AT), indicating that AN fails to interact with Rb (right panel). As a positive control, Rb was shown to interact with Cyclin D3 (right panel) (GIF 29.2 KB)

High-resolution image file (TIFF 5.09 MB)

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Stern, M.D., Aihara, H., Cho, KH. et al. Structurally related Arabidopsis ANGUSTIFOLIA is functionally distinct from the transcriptional corepressor CtBP. Dev Genes Evol 217, 759–769 (2007). https://doi.org/10.1007/s00427-007-0186-8

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