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Biochem Biophys Res Commun
2008 Dec 05;3771:73-8. doi: 10.1016/j.bbrc.2008.09.116.
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The function of the Aristaless-related homeobox (Arx) gene product as a transcriptional repressor is diminished by mutations associated with X-linked mental retardation (XLMR).
Fullenkamp AN
,
El-Hodiri HM
.
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The Aristaless-related homeobox (Arx) is mutated in patients with X-linked mental retardation and a range of other neurological diseases. The molecular consequences of these mutations are unclear. Here, we show that two disease-associated mutations disrupt the function of Arx as a transcriptional repressor. We found that Arx contains two independent repression domains: an N-terminal octapeptide motif/engrailed homology domain and a novel domain located in the C-terminus. The octapeptide motif functions through interaction with members of the Groucho family of co-repressors. The C-terminal domain functions through interaction with C-terminal binding protein (CtBP).
Fig. 1.
Xenopus Arx functions as a transcriptional repressor. A. Schematic representation of Arx conserved domains. OP, octapeptide motif; HD, homeodomain; OAR, orthopedia-Aristaless-Rx domain; CR, conserved Region. (B–D) Luciferase assays performed using lysates from Cos 7 cells co-transfected (B–D) or Xenopus embryos injected (C) with wt or mutated Gal4 DBD-Arx expression plasmid, effectors as shown, and a Gal4-UAS-Luc reporter plasmid. (B) Luciferase assay performed using lysates from stage 10 Xenopus embryos micro-injected with 10 pg of RNA encoding Gal4-DBD-Arx-OP or L33P and a Gal4-UAS-Luc reporter plasmid. The graph represents a trend observed from three experiments. (C) Disruption of the OP by deletion or mutation (L33P) results in reduced repression activity. (D) Deletion of portions of the Arx C-terminus results in reduced repression activity. ∗p < 0.05.
Fig. 2.
The Octapeptide mediates repression via the Groucho family of co-repressors. (A–D) Whole mount in situ hybridization showing expression of Xenopus Groucho homolog Esg-1 in the brain of varying stages of Xenopus embryos at stages 21, 27, 32, and 35. Ey, eye; fb, forebrain; mb, midbrain. (E) The Xenopus Groucho homolog Grg4 enhances the repression of Arx. Luciferase assay performed using lysates from stage 10 Xenopus embryos that were co-injected with RNA encoding Gal4-DBD-Arx and xGrg4 and a Gal4-UAS-Luc reporter plasmid. The graph represents the trend observed from three experiments. (F) Luciferase assay performed using lysates from Cos7 cells co-transfected with Gal4 DBD-Arx, Gal4 DBD-Arx L33P, or Gal4 DBD-Arx-OP and xGrg4 expression plasmids along with a Gal4-UAS-Luc reporter plasmid. ∗p < 0.05.
Fig. 3.
C-terminal binding protein 1 (xCtBP1) mediates repression of Arx. (A–C) Double whole mount in situ hybridization showing overlapping expression of Arx and and xCtBP in the developing brain of Xenopus embryos at stages 21, 32, and 35. Ey, eye; fb, forebrain. Arx is stained magenta, xCtBP is stained turquoise, and areas of overlap are purple. (D) Luciferase assay performed using lysates from Cos7 cells co-transfected with Gal4 DBD-Arx and xCtBP expression plasmids and a Gal4-UAS-Luc reporter plasmid. ∗p < 0.05. (E) Co-immunoprecipitation of CtBP performed using lysates prepared from Cos7 cells transfected with HA-Arx. Control IP was performed using mouse IgG.
Fig. 4.
The ORD mediates repression by CtBP1. (A) Luciferase assay performed using lysates prepared from Cos7 cells co-transfected with Gal4 DBD-Arx, Gal4 DBD-Arx-OP/Ex5 truncation, or Gal4 DBD-Arx-OP/398 truncation and xCtBP expression plasmids along with a Gal4-UAS-Luc reporter plasmid. ∗p < 0.05. (B) Co-immunoprecipitation of Groucho performed using lysates prepared from Cos7 cells transfected with HA-Arx. Control IP was performed using mouse IgG.
Supplementary Fig. 1.
(A) Arx functions as a transcriptional repressor. Reporter activity was significantly repressed at all doses (p < 0.05). (B) Deletion of a portion of the Arx C-terminus results in reduced repression activity. ∗p < 0.05.
Supplementary Fig. 2.
(A) Schematic representation of mutation constructs used in luciferase assays. (B) Conservation of Arx protein sequence in human, mouse, Xenopus and Zebrafish indicating the region containing the ORD and the putative CtBP binding site within it.
