Zeng W et al. (2010), Xenopus RCOR2 (REST corepressor 2) interacts wi...

XB-ART-41362

Biochem Biophys Res Commun 2010 Apr 16;3944:1024-9. doi: 10.1016/j.bbrc.2010.03.115.

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Xenopus RCOR2 (REST corepressor 2) interacts with ZMYND8, which is involved in neural differentiation.

Zeng W , Kong Q , Li C , Mao B .

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Regulation of neuronal gene expression is critical to nervous system development. REST (RE1-silencing transcription factor) regulates neuronal gene expression through interacting with a group of corepressor proteins including REST corepressors (RCOR). Here we show that Xenopus RCOR2 is predominantly expressed in the developing nervous system. Through a yeast two-hybrid screen, we isolated Xenopus ZMYND8 (Zinc finger and MYND domain containing 8) as an XRCOR2 interacting factor. XRCOR2 and XZMYND8 bind each other in co-immunoprecipitation assays and both of them can function as transcriptional repressors. XZMYND8 is co-expressed with XRCOR2 in the nervous system and overexpression of XZMYND8 inhibits neural differentiation in Xenopus embryos. These data reveal a RCOR2/ZMYND8 complex which might be involved in the regulation of neural differentiation.

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Species referenced: Xenopus laevis
Genes referenced: lgals4.2 myc prl.2 rcor2 snai2 sox2 uqcc6 zmynd8

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	Fig. 1. Temporal and spatial expression patterns of Xenopus RCOR2. (A) The schematic structures of XRCOR1 and XRCOR2. (B) RT-PCR analysis of the developmental expression of XRCOR2. E, unfertilized egg; N, negative control. (Câ€“G) Whole-mount in situ hybridization analysis of XRCOR2 expression. (C) Four-cell stage, animal pole to the top. (D) Stage 8, animal pole to the top. (E) Stage 15, dorsal view, anterior to the top. np, neural plate. (F) Stage 25, lateral view, anterior to the left. (G) Stage 30, lateral view, anterior to the left. br, branchial arches; op, optic vesicle; ot, otic vesicle.
	Fig. 2. XRCOR2 interacts with XZMYND8 and XREST. (A) The schematic structure of XZMYND8. P, PHD domain; BR, bromodomain; PW, PWWP domain; M, MYND domain. (B) The wild-type XZMYND8 and the XZMYND8âMYND can be immunoprecipitated by XRCOR2, but not the XZMYND8â4MYND. WCL, whole cell lysate; IP, immunoprecipitation; IB, immunoblot. (C) In the reverse co-IP assay, XRCOR2 is also pulled-down by XZMYND8 and the MYND domain, but not XZMYND8â4MYND. (D) Co-IP result showing that XZMYND8 can be pulled-down by XRCOR1. (E) Co-IP result showing that the C-terminus of XREST interacts with XRCOR2. (F) XRCOR2 (red) colocalizes with XZMYND8 (green) in the nuclei of transfected HeLa cells. The nuclei were stained using DAPI. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
	Fig. 3. XRCOR2 and XZMYND8 have repressor activities in 293T cells. (A) Schematic diagram of the pGAL4-TK-Luc reporter construct. GAL4 BE, GAL4 binding elements. (B) Luciferase reporter assay showing the repressor activities of XRCOR2 and XZMYND8. 293T cells were transiently transfected with 100 ng pGAL4-TK-Luc, 10 ng pRL-TK and increasing amounts (1 ng, 5 ng, and 50 ng) of GAL4-ZMYND8, GAL4- RCOR2, and GAL4-ZMYND8â4MYND (4M in the figure) plasmids. Rep, reporter plasmids alone; Co, control using the empty pCMV-BD vector (100 ng); RLU, relative light units. (C) Luciferase reporter assay showing the co-operative effect between XRCOR2 and XZMYND8. 293T cells were transiently transfected with plasmids as indicated. Doses used: pGAL4-TK-Luc, 100 ng; pRL-TK, 10 ng; GAL4-ZMYND8 and GAL4-ZMYND8â4MYND, 5 ng; Myc-RCOR2, 50 ng.
	Fig. 4. XZMYND8 is involved in neural differentiation. (A) RT-PCR analysis of the temporal expression of Xenopus ZMYND8. E, unfertilized egg; N, negative control. (Bâ€“F) Whole-mount in situ hybridization analysis of XZMYND8. (B) Four-cell stage, animal pole to the top. (C) Stage 8, animal pole to the top. (D) Stage 15, dorsal view, anterior to the top. np, neural plate. (E) Stage 25, lateral view, anterior to the left. br, branchial arches; op, optic vesicle; ot, otic vesicle. (F) Stage 30, lateral view, anterior to the left. (Gâ€“L) Overexpression of ZMYND8 in Xenopus laevis embryos leads to neural tube defects. Two hundred picograms of XZMYND8 mRNA was injected into 1 dorsal blastomere at fourcell stage. (G) A stage 17 embryo showing that the neural plate of the injected side (right) fails to fold towards the midline. Dorsal view, anterior to the top. (H) Neural tube defects in stage 21 embryos injected with XZMYND8 mRNA. Dorsal view, anterior to the top. (I) The expression domain of Sox2 expanded on the ZMYND8 mRNA injected side (right). (Jâ€“L) The expressions of N-tubulin (J), Slug (K), and Krox-20 (L) were all down-regulated on the injected sides (white arrowheads). The black arrowheads show the normal expression domains of the marker genes on the control sides. LacZ mRNA was co-injected for tracing and the injected sides were stained in red (right sides in (Gâ€“L)). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
	zmynd8 (zinc finger, MYND-type containing 8) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, horizontal view, animal up.
	zmynd8 (zinc finger, MYND-type containing 8) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 8, horizontal view, animal up.
	zmynd8 (zinc finger, MYND-type containing 8) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior up.
	zmynd8 (zinc finger, MYND-type containing 8) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
	zmynd8 (zinc finger, MYND-type containing 8) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.