Hwang YS et al. (2003), Active repression of organizer genes by C-termi...

XB-ART-4927

Biochem Biophys Res Commun 2003 Aug 15;3081:79-86. doi: 10.1016/s0006-291x(03)01321-4.

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Active repression of organizer genes by C-terminal domain of PV.1.

Hwang YS , Lee HS , Roh DH , Cha S , Lee SY , Seo JJ , Kim J , Park MJ .

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PV.1, a homeotic protein, ventralizes dorsal mesoderm and inhibits neuralization by mediating BMP-4 signaling in Xenopus embryo. In our previous report antimorphic PV.1 causes a secondary axis by inducing the ectopic organizer. We analyzed the structure of this transcription factor through domain level assessment. In a phenotype-inducing test, half of the N-terminus at the N-terminal side was unessential for inducing ventralization of embryos. We examined the transacting activity of several regions of PV.1 utilizing GAL4 hybrid system. The C-terminal region/GAL4DBD (DNA binding domain) exhibited strong repressive activity on a reporter gene (operator/promoter/reporter; Gal4-TK-luc) as much as the whole polypeptide/GAL4DBD, whereas the N-terminal region/GAL4DBD showed only modest repression. The results suggest that PV.1 functions as a transcriptional repressor and this repressive activity is localized mostly to the C-terminal region. Additional characterizations of N- and C-terminus with respect to the effects on the expression of other genes are described.

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Species referenced: Xenopus laevis
Genes referenced: lgals4.2 nog ventx1

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	Fig. 1. Schematic diagram of deletion mutants of PV.1 To characterize the functional domain of PV.1, we constructed several deletion mutants and numbered them (1)–(7). (A) Although no region of PV.1 (as indicated; numbers represent amino acids) but homeodomain (HD) shows meaningful amino acid sequence identity in conserved domain database, there are three characteristic regions. N-terminus comprises two distinct regions, the upper half region (upper 1/2N has PHIPCAPQPLPPNKYAKE sequence at its C-terminal side, which includes six prolines within 18 amino acids.) and the other serine-rich acidic down half region (19 serines within 68 amino acids). The whole C-terminal arm is rich in proline (21 prolines within 94 amino acids). (B) Representation of different portions of PV.1 which are encoded by the injected RNAs.
	Fig. 2. The phenotype-inducing activities of deletion mutants of PV.1. Four-cell stage embryos were dorsally microinjected (ventral injections had little effects; described only in Table 1) with indicated mRNAs and allowed to grow to stage 28–29. b-Galactosidase (0.125 ng/blstmr; same below, otherwise described) injected control (I) embryos were normal. The dorsal injection of PV.1 (A), 1/2NHC-PV1 (C), and HC-PV1 (E) ventralized embryos like BMP-4 injection (H) whereas dorsal injection of NH-PV1 (B), 1/2NH-PV1 (D; 0.5 ng/blstmr), H-PV1(F; 0.5 ng/ blstmr), and N-PV1(G) did not. (D) and (F) show the small portion exhibiting the most extreme phenotype among resulted embryos (see Table 1).
	Fig. 3. Schematic diagram of effector constructs and relative luciferase activity of GAL4-TK-luc. (A) We generated fusion proteins by attaching G4DBD (GAL4 DNA binding domain) to deletion constructs of PV.1. We then named and numbered (noted in parentheses) them after the deletion constructs in Fig. 1. G4DBD-18a.a (10) (18 random amino acid-peptide; EFIHSRSALYSPAAPPPYstop) was introduced to count out a probable transacting event imposed by any polypeptide fused to G4DBD and, consequently, to confirm the ‘active repression’ mechanism. Each effector mRNA (250 pg/blstmr) was co-injected with GAL4-TK-luc (25 pg/blastmr) plasmid DNA at the animal pole of each blastomere of two-cell stage embryos. (C) Four repeated copies of a GAL4-binding upstream activating sequence (UASG; 50-CGACGGAGTACTGTCCTCCGAGCT) were cloned into a TK promoter/luciferase (TK-luc) reporter that contains the herpes virus thymidine kinase promoter ()105/+51) to enhance the basal transcription level of luciferase. (B) At stage 8.5, animal caps were explanted and cultured for four more hours and then harvested for luminescence measurement. Those G4DBD-unfused effector RNAs (PV1, NH-PV1, HC-PV1 and N-PV1) served as controls to count out the nonspecific DNA binding or toxicity of effector proteins. GAL4-TK-luc exhibited highly enhanced reporter gene expression. G4DBD-PV1 (1), G4DBD-1/2NHC-PV1 (3), G4DBD-HC-PV1 (5), and G4DBD-C-PV.1 (8) significantly reduced this enhancement when compared to G4DBD-18a.a while G4DBD-NHPV1 (2), G4DBD-N-PV1 (7), and G4DBD-1/2N-PV1 (9) did not.
	Fig. 4. HC-PV1 ventralizes dorsal mesoderm. Four-cell stage embryos were injected with PV.1, NH-PV1, and HC-PV1 mRNAs at their dorsal equatorial region. Dorsal marginal zone tissue was dissected at an early gastrula stage and cultured until the siblings reached stage 11 or stage 25 for RNA extraction. PV.1 and HC-PV1 overexpression induces ventral marker genes (A) and suppresses dorsal specific early genes (B) while NH-PV1 fails to do this.
	Fig. 5. Luciferase assay for XFD-10 promoter and Noggin promoter in response to deletion mutants of PV.1. mRNAs encoding NH-PV1, HC-PV1, and wild type PV.1 (125 pg/blastomere, respectively) were co-injected with a reporter construct DNA (XFD-luc: 20 pg/blastomere, Nog-luc: 10 pg/ blastomere; for details see Materials and methods) and reference plasmid DNA (pRL-TK; 10 pg/blastomere) at the dorsal equatorial region of fourcell stage embryos. Luciferase activity was measured at stage 12. Wild type PV.1 reduced XFD-10 promoter (A) and noggin promoter (B) activity. HCPV1 also reduced both promoter activities to the same level in noggin promoter assay (B) or to the lower level in XFD-luc promoter assay (A) as to wild type PV.1.