Hiratani I et al. (2003), Selective degradation of excess Ldb1 by Rnf12/R...

XB-ART-4965

Development 2003 Sep 01;13017:4161-75. doi: 10.1242/dev.00621.

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Selective degradation of excess Ldb1 by Rnf12/RLIM confers proper Ldb1 expression levels and Xlim-1/Ldb1 stoichiometry in Xenopus organizer functions.

Hiratani I , Yamamoto N , Mochizuki T , Ohmori SY , Taira M .

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The Xenopus LIM homeodomain (LIM-HD) protein, Xlim-1, is expressed in the Spemann organizer and cooperates with its positive regulator, Ldb1, to activate organizer gene expression. While this activation is presumably mediated through Xlim-1/Ldb1 tetramer formation, the mechanisms regulating proper Xlim-1/Ldb1 stoichiometry remains largely unknown. We isolated the Xenopus ortholog (XRnf12) of the RING finger protein Rnf12/RLIM and explored its functional interactions with Xlim-1 and Ldb1. Although XRnf12 functions as a E3 ubiquitin ligase for Ldb1 and causes proteasome-dependent degradation of Ldb1, we found that co-expression of a high level of Xlim-1 suppresses Ldb1 degradation by XRnf12. This suppression requires both the LIM domains of Xlim-1 and the LIM interaction domain of Ldb1, suggesting that Ldb1, when bound to Xlim-1, escapes degradation by XRnf12. We further show that a high level of Ldb1 suppresses the organizer activity of Xlim-1/Ldb1, suggesting that excess Ldb1 molecules disturb Xlim-1/Ldb1 stoichiometry. Consistent with this, Ldb1 overexpression in the dorsal marginal zone suppresses expression of several organizer genes including postulated Xlim-1 targets, and importantly, this suppression is rescued by co-expression of XRnf12. These data suggest that XRnf12 confers proper Ldb1 protein levels and Xlim-1/Ldb1 stoichiometry for their functions in the organizer. Together with the similarity in the expression pattern of Ldb1 and XRnf12 throughout early embryogenesis, we propose Rnf12/RLIM as a specific regulator of Ldb1 to ensure its proper interactions with LIM-HD proteins and possibly other Ldb1-interacting proteins in the organizer as well as in other tissues.

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Species referenced: Xenopus
Genes referenced: cer1 chrd dkk1 foxa4 foxp1 gal.2 gsc ldb1 lhx1 lhx3 lhx5 lmo2 lmo4.2 mix1 not otx2 pcdh8 rlim rnf13 tbx2 tbxt tubb2b

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	Fig. 2. Temporal and spatial expression patterns of XRnf12. (A) Northern analysis. The 3.5 kb transcript is expressed throughout early embryogenesis, while the 3.0 kb transcript disappears at stage 11. 18S rRNA stained with ethidium bromide served as a loading control. Numbers at the top are developmental stages. (B-D) Overlapping expression of Xlim-1, XRnf12 and Ldb1 in the gastrula mesoderm as revealed by whole-mount in situ hybridization of sagittally bisected gastrula embryos (stage 10.5). Animal side is to the top, dorsal to the right. Arrowhead, blastopore. (E-J) Co-expression of XRnf12 and Ldb1 in neurula and tailbud stage embryos. Dorsal is to the top, anterior to the left. Arrowhead indicates profundal-trigeminal placodal area; numbers in the bottom right of each panel are the developmental stages. (K-P) Co-expression of XRnf12 and Ldb1 as revealed by cross sections of stained tailbud embryos (stage 26). Upper, or left, panels are bright-field images; lower, or right, panels show DAPI staining. XRnf12 (EG,K-M) and Ldb1 (H-J,N-P) are similarly expressed throughout early embryogenesis. Turquoise staining inside of the embryos shown in the cross sections resulted from BCIP staining as NBT was depleted by strong staining in the epidermis. Both genes are expressed similarly in the neural tube along the AP axis. ba, branchial arches; dc, diencephalon; ey, eye; hb, hindbrain; hm, head mesenchyme; nc, neural crest; ov, otic vesicle; p, pronephros; pf, pronephric field; sc, spinal cord.
	Fig. 8. Effects of Ldb1 or XRnf12 overexpression on organizer gene expression. Embryos injected dorsally with the mRNAs indicated were scored for expression of various marker genes in the organizer as assayed by whole-mount in situ hybridization. Genes categorized in group 1 (A-E, gsc, chd, Xotx2, XPAPC and cer) are downregulated by overexpression of either Ldb1 or XRnf12, whereas genes categorized in group 2 (F,G, XFKH1 and Xnot) are downregulated only by XRnf12, and those in group 3 (H,I, dkk1 and Mix.1) are downregulated only by Ldb1. Xbra (J) is only slightly affected by Ldb1 overexpression. Numbers indicate the frequency of the phenotype observed: numbers in red indicates downregulation, whereas numbers in black or white indicates normal expression. Note that downregulation of gene expression by either Ldb1 or XRnf12 overexpression was restored upon co-expression of both, and that the rescuing effect of XRnf12 co-expression was RING-dependent (except for dkk1, see Discussion). nβ-gal mRNA was coinjected as a lineage tracer, stained in red. Amounts of mRNAs (ng/embryo): nβ-gal, 0.06; β-globin, 4.0; Ldb1, 4.0; XRnf12, 2.0 or 4.0; XRnf12 constructs coinjected with Ldb1, 1.0 or 2.0.
	A high level of Xlim-1 suppresses XRnf12-mediated degradation of Ldb1 through interaction with Ldb1. (A) Effects of Xlim-1 on XRnf12-mediated degradation of Ldb1. The experimental design is the same as in Fig. 4A,B. Xlim-1 increases the expression level of FLAG-Ldb1 dose-dependently in the presence (lanes 3-7) or absence (lanes 8-12) of XRnf12. Comparison between lanes 6 and 7, and lanes 11 and 12 suggests that Xlim-1 suppresses Ldb1 degradation by XRnf12. See text for details. Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; XRnf12, 0.25; Xlim-1, 0.25 (lanes 4,9), 0.5 (lanes 5,10), 1.0 (lanes 6,11), 2.0 (lanes 7,12). (B) The LIM domain-containing fragment (ABL60) of Xlim-1 is sufficient for the suppression of XRnf12-mediated degradation of Ldb1. A series of Xlim-1 constructs depicted in E were tested at the highest dose used in A (lanes 7,12) for their ability to block Ldb1 degradation by XRnf12. ABL60, which contains the LIM domains, efficiently blocks Ldb1 degradation whereas other constructs does not. Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; XRnf12, 0.25; Xlim-1 constructs, 2.0. (C) LIM-only protein LMO2 also efficiently blocks Ldb1 degradation in a different set of experiments designed as in B. (D) Interactions between 35S-labeled XRnf12 and a series of GST-Xlim-1 constructs depicted in E were analyzed by GST pull-down assay. XRnf12 shows weak interactions with GST-HD27 and GST-ΔNA, while other GST-Xlim-1 constructs shows little or no interaction with XRnf12. GST-Ldb1 shows stronger interaction with XRnf12 than GST-Xlim-1 does. XRnf12 also shows weak self-interaction with GST-XRnf12ΔN (aa 282-616). GST alone serves as a negative control. Coomassie brilliant blue staining (lower panel) shows comparable amounts of GST fusion proteins (indicated by dots) used in the assay. (E) Representation of the GST-Xlim-1 constructs used for mapping experiments and the summary of the results shown in D. The homeodomain-containing region (aa 178-265) of Xlim-1 is necessary and sufficient for the interaction with XRnf12. A, B, LIM domains A and B; HD, homeodomain; n.d., not done; numbers, amino acid positions. (F) The LIM interaction domain of Ldb1 is required for the suppression of XRnf12-mediated Ldb1 degradation by Xlim-1 as revealed by the use of FLAG-Ldb1ΔC. XRnf12 causes degradation of Ldb1ΔC in a RING-dependent manner, which is not suppressed by Xlim-1 or ABL60. To avoid an overlap with a non-specific band, we used anti-Ldb1/CLIM2 (N-18) antibody in F to detect FLAG-Ldb1ΔC instead of anti-FLAG antibody used in the rest of the experiments in Fig. 5. Amounts of mRNAs injected (ng/embryo): FLAG-Ldb1ΔC, 0.5; XRnf12 constructs, 0.5; Xlim-1 constructs, 2.0.
	Characterization of XRnf12. (A) Amino acid sequence alignment of XRnf12, XRnf12B, and XRnf12C, as well as mouse and chick Rnf12. Note the repetitive heptamers present in XRnf12B and XRnf12C. A PDZ domain-binding motif is present in the C terminus. NLS, nuclear localization signal; asterisks, sites of mutation in the RING mutant XRnf12(HC>AA). DDBJ Accession Numbers are: XRnf12 (AB114039), XRnf12B (AB114040) and XRnf12C (AB114041). (B) Schematic representation of mRnf12, XRnf12, XRnf12B, and XRnf12C. (C) The N-terminal region (aa 1-283) of XRnf12 interacts with LIM homeodomain proteins and Ldb1. GST pull-down assay was performed with 35S-labeled Xlim-1, Xlim-3, Xlim-5, Ldb1 and Ldb1ΔC. While GST XRnf12ΔC interacts with all of the proteins tested, GST XRnf12ΔN shows little interaction. GST and GST Ldb1 serves as negative and positive controls, respectively.
	XRnf12 suppresses axis duplication elicited by Xlim-1/Ldb1 and antagonizes head organizer activity in a RING-dependent manner. (A) XRnf12 blocks secondary axis formation elicited by Xlim-1/Ldb1, whereas the RING mutant XRnf12(HC>AA) does not. Embryos injected with the mRNAs indicated were scored for axis development at the tailbud stage and categorized as secondary axis (dark blue bars), normal (white bars), or others (hatched bars).β -globin serves as a negative control. n, total number of injected embryos; expt, number of independent experiments. Amounts of mRNAs injected (ng/embryo): Xlim-1, 0.25; Ldb1, 0.5; XRnf12 constructs, 0.25; β-globin (coinjected), 0.25; β-globin (alone), 1.0. (B-D) Overexpression of XRnf12 in the dorsal marginal zone leads to head defects. While XRnf12(HC>AA) has little or no effect (B), XRnf12 overexpression in the dorsal region results in reduced head structures (C). β-globin has no effect (D). Amounts of mRNAs injected were 2.0 or 4.0 ng/embryo.
	Fig 5. XRnf12 causes ubiquitination and proteasome-dependent degradation of Ldb1 in a RING-dependent manner. (A) XRnf12 does not affect the steady-state level of Xlim-1-FLAG, either in the presence or absence of Ldb1. The indicated mRNAs were injected into the ventral region and the expression levels of the FLAG-tagged proteins were examined at the gastrula stage. Note that the levels of Xlim-1-FLAG are increased in the presence of Ldb1. β-tubulin, loading control. Amounts of mRNAs injected (ng/embryo): Xlim-1-FLAG, 0.25; Ldb1, 0.5; XRnf12, 0.25. (B) XRnf12 decreases the steady-state level of FLAG-Ldb1 both in the presence and absence of Xlim-1 in a RING-dependent manner. Note the increase in the expression level of FLAG-Ldb1 by Xlim-1 co-expression. Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; Xlim-1, 0.25; XRnf12 constructs, 0.25. (C) XRnf12 enhances ubiquitination of Ldb1. Embryos were injected ventrally with the mRNAs indicated and the cell lysates were immunoprecipitated (IP) with anti-FLAG antibody followed by either anti-FLAG or anti-HA immunoblotting (IB) to detect non-ubiquitinated Ldb1 or ubiquitinated proteins, respectively. Co-expression of XRnf12 results in downregulation of non-ubiquitinated FLAG-Ldb1 expression levels (lower panel). While weak ubiquitination is observed in the absence of XRnf12 (lane 4), strong ladder-like ubiquitination signals appear in the presence of XRnf12 (lane 7). XRnf12(HC>AA) does not enhance ubiquitination (lane 8). Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 2.0; HA-Ub, 1.0; XRnf12 constructs, 1.0. (D) The N-terminal region (aa 1-291) of Ldb1 is sufficient for ubiquitination by XRnf12. By using FLAG-Ldb1ΔC instead of FLAG-Ldb1, smaller-sized ubiquitinated protein bands are detected, confirming that the ubiquitinated proteins in C are indeed Ldb1 and not some other proteins associated with Ldb1. Co-expression of XRnf12 also results in downregulation of non-ubiquitinated FLAG-Ldb1ΔC. The amounts of mRNAs used are the same as in C. Arrowhead indicates the position of IgG. (E) XRnf12 causes proteasome-dependent degradation of Ldb1. After mRNA injection, cells were dispersed and cultured in the presence or absence of MG-132 until the gastrula stage. Decrease of FLAG-Ldb1 levels by XRnf12 (lane 5) is suppressed in the presence of MG-132 (lane 6). MG-132 does not affect the expression of FLAG-Ldb1 (lanes 3,4). Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; Xlim-1, 0.25; XRnf12, 0.25. (F) The steady-state level of FLAG-Ldb1 is downregulated by hRNF6 and, to a lesser extent, by hRNF38, but not by hRNF13. The experimental design is the same as in A and B. β-tubulin, loading control. Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; RING finger proteins, 0.5. (G) RING finger proteins that cause reduction in the steady-state level of Ldb1 interact with Ldb1. GST pull-down assay was performed with 35S-labeled XRnf12, hRNF6, hRNF13 and hRNF38. Human RNF13 does not interact with GST-Ldb1, while other RING finger proteins do. GST serves as a negative control.
	Analysis of endogenous Ldb1 protein levels. (A) XRnf12 downregulates endogenous Ldb1 expression level in a RING-dependent manner. Animal caps were dissected at the blastula stage (stage 8-9) from embryos injected with the mRNAs (2 ng/embryo) indicated, and collected at the gastrula stage (stage 11). Endogenous Ldb1 expression levels were analyzed by western blotting using anti-Ldb1/CLIM2 (N-18) antibody. (B) Ldb1 does not exhibit dorsal-to-ventral (DV) difference in protein expression levels as revealed by western blot analysis. DMZ and VMZ explants dissected at the gastrula stage (stage 11) were compared for Ldb1 expression using anti-Ldb1/CLIM2 (N-18) antibody. (C) Ldb1 is uniformly expressed in the bisected mid gastrula embryo as revealed by immunostaining using anti-Ldb1/NLI antibody. Animal side is to the top, dorsal to the right. Arrowhead indicates blastopore.
	Disturbing Xlim-1/Ldb1 stoichiometry affects their axis inducing activity. Ldb1 dose-dependently suppresses axis duplication by Xlim-1, which is rescued by co-expression of a higher dose (1 ng) of Xlim-1. Embryos injected with the mRNAs indicated were scored for axis development at the tailbud stage as in Fig. 3A. β-globin (2.0 ng/embryo) serves as a negative control. n, total number of injected embryos; expt, number of independent experiments. Amounts of mRNAs injected are indicated in parentheses (ng/embryo).
	Ldb1 overexpression affects the maintenance phase of gsc expression. Embryos injected dorsally with the mRNAs (4.0 ng/embryo) indicated were collected at stages 9.5, 10, 10.5 and 11 (late blastula to mid gastrula) and the expression of gsc was analyzed by RT-PCR. Downregulation of gsc by Ldb1 overexpression was not prominent at early-gastrula stages (st 9.5, 10) but became prominent after mid gastrula (stage 10.5, 11). Co-expression of XRnf12 restored gsc expression. EF-1α serves as a loading control.
	A model for the role of XRnf12 in the establishment of proper Xlim-1/Ldb1 stoichiometry in the Spemann organizer. In the organizer, tetramer formation of Xlim-1/Ldb1 is required for their activity. XRnf12 selectively degrades excess Ldb1 unbound to Xlim-1, which interferes with organizer gene expression presumably by disturbing Xlim-1/Ldb1 tetramer formation. Excess Ldb1 may also possibly interfere with LIM domain-dependent association of Xlim-1 with other proteins. In this way, proper stoichiometry and maximal activity of Xlim-1/Ldb1 is assured in the presence of XRnf12 in the organizer. In the ventrolateral mesoderm, Ldb1 may escape degradation by XRnf12 through interaction with Ldb1-interacting proteins, one of which may be XLMO4 (J. L. Gomez-Skarmeta, personal communication). The putative Ldb1/LMO complex may contribute to complete suppression of Xlim-1/Ldb1 activity in the ventrolateral region, and may participate in a distinct transcriptional regulatory complex. Xlim-1 unbound to Ldb1 may be subject to proteasome-dependent degradation by an unidentified ubiquitin ligase, similar to the case of Drosophila Apterous (Weihe et al., 2001).