Developmental Biology

The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability

Huishan Wang
Xingyan Liu
Yamin Liu
Chencheng Yang
Yaxin Ye
Xiaomei Yu
Nengyin Sheng
Shihua Zhang author has email address
Bingyu Mao author has email address
Pengcheng Ma author has email address

Key Laboratory of Genetic Evolution and Animal Models, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China
Academy of Mathematics and Systems Science, Chinese Academy of Science, Beijing, China
School of Mathematical Sciences, University of Chinese Academy of Sciences, Beijing, China
Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China
National Resource Center for Non-Human Primates, Kunming Primate Research Center and National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China
Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, China
Key Laboratory of Animal Models and Human Disease Mechanisms of Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China
Key Laboratory of Systems Biology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Hangzhou, China

https://doi.org/10.7554/eLife.94657.2

Open access
Copyright information

Figures and data

Hox genes up-regulated in pons of Rnf220^+/− mice.
(A) The heatmap of RNA-seq data showing Hox genes expression in pons of WT or Rnf220^+/− mice (n=2 mice per group). (B-C) UMAP diagram showing 15 identified cell clusters annotated by snRNA-seq analysis of pons. Each dot represents a single cell, and cells are laid out to show similarities (n=3 mice per group). (D) Heatmap of snRNA-seq data showing Hox expression changes in each cell cluster. (E) Quantitative real time polymerase chain reaction (qRT-PCR) analysis showing mRNA levels of indicated Hox genes in P19 cells when endogenous Rnf220 was knocked down by siRNAs in the presence of RA. WT, wild-type. HE, heterozygote. RA, retinoic acid. **p < 0.01, ***p < 0.001.

Differently expressed genes identified using microarray between WT and Rnf220^+/− mice.

Differently expressed genes identified using microarray between WT and Rnf220^+/− mice.

Differently expressed genes identified using microarray between WT and Rnf220^−/− mice.

Differently expressed genes identified using microarray between WT and Rnf220^−/− mice.

Uniquely and highly expressed markers of each cluster in snRNA-seq.

Uniquely and highly expressed markers of each cluster in snRNA-seq.

Hox gene expression was dysregulated and motor cortex projections were disorganized in PN of Rnf220^+/− mice.
(A) qRT-PCR analysis of relative expression levels of Hox3, Hox4, and Hox5 in rostral, middle, and caudal sections of PN in WT and RNF220^+/− mice. Expression level of each gene in rostral section of WT PN was set to 1. (n=5 mice per group). Bar graphs show the relative levels normalized against rostral group in the respective wild-type mice. (B) Diagram of experimental stereotactic injections. (C) Green fluoresce showed the projection from motor cortex to PNs in adult (2 months) WT (n=10 mice) and Rnf220^+/− mice (n=9 mice). (D-E) The diameter (D) and area (E) sizes of PN in WT and Rnf220^+/− mice. Each data presents the average diameter and area sizes of PN from four consecutive slices for completely presenting circular fluorescence projections. (F) The area of fluorescence projection from motor cortex to PN in WT and Rnf220^+/− mice. The sample used for statistics is consistent with the one selected in Figure D-E. Each data represents the average fluorescence area of four consecutive slices. (G) The proportion of projected-fluorescence area from motor cortex to PN area. The sample used for statistics is consistent with the one selected in Figure D-E. WT, wild-type. HE, heterozygote. n.s., not significant. **p < 0.01.

RNF220 mediates WDR5 degradation.
(A-D) Western blots analysis showing the protein level of WDR5 in the indicated brain tissues of mice with different genotypes at different ages. (E) Western blots showed WDR5 levels of in the pons of adult mice with indicated genotypes. (F) Western blot analysis of protein levels of WDR5 in P19 cells with Rnf220 knockdown or not in the presence or absence of RA. IB, immunoblot. WT, wild-type. HE, heterozygote. KO, knockout. PN, pontine nuclei. NC, negative control. RA, retinoic acid.

RNF220 interacts with and targets WDR5 for K48-linked polyubiquitination.
(A-B) Co-immunoprecipitation (co-IP) analysis of interactions between RNF220 and WDR5 in HEK293 cells. HEK293 cells were transfected with indicated plasmids and harvested after 48 h. Cell lysates were immunoprecipitated with anti-FLAG beads. Whole-cell lysate and immunoprecipitates were subjected to western blot analysis using indicated antibodies. (C) Endogenous co-immunoprecipitation analysis showing the interaction between RNF220 and WDR5 in hindbrains of WT mice. (D) Western blots analysis shows the protein level of WDR5 when co-expressed with wild-type or mutated RNF220 in HEK293 cells. (E) Western blots analysis shows the protein level of WDR5 when co-expressed with RNF220 in HEK293 cells in the presence of MG132 (10 mM) or not. (F) In vivo ubiquitination assays showing the ubiquitination status of WDR5 when co-expressed with WT or mutated RNF220 in HEK293 cells. (G) In vivo ubiquitination assays showing the ubiquitination status of WDR5 in hindbrains of WT and Rnf220^+/− mice. (H) In vivo ubiquitination assays showing RNF220-induced polyubiquitination of WDR5 when the indicated ubiquitin mutations were used in HEK293 cells. (I) In vivo ubiquitination assays showing the ubiquitination status of the indicated WDR5 mutants when co-expressed with WT or ligase dead RNF220 in HEK293 cells. WT, wild-type. HE, heterozygote. KO, knockout. IB, immunoblot. IP, immunoprecipitation. UB, ubiquitin. WCL, whole cell lysate. △Ring, RNF220 Ring domain deletion. W539R, RNF220 ligase dead mutation. K48, ubiquitin with all lysines except the K48 mutated to arginine. K48R, ubiquitin with the K48 was substituted by an arginine. 3KR, substitution of lysines at the positions of 109, 112, and 120 in WDR5 with arginines simultaneously.

WDR5 recovered Rnf220 deficiency-induced up-regulation of Hox genes in P19 cell line.
(A-B) qRT-PCR analysis showing the expression levels of Rnf220 (A) and Wdr5 (B) when transfected the indicated combinations of siRNAs against Rnf220 or Wdr5 in the presence or absence of RA. Bar graphs show the relative levels normalized against control group without siRNA or RA treatment. (C) qRT-PCR analysis showing the expression levels of HoxA1, HoxB1, HoxA9, HoxB9 when transfected siRnf220 or both siRnf220 and siWdr5 when treated with RA. RA: retinoic acid. n.s., not significant. ***p < 0.001.

Genetic and pharmacological ablation of WDR5 recovered Rnf220 deficiency-induced up-regulation of Hox genes.
(A) Diagram of experimental strategy for in utero local injection of WDR5 inhibitors. (B-C) qRT-PCR analysis of expression levels of Rnf220 (B), Hox3-Hox5 (C) in hindbrains of WT and Rnf220^+/−mouse embryos treated with WDR5 inhibitors or not at E18.5 (n=3 mice per group). Actin was used as the internal controls. Heatmap of Hox expression showed the relative levels normalized against WT group. (D-E) qRT-PCR analysis of expression levels of Rnf220 (D), Wdr5 (D), Hox3-Hox5 (E) in pons of P15 mice with indicated genotypes (n=2 mice per group). GAPDH was used as the internal controls. Heatmap of Hox expression showed the relative levels normalized against WT group. WT, wild-type. HE, heterozygote.

Primers used for quantitative real time polymerase chain reaction (qRT-PCR)

Primers used for quantitative real time polymerase chain reaction (qRT-PCR)

Primers used for ChIP-qPCR

Primers used for ChIP-qPCR

Sign up for email alerts