The E3 ubiquitin ligase RNF220 maintains hindbrain Hox expression patterns through regulation of WDR5 stability
- Key Laboratory of Genetic Evolution and Animal Models, Kunming Institute of Zoology, Chinese Academy of Sciences, China
- Academy of Mathematics and Systems Science, Chinese Academy of Science, China
- School of Mathematical Sciences, University of Chinese Academy of Sciences, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, 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, China
- Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, China
- Key Laboratory of Animal Models and Human Disease Mechanisms of Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, China
- Key Laboratory of Systems Biology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, China
Figures
Figure 1 with 3 supplements
Hox genes up-regulated in pons of Rnf220+/- mice.
(A) The heatmap of RNA sequencing (RNA-seq) data showing Hox genes expression in pons of WT or Rnf220+/- mice (n=2 mice per group). (B–C) Uniform manifold approximation and projection (UMAP) diagram …
Figure 1—figure supplement 1
Hox genes exhibited de-repression in hindbrain of Rnf220-/- embryos at late developmental stages.
Heatmap of Hox expression in hindbrain of Rnf220-/- mouse embryos at E16.5 and E15.5. Expression of each Hox gene was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) against Ga…
Figure 1—figure supplement 2
Hox genes were up-regulated in brainstem of Rnf220+/- mice.
(A) Diagram of central nervous system (CNS) in mice. (B) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of Rnf220 expression in CNS of Rnf220+/- and control mice. (C–D) qRT-PCR …
Figure 1—figure supplement 3
Expression levels of Hox genes were not affected by Rnf220 knockdown in P19 cell line without RA induction.
(A) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of Rnf220 and indicated Hox genes expression after RA treatment in P19 cells. (B) qRT-PCR analysis of the expression levels of …
Figure 2 with 2 supplements
Hox gene expression was dysregulated and motor cortex projections were disorganized in pontine nuclei (PN) of Rnf220+/- mice.
(A) Quantitative real-time polymerase chain reaction (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. …
Figure 2—figure supplement 1
Hox genes were up-regulated in pontine nuclei (PN) of Rnf220+/- mice.
(A) Heatmap of single-nucleus RNA sequencing (snRNA-seq) data showing expression levels of PN markers (Pax6, Barhl1, Unc5b, and Nfib) among 15 identified cell clusters (n=3 mice per group). (B– C) …
Figure 2—figure supplement 2
PN showed no structure difference but disorganized projection pattern from motor cortex between WT and Rnf220+/- mice.
(A) Nissl staining showed the pons structure in adult (2 months) WT and Rnf220+/- mice. Scale bars, 500 μm. (B) DAPI labeling PN structure in adult (2 months) WT and Rnf220+/- mice. Scale bars, 500 …
Figure 3 with 2 supplements
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 blot analysis showing WDR5 levels in the …
Figure 3—figure supplement 1
Repressive epigenetic modification was down-regulated while activated epigenetic modification was up-regulated in promoter regions of indicated Hox genes in hindbrains of Rnf220+/- mice.
(A–B) ChIP-qRT-PCR analysis of repressive epigenetic modification (H3K27me3) (A) and activated epigenetic modification (H3K4me3) (B) levels in promoter regions of indicated Hox genes in hindbrains …
Figure 3—figure supplement 2
Protein levels of the indicated core components of PRC1 and PRC2 complex in indicated mouse brain tissues of different genotypes.
(A) Western blot analysis of protein levels of core components of PRC1 and PRC2 complexes in hindbrain of WT, Rnf220+/-, and Rnf220-/- mouse embryos at E18.5. (B) Western blot analysis of protein …
Figure 4 with 1 supplement
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 hr. Cell lysates …
Figure 4—figure supplement 1
RNF220 interacted with and targeted WDR5 for polyubiquitination at multiple lysine sites.
(A) Western blot analysis of the levels of three WDR5 truncated proteins in HEK293 cells when co-transfected with RNF220 or not. (B) In vivo ubiquitination analysis of ubiquitination status of …
Figure 5 with 1 supplement
WDR5 recovered Rnf220 deficiency-induced up-regulation of Hox genes in P19 cell line.
(A–B) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showing the expression levels of Rnf220 (A) and Wdr5 (B) when transfected the indicated combinations of small interfering …
Figure 5—figure supplement 1
Wdr5 knockdown had no effect on Hox genes expression in P19 cells in the presence of RA or not.
(A) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showing the expression levels of Hoxa1, Hoxb1, Hoxa9, Hoxb9 when transfected siRnf220 or both siRnf220 and siWdr5 without RA …
Figure 6 with 1 supplement
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) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of expression levels of Rnf220 (B), Hox…
Figure 6—figure supplement 1
Rnf220 and Wdr5 co-suppression recovered Hox epigenetic modification to a certain degree.
(A–B) ChIP-qRT-PCR analysis of repressive epigenetic modification (H3K27me3) (A) and activated epigenetic modification (H3K4me3) (B) levels in promoter regions of indicated Hox genes in P19 cell …
Author response image 1
Some Hox8 and Hox9 expression pattern in E9.
5 embryos.
Author response image 2
The effect of Rnf220 on RA synthesis and degradation pathways.
Tables
Table 1
Primers used for quantitative real-time polymerase chain reaction (qRT-PCR).
| Genes | Forward primers | Reverse primers |
|---|---|---|
| Actin | GCCAACCGTGAAAAGATGAC | GAGGCATACAGGGACAGCAC |
| Gapdh | AGGTCGGTGTGAACGGATTTG | TGTAGACCATGTAGTTGAGGTCA |
| Rnf220 | GTCTCAGTAGACAAGGACGTTCACA | GGGGTGGAGGTGTAGTAAGGAAG |
| Wdr5 | CGTGACAGGCGGGAAGTGGA | CGGGTGACAAGCCGTGGAAAT |
| Hoxa1 | AGCTCTGTGAGCTGCTTGGT | AAAAGAAACCCTCCCAAAACA |
| Hoxa2 | TGCCATCAGCTATTTCCAGG | GATGAAGGAGAAGAAGGCGG |
| Hoxa3 | TCTTAACATGGAGGGAGCCA | TCTGAAGGCTACGTGTGCTG |
| Hoxa4 | ACGCTGTGCCCCAGTATAAG | ACCTTGATGGTAGGTGTGGC |
| Hoxa5 | CAGGGTCTGGTAGCGAGTGT | CTCAGCCCCAGATCTACCC |
| Hoxa6 | GTCTGGTAGCGCGTGTAGGT | CCCTGTTTACCCCTGGATG |
| Hoxa7 | CTTCTCCAGTTCCAGCGTCT | AAGCCAGTTTCCGCATCTAC |
| Hoxa9 | GTAAGGGCATCGCTTCTTCC | ACAATGCCGAGAATGAGAGC |
| Hoxa10 | TCTTTGCTGTGAGCCAGTTG | CTCCAGCCCCTTCAGAAAAC |
| Hoxa11 | CCTTTTCCAAGTCGCAATGT | AGGCTCCAGCCTACTGGAAT |
| Hoxa13 | CGGTGTCCATGTACTTGTCG | AGCGGCTACTACCCGTGC |
| Hoxb1 | GGTGAAGTTTGTGCGGAGAC | TTCGACTGGATGAAGGTCAA |
| Hoxb2 | GAACCAGACTTTGACCTGCC | GAGCTGGAGAAGGAGTTCCA |
| Hoxb3 | ATCTGTTTGGTGAGGGTGGA | CCGCACCTACCAGTACCACT |
| Hoxb4 | GACCTGCTGGCGAGTGTAG | CTGGATGCGCAAAGTTCAC |
| Hoxb5 | CTGGTAGCGAGTATAGGCGG | AGGGGCAGACTCCACAGATA |
| Hoxb6 | TAGCGTGTGTAGGTCTGGCG | AGCAGAAGTGCTCCACGC |
| Hoxb7 | CTTTCTCCAGCTCCAGGGTC | AACTTCCGGATCTACCCCTG |
| Hoxb8 | GAACTCCTTCTCCAGCTCCA | CACAGCTCTTTCCCTGGATG |
| Hoxb9 | TCCAGCGTCTGGTATTTGGT | GAAGCGAGGACAAAGAGAGG |
| Hoxb13 | TGCCCCTTGCTATAGGGAAT | ATTCTGGAAAGCAGCGTTTG |
| Hoxc4 | CTAATTCCAGGACCTGCTGC | AAAAATTCACGTTAGCACGGT |
| Hoxc5 | TTCTCGAGTTCCAGGGTCTG | ATTTACCCGTGGATGACCAA |
| Hoxc6 | CAGGGTCTGGTACCGAGAGTA | TCCAGATTTACCCCTGGATG |
| Hoxc8 | CAAGGTCTGATACCGGCTGT | ATCAGAACTCGTCTCCCAGC |
| Hoxc9 | AATCTGTCTCTGTCGGCTCC | AGTCTGGGCTCCAAAGTCAC |
| Hoxc10 | ACCTCTTCTTCCTTCCGCTC | ACTCCAGTCCAGACACCTCG |
| Hoxc11 | AAATGAAGGCTCCTACGGCG | TGTCGAAGAAGCGGTCGAAA |
| Hoxc12 | AATACGGCTTGCGCTTCTT | GACCCTGGCTCTCTGGTTTC |
| Hoxc13 | CTCACTTCGGGCTGTAGAGG | TCAGGTGTACTGCTCCAAGG |
| Hoxd1 | TCTGTCAGTTGCTTGGTGCT | TGAAAGTGAAGAGGAACGCC |
| Hoxd3 | ACCAGCTGAGCACTCGTGTA | AGAACAGCTGTGCCACTTCA |
| Hoxd4 | CTCCCTGGGCTGAGACTGT | CCCTGGGAACCACTGTTCT |
| Hoxd8 | GCCCGCGAAGTTTTACGGAT | TAAGTGGTCTGGGTCCTCGC |
| Hoxd9 | TTGTTTGGGTCAAGTTGCTG | CTCAGCTTGCAGCGATCA |
| Hoxd10 | TCTCCTGCACTTCGGGAC | GGAGCCCACTAAAGTCTCCC |
| Hoxd11 | AGTGAGGTTGAGCATCCGAG | ACACCAAGTACCAGATCCGC |
| Hoxd12 | TGCTTTGTGTAGGGTTTCCTCT | CTTCACTGCCCGACGGTA |
| Hoxd13 | TGGTGTAAGGCACCCTTTTC | CCCATTTTTGGAAATCATCC |
| Unc5b | CGTGACAGGCGGGAAGTGGA | CGGGTGACAAGCCGTGGAAAT |
| Pax6 | AGACAACAACAAAGCGGACT | CTTCGCAAATGACAACTGAC |
| Barhl1 | TACCAGAACCGCAGGACTAAAT | AGAAATAAGGCGACGGGAACAT |
| Nfib | AGAAGCCCGAAATCAAGCAG | CCAGTCACGGTAAGCACAAA |
| Neph2 | ACAAGGTTCGGAAATGAAGTCG | GTTGCCATTAGGACGAGGAA |
Table 2
Primers used for ChIP-qPCR.
| Genes | Forward primers | Reverse primers |
|---|---|---|
| Hoxa1 | GCAGGACAAGGTTGATGGG | GCAGGTGGGAGGGACAGAT |
| Hoxa3 | GGACAGACTCGGTGGTAAGA | AGTTCATGTTCACGGTTCCTAT |
| Hoxa9 | GCAGGAAACACTTTGCCAGA | GCCCGAGTTAGGACCCGTA |
| Hoxa10 | CTCCTTGCCTCCTTCTTCC | CCTGGGTATCTGAGCATCTAA |
| Hoxb3 | CCGAGGACGGACCGAAGAT | CCCTGAACTGGACCACCAT |
| Hoxb4 | GAAGAACGCACGGAAAGTAAG | GGGAAAGAATATGAGCGGAGT |
| Hoxb7 | CCTTAGGGACGCCTTGGTC | ACGCAGGGATTGAATGTTCG |
| Hoxb8 | GCCATTGAATTTCTATCCCAC | GGTGAGGCAAGCTAAAGCAG |
| Hoxc6 | CTTCTCCTCTGCCCTCTTC | GTTAGTTAATACATGGACCTCT |
| Hoxc8 | GTCGTGGATTGATGAACGCG | TCTGCTCACTGTCGGTAGG |
| Hoxc9 | TGTGCCTTGAGTCACTTTGC | CTTGCTCCACTTCTCCAGAT |
| Hoxc10 | TTTTCTTTGGGTCCTCGTAAA | AGTCTAGGGAGCCATTTGTC |
| Hoxd3 | TTTTCCGAGTCCTATTGCTTG | CTGTATCATCTGCCCTCTATC |
| Hoxd8 | AGGACTTTGATTCGCTTTGATA | CGAGGTTGACGGATTGATTG |
| Hoxd9 | AACCTACCCTCGGAGATGC | GCACTGGAGTCCCAAGGAG |
| Hoxd10 | GGAGGGATGTTTCCGAACT | CACATACCCAGGCAGAACG |
Additional files
-
Supplementary file 1
Differently expressed genes identified using microarray between wild-type (WT) and Rnf220+/- mice.
Whole-mount brain from E18.5 mice were used (n=2 in WT group and n=3 in Rnf220+/- group).
- https://cdn.elifesciences.org/articles/94657/elife-94657-supp1-v1.docx
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Supplementary file 2
Differently expressed genes identified using microarray between wild-type (WT) and Rnf220-/- mice.
Whole-mount brain from E18.5 mice were used (n=2 in WT group and n=3 in Rnf220-/- group).
- https://cdn.elifesciences.org/articles/94657/elife-94657-supp2-v1.docx
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Supplementary file 3
Uniquely and highly expressed genes of each cluster in single-nucleus RNA sequencing (snRNA-seq).
The pons from 2 months’ mice were used (n=3 mice per group).
- https://cdn.elifesciences.org/articles/94657/elife-94657-supp3-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/94657/elife-94657-mdarchecklist1-v1.docx
