. Shanghai Institute of Hematology, State Key Laboratory of Omics and Diseases, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200025, China . CAS Key Laboratory of Tissue Microenvironment and Tumor, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China . Department of Clinical Laboratory, The First People's Hospital of Lianyungang, The Affiliated Lianyungang Hospital of Xuzhou Medical University, Lianyungang 222000, China . Sylvester Comprehensive Cancer Center and Department of Medicine, University of Miami Miller School of Medicine, Miami, FL 33136, USA . Department of Biomedical Science, Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL 33431, USA
SETD2 is the only enzyme responsible for transcription-coupled histone H3 lysine 36 trimethylation (H3K36me3). Mutations in SETD2 cause human diseases including cancer and developmental defects. In mice, Setd2 is essential for embryonic vascular remodeling. Given that many epigenetic modifiers have recently been found to possess noncatalytic functions, it is unknown whether the major function(s) of Setd2 is dependent on its catalytic activity or not. Here, we established a site-specific knockin mouse model harboring a cancer patient-derived catalytically dead Setd2 (Setd2-CD). We found that the essentiality of Setd2 in mouse development is dependent on its methyltransferase activity, as the Setd2CD/CD and Setd2−/− mice showed similar embryonic lethal phenotypes and largely comparable gene expression patterns. However, compared with Setd2−/−, the Setd2CD/CD mice showed less severe defects in allantois development, and single-cell RNA-seq analysis revealed differentially regulated allantois-specific 5′ Hoxa cluster genes in these two models. Collectively, this study clarifies the importance of Setd2 catalytic activity in mouse development and provides a new model for comparative study of previously unrecognized Setd2 functions.
Just Accepted Date: 09 July 2024Online First Date: 05 August 2024Issue Date: 29 October 2024
Cite this article:
Shubei Chen,Dianjia Liu,Bingyi Chen, et al. Catalytic activity of Setd2 is essential for embryonic development in mice: establishment of a mouse model harboring patient-derived Setd2 mutation[J]. Front. Med.,
2024, 18(5): 831-849.
Fig.1 Identification of a cancer patient-derived catalytically dead (CD) SETD2 mutation. (A) Lollipop plots of the mutations in SETD2 identified in patients with clear cell renal cell carcinoma (ccRCC). Data are retrieved from the Catalogue of Somatic Mutations (COSMIC) database. The top panel shows various types of mutations, whereas the bottom panel shows the missense mutations. The Lollipop plots were generated with the OmicStudio tools. Red arrow denotes the C1685F mutation. Residue positions are according to GenBank protein accession number NP_054878.5. (B) Amino acid sequence alignment of the catalytic domains of SETD2 and its orthologs in various species, as well as its close paralogs in human. Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xt, Xenopus tropicalis; Dr, Danio rerio; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae. Red arrow denotes the conserved C1685 in human SETD2, corresponding to C1659 in mouse Setd2. (C) Structure of SETD2 catalytic domains in complex with a nucleosome containing H3.3K36M (PDB: 7EA8). Note that the C1685 residue (yellow) interacts with a zinc ion (blue) and locates close to the catalytic site and the substrate histone H3 tail (orange). (D) Experimental design, showing the domain architectures of the GST-tagged wild-type and CD SETD2 fragments used in the in vitro histone methyltransferase activity assays, and a summary of the experimental results. Note that, while only human SETD2 is illustrated, the experiments using the human and mouse versions of the enzymes produce the same results. The fragment containing the AWS-SET-PostSET domains is abbreviated as ASP, and the mutation is denoted with an asterisk. (E, F) Results of the in vitro histone methyltransferase activity assays showing that the CD mutants of human SETD2 (E) and mouse Setd2 (F) completely lose their activities to catalyze mono-, di- and trimethylation of H3K36 in polynucleosomes. The H3K36 methylation states were determined by specific antibodies and the loading controls were visualized by Coomassie blue staining. (G) Experimental design, showing the domain architectures of the FLAG-tagged wild-type (WT) and CD SETD2 fragments used in the coimmunoprecipitation (co-IP) and H3K36me3 restoration assays, and a summary of the experimental results. The fragment containing the AWS-SET-PostSET, WW, and SRI domains is abbreviated as ASPWS, and the mutation is denoted with an asterisk. (H) Results of the co-IP assays showing that the WT and CD SETD2(ASPWS) proteins similarly interact with the hyperphosphorylated, but not the unphosphorylated, RNA polymerase II (Pol II). (I) Immunoblot analysis of H3K36 methylation states in the SETD-knockout HEK293T cells transfected with WT or CD SETD2(ASPWS) proteins. Note the failure of the CD SETD2(ASPWS) protein to restore the H3K36me3 in the cells. Total H3 was used as a loading control.
Fig.2 Generation of the site-specific Setd2-CD knockin mouse model. (A) Schematic diagram of the targeting strategy. Asterisk denotes the G-to-T mutation in exon 9, which causes the C1659F mutation in mouse Setd2 protein. A Neo cassette flanked with two LoxP sites and a reversed Tk cassette are used as positive and negative selection markers in the targeted embryonic stem (ES) cells. Green and orange arrows indicate the positions of PCR primers for genotyping the targeted ES cells and mice, respectively, and the corresponding numbers indicated the expected sizes of PCR products. (B) Genomic PCR experiments confirming the targeted ES cell clones. Clone #9 was used for blastocyst microinjection. (C) Sequencing results of the genomic PCR products of the clone #9 ES cells, showing the junction sites between the genomic and inserted DNA sequences. (D) Genomic PCR for genotyping the newborn mice produced by heterozygous parents. Note that there was no homozygous mouse born, while the WT (+/+) and heterozygous (+/CD) littermates showed a normal ratio of roughly 1:2. (E) Representative genomic PCR products containing exon 9 of the WT and heterozygous mice. (F) Sequencing results of the PCR products of the WT and heterozygous mice as shown in panel (E). Note the double-peaks at the G-to-T mutation site in the heterozygous mice. Deduced amino acid sequences are written beneath, and the region of the PostSET domain is indicated. (G) Genotype statistics of the littermates produced by heterozygous intercrosses. Note the absence of homozygous (Setd2CD/CD) offspring.
Fig.3 Comparable embryonic lethality phenotypes and reduced H3K36me3 levels in the Setd2CD/CD and Setd2?/? mice. (A, B) Bright-field microscopy images of the wild-type, heterozygous and homozygous Setd2-CD (A) and Setd2 knockout (B) embryos at E10.5. The top panels show embryos enclosed in the yolk sacs, and the bottom panels show isolated embryos proper. Scale bar = 1 mm. (C, D) Immunoblot analysis of Setd2 protein levels in the wild-type, heterozygous and homozygous Setd2-CD (C) and Setd2 knockout (D) embryos at E10.5. Each sample was loaded in 2-fold serial dilution to facilitate quantification. Gapdh was used as a loading control. (E, F) Immunoblot analysis of the different H3K36 methylation states of the wild-type, heterozygous and homozygous Setd2-CD (E) and Setd2 knockout (F) embryos at E10.5. Each sample was loaded in 2-fold serial dilution to facilitate quantification. Total H3 was used as a loading control. (G, H) Quantification of H3K36 methylation levels in the heterozygous and homozygous Setd2-CD (G) and Setd2 knockout (H) embryos relative to their wild-type littermates. Note that the Setd2CD/CD and Setd2?/? embryos similarly show dramatic reduction of H3K36me3 but not H3K36me1/2 levels.
Fig.4 Similar vascular remodeling defects in the Setd2CD/CD and Setd2?/? mice. (A) Principal component analysis (PCA) of the highly variable genes in the wild-type (WT), heterozygous (HE) and homozygous (HO) Setd2-CD and Setd2 knockout (KO) yolk sacs at E9.5. Note that the Setd2-CD and Setd2-KO HO samples were closely clustered, whereas all the HE and WT samples were clustered into another group. (B) Gene set enrichment analysis (GSEA) of the RNA-seq data showing that the angiogenesis hallmark genes are similarly downregulated in the Setd2CD/CD and Setd2?/? yolk sacs compared with their WT littermates. (C) Unsupervised hierarchical clustering analysis of the 12 indicated yolk sac samples and the angiogenesis hallmark genes used in panel (B), validating the consistent downregulation of these genes in the 4 HO samples. (D, E) Applying RNA-seq data to re-analyze the previously reported cDNA microarray gene signature of the Setd2-KO yolk sacs. The left and right panels show the downregulated and upregulated genes, respectively, in the previous signatures which are largely recapitulated by the RNA-seq analysis of the Setd2-CD (D) and Setd2-KO (E) yolk sacs. (F, G) Microscopy images of the hematoxylin and eosin (H&E) stained yolk sac sections of the Setd2CD/CD (F) and Setd2?/? (G) mice at E9.5 in comparison with their WT littermates. Scale bars in the top panels represent 100 μm, and those in the bottom panels represent 10 μm.
Fig.5 Global gene expression profiling of the homozygous, heterozygous and wild-type (WT) Setd2-CD and Setd2 knockout yolk sacs. (A) A summary of gene set enrichment analysis (GSEA) of the homozygous and heterozygous Setd2-CD and Setd2 knockout yolk sacs compared with their WT littermates, showing the significantly enriched and depleted hallmark gene sets, as well as their nominal P-values. Dashed vertical lines indicate the threshold of nominal P-values at 0.05. The dashed horizontal line divides between enriched and depleted gene sets in the Setd2CD/CD versus WT data analysis. Note that these two models show comparable global tendencies with relatively minor differences. (B) GSEA results showing that the collagen assembly related genes are similarly downregulated in the Setd2CD/CD and Setd2?/? yolk sac compared with their WT littermates. (C) Unsupervised hierarchical clustering analysis of the 12 indicated yolk sac samples and the collagen assembly related genes used in panel (B), validating the consistent downregulation of these genes in the 4 HO samples.
Fig.6 The Setd2-CD mice exhibit slightly milder developmental defects than the Setd2 knockout mice. (A) Venn diagram showing the numbers and overlapping of the upregulated and downregulated genes in the Setd2CD/CD and Setd2?/? embryos compared with their WT littermates. (B) Comparisons of gene expression alteration levels of the shared upregulated (left) and downregulated (right) genes between the Setd2CD/CD and Setd2?/? embryos compared with their WT littermates. The genes are ranked along their fold changes in the Setd2?/? embryos. Note that the fold changes in the Setd2CD/CD embryos are generally less dramatic than those in the Setd2?/? embryos. (C) Bright-field microscopy images of the Setd2CD/CD and Setd2?/? embryos at E10.5, showing different ratios of incomplete chorioallantoic attachment. Scale bar = 1 mm. (D) Representative images of histological section analysis showing different levels of severeness of the developmental defects in the Setd2CD/CD and Setd2?/? placentas. Note the normal and thinner labyrinthine layers in the wild-type and Setd2CD/CD embryos, respectively, and the lack of labyrinthine layer in a representative Setd2?/? embryo. Scale bar = 100 μm. (E) Statistics of the frequencies of chorioallantoic attachment defects in the Setd2CD/CD and Setd2?/? embryos.
Fig.7 Single cell RNA sequencing (scRNA-seq) analysis of the Setd2CD/CD and Setd2?/? E8.5 embryos reveals differential regulation of allantois-specific 5′ Hoxa cluster genes. (A) Uniform manifold approximation and projection (UMAP) analysis of the scRNA-seq data of wild-type (WT) E8.5 embryos leading to identification of 20 distinct cell clusters. (B) Hierarchical clustering heatmap illustrating the relative expression levels of marker genes in each identified cell type. (C) Single cells of the Setd2CD/CD and Setd2?/? embryos projected to the reference map using the mutual nearest neighbors (MNNs) algorithm. The cells are assigned to, and colored as, one of the 20 WT cell types as shown in panel (A). WT cells are shown as background in gray. Red arrows denote allantois cells. Two asterisks in each panel denote the clusters of erythrocytes (lower) and blood progenitors (upper), which tend to be lost during harvesting the samples so that they are not included in calculating frequencies as shown in panel (D). (D) A scatter plot showing the frequencies of the 18 clusters of cells in the Setd2CD/CD versus Setd2?/? embryos. Red arrow denotes the allantois cells. Two dashed lines denote the degree of 50% similarity, indicating that the frequencies of all these clusters share higher similarities than this degree. (E) Results of scRNA-seq analysis of WT embryos showing that the 5′ Hoxa cluster genes are relatively highly expressed in the allantois cells. (F) Violin plots of expression levels of the 5′ Hoxa cluster genes in the Setd2CD/CD and Setd2?/? allantois cells compared with their WT littermates. Note that these genes are dramatically downregulated in the Setd2?/? but not the Setd2CD/CD allantois cells.
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