Skip to main content
Download PDF

Zmynd11 is essential for neurogenesis by coordinating H3K36me3 modification of Epha2 and PI3K signaling pathway

Cell & Bioscience volume 15, Article number: 55 (2025) Cite this article

Abstract

10p15.3 deletion syndrome is caused by the deficiency of MYND-type zinc finger domain-containing protein 11 (ZMYND11) and featured by global developmental delay, intellectual disability, behavioral abnormalities, etc. Although the roles of Zmynd11 is intensively studied in cancer, the function and associated mechanisms of Zmynd11 in neurodevelopment remain largely unknown. Here, we show that Zmynd11 displays abundant and dynamic expression pattern during embryonic neurodevelopment. Zmynd11 deficiency impairs embryonic neurogenesis and neurodevelopment in vitro and in vivo, and inhibits morphological maturation of neurons. Mechanistically, Zmynd11 deficiency leads to decreased Epha2 and disrupts PI3K signaling pathway. Under Zmynd11 deficient condition, H3K36me3 modification on Epha2 promoter abnormally increases and the binding of RNA polymerase II decreases. The restoration of PI3K signaling pathway by exogenous Epha2 can rescue aberrant neurogenesis induced by Zmynd11 depletion in vitro and in vivo. Collectively, our study reveals the essential function of Zmynd11 in neurogenesis via coordinating H3K36me3 modification of Epha2 and PI3K signaling pathway.

Introduction

MYND-type zinc finger domain-containing protein 11 (Zmynd11) primarily represses gene expression by specifically recognizing histone modifications H3.3K36me3 [1,2,3,4]. Previous studies have shown that ZMYND11 plays critical function as tumor suppressor [3, 5,6,7,8,9], and decreased ZMYND11 promotes tumor cell growth and migration, which is correlated with poor outcomes and lower survival rate [6,7,8,9]. In addition, ZMYND11 was remarkably decreased in glioblastoma multiform (GBM) tissues, while exogenous Zmynd11 significantly reduced tumor cell proliferation and invasion, and promoted cell cycle arrest and apoptosis [10]. In pediatric high-grade gliomas (pHGGs), ZMYND11 binding to H3.3-G34R maintained the expression of key neural progenitor genes and promoted tumorigenesis [6, 11]. A recent study showed that Zmynd11 exhibits tumor repressive function by interacting with arginine methyltransferase PRMT5 and prohibiting the formation of HNRNPA1-mediated stress granule [9]. Collectively, these studies have uncovered multifaced function and associated mechanisms of Zmynd11 in tumor.

ZMYND11 is highly expressed in fetal brain [12, 13], and ZMYND11 haploinsufficiency causes 10p15.3 deletion syndrome in human, which is featured by global developmental delay, intellectual disability, behavioral abnormalities, etc. [14,15,16,17,18,19]. In a cellular model, Zmynd11 deficiency impaired neuronal generation through regulating brain-specific isoform splicing [20]. However, the exact function and associated mechanisms of Zmynd11 in regulating neurodevelopment remain underexplored.

Here, we show that Zmynd11 displays abundant and dynamic expression pattern during embryonic neurodevelopment. Zmynd11 deficiency inhibits proliferation and induces aberrant neuronal differentiation of embryonic neuronal progenitor cells (eNPCs) in vitro and in vivo, and impairs morphological maturation of neurons. Mechanistically, Zmynd11 deficiency leads to decreased Epha2 and disrupts PI3K signaling pathway. Under Zmynd11 deficiency condition, H3K36me3 modification at Epha2 promoter abnormally increases and the binding of RNA polymerase II decreases. The restoration of PI3K signaling pathway by exogenous Epha2 can rescue aberrant neurogenesis induced by Zmynd11 depletion in vitro and in vivo. Collectively, our study reveals the essential function of Zmynd11 in neurogenesis and neurodevelopment through regulating PI3K signaling pathway.

Results

Zmynd11 deficiency inhibits the proliferation and induces aberrant neuronal differentiation of NPCs

To determine the roles of Zmynd11 in neurodevelopment, we first examined its expression pattern in the brain of mice. Immunofluorescence staining of brain sections collected from embryonic day 14 (E14), E17 and postnatal day 0 (P0) mice revealed that Zmynd11 signal was colocalized with embryonic neural progenitor cells (eNPCs) marker Nestin and neuronal marker β-III tubulin (Tuj1) (Figure S1A). The intensity of Zmynd11 signal was increased from E14 to P0 (Figure S1A). In consistency, Western blot (WB) and qRT-PCR assays showed that the level of Zmynd11 was increased during neurodevelopment (Figure S1B-1D).

Next, we isolated eNPCs and neurons from embryonic mice brains (E14), and astrocytes from the brains of new born pups (P0). Immunostaining, WB and qRT-PCR assays results showed that the expression of Zmynd11 was remarkably elevated upon the differentiation of eNPCs (Figure S1E-1H). Notably, Map2+ Neurons and Gfap+ astrocytes exhibited the greater level of Zmynd11 compared to that of Nestin+ eNPCs (Figure S1I-1L). Collectively, these results suggest that Zmynd11 displays a dynamic expression pattern during neurodevelopment.

To examine the effects of Zmynd11 on neurogenesis, we performed Zmynd11 knockdown (KD) with lentivirus expressing short hairpin RNA against Zmynd11 in eNPCs. Zmynd11 KD significantly reduced protein levels of Zmynd11 in proliferating eNPCs (Fig. 1A, B). Upon the differentiation of eNPCs, Zmynd11 KD significantly reduced the level of actrocytic marker Gfap, but enhanced the level of neuronal marker Tuj1 (Fig. 1A, C, D). Immunostaining and quantification results showed that the percentages of Ki67+GFP+/GFP+ and BrdU+GFP+/GFP+ cells were significantly decreased in Zmynd11 KD group compared to scramble group (Fig. 1E–H). Upon the differentiation of eNPCs, the percentage of Tuj1+GFP+/GFP+ was significantly increased (Fig. 1I, J), but the percentage of Gfap+GFP+/GFP+ cells was significantly decreased in Zmynd11 KD group compared to scramble group (Fig. 1K, L). qRT-PCR assay results also showed that Zmynd11KD significantly reduced Gfap expression, but increased Tuj1 expression (Fig. 1M–P).

Fig. 1
figure 1

Zmynd11 depletion impairs the proliferation and differentiation of eNPCs in vitro. A–D Representative Western blot (WB) images of Zmynd11, neuronal marker Tuj1 and astrocytic marker Gfap (A). Zmynd11 KD significantly reduced the level Zmynd11 protein in proliferating eNPCs (B). Under differentiation condition, Zmynd11 KD significantly reduced Gfap (C), but increased Tuj1 protein compared to scramble group (Ctrl) (D). n = 3 biologically independent experiments for each group. Data represent mean ± SEM, unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. no significance. E Representative images of Ki67 immunofluorescence staining with eNPCs infected with lentivirus expressing scramble and shRNA against Zmynd11 (shZmynd11) compared to scramble group (Ctrl). eNPCs were infected with lentivirus for 72 h. Scale bar, 50 μm. F Quantification results showed that Zmynd11 knockdown (KD) led to a decreased percentage of Ki67+ cells. n = 4 biologically independent experiments for each group. Data represent mean ± SEM, unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. no significance. G Representative images of BrdU immunofluorescence staining with eNPCs infected with lentivirus expressing scramble and shZmynd11. eNPCs were infected with lentivirus for 72 h, and BrdU was incorporated for 6 h at a final concentration of 5 Î¼M. Scale bar, 50 μm. H Quantification results showed that Zmynd11 KD reduced the percentage of BrdU+ cells. n = 4 biologically independent experiments for each group. Data represent mean ± SEM, unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. no significance. I Representative images of Tuj1 immunofluorescence staining with eNPCs infected with lenti-scramble and lenti-shZmynd11. After eNPCs were infected with lentivirus for 72 h, eNPCs differentiation was induced for 48 h. Scale bar, 50 μm. J Quantification results showed that Zmynd11 KD enhanced the percentage of Tuj1+ cells. n = 4 biologically independent experiments for each group. Data represent mean ± SEM, unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. no significance. K Representative images of Gfap immunofluorescence staining with eNPCs infected with lenti-scramble and lenti-shZmynd11. After eNPCs were infected with lentivirus for 72 h, eNPCs differentiation was induced for 48 h. Scale bar, 50 μm. L Quantification results showed that Zmynd11 KD reduced the percentage of Gfap+ cells. n = 4 biologically independent experiments for each group. Data represent mean ± SEM, unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. no significance. M–P qRT-PCR assay results showed that Zmynd11 KD significantly reduced the mRNA levels of Zmynd11 (M), Gfap (N), s100β (O) and increased the mRNA level of Tuj1 (P). n = 3 independent experiments for each group. Data represent mean ± SEM, unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. no significance

Full size image

Next, we generated conditional knockout (cKO) mice (Zmynd11loxp/loxp; Nestin-Cre) by crossing Zmynd11loxp/loxp mice with Nestin-Cre mice (Figure S2 A). cKO pups (P0) displayed less body weight compared to Control (Ctrl) group (Zmynd11loxp/loxp) (Figures S2B, S2 C). WB assay and qRT-PCR results showed that the protein and mRNA levels of Zmynd11 was significantly decreased in the brain of cKO pups (P0) (Fig. 2A–C). In consistency with in vitro study, the protein level of Gfap was significantly decreased, but the level of Tuj1 was significantly increased in the brain of cKO pups compared to Ctrl (P0) (Fig. 2D, E). Ctrl and cKO pups were administrated with 5-bromo-2’-deoxyuridine (BrdU), and immunofluorescence staining showed that cKO mice displayed remarkable decrease of BrdU+ cells in ventricular zone/subventricular zone (VZ/SVZ) regions compared to that of Ctrl group (Fig. 1F, G). The number of PAX6+ radial glial cells (RGCs), one type of NPC, was also significantly decreased in VZ/SVZ of cKO mice (Fig. 1H, I). Of note, the number of Tbr2+ cells, one type of neuronal progenitor cells, was significantly increased in VZ/SVZ of cKO mice compared to Ctrl (Fig. 1J, K).

Fig. 2
figure 2

Zmynd11 deficiency disrupts neurogenesis in embryonic brain. A Representative WB images of Zmynd11, Tuj1 and Gfap in the brain of Zmynd11loxp/loxp (Ctrl) and Nestin-Cre;Zmynd11−/− (cKO) pups at the age of postnatal day 0 (P0). B, C Quantification results showed that cKO mice had decreased protein and mRNA levels of Zmynd11. n = 4 independent experiments for each group in (B) and n = 3 biologically independent experiments for each group in (C). Data represent mean ± SEM, unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. no significance. D, E Quantification results showed that cKO mice had decreased Gfap (D), but increased Tuj1 (E). n = 4 independent experiments for each group. Data represent mean ± SEM, unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. no significance. F Representative images of BrdU immunostaining with brain sections of Ctrl and cKO mice (P0). BrdU was injected (100 mg/kg, intraperitoneally [i.p.]) 1.5 h prior to the sacrifice. Scale bar, 50 μm. G Quantification results showed that the number of BrdU+ cells was significantly decreased in VZ/SVZ of cKO mice compared to Ctrl. n = 5 pups for each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, unpaired Student’s t test. H Representative images of radial glial marker PAX6 immunostaining with brain sections of Ctrl and cKO mice at the age of P0. Scale bar, 50 μm. I Quantification results showed that the number of PAX6+ cells in VZ/SVZ was significantly decreased in VZ/SVZ of cKO mice compared to Ctrl. n = 5 pups for each group. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01; ***p < 0.001, unpaired Student’s t test. J Representative images of intermediate progenitor cell marker Tbr2 immunostaining with brain sections of Ctrl and cKO mice (P0). Scale bar, 50 μm. K Quantification results showed the number of Tbr2+ cells was significantly increased in VZ/SVZ of cKO mice compared to Ctrl. n = 5 pups for each group. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, unpaired Student’s t test. L Representative images of BrdU immunostaining with scramble and Zmynd11 KD brain sections. Scramble and shZmynd11 plasmids were delivered into E14 fetal brain via in utero electroporation, and animals were sacrificed for assay at E17. BrdU was injected (100 mg/kg, i.p.) 1.5 h prior to the sacrifice. Scale bar, 50 μm. M Quantification results showed the percentage of BrdU+GFP+/GFP+ in VZ/SVZ was significantly decreased in Zmynd11 KD group compared to Ctrl. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, unpaired Student’s t test. N Representative images of Tuj1 immunostaining with scramble and Zmynd11 KD brain sections as in (L). Scale bar, 50 μm. O Quantification results showed the percentage of Tuj1+GFP+/GFP+ was significantly increased in cortical plate (CP) layer of Zmynd11 KD group compared to Ctrl. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, unpaired Student’s t test

Full size image

Next, we adopted in utero electroporation (IUE) to deliver scramble and shZmynd11 plasmids into the brain of E14 embryonic fetus, respectively, which were analyzed on E17. Immunostaining showed that the percentages of BrdU+GFP+/GFP+ and Pax6+GFP+/GFP+ cells were significantly decreased in VZ/SVZ of Zmynd11 KD group compared to scramble group (Fig. 2L, M, S2D, S2E). The percentages of Tuj1+GFP+/GFP+ cells and Tbr2+GFP+/GFP+ cells were significantly increased in VZ/SVZ of Zmynd11 KD group compared to scramble group (Fig. 2N, O, S2F, S2G). Zmynd11 KD also disrupted the migration of new born neurons, indicated by the decreased percentage of GFP+ cells in VZ/SVZ and cortical plate (CP), and increased percentage of GFP+ cells in intermediate zone (IZ) (Figures S2H-S2K). Collectively, these results suggest that Zmynd11 deficiency leads to abnormal neurogenesis in vitro and in vivo.

Zmynd11 depletion inhibits the morphological development of neurons

Next, we isolated primary neurons from cortical tissues of embryonic brain (E14), and performed Zmynd11 KD assay at days in vitro (DIV) 14 and DIV18, and analyzed at DIV 17 and DIV 21, respectively. We observed that Zmynd11 KD reduced the length of dendrites, number of intersections at DIV 17 (Fig. 3A–C). Sholl analysis showed that Zmynd11 KD led to decreased complexity of neurons (Fig. 3D). In addition, at DIV 21, neurons exhibited fewer spine density in Zmynd11 KD group (Fig. 3E, F). Zmynd11 KD also led to reduced mushroom spines, and increased stubby spines, thin spines, filopodia spines (Fig. 3E, G–J). Collectively, these results suggest that Zmynd11 depletion impairs morphological development of neurons.

Fig. 3
figure 3

Zmynd11 deficiency inhibits morphological development of neurons. A Representative images of cultured cortical neurons at DIV 17. Primary neurons were isolated from the cortical tissues of E14 fetal brains. Scramble and shZmynd11 plasmids were transfected at DIV 14, and cells were collected for assay at DIV 17. Scale bar, 50 μm. B, C Quantification results showed that Zmynd11 KD significantly reduced the dendritic length (B) and the number of branch points (C) of cortical neurons compared to scramble group. n = 30 cells were analyzed for each group. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test. D Sholl analysis results showed that Zmynd11 KD reduced the dendritic complexity of cortical neurons compared to scramble group. n = 30 cells were analyzed for each group. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test. E Representative images of neurons cultured for 21 days in vitro. Scramble and shZmynd11 plasmids were transfected at DIV 18, and cells were collected for assays at DIV 21. Scale bar, 5 Î¼m. F–J Quantification results showed that Zmynd11 KD reduced the spine density (F) and the proportion of mushroom spines (G), but increased the proportion of stubby (H), thin (I), filopodium (J) spines. n = 30 cells were analyzed for each group. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; unpaired Student’s t test

Full size image

Zmynd11 depletion leads to the dysregulated PI3K signaling pathway

To uncover the molecular mechanism of Zmynd11 in regulating neurogenesis, we performed RNA sequencing (RNA-seq) with eNPCs infected with lentivirus-scramble and lenti-shZmynd11, respectively. RNA-seq data analysis identified 536 differentially expressed genes (DEGs), 355 genes downregulated and 181 genes upregulated, in Zmynd11 KD group (Fig. 4A, Table S1). Gene Ontology (GO) enrichment analysis of DEGs showed that the upregulated genes were significantly enriched in terms including T cell activation and proliferation, etc., while downregulated DEGs showed a high enrichment in neurogenesis, neural precursor/stem cell proliferation, etc. (Fig. 4B, C). In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the upregulated DEGs were implicated in pathways associated with T cell signaling and cytokine interaction, etc. (Fig. 4D). Notably, the downregulated DEGs were implicated in pathways associated with the phosphoinositide 3-kinase (PI3K) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, etc. (Fig. 4E). Gene set enrichment analysis (GSEA) further showed that Zmynd11 KD significantly downregulated the expression of genes involved in PI3K signaling pathway (Fig. 4F). qRT-PCR assay results showed that Zmynd11 KD bona fide resulted in the decreased expression of genes associated with PI3K signaling pathway including Epha2, Osmr, Ngf, Lama3, Col6a1, Ccnd2, etc. (Fig. 4G–L, S3A-S3I). WB assay results showed that Zmynd11 KD significantly reduced the protein levels of total Epha2 (Figure S3J, S3K). Although the protein levels of total IRS1, PI3K, PDPK1 and SGK1 showed no difference between Ctrl and Zmynd11 KD groups, the levels of phosphorylated-IRS1 (p-IRS1), phosphorylated-PI3K (p-PI3K), phosphorylated-PDPK1 (p-PDPK1) and phosphorylated-SGK1 (p-SGK1) was significantly decreased in Zmynd11 KD NPCs compared to Ctrl group (Figure S3J, S3L-S3S).

Fig. 4
figure 4figure 4

Zmynd11 deficiency alters the transcriptome and disrupts PI3K signaling pathway. A Heatmap shows differentially expressed genes (DEGs) between scramble and Zmynd11 KD eNPCs. B Gene ontology (GO) analysis showed that upregulated genes were highly enriched for the terms related with T cell activation and proliferation in Zmynd11 KD eNPCs. C GO analysis showed that downregulated genes were highly enriched for the terms related with neuronal development in Zmynd11 KD eNPCs. D KEGG analysis showed the upregulated pathways in Zmynd11 KD eNPCs. E KEGG analysis showed the downregulated pathways including PI3K signaling pathway in Zmynd11 KD eNPCs. F Gene Set Enrichment Analysis (GSEA) map of influenced genes in PI3K signaling pathway. G–L qRT-PCR assay results showed that Zmynd11 KD significantly reduced the mRNA level of Epha2 (G), Osmr (H), Ngf (I), Lama3 (J), Col6a1 (K), Ccnd2 (L), key components of PI3K signaling pathway. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test. M Representative WB images of Zmynd11, Epha2, IRS1, p-IRS1, PI3K, p-PI3K, PDPK1, p-PDPK1, SGK1, p-SGK1 in the cortical tissues of Ctrl and cKO mice. Tubulin and GAPDH were used as internal controls. N–V Quantification results showed that level of Epha2 was significantly decreased in the brain of cKO mice compared to Ctrl (N). The levels of total IRS1 (O), PI3K (P), PDPK1 (Q), and SGK1 (R) showed no difference between Ctrl and cKO mice, but the levels of p-IRS1 (S), p-PI3K (T), p-PDPK1 (U), and p-SGK1 (V) were remarkably decreased in cKO mice compared to Ctrl. n = 4 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test

Full size image

Furthermore, we examined PI3K pathway components with cortical brain tissues of Ctrl and cKO mice (P0) and found that Zmynd11 deficiency resulted in significant reduction of total Epha2, p-IRS1, p-PI3K, p-PDPK1 and p-SGK1, but not altered the protein levels of total IRS1, PI3K, PDPK1 and SGK1 (Fig. 4M–V), which was consistent with in vitro results. Together, these results suggest that Zmynd11 depletion leads to the dysregulation of PI3K signaling pathway.

Zmynd11 regulates Epha2 expression through recognizing H3K36me3 modification

Previous studies have shown that Zmynd11 regulates gene expression as an H3K36me3 reader [1,2,3,4, 21,22,23,24]. We then performed immunoprecipitation (IP) with cortical brain tissues of wild-type mice using Zmynd11 antibody followed by WB assays, and observed that Zmynd11 antibody easily precipitated H3K36me3, and vice versa (Fig. 5A), and similar results were observed in eNPCs (Figure S4 A). Of note, we did not observe a direct interaction between Zmynd11 and Epha2 or RNA polymerase II (Fig. 5A). ChIP-qPCR results showed that the enrichment of H3K36me3 modification was remarkably increased at the promoter region of Epha2 in cortical brain tissues of cKO mice (Fig. 5B, C) and Zmynd11 KD eNPCs (Figs. 5D, S4B) compared to Ctrl group, while the level of total H3K36me3 showed no difference between groups (Figures S4C-S4E). In addition, the binding of RNA polymerase II (Pol II) to Epha2 promoter regions was significantly decreased in cortical brain tissues of cKO mice (Fig. 5E) and Zmynd11 KD NPCs (Figs. 5F, S4F) compared to Ctrl groups, but the level of total Pol II showed no difference between groups (Figures S4G, S4H). DNA IP with a biotin probe targeting to Epha2 promoter showed that the enrichment of H3K36me3 increased, but the enrichment of RNA Pol II decreased at Epha2 promoter regions in cortical brain tissues of cKO mice compared to Ctrl group (Fig. 5G–I). Collectively, these results suggest that Zmynd11 regulates Epha2 expression through effecting H3K36me3 modification and RNA Pol II binding at Epha2 promoter.

Fig. 5
figure 5

Zmynd11 deficiency results in abnormal Epha2 transcription in vivo. A Immunoprecipitation (IP) followed by WB assay showed that Zmynd11 easily precipitated H3K36me3, and vice versa. Zmynd11 did not directly interact with Epha2 and RNA Polymerase II. B Schematic illustration of primers design for analyzing Epha2 promoter and DNA pull down assay. C Chromatin immunoprecipitation-qPCR (ChIP-qPCR) and quantification results showed that the enrichment of H3K36me3 at Epha2 promotor was significantly increased in cKO brain tissues compared to Ctrl. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test. D ChIP-qPCR and quantification results showed that the enrichment of H3K36me3 at Epha2 promotor was significantly increased in Zmynd11 KD eNPCs compared to Ctrl. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test. E ChIP-qPCR and quantification results showed that the binding of RNA Polymerase II to Epha2 promotor was significantly decreased in cKO brain tissues compared to Ctrl. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Student’s t test. F ChIP-qPCR and quantification results showed that the binding of RNA Polymerase II to Epha2 promotor was significantly decreased in Zmynd11 KD eNPCs compared to Ctrl. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test. G–I DNA pull down followed by WB assay and quantification results showed that the enrichment of H3K36me3 at Epha2 promotor was significantly increased, but RNA Polymerase II binding was significantly decreased in cKO brain tissues compared to Ctrl. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test

Full size image

Exogenous Epha2 rescues neurodevelopmental deficits induced by Zmynd11 deficiency

Next, we aim to determine whether exogenous Epha2 could rescue neurodevelopmental deficits induced by Zmynd11 deficiency. Exogenous Epha2 was delivered into eNPCs under scramble and Zmynd11 KD conditions, respectively. Under the proliferating condition, exogenous Epha2 restored the proliferating capability of eNPCs indicated by the increased percentage of BrdU+ cells in Zmynd11 KD group (Fig. 6A, B). Under the differentiation condition, exogenous Epha2 reduced the percentage of Tuj1+ neurons, but increased the percentage of Gfap+ astrocytes in Zmynd11 KD group (Fig. 6C–F). In addition, exogenous Epha2 restored morphological deficits of cultured neurons induced by Zmynd11 KD (Figures S5A-S5D).

Fig. 6
figure 6figure 6

Exogenous Epha2 rescues neurogenic deficits and restores PI3K signaling in Zmynd11 KD eNPCs. A Representative images of BrdU immunostaining of WT eNPCs infected with lenti-scramble-GFP, lenti-shZmynd11-GFP, and lenti-shZmynd11-GFP + lenti-Epha2-RFP, respectively. BrdU was incorporated for 6 h at a final concentration of 5 Î¼M. Scale bar, 50 μm. B Quantification results showed that ectopic Epha2 significantly enhanced the percentage of BrdU+ cells in Zmynd11 KD eNPCs compared to Ctrl. n = 4 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 9) = 13.73. C Representative images of Tuj1 immunostaining of WT eNPCs infected with lenti-scramble-GFP, lenti-shZmynd11-GFP, and lenti-shZmynd11-GFP + lenti-Epha2-RFP, respectively. Scale bar, 50 μm. D Quantification results showed that ectopic Epha2 significantly reduced the percentage of Tuj1+ cells in Zmynd11 KD group compared to Ctrl. n = 4 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 9) = 154.6. E Representative images of immunostaining of WT eNPCs infected with lenti-scramble-GFP, lenti-shZmynd11-GFP, and lenti-shZmynd11-GFP + lenti-Epha2-RFP, respectively. Scale bar, 50 μm. F Quantification results showed that ectopic Epha2 significantly enhanced the percentage of Gfap+ cells in Zmynd11 KD group compared to Ctrl. n = 4 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 9) = 59.39. G–M Representative WB images (G) and quantification results showed that exogenous Epha2 did not affect the protein level of Zmynd11 (H), but significantly enhanced the level of Epha2 (I) in Zmynd11 KD eNPCs compared to Ctrl. Exogenous Epha2 significantly enhanced p-PI3K/PI3K (J), p-SGK1/SGK1 (K), and Gfap (L), but reduced the level of Tuj1 (M) in Zmynd11 KD eNPCs compared to Ctrl. n = 4 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 12) = 30.36 for (H), F (2, 12) = 61.28 for (I), F (2, 12) = 12.68 for (J), F (2, 12) = 50.39 for (K), F (2, 12) = 40.36 for (L), F (2, 12) = 15.66 for (M)

Full size image

We next examined the effects of exogenous Epha2 on PI3K signaling pathway. qRT-PCR assay results showed that exogenous Epha2 significantly increased mRNA levels of Epha2 and Gfap, but reduced Tuj1 mRNA in Zmynd11 KD eNPCs, while the level of Zmynd11 mRNA was not changed (Figures S5E-S5H). WB assay results showed that exogenous Epha2 significantly restored the decreased levels of Epha2, p-PI3K, p-SGK1 in Zmynd11 KD eNPCs (Fig. 6G–K). In addition, exogenous Epha2 significantly increased the level of Gfap protein, but reduced the level of Tuj1 protein in Zmynd11 KD eNPCs (Fig. 6G, L, M).

Finally, we determined whether exogenous Epha2 could rescue neurodevelopmental deficits in vivo. Epha2 was delivered into Ctrl and cKO E14 embryonic brains via IUE, respectively, and immunostaining assays were performed at E17. We observed that exogenous Epha2 significantly enhanced the percentages of BrdU+GFP+ cells and PAX6+GFP+ cells in VZ/SVZ of cKO fetal brains (Fig. 7A–D), but reduced the percentages of Tbr2+GFP+ in VZ/SVZ and Tuj1+GFP+ in CP of cKO fetal brains (Fig. 7E–H). Collectively, these results suggest that exogenous Epha2 could rescue neurodevelopmental deficits induced by Zmynd11 deficiency.

Fig. 7
figure 7

Exogenous Epha2 rescues neurogenic deficits in vivo. A Representative images of BrdU immunostaining with Ctrl, cKO + GFP and cKO + Epha2-GFP brain sections. Venus GFP and Epha2 plasmids were delivered into fetal brain via in utero electroporation at E14, and animals were sacrificed for assay at E17. BrdU was injected (100 mg/kg, i.p.) 1.5 h prior to the sacrifice. Scale bar, 50 μm. B Quantification results showed the percentage of BrdU+GFP+/GFP+ in VZ/SVZ was significantly increased in cKO + Epha2-GFP group compared to cKO-GFP group. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 12) = 59.67. C Representative images of PAX6 immunostaining with Ctrl, cKO + GFP and cKO + Epha2-GFP brain sections. Scale bar, 50 μm. D Quantification results showed the percentage of PAX6+GFP+/GFP+ in VZ/SVZ was significantly increased in cKO + Epha2-GFP group compared to cKO-GFP group. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2,12) = 64.34. E Representative images of Tbr2 immunostaining with Ctrl, cKO + GFP and cKO + Epha2-GFP brain sections. Scale bar, 50 μm. F Quantification results showed the percentage of Tbr2+GFP+/GFP+ in VZ/SVZ was significantly decreased in cKO + Epha2-GFP group compared to cKO-GFP group. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 12) = 19.6. G Representative images of Tuj1 immunostaining with Ctrl, cKO + GFP and cKO + Epha2-GFP brain sections. Scale bar, 50 μm. H Quantification results showed the percentage of Tuj1+GFP+/GFP.+ in VZ/SVZ was significantly decreased in cKO + Epha2-GFP group compared to cKO-GFP group. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 12) = 11.81

Full size image

Discussion

Embryonic and postnatal neurodevelopment in mammalian is intensively regulated by diverse mechanisms, including environment, genetics and epigenetics [25,26,27,28,29]. In the present study, we have found that Zmynd11 widely presents in embryonic brain and shows increased expression along neurodevelopment. Zmynd11 deficiency impairs the proliferating capability of neuronal progenitor cells, but aberrantly promotes production of neurons and inhibits glial differentiation. Zmynd11 deficiency promotes the binding of SETD2, a methyltransferase for depositing H3K36me3 modification, and enhances H3K36me3 modification at Epha2 promoter region, which consequently inhibits PI3K signaling pathway and leads to neurodevelopmental deficits. Exogenous Epha2 restores the inhibited PI3K signaling pathway and aberrant neurogenesis and neurodevelopment. Collectively, our study has revealed the important function of Zmynd11 in regulating neurogenesis and associated mechanisms.

Zmynd11 haploinsufficiency induces 10p15.3 deletion syndrome in human, and patients display neurological deficits including intellectual disability, behavioral abnormalities, and seizures [14, 18, 19, 30,31,32,33]. However, the knowledge about underlying mechanism for these phenotypes are very limited. Ectopic Zmynd11 and Zmynd11 deficiency both impairs neurogenesis and neurodevelopment in vitro, suggesting a complicated function of Zmynd11 in brain [20, 34]. Our present data show that Zmynd11 deficiency not only disturbs the proliferation of embryonic neural progenitor cells, but leads to aberrant neuronal production and prohibits neuronal maturation in vitro and in vivo. In addition, our data show that Zmynd11 deficiency reduced Epha2, which led to the dysregulation of PI3K signaling pathway. The restoration of PI3K signaling pathway by ectopic Epha2 partially rescues neurodevelopmental deficits. PI3K signaling pathway is regulated by Epha2 [35,36,37,38], and is essential for neurogenesis and neurodevelopment [26, 39, 40]. Altogether, our study reveals the important function of Zmynd11 regulates neurodevelopment via Epha2-PI3K pathway.

Previous studies have shown that Zmynd11 recognizes H3K36me3 on H3.3 (H3.3K36 me3) modification and serves as a transcription repressor [1,2,3,4, 21,22,23,24]. However, some result also indicate that H3K36me3 inhibits gene expression [41,42,43]. Our data show that Zmynd11 deficiency results in the increased expression of some genes while the expression of some genes was decreased in neuronal progenitor cells (NPCs), suggesting that Zmynd11 plays multifacet function in regulating gene expression during neurodevelopment. In addition, Zmynd11 deficiency led to increased H3K36me3 modification at the Epha2 promoter, while the total level of H3K36me3 modification showed no difference between Ctrl and cKO in vitro and in vivo. It would be intriguing to examine whether Zmynd11 deficiency affected the binding of SETD2 to Epha2 promoter region and associated mechanism. Collectively, these results suggest that the function of H3K36me3 on gene expression could be context dependent, i.e., where and when H3K36me3 mark is placed, and what reader protein binds to this modification and the additional surrounding marks leading to different biological outcomes.

Although it has been shown that Zmynd11 regulates RNA polymerase II elongation in tumor, we observed that Zmynd11 deficiency reduced the binding of RNA Polymerase II at Epha2 promoter in eNPCs and brain samples. These data suggest that Zmynd11 maintains gene expression by effecting SETD2-catalyzed H3K36me3 and Pol II binding to specific genes in brain. Of note, considering that Zmynd11 deficiency alters the expression of a set of genes including Epha2, further studies are requisite to uncover their function in neurodevelopment. Collectively, our study reveals a new layer how Zmynd11 regulates gene expression during neurodevelopment and provides novel insights for the mechanisms underlying the neurological deficits of 10p15.3 deletion syndrome in human.

Materials and methods

Animals

Zmynd11loxp/loxp mice were obtained from GemPharmatech (Nanjing, China), and crossed with Nestin-Cre mice to generate conditional knockout mice (Nestin-Cre;Zmynd11loxp/loxp, cKO). Wild-type (WT) C57BL/6 pregnant mice were purchased from Shanghai SLAC Laboratory Animal Center, and gestational day 0.5 was determined by observation of vaginal plug. All animals were housed under a 12-h light/dark cycle at the Experimental Animal Center of Zhejiang University with free access to food and water. The mice were genotyped by PCR, and the sequences of used primers was included in Table S2.

Isolation and culture of embryonic neuronal progenitor cells (eNPCs)

The isolation and culture of eNPCs were performed as described previously [44]. The cortical tissues of E14 fetal brains were dissected and cut into pieces followed by the digestion with 0.1% Trypsin (25200072, GIBCO) for 5 min at 37 °C. Samples were centrifuged at a speed of 1500 rpm, and pellets were collected and washed with DMEM/F-12 medium (D8437, Sigma). Cells were cultured with DMEM/F-12 medium containing 2% B27 (minus vitamin A) (17504044, Thermo Fisher Scientific), 20 ng/mL FGF2 (100-18B, Peprotech), 2 mM L-Glutamine (25030149, GIBCO), 20 ng/mL EGF (AF-100–15, Peprotech) and 1% antibiotic–antimycotic (15140-122, Thermo Fisher Scientific) in a 5% CO2 incubator at 37 °C.

For the proliferation and differentiation assays, eNPCs were plated onto the coverslips (354087, BD), and BrdU at a final concentration of 5 Î¼M was added to the medium for 6 h. At the scheduled time point, cells were fixed with 4% PFA and immunofluorescence staining was performed. The antibody information was included in Table S3.

Isolation and culture of primary neurons and astrocytes

Primary neuron were isolated from cortical tissues of E14 fetal brains as described previously [44]. Briefly, tissues were digested with 0.25% trypsin at 37°C for 30 min followed by a centrifuge at a speed of 1500 rpm. Around 1.5 × 105 neurons were plated onto a slice (Corning) coated with poly-D-lysine (5 mg/mL, Sigma, P0899-10). Neurons were plated in the plating medium containing DMEM (Gibco, 11095-080), 10% FBS (Gibco, 10091-148), 1% L-Glutamine (Gibco, 5030-149), and 1% sodium pyruvate (Gibco, 11360-070). 4 h later, the medium was replaced with growing medium containing neurobasal (Gibco 21103-049), 2% B27 (Gibco 25030-149), 0.25% L-Glutamine and 0.125% Glutamax (Thermo 35050061).

For dendritic spine analysis was performed as described previously [45]. Spine numbers were counted on the entire dendritic segment and spine density per unit of dendritic segment length was calculated. The shape of each dendritic spine was analyzed by calculating the ratio of the width/the length of each spine. All experiments were from a minimum of three independent cultures and at least ten transfected neurons per slice. Dendritic spine subgroups classified on the basis of the following morphology types: stubby spines (< 0.5 µm in length, lacking a clear head), mushroom spines (mushroom-shaped head, approximately 1 Âµm in length), thin spines (with an elongated narrow neck with a distinctive head), and filopodia (longer than 5 Âµm in length).

Astrocytes were isolated as previous description [44]. In brief, cortical and hippocampal tissues of postnatal day 1 pups were dissected. The tissues samples were digested with 0.25% trypsin for 25 min at 37°C followed by a centrifuge at a speed of 1500 rpm. The pellets were resuspended and around 1 × 107 cells were plated into T25 culture flask coated with DMEM medium supplemented with 10% FBS, 1% antibiotic–antimycotic, and 2 mM L-Glutamine. The medium was replaced every 2 days. Upon cell growth proportion reaching to 80% area of the T25 flask, the flasks were put on the shaker (240 rpm) overnight at 37°C, and cells were further cultured with flesh culture medium.

BrdU labeling and immunostaining

For in vivo neurogenesis assay, pregnant mice and postnatal day 1 pups were injected with BrdU (B5002, Sigma) intraperitoneally (i.p.) at a dosage of (100 mg/kg body weight). 1.5 h later, animals were deeply anesthetized with isoflurane and transcardically perfused with cold PBS followed by cold 4% PFA. Fetal brains were removed and postfixed in 4% PFA overnight. After dehydrated with 30% sucrose at 4°C, brain samples were embedded with optimal cutting temperature (OCT) compound and coronal sections were prepared with cryostat (Leica) at 20 μm-thickness.

To perform immunofluorescence staining, brain sections and cell samples were washed with PBS, and then incubated with blocking buffer (PBS containing 3% goat serum and 0.1% Triton X-100) for 1 h at room temperature followed by incubation with primary antibodies at 4°C overnight. The second day, sections and cell sample slides were applied with DAPI (4′,6-diamidino-2-phenylindole) (D8417, Sigma) and fluorescence-labeled secondary antibodies for 1 h. For BrdU immunostaining, the sections and cell samples were pretreated with 1 M HCl in at 37°C for 30 min before blocking. The samples were viewed and images were taken with Olympus confocal microscope (Olympus IX83-FV3000). The antibody information was included in Table S3.

Plasmids construction, transfection and lentivirus transduction

Mouse EphA2 cDNA, Scramble (5’-TTCTCCGAACGTGTCACGT-3’) and shRNAs targeting mouse Zmynd11 (5’-TTGCCAACATTGATCGTATTA-3’) were cloned into pSicoR lentiviral vectors (pSicoR-TOMATO or pSicoR-GFP) for lentivirus packaging or pLKO.1 vector, respectively. Lentivirus was added to cultured eNPCs and neurons (DIV 4) (MOIs: 10), respectively. eNPCs were induced for differentiation 3 days post virus infection, and harvested for assays 48 h later. Neurons were harvested for assays at DIV 7.

For transfection of cultured neurons at DIV 14 and DIV 18, 2 Î¼g plasmids, 2 Î¼L Lipo2000 were mixed with 600 μL neurobasal medium. After incubated for 30 min, the mixture of 200 μL was added to each well in 24-well plate. The medium was replaced with fresh maintaining medium 4 h later and cells were harvested for assays 3 days later.

Co-immunoprecipitation assay

Cells were lysed with lysis buffer (50 mM Tris–HCl pH 7.8, 150 mM KCl, 0.1% Triton X-100, 5 mM EDTA, 1 tablet of protease inhibitor/50 mL) for 30 min on ice followed by a centrifugation at 4°C. The supernatants were incubated with primary antibody at 4°C overnight. The second day, Protein A/G magnetic beads were added into samples and incubated for 6 h at 4°C. Ater washed with washing buffer (50 mM Tris–HCl pH 7.8, 150 mM KCl, 0.01% Triton X-100, 5 mM EDTA, 1 tablet of protease inhibitor/50 mL) for 10 min 3 times, the beads were resuspended with loading buffer. After incubated at 100°C for 5 min and cool down for 5 min on ice, the samples were applied for immunoblotting assay.

In utero electroporation

In utero electroporation (IUE) was performed as described previously [44, 46]. C57/BL6 pregnant mice (E14.5) was anesthetized with isoflurane (RWD). Plasmids were mixed with Venus-GFP (ratio of 3:1) and 0.01% fast green, and manually microinjected into fetal lateral ventricle with a glass capillary (Hirchmann DE-M 16). Five 100-μs pulses of 35 V at 900-μs intervals were delivered across the uterus using an electroporator (BEX, SN. 101438). At E17, fetuses were dissected out and perfused with cold PBS followed by cold 4% PFA. Brain samples were removed out and fixed with 4% PFA overnight at 4°C. After dehydrated with 30% sucrose solution, brain samples were embedded with OCT, and sectioned coronally at a thickness of 15 μm. Brain sections were viewed and images were captured with confocal microscopy (OLYMPUS IX83-FV3000). Images were analyzed with ImageJ software.

RNA extraction and qRT-PCR

Total RNA of brain tissues and cells were extracted with TRIzol reagent following manufacturer’s protocol (15596018, Invitrogen), respectively. The concentration of RNA was measured by NanoDrop spectrophotometer 2000 (Thermo Fisher Scientific) and 500 ng of total RNA was used to generate cDNA. qRT-PCR was made using SYBR Green qPCR mix (Q711, Vazyme) in triplicate, and measured by Applied Biosystems Viiia 7. The final results were calculated in 2−△△ct method. The used primers for qRT-PCR were shown in Table S2.

RNA sequencing

Samples used for the cDNA library were assessed by NanoDrop 2000 (Thermo Fisher Scientific). The RNA integrity value (RIN) was determined with the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies Inc.). A total amount of 3 Î¼g RNA was used for each RNA sample preparations. Sequencing libraries were generated following manufacturer’s recommendations and index codes were added to attribute sequences to each sample using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB).

Raw reads of fastq format were processed to analyze RNA-seq data. Clean reads were gained from raw data by removing reads containing adapter and ploy-N. Then the remained clean reads were mapped to the Mus musculus genome (mm10) using Hisat2 v2.2.1. FeatureCounts v2.0.1 was used to count the read numbers mapped to each gene to quantify the gene expression level. Fragments per kilobase of transcript per million mapped reads (FPKM) of genes were calculated based on the length of the genes and the genes mapping of read counts. The analysis of differential expression was performed using the edgeR package (3.32.1). The p-values were adjusted with the Benjamini and Hochberg’s approach for controlling the false discovery rate. A p-value of 0.05 and absolute fold-change of 2 were set as the threshold for significantly differential expression.

Histone extraction

Cortical tissues of new born pups (P0) were dissected and cut into small pieces. Tissues and cultured eNPCs were resuspended with 1 mL hypotonic lysis buffer (10 mM Tris–HCl, 1 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 10 mM phosphatase inhibitor). The mixture was incubated at 4°C for 30 min and centrifuged for 10 min at a speed of 10000 g. The precipitation was resuspended with 200 μL 0.4 M H2SO4 was added and incubated at 4°C for 2 h. After centrifuged (16000 g for 30 min), the supernatants were collected, and 132 μL trichloroacetic acid was added followed by incubation at 4°C overnight. The second day, the mixture was centrifuged (16000 g, 30 min), and the precipitation was resuspended with 1 mL acetone followed by a centrifugation (16000 g, 4 min). The supernatants were discarded and 1 mL 95% ethanol was added followed by a centrifugation (16000 g, 5 min). The precipitation was dried at room temperature, and dissolved with nuclease free water followed by WB assays.

H3K36me3 co-immunoprecipitation assay

eNPCs and cortical tissues were collected and lysed with 500 μL NP40 buffer 1 (150 mM NaCl, 1.5 mM MgCl2, 0.5% NP40, 50 mM Tris–HCl at pH8.0, 1 tablet protease inhibitor cocktail (P8340, Sigma) on ice for 30 min. Cell lysates were used for immunoprecipitation with H3K36me3 antibody (PTM-625, PTMab) and the corresponding IgG (B900610, Proteintech). Protein A/G magnetic beads (10002D, Thermo Fisher Scientific) were used for H3K36me3 IP. Protein A/G magnetic beads were washed 3 times with IP washing buffer (10 mM Tris–HCl pH7.5, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Triton-X, 0.2 mM sodium orthovanadate) and analyzed with western blotting.

Chromatin immunoprecipitation-qPCR

ChIP experiment was performed following the manufacture’s protocol of the ChIP Kit (P2078, Biyuntian). The experimental procedure was conducted as follows: Freshly harvested cells were crosslinked with 1% formaldehyde in complete medium for 10 min at room temperature to stabilize protein-DNA interactions. The crosslinking reaction was quenched by adding 125 mM glycine for 5 min. Cells were subsequently washed three times with ice-cold phosphate-buffered saline (PBS, pH 7.4) and pelleted by centrifugation at 300 g for 5 min at 4 Â°C. Nuclear fractions were isolated using SDS-lysis buffer (50 mM Tris–HCl pH 8.1, 10 mM EDTA, 1% SDS) supplemented with protease inhibitor cocktail. Chromatin was fragmented to 200–500 bp fragments. Fragment size distribution was verified by 1.5% agarose gel electrophoresis. For immunoprecipitation, chromatin lysates were pre-cleared with Protein A/G agarose beads for 1 h at 4 Â°C. Subsequently, 2 Î¼g of specific primary antibodies were added to chromatin lysate and incubated with rotation at 4 Â°C overnight. Antibody-chromatin complexes were captured by incubation with Protein A/G magnetic beads for 2 h at 4 Â°C. Immune complexes were sequentially washed. Bound chromatin was eluted using elution buffer (1% SDS, 100 mM NaHCO3) and reverse crosslinked at 65 °C overnight. Purified DNA was analyzed by quantitative PCR (qPCR) using SYBR Green Master Mix on a Real-Time PCR System. Primer sequences targeting specific genomic regions of interest were validated for amplification efficiency. Data were normalized to input controls and presented as percentage input using the ΔΔCt method. All experiments were performed in triplicate biological replicates.

DNA pull-down

DNA template was labeled with Biotin-11-dUTP (86303-25-5, MedChemExpress) as manufacturer’s protocol (R001 A, Takara). DNA template labeled with biotin was verified by DNA gel electrophoresis. Desired DNA was extracted by VAHTS DNA clean beads (N411-01, Vazyme). Nucleic proteins were extracted from fresh cortical tissues with NE-PER Nuclear and cytoplasmic Extraction Reagents (78833, Thermo Scientific). 1 Î¼g biotin-labeled DNA template was mixed with 70 μg nucleic protein samples and 50 μL Mag-SA beads (M1000S, BIOEAST). The mixture was incubated at 4°C for 1 h. The mixture was washed with ice-cold PBS for 3 times, and the beads were centrifuged (5000 g, 1 min). The beads were mixed with loading buffer and denatured at 100°C for 10 min followed by WB assays. The template probe primers could be found in Table S2.

Western blot assay

Cells and brain tissues were homogenized in RIPA buffer (ab156034, Abcam) containing 1X protease inhibitor cocktail (P8340, Sigma) and centrifuged at 14,000 rpm for 30 min at 4 Â°C. The supernatants were collected and the concentration of protein was measured with Biophotometer (Eppendorf). 20–40 μg proteins of each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) according to the molecular size and then transferred onto a polyvinylidene fluoride membrane. The bands were blocked in 5% BSA (MB4219, Meilunbio) in TBST at 25 °C for 1 h and incubated with antibodies at 4 Â°C overnight. The signals of the targeted bands were incubated with UltraSignal ECL (4 AW001, 4 A Biotech), and detected by Tanon Detection system (Tanon 5200). The intensity was analyzed with Adobe Photoshop software.

Statistical analysis

All data are expressed as mean ± SEM using GraphPad Prism 10 (GraphPad Software, CA). The number of independent biological replicates was described in figure legends. Quantitative RT-PCR, immunofluorescence staining and western blot assays were replicated at least three times. The differences were assessed using an unpaired Student’s t test between two groups; one-way and two-way ANOVA analysis followed by Tukey’s post hoc analysis were used to determine differences for comparison of multiple groups. Statistical significance was considered when p < 0.05 (statistical significance: n.s, no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001).

Availability of data and materials

The plasmids and related data are available from the corresponding authors on reasonable request.

References

  1. Guo R, et al. BS69/ZMYND11 reads and connects histone H3.3 lysine 36 trimethylation-decorated chromatin to regulated pre-mRNA processing. Mol Cell. 2014;56:298–310. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.molcel.2014.08.022.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wen H, Shi X. H3.3S31 phosphorylation: linking transcription elongation to stimulation responses. Signal Transduct Target Ther. 2020;5:176. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41392-020-00293-6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Wen H, et al. ZMYND11 links histone H3.3K36me3 to transcription elongation and tumour suppression. Nature. 2014;508:263–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/nature13045.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Armache A, et al. Histone H3.3 phosphorylation amplifies stimulation-induced transcription. Nature. 2020;583:852–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41586-020-2533-0.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Zhu J, et al. microRNA-10a-5p from gastric cancer cell-derived exosomes enhances viability and migration of human umbilical vein endothelial cells by targeting zinc finger MYND-type containing 11. Bioengineered. 2022;13:496–507. https://doiorg.publicaciones.saludcastillayleon.es/10.1080/21655979.2021.2009962.

    Article  CAS  PubMed  Google Scholar 

  6. Bressan RB, et al. Regional identity of human neural stem cells determines oncogenic responses to histone H3.3 mutants. Cell Stem Cell. 2021;28:877–93. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.stem.2021.01.016.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhang Z, Xu L, Hu Z, Yang B, Xu J. MicroRNA-196b promotes cell growth and metastasis of ovarian cancer by targeting ZMYND11. Minerva Med. 2022;113:707–17. https://doiorg.publicaciones.saludcastillayleon.es/10.23736/s0026-4806.20.06654-9.

    Article  PubMed  Google Scholar 

  8. Meng X, et al. USP53 affects the proliferation and apoptosis of breast cancer cells by regulating the ubiquitination level of ZMYND11. Biol Proced Online. 2024;26:24. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12575-024-00251-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lian C, et al. Epigenetic reader ZMYND11 noncanonical function restricts HNRNPA1-mediated stress granule formation and oncogenic activity. Signal Transduct Target Ther. 2024;9:258. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41392-024-01961-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Yang JP, et al. Downregulation of ZMYND11 induced by miR-196a-5p promotes the progression and growth of GBM. Biochem Biophys Res Commun. 2017;494:674–80. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.bbrc.2017.10.098.

    Article  CAS  PubMed  Google Scholar 

  11. Salloum R, et al. Characterizing temporal genomic heterogeneity in pediatric high-grade gliomas. Acta Neuropathol Commun. 2017;5:78. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40478-017-0479-8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Deloukas P, et al. The DNA sequence and comparative analysis of human chromosome 10. Nature. 2004;429:375–81. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/nature02462.

    Article  CAS  PubMed  Google Scholar 

  13. Uhlén M, et al. Proteomics. Tissue-based map of the human proteome. Science. 2015;347:1260419. https://doiorg.publicaciones.saludcastillayleon.es/10.1126/science.1260419.

    Article  CAS  PubMed  Google Scholar 

  14. Coe BP, et al. Refining analyses of copy number variation identifies specific genes associated with developmental delay. Nat Genet. 2014;46:1063–71. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/ng.3092.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lindstrand A, et al. Molecular and clinical characterization of patients with overlapping 10p deletions. Am J Med Genet A. 2010;152a:1233–43. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/ajmg.a.33366.

    Article  PubMed  Google Scholar 

  16. Tumiene B, et al. Phenotype comparison confirms ZMYND11 as a critical gene for 10p15.3 microdeletion syndrome. J Appl Genet. 2017;58:467–74. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s13353-017-0408-3.

    Article  CAS  PubMed  Google Scholar 

  17. DeScipio C, et al. Subtelomeric deletion of chromosome 10p15.3: clinical findings and molecular cytogenetic characterization. Am J Med Genet A. 2012;158a:2152–61. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/ajmg.a.35574.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Oates S, et al. ZMYND11 variants are a novel cause of centrotemporal and generalised epilepsies with neurodevelopmental disorder. Clin Genet. 2021;100:412–29. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/cge.14023.

    Article  CAS  PubMed  Google Scholar 

  19. Yates TM, et al. ZMYND11-related syndromic intellectual disability: 16 patients delineating and expanding the phenotypic spectrum. Hum Mutat. 2020;41:1042–50. https://doiorg.publicaciones.saludcastillayleon.es/10.1002/humu.24001.

    Article  CAS  PubMed  Google Scholar 

  20. Chang X, et al. ZMYND11 functions in bimodal regulation of latent genes and brain-like splicing to safeguard corticogenesis. bioRxiv. 2024. https://doiorg.publicaciones.saludcastillayleon.es/10.1101/2024.10.15.618524.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Barral A, et al. SETDB1/NSD-dependent H3K9me3/H3K36me3 dual heterochromatin maintains gene expression profiles by bookmarking poised enhancers. Mol Cell. 2022;82:816–32. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.molcel.2021.12.037.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zhu K, et al. SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing. Nucleic Acids Res. 2017;45:92–105. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/nar/gkw814.

    Article  CAS  PubMed  Google Scholar 

  23. Edmunds JW, Mahadevan LC, Clayton AL. Dynamic histone H3 methylation during gene induction: HYPB/Setd2 mediates all H3K36 trimethylation. EMBO J. 2008;27:406–20. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/sj.emboj.7601967.

    Article  CAS  PubMed  Google Scholar 

  24. Yoh SM, Lucas JS, Jones KA. The Iws1:Spt6:CTD complex controls cotranscriptional mRNA biosynthesis and HYPB/Setd2-mediated histone H3K36 methylation. Genes Dev. 2008;22:3422–34. https://doiorg.publicaciones.saludcastillayleon.es/10.1101/gad.1720008.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhou L, et al. Mina53 demethylates histone H4 arginine 3 asymmetric dimethylation to regulate neural stem/progenitor cell identity. Nat Commun. 2024;15:10227. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41467-024-54680-6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Guo H, et al. Fucosyltransferase 8 regulates adult neurogenesis and cognition of mice by modulating the Itga6-PI3K/Akt signaling pathway. Science China Life sciences. 2024;67:1427–40. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s11427-023-2510-0.

    Article  CAS  PubMed  Google Scholar 

  27. Li L, et al. Fat mass and obesity-associated (FTO) protein regulates adult neurogenesis. Hum Mol Genet. 2017;26:2398–411. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/hmg/ddx128.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhang J, et al. Ogt deficiency induces abnormal cerebellar function and behavioral deficits of adult mice through modulating RhoA/ROCK signaling. J Neurosci. 2023;43:4559–79. https://doiorg.publicaciones.saludcastillayleon.es/10.1523/JNEUROSCI.1962-22.2023.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kempermann G. Environmental enrichment, new neurons and the neurobiology of individuality. Nat Rev Neurosci. 2019;20:235–45. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41583-019-0120-x.

    Article  CAS  PubMed  Google Scholar 

  30. Cobben JM, et al. A de novo mutation in ZMYND11, a candidate gene for 10p15.3 deletion syndrome, is associated with syndromic intellectual disability. Eur J Med Genet. 2014;57:636–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ejmg.2014.09.002.

    Article  CAS  PubMed  Google Scholar 

  31. Moskowitz AM, et al. A de novo missense mutation in ZMYND11 is associated with global developmental delay, seizures, and hypotonia. Cold Spring Harb Mol Case Stud. 2016;2:a000851. https://doiorg.publicaciones.saludcastillayleon.es/10.1101/mcs.a000851.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Iossifov I, et al. De novo gene disruptions in children on the autistic spectrum. Neuron. 2012;74:285–99. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.neuron.2012.04.009.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bodetko A, et al. Further delineation of clinical phenotype of ZMYND11 variants in patients with neurodevelopmental dysmorphic syndrome. Genes (Basel). 2024. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/genes15020256.

    Article  PubMed  Google Scholar 

  34. Yu B, et al. BS69 undergoes SUMO modification and plays an inhibitory role in muscle and neuronal differentiation. Exp Cell Res. 2009;315:3543–53. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.yexcr.2009.09.011.

    Article  CAS  PubMed  Google Scholar 

  35. Fu H, et al. Dihydroartemisinin inhibits EphA2/PI3K/Akt pathway-mediated malignant behaviors and vasculogenic mimicry in glioma stem cells. Heliyon. 2025;11:e42095. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.heliyon.2025.e42095.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Liu B, et al. microRNA-451a promoter methylation regulated by DNMT3B expedites bladder cancer development via the EPHA2/PI3K/AKT axis. BMC Cancer. 2020;20:1019. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12885-020-07523-8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Aoki M, Yamashita T, Tohyama M. EphA receptors direct the differentiation of mammalian neural precursor cells through a mitogen-activated protein kinase-dependent pathway. J Biol Chem. 2004;279:32643–50. https://doiorg.publicaciones.saludcastillayleon.es/10.1074/jbc.M313247200.

    Article  CAS  PubMed  Google Scholar 

  38. Li A, et al. USP3 promotes osteosarcoma progression via deubiquitinating EPHA2 and activating the PI3K/AKT signaling pathway. Cell Death Dis. 2024;15:235. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41419-024-06624-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Rajebhosale P, et al. Neuregulin1 nuclear signaling influences adult neurogenesis and regulates a schizophrenia susceptibility gene network within the mouse dentate gyrus. J Neurosci. 2024. https://doiorg.publicaciones.saludcastillayleon.es/10.1523/JNEUROSCI.0063-24.2024.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Jin Y, et al. Serum-tolerant polymeric complex for stem-cell transfection and neural differentiation. Nat Commun. 2025. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41467-025-57278-8.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Strahl BD, et al. Set2 is a nucleosomal histone H3-selective methyltransferase that mediates transcriptional repression. Mol Cell Biol. 2002;22:1298–306. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/MCB.22.5.1298-1306.2002.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Connacher J, et al. H3K36 methylation reprograms gene expression to drive early gametocyte development in Plasmodium falciparum. Epigenetics Chromatin. 2021;14:19. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13072-021-00393-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wagner EJ, Carpenter PB. Understanding the language of Lys36 methylation at histone H3. Nat Rev Mol Cell Biol. 2012;13:115–26. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/nrm3274.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Li Q, et al. RYBP modulates embryonic neurogenesis involving the Notch signaling pathway in a PRC1-independent pattern. Stem cell reports. 2021. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.stemcr.2021.10.013.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Shen H, et al. O-GlcNAc transferase Ogt regulates embryonic neuronal development through modulating Wnt/beta-catenin signaling. Hum Mol Genet. 2021;31:57–68. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/hmg/ddab223.

    Article  CAS  PubMed  Google Scholar 

  46. Li Y, Jiao J. Deficiency of TRPM2 leads to embryonic neurogenesis defects in hyperthermia. Sci Adv. 2020;6:eaay6350. https://doiorg.publicaciones.saludcastillayleon.es/10.1126/sciadv.aay6350.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was supported in part by the National Natural Science Foundation of China (grants 82371182 to X.L., 32200816 to R.Z.), and Natural Science Foundation of Zhejiang Province (LD25H090001 to X.L.). J.Z. was supported by China Postdoctoral Science Foundation (2024M762895).

Author information

Authors and Affiliations

  1. Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, China

    Xu Yang, Lan Li, Wenzheng Qu, Xuejun Cheng, Jinyu Zhang, Rui Zheng & Xuekun Li

  2. The Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China

    Xu Yang, Lan Li & Xuekun Li

  3. Binjiang Institute of Zhejiang University, Hangzhou, China

    Xu Yang, Jinyu Zhang & Xuekun Li

  4. Department of Neurology, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Yan Sun & Guoping Peng

  5. Center for Reproductive Medicine, Department of Obstetrics, Zhejiang Provincial People’s Hospital, Hangzhou, China

    Suxiao Liu

Authors
  1. Xu Yang
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Lan Li
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Wenzheng Qu
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Xuejun Cheng
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Jinyu Zhang
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Yan Sun
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Suxiao Liu
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Guoping Peng
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Rui Zheng
    View author publications

    You can also search for this author inPubMed Google Scholar

  10. Xuekun Li
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

X.L. conceptualized the project. X.Y. performed immunofluorescence staining, qRT-PCR and Western blot with the help of L.L., S.L. and Y.S. W.Q. analyzed RNA sequencing data. X.Y., X.C. and R. Z. conducted the isolation and culture of eNPCs and neurons. X.L. wrote the manuscript with the help from R.Z. and G. P.

Corresponding authors

Correspondence to Guoping Peng, Rui Zheng or Xuekun Li.

Ethics declarations

Ethics approval and consent to participate

All animal experiments were carried out in accordance with the protocols approved by the Zhejiang University Animal Care and Ethics Committee.

Consent for publication

All authors reviewed and approved the final manuscript for publication.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

13578_2025_1392_MOESM1_ESM.pdf

Supplementary material 1: Figure S1. Dynamic and abundant expression of Zmynd11 in embryonic brain and neuronal progenitor cells.Representative images of Zmynd11 and embryonic neuronal progenitor cellsmarker Nestin, neuronal cell marker Tuj1 immunostaining with brain sections of embryonic miceand postnatal day 0 mice. Scale bars, 50 μm. SVZ, subventricular zone; VZ, ventricular zone; IZ, intermediate zone. Representative images of WBand quantification resultsshowed that the level of Zmynd11 was significantly increased in the cortex with neuronal development of mice. GAPDH was used as an internal control. n = 5 mice in each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 62.19. qRT-PCR assay results showed the level of Zmynd11 was significantly increased in the cortex of mice with neuronal development. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 116.3. Representative images of Zmynd11, Nestin and Tuj1 immunostaining with proliferating and differentiated eNPCs, respectively. Scale bars, 50 μm. Representative images of WB and quantification results showed that the level of Zmynd11 was significantly increased upon the differentiation of eNPCs. GAPDH was used as an internal control. n = 5 mice in each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, unpaired Student’s t test.qRT-PCR assay results showed the level of Zmynd11 was significantly increased upon the differentiation of eNPCs. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test.Representative images of Zmynd11, Nestin, neuronal cell marker MAP2 and astrocyte cell marker GFAP immunostaining with proliferating eNPCs, neurons and astrocytes respectively. Scale bars, 50 μm.Representative images of WBand quantification resultsshowed that the level of Zmynd11 in neurons and astrocytes was significantly increased compared to proliferating eNPCs. GAPDH was used as an internal control. n = 5 mice in each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 61.14.qRT-PCR assay results showed the level of Zmynd11 in neurons and astrocytes was significantly increased compared to proliferating eNPCs. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 106.2. Figure S2. Specific deficiency of Zmynd11 in Nestin+ cells results in developmental deficits of mice. Schematic diagram of the generation of Zmynd11 cKO mice.Representative images of Ctrl and cKO mice at the age of P0. Quantification of body weight of Ctrl and cKO mice at P0. n = 6 mice for each group. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test. Representative images of radial glial marker PAX6 immunostaining with scramble and Zmynd11 KD brain sections. Scramble and shZmynd11 plasmids were delivered into the brain via in utero electroporation at E14, and mice were sacrificed for assay at E17. Scale bar, 50 μm. Quantification results showed the percentage of PAX6+GFP+/GFP+ in VZ/SVZ layer was significantly decreased in Zmynd11 KD group compared to Ctrl. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01, unpaired Student’s t test.Representative images of intermediate progenitor marker Tbr2 immunostaining with scramble and Zmynd11 KD brain sections. Scale bar, 50 μm. Quantification results showed the percentage of Tbr2+GFP+/GFP+ in VZ/SVZ layer was significantly increased in Zmynd11 KD group compared to Ctrl. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01, ***p < 0.001, unpaired Student’s t test. Representative images of the distribution of GFP+ cells in the cortex of E17 fetal brain. Scramble and shZmynd11 plasmids were delivered into E14 mice brains via IUE, and mice were sacrificed for assays at E17. Scale bar, 50 μm. Quantification results showed that Zmynd11 KD induced fewer GFP+ cells in VZ/SVZ and CP regions, but more cells in IZ compared to scramble group. n = 5 fetuses at least from 4 pregnant mice for each group. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01, ***p < 0.001, unpaired Student’s t test. Figure S3. Zmynd11 KD inhibits PI3K signaling pathway in eNPCs. Schematic diagram of genes relating to PI3K signaling pathway.qRT-PCR assay results showed that Zmynd11 KD significantly reduced the mRNA level of multiple key components of PI3K signaling pathway in eNPCs. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance; unpaired Student’s t-test. Representative images of WB of PI3K components in proliferating eNPCs infected with lenti-scramble and lenti-shZmynd11, respectively. Cells were collected for assays 3 days post the virus infection. Quantification results showed that Zmynd11 KD significantly reduced the level of Epha2. Although the levels of total IRS1, PI3K, PDPK1, and SGK1were not changed, the levels of Epha2, p-IRS1, p-PI3K, p-PDPK1, and p-SGK1were significantly decreased in eNPCs. n = 4 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test. Figure S4. Zmynd11 deficiency did not affect total levels of H3K36me3 and Pol II. Immunoprecipitation followed by WB assays showed that Zmynd11 easily precipitated H3K36me3 in eNPCs, and vice versa. ChIP followed by WB assays showed the enrichment of H3K36me3 at Epha2 promotor in eNPCs.WB assays and quantification results showed that the level of total H3K36me3 was not changed in cKO brain tissues and Zmynd11 KD eNPCs. H3 was used as internal control. n = 4 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test.ChIP followed by WB assay showed the binding of RNA Polymerase II at Epha2 promotor in eNPCs. WB assay and quantification results showed that levels of Pol II was not changed in cKO brain tissues. Tubulin was used as internal control. n = 4 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test. Figure S5. Exogenous Epha2 rescues neuronal deficits induced by Zmynd11 KD. Cortical neurons from E14 fetal mice were infected with lentivirus at DIV 4, and MAP2 immunostaining was performed at DIV 7. Lenti-scramble-GFP, Lenti-RFP, Lenti-shZmynd11-GFP, and lenti-Epha2-RFP viruses were used for infecting cells. Scale bar, 50 μm. Quantification results showed that exogenous Epha2 significantly enhanced the dendritic lengthand the number of branch pointsof cortical neurons. n = 30 cells for each group. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 44.25 for, F= 42.61 for. Sholl analysis results showed that exogenous Epha2 significantly increased the dendritic complexity of cortical neurons. n = 30 cells for each group. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 10.53. qRT-PCR assay results showed that exogenous Epha2 did not affect the level of Zmynd11 mRNA, but significantly enhanced mRNA levels of Epha2 and Gfap, and reduced the mRNA level of Tuj1. n = 3 biologically independent experiments. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F= 82.44 for, F= 5374 for, F= 71.21 for, F= 107.8 for.

Supplementary material 2: Table S1. Differentially expressed genesin Zmynd11 KD eNPCs

Supplementary material 3: Table S2. The used primers

Supplementary material 4: Table S3. The used antibodies

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, X., Li, L., Qu, W. et al. Zmynd11 is essential for neurogenesis by coordinating H3K36me3 modification of Epha2 and PI3K signaling pathway. Cell Biosci 15, 55 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13578-025-01392-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13578-025-01392-z

Keywords

Cell & Bioscience

ISSN: 2045-3701

Contact us