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Ezh2 shapes T cell plasticity to drive atherosclerosis

ABSTRACT: The activation and polarization of T cells play a crucial role in atherosclerosis and dictate athero-inflammation. The epigenetic enzyme enhancer of zeste homolog 2 (EZH2) mediates the tri-methylation of lysine27 on histone3 (H3K27me3) and is pivotal in controlling T cell responses. To detail the role of T cell-EZH2 in atherosclerosis, we used human carotid endarterectomy specimens to reveal plaque expression and geography of EZH2. Atherosclerosis-prone Apoe-deficient mice with CD4+ and/or CD8+ T cell-specific Ezh2 deletion (Ezh2cd4-KO, Ezh2cd8-KO) were analyzed to unravel T cell-Ezh2’s role in atherosclerosis and T cell-associated immune status.EZH2 expression is elevated in advanced human atherosclerotic plaques and primarily expressed in the T cell nucleus, suggesting the importance of canonical EZH2 function in atherosclerosis. Ezh2cd4-KO, but not Ezh2cd8-KO mice showed reduced atherosclerosis with less advanced plaques, which contained less collagen and macrophages, indicating that Ezh2 in CD4+ T cells drives atherosclerosis. In-depth analysis of CD4+ T cells of Ezh2cd4-KO mice revealed that absence of Ezh2 results in a type II immune response with increased Il-4 gene and protein expression in the aorta and lymphoid organs. In vitro, Ezh2-deficient T cells polarized macrophages towards an anti-inflammatory phenotype. scRNAseq of splenic T cells revealed that Ezh2-deficiency reduced naive, Ccl5+ and regulatory T cell populations and increased the frequencies of memory T cells and invariant natural killer T (iNKT) cells. Flow cytometric analysis identified a shift towards T helper 2 (Th2) effector CD4+ T cells in Ezh2cd4-KO mice and confirmed a profound increase in splenic iNKT cells with high expression of Plzf, the characteristic marker of the iNKT2 subset. Likewise, Zbtb16 (Plzf-encoding gene) transcripts were elevated in the aorta of Ezh2cd4-KO mice, suggesting an accumulation of iNKT2 cells in the plaque. H3K27me3-chromatin immunoprecipitation followed by qPCR showed that T cell-Ezh2 regulates the transcription of the Il-4 and Zbtb16 genes. Our study uncovers the importance of T cell-EZH2 in human and mouse atherosclerosis. Inhibition of Ezh2 in CD4+ T cells drives type II immune responses, resulting in an accumulation of iNKT2 and Th2 cells, memory T cells and anti-inflammatory macrophages that limit the progression of atherosclerosis. We performed scRNAseq of splenic T cells isolated from Ezh2cd4-KO and respective controls fed with Western type diet, to study T cells during atherosclerosis progression.

ORGANISM(S): Mus musculus

PROVIDER: GSE283002 | GEO | 2025/02/13

REPOSITORIES: GEO

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Project description:The activation and polarization of T cells play a crucial role in atherosclerosis and dictate athero-inflammation. The epigenetic enzyme enhancer of zeste homolog 2 (EZH2) mediates the tri-methylation of lysine27 on histone3 (H3K27me3) and is pivotal in controlling T cell responses. To detail the role of T cell-EZH2 in atherosclerosis, we used human carotid endarterectomy specimens to reveal plaque expression and geography of EZH2. Atherosclerosis-prone Apoe-deficient mice with CD4+ and/or CD8+ T cell-specific Ezh2 deletion (Ezh2cd4-KO, Ezh2cd8-KO) were analyzed to unravel T cell-Ezh2’s role in atherosclerosis and T cell-associated immune status.EZH2 expression is elevated in advanced human atherosclerotic plaques and primarily expressed in the T cell nucleus, suggesting the importance of canonical EZH2 function in atherosclerosis. Ezh2cd4-KO, but not Ezh2cd8-KO mice showed reduced atherosclerosis with less advanced plaques, which contained less collagen and macrophages, indicating that Ezh2 in CD4+ T cells drives atherosclerosis. In-depth analysis of CD4+ T cells of Ezh2cd4-KO mice revealed that absence of Ezh2 results in a type II immune response with increased Il-4 gene and protein expression in the aorta and lymphoid organs. In vitro, Ezh2-deficient T cells polarized macrophages towards an anti-inflammatory phenotype. scRNAseq of splenic T cells revealed that Ezh2-deficiency reduced naive, Ccl5+ and regulatory T cell populations and increased the frequencies of memory T cells and invariant natural killer T (iNKT) cells. Flow cytometric analysis identified a shift towards T helper 2 (Th2) effector CD4+ T cells in Ezh2cd4-KO mice and confirmed a profound increase in splenic iNKT cells with high expression of Plzf, the characteristic marker of the iNKT2 subset. Likewise, Zbtb16 (Plzf-encoding gene) transcripts were elevated in the aorta of Ezh2cd4-KO mice, suggesting an accumulation of iNKT2 cells in the plaque. H3K27me3-chromatin immunoprecipitation followed by qPCR showed that T cell-Ezh2 regulates the transcription of the Il-4 and Zbtb16 genes. Our study uncovers the importance of T cell-EZH2 in human and mouse atherosclerosis. Inhibition of Ezh2 in CD4+ T cells drives type II immune responses, resulting in an accumulation of iNKT2 and Th2 cells, memory T cells and anti-inflammatory macrophages that limit the progression of atherosclerosis. We performed RNAseq on splenic CD4+ T cells isolated from Ezh2cd4-KO mice and Ezh2cd4-WT controls using an efficient sequencing method (Prime-seq).

Project description:Spermatogonial stem cells (SSCs) balance self-renewal versus differentiation/spermatogenesis to ensure continuous sperm production. Here, we uncover multiple roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG). ZBTB16 activates genes in uSPG promoting self-renewal and cell cycle progression (Ccnd1) to maintain uSPG and transit-amplifying states. Remarkably, in uSPG, ZBTB16, SALL4, SOX3 all co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions also feature broad H3K4me3 and H3K27ac marks, DNA hypomethylation, and often CTCF binding. Hi-C analyses reveal robust promoter-promoter physical interactions, revealing a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation. Therefore, ZBTB16 ensures uSPG cell cycle progression and colocalizes with SALL4, SOX3 and often CTCF to establish a novel chromatin poising network.Spermatogonial stem cells (SSCs) balance self-renewal and differentiation to ensure continuous sperm production in the testis. The transcription factor Zbtb16 (PLZF) supports undifferentiated SSC maintenance through partly unknown mechanisms. We combined genomics (RNA-seq and ChIP-seq) and genetic approaches to reveal multiple functions of Zbtb16 in juvenile mouse SSCs. Zbtb16-bound loci show a striking correlation with active promoters bearing H3K4me3 and the activator Sall4. Zbtb16 activates genes that support SSC self-renewal and cell cycle progression (e.g., Ccnd1) that help maintain undifferentiated SSC pools, including both self-renewing SSCs and transit-amplifying progenitors. Zbtb16 also attenuates certain genes, including meiotic genes and specific retrotransposons that confer genome instability. Notably, Zbtb16 genome localization and its impact on the transcriptome are dynamic, displaying mesenchymal gene targets in vivo, which are not maintained in cultured SSCs. Our data reveal dynamic roles for Zbtb16 in ensuring SSC identity, amplification, and maintenance in vivo.

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Ezh2 shapes T cell plasticity to drive atherosclerosis

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