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Critical role of histone demethylase Jmjd3 in the regulation of CD4+ T-cell differentiation.

Li Q, Zou J, Wang M, Ding X, Chepelev I, Zhou X, Zhao W, Wei G, Cui J, Zhao K, Wang HY, Wang RF - Nat Commun (2014)

Bottom Line: The skewing of T-cell differentiation is concomitant with changes in the expression of key transcription factors and cytokines.H3K27me3 and H3K4me3 levels in Jmjd3-deficient cells are correlated with altered gene expression through interactions with specific transcription factors.Our results identify Jmjd3 as an epigenetic factor in T-cell differentiation via changes in histone methylation and target gene expression.

View Article: PubMed Central - PubMed

Affiliation: Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, Texas 77030, USA.

ABSTRACT
Epigenetic factors have been implicated in the regulation of CD4(+) T-cell differentiation. Jmjd3 plays a role in many biological processes, but its in vivo function in T-cell differentiation remains unknown. Here we report that Jmjd3 ablation promotes CD4(+) T-cell differentiation into Th2 and Th17 cells in the small intestine and colon, and inhibits T-cell differentiation into Th1 cells under different cytokine-polarizing conditions and in a Th1-dependent colitis model. Jmjd3 deficiency also restrains the plasticity of the conversion of Th2, Th17 or Treg cells to Th1 cells. The skewing of T-cell differentiation is concomitant with changes in the expression of key transcription factors and cytokines. H3K27me3 and H3K4me3 levels in Jmjd3-deficient cells are correlated with altered gene expression through interactions with specific transcription factors. Our results identify Jmjd3 as an epigenetic factor in T-cell differentiation via changes in histone methylation and target gene expression.

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Jmjd3 regulates Th1 and Th2 differentiation by facilitating the T-bet-RbBP5 and Smad3-Ash2L interaction(a) 293T cells were cotransfected with HA-Jmjd3 and FLAG-tagged T-bet, GATA-3, Foxp3, and RORγT plasmids. Whole cell lysates (WCL) were immunoprecipitated with anti-T-bet, anti-GATA-3, anti-Foxp3, and anti-RORγT antibodies and immunoblotted with anti-FLAG antibody. (b) 293T cells were cotransfected with HA-Jmjd3 and FLAG-tagged Wdr5, Ash2L, or RbBP5. WCL were immunoprecipitated with anti-HA beads and immunoblotted with anti-FLAG antibody. (c) 293T cells were cotransfected with T-bet and FLAG-tagged Wdr5, Ash2L, Dpy30 or RbBP5. WCL were immunoprecipitated with anti-T-bet antibody and protein (A+G), and immunoblotted with anti-FLAG antibody. (d) Cell lysates were obtained from WT and Jmjd3 cKO Th1 cells and immunoprecipitated with anti-Jmjd3 antibody and protein (A+G). The immunoprecipitated product was immunoblotted with anti-Jmjd3, anti-T-bet, and anti-Ash2L antibodies. (e) WCL were obtained from WT and Jmjd3 cKO Th1 cells and immunoprecipitated with anti-T-bet antibody and protein (A+G). The immunoprecipitated product was immunoblotted with anti-RbBP5 antibody. (f) ChIP-qPCR analysis of T-bet binding to the promoter regions of Cxcr3 and Ifng genes in WT and Jmjd3 cKO Th1 cells. (g) WCL obtained from 293T cells cotransfected with HA-Jmjd3 and FLAG-Smad1, FLAG-Smad2, or FLAG-Smad3 plasmids were immunoprecipitated with anti-HA beads. The immunoprecipitated product was immunoblotted with anti-FLAG and anti-HA antibodies. (h) WCL and protein isolates from WT and Jmjd3cKO CD4+ T cells derived from thymus. (i) 293T cells were cotransfected with HA-tagged Smad3 and FLAG-tagged Wdr5, Ash2L, Dpy30 or RbBP5. WCL were immunoprecipitated with anti-FLAG beads and immunoblotted with anti-HA antibody. (j) Cell lysates were obtained from WT and Jmjd3 cKO Treg cells and immunoprecipitated with anti-Smad3 antibody. The immunoprecipitated product was immunoblotted with anti-Jmjd3, anti-Smad3 and anti-Ash2L antibodies. (k) ChIP-qPCR analysis of Smad3 binding to the promoter regions of Foxp3 gene in WT and Jmjd3 cKO Treg cells.
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Figure 7: Jmjd3 regulates Th1 and Th2 differentiation by facilitating the T-bet-RbBP5 and Smad3-Ash2L interaction(a) 293T cells were cotransfected with HA-Jmjd3 and FLAG-tagged T-bet, GATA-3, Foxp3, and RORγT plasmids. Whole cell lysates (WCL) were immunoprecipitated with anti-T-bet, anti-GATA-3, anti-Foxp3, and anti-RORγT antibodies and immunoblotted with anti-FLAG antibody. (b) 293T cells were cotransfected with HA-Jmjd3 and FLAG-tagged Wdr5, Ash2L, or RbBP5. WCL were immunoprecipitated with anti-HA beads and immunoblotted with anti-FLAG antibody. (c) 293T cells were cotransfected with T-bet and FLAG-tagged Wdr5, Ash2L, Dpy30 or RbBP5. WCL were immunoprecipitated with anti-T-bet antibody and protein (A+G), and immunoblotted with anti-FLAG antibody. (d) Cell lysates were obtained from WT and Jmjd3 cKO Th1 cells and immunoprecipitated with anti-Jmjd3 antibody and protein (A+G). The immunoprecipitated product was immunoblotted with anti-Jmjd3, anti-T-bet, and anti-Ash2L antibodies. (e) WCL were obtained from WT and Jmjd3 cKO Th1 cells and immunoprecipitated with anti-T-bet antibody and protein (A+G). The immunoprecipitated product was immunoblotted with anti-RbBP5 antibody. (f) ChIP-qPCR analysis of T-bet binding to the promoter regions of Cxcr3 and Ifng genes in WT and Jmjd3 cKO Th1 cells. (g) WCL obtained from 293T cells cotransfected with HA-Jmjd3 and FLAG-Smad1, FLAG-Smad2, or FLAG-Smad3 plasmids were immunoprecipitated with anti-HA beads. The immunoprecipitated product was immunoblotted with anti-FLAG and anti-HA antibodies. (h) WCL and protein isolates from WT and Jmjd3cKO CD4+ T cells derived from thymus. (i) 293T cells were cotransfected with HA-tagged Smad3 and FLAG-tagged Wdr5, Ash2L, Dpy30 or RbBP5. WCL were immunoprecipitated with anti-FLAG beads and immunoblotted with anti-HA antibody. (j) Cell lysates were obtained from WT and Jmjd3 cKO Treg cells and immunoprecipitated with anti-Smad3 antibody. The immunoprecipitated product was immunoblotted with anti-Jmjd3, anti-Smad3 and anti-Ash2L antibodies. (k) ChIP-qPCR analysis of Smad3 binding to the promoter regions of Foxp3 gene in WT and Jmjd3 cKO Treg cells.

Mentions: We next determined whether Jmjd3 recruitment to specific gene promoters was mediated through interactions with transcription and epigenetic factors involved in T cell differentiation. Co-immunoprecipitation analyses revealed that Jmjd3 interacts with T-bet, but not Gata3, Foxp3, or RoRγt, in 293T cells (Fig. 7a and Supplementary Fig. 3b). Because H3K4 methylation levels were altered at many gene loci in Jmjd3-deficient T cells, we determined its interaction with key components of the H3K4 methyltransferase complex (Ash2L, RbBP5, and Wdr5). We found that Jmjd3 interacted with Ash2L, but not with RbBP5 or Wdr5, in 293T cells (Fig. 7b and Supplementary Fig. 3c). Co-IP in 293T cells, co-transfected with T-bet and FLAG-tagged Ash2L, RbBP5 and Wdr5 plasmids, revealed that T-bet physically interacted with RbBP5 (Fig. 7c and Supplementary Fig. 3d). The endogenous interaction of Jmjd3 with Ash2L and T-bet was observed in WT but not in Jmjd3 cKO T cells (Fig. 7d and Supplementary Fig. 3e). Although T-bet interacts with Jmjd3 and RbBP5, a core protein of the H3K4 methyltransferase complex39, it is unclear whether Jmjd3 is required for T-bet-RbBP5 interaction. Immunoprecipitation and immunoblot analysis revealed that T-bet interacted with RbBP5 in the presence of Jmjd3, but failed to interact in Jmjd3-deficient T cells (Fig. 7e and Supplementary Fig. 3f). To determine the functional relevance, we performed ChIP-quantitative PCR (qPCR) analysis of Cxcr3 and Ifng promoter regions in WT and Jmjd3 cKO Th1 cells and CD4+ SP thymocytes. Consistent with our previous observations, Jmjd3-deficient T cells disrupted the endogenous T-bet-RbBP5 interaction and markedly reduced T-bet binding to the promoter regions of Cxcr3, Ifng, and Cd44 genes (Fig. 7f and Supplementary Fig. 6a). These results suggest that Jmjd3 is required for T-bet and Ash2L to form a stable complex capable of binding to specific target gene promoters. Jmjd3 deficiency might destabilize this complex formation and reduce T-bet binding to Cxcr,Ifng, and Cd44 promoters, leading Cxcr3, Ifng, and Cd44 gene expression downregulation.


Critical role of histone demethylase Jmjd3 in the regulation of CD4+ T-cell differentiation.

Li Q, Zou J, Wang M, Ding X, Chepelev I, Zhou X, Zhao W, Wei G, Cui J, Zhao K, Wang HY, Wang RF - Nat Commun (2014)

Jmjd3 regulates Th1 and Th2 differentiation by facilitating the T-bet-RbBP5 and Smad3-Ash2L interaction(a) 293T cells were cotransfected with HA-Jmjd3 and FLAG-tagged T-bet, GATA-3, Foxp3, and RORγT plasmids. Whole cell lysates (WCL) were immunoprecipitated with anti-T-bet, anti-GATA-3, anti-Foxp3, and anti-RORγT antibodies and immunoblotted with anti-FLAG antibody. (b) 293T cells were cotransfected with HA-Jmjd3 and FLAG-tagged Wdr5, Ash2L, or RbBP5. WCL were immunoprecipitated with anti-HA beads and immunoblotted with anti-FLAG antibody. (c) 293T cells were cotransfected with T-bet and FLAG-tagged Wdr5, Ash2L, Dpy30 or RbBP5. WCL were immunoprecipitated with anti-T-bet antibody and protein (A+G), and immunoblotted with anti-FLAG antibody. (d) Cell lysates were obtained from WT and Jmjd3 cKO Th1 cells and immunoprecipitated with anti-Jmjd3 antibody and protein (A+G). The immunoprecipitated product was immunoblotted with anti-Jmjd3, anti-T-bet, and anti-Ash2L antibodies. (e) WCL were obtained from WT and Jmjd3 cKO Th1 cells and immunoprecipitated with anti-T-bet antibody and protein (A+G). The immunoprecipitated product was immunoblotted with anti-RbBP5 antibody. (f) ChIP-qPCR analysis of T-bet binding to the promoter regions of Cxcr3 and Ifng genes in WT and Jmjd3 cKO Th1 cells. (g) WCL obtained from 293T cells cotransfected with HA-Jmjd3 and FLAG-Smad1, FLAG-Smad2, or FLAG-Smad3 plasmids were immunoprecipitated with anti-HA beads. The immunoprecipitated product was immunoblotted with anti-FLAG and anti-HA antibodies. (h) WCL and protein isolates from WT and Jmjd3cKO CD4+ T cells derived from thymus. (i) 293T cells were cotransfected with HA-tagged Smad3 and FLAG-tagged Wdr5, Ash2L, Dpy30 or RbBP5. WCL were immunoprecipitated with anti-FLAG beads and immunoblotted with anti-HA antibody. (j) Cell lysates were obtained from WT and Jmjd3 cKO Treg cells and immunoprecipitated with anti-Smad3 antibody. The immunoprecipitated product was immunoblotted with anti-Jmjd3, anti-Smad3 and anti-Ash2L antibodies. (k) ChIP-qPCR analysis of Smad3 binding to the promoter regions of Foxp3 gene in WT and Jmjd3 cKO Treg cells.
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Figure 7: Jmjd3 regulates Th1 and Th2 differentiation by facilitating the T-bet-RbBP5 and Smad3-Ash2L interaction(a) 293T cells were cotransfected with HA-Jmjd3 and FLAG-tagged T-bet, GATA-3, Foxp3, and RORγT plasmids. Whole cell lysates (WCL) were immunoprecipitated with anti-T-bet, anti-GATA-3, anti-Foxp3, and anti-RORγT antibodies and immunoblotted with anti-FLAG antibody. (b) 293T cells were cotransfected with HA-Jmjd3 and FLAG-tagged Wdr5, Ash2L, or RbBP5. WCL were immunoprecipitated with anti-HA beads and immunoblotted with anti-FLAG antibody. (c) 293T cells were cotransfected with T-bet and FLAG-tagged Wdr5, Ash2L, Dpy30 or RbBP5. WCL were immunoprecipitated with anti-T-bet antibody and protein (A+G), and immunoblotted with anti-FLAG antibody. (d) Cell lysates were obtained from WT and Jmjd3 cKO Th1 cells and immunoprecipitated with anti-Jmjd3 antibody and protein (A+G). The immunoprecipitated product was immunoblotted with anti-Jmjd3, anti-T-bet, and anti-Ash2L antibodies. (e) WCL were obtained from WT and Jmjd3 cKO Th1 cells and immunoprecipitated with anti-T-bet antibody and protein (A+G). The immunoprecipitated product was immunoblotted with anti-RbBP5 antibody. (f) ChIP-qPCR analysis of T-bet binding to the promoter regions of Cxcr3 and Ifng genes in WT and Jmjd3 cKO Th1 cells. (g) WCL obtained from 293T cells cotransfected with HA-Jmjd3 and FLAG-Smad1, FLAG-Smad2, or FLAG-Smad3 plasmids were immunoprecipitated with anti-HA beads. The immunoprecipitated product was immunoblotted with anti-FLAG and anti-HA antibodies. (h) WCL and protein isolates from WT and Jmjd3cKO CD4+ T cells derived from thymus. (i) 293T cells were cotransfected with HA-tagged Smad3 and FLAG-tagged Wdr5, Ash2L, Dpy30 or RbBP5. WCL were immunoprecipitated with anti-FLAG beads and immunoblotted with anti-HA antibody. (j) Cell lysates were obtained from WT and Jmjd3 cKO Treg cells and immunoprecipitated with anti-Smad3 antibody. The immunoprecipitated product was immunoblotted with anti-Jmjd3, anti-Smad3 and anti-Ash2L antibodies. (k) ChIP-qPCR analysis of Smad3 binding to the promoter regions of Foxp3 gene in WT and Jmjd3 cKO Treg cells.
Mentions: We next determined whether Jmjd3 recruitment to specific gene promoters was mediated through interactions with transcription and epigenetic factors involved in T cell differentiation. Co-immunoprecipitation analyses revealed that Jmjd3 interacts with T-bet, but not Gata3, Foxp3, or RoRγt, in 293T cells (Fig. 7a and Supplementary Fig. 3b). Because H3K4 methylation levels were altered at many gene loci in Jmjd3-deficient T cells, we determined its interaction with key components of the H3K4 methyltransferase complex (Ash2L, RbBP5, and Wdr5). We found that Jmjd3 interacted with Ash2L, but not with RbBP5 or Wdr5, in 293T cells (Fig. 7b and Supplementary Fig. 3c). Co-IP in 293T cells, co-transfected with T-bet and FLAG-tagged Ash2L, RbBP5 and Wdr5 plasmids, revealed that T-bet physically interacted with RbBP5 (Fig. 7c and Supplementary Fig. 3d). The endogenous interaction of Jmjd3 with Ash2L and T-bet was observed in WT but not in Jmjd3 cKO T cells (Fig. 7d and Supplementary Fig. 3e). Although T-bet interacts with Jmjd3 and RbBP5, a core protein of the H3K4 methyltransferase complex39, it is unclear whether Jmjd3 is required for T-bet-RbBP5 interaction. Immunoprecipitation and immunoblot analysis revealed that T-bet interacted with RbBP5 in the presence of Jmjd3, but failed to interact in Jmjd3-deficient T cells (Fig. 7e and Supplementary Fig. 3f). To determine the functional relevance, we performed ChIP-quantitative PCR (qPCR) analysis of Cxcr3 and Ifng promoter regions in WT and Jmjd3 cKO Th1 cells and CD4+ SP thymocytes. Consistent with our previous observations, Jmjd3-deficient T cells disrupted the endogenous T-bet-RbBP5 interaction and markedly reduced T-bet binding to the promoter regions of Cxcr3, Ifng, and Cd44 genes (Fig. 7f and Supplementary Fig. 6a). These results suggest that Jmjd3 is required for T-bet and Ash2L to form a stable complex capable of binding to specific target gene promoters. Jmjd3 deficiency might destabilize this complex formation and reduce T-bet binding to Cxcr,Ifng, and Cd44 promoters, leading Cxcr3, Ifng, and Cd44 gene expression downregulation.

Bottom Line: The skewing of T-cell differentiation is concomitant with changes in the expression of key transcription factors and cytokines.H3K27me3 and H3K4me3 levels in Jmjd3-deficient cells are correlated with altered gene expression through interactions with specific transcription factors.Our results identify Jmjd3 as an epigenetic factor in T-cell differentiation via changes in histone methylation and target gene expression.

View Article: PubMed Central - PubMed

Affiliation: Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, Texas 77030, USA.

ABSTRACT
Epigenetic factors have been implicated in the regulation of CD4(+) T-cell differentiation. Jmjd3 plays a role in many biological processes, but its in vivo function in T-cell differentiation remains unknown. Here we report that Jmjd3 ablation promotes CD4(+) T-cell differentiation into Th2 and Th17 cells in the small intestine and colon, and inhibits T-cell differentiation into Th1 cells under different cytokine-polarizing conditions and in a Th1-dependent colitis model. Jmjd3 deficiency also restrains the plasticity of the conversion of Th2, Th17 or Treg cells to Th1 cells. The skewing of T-cell differentiation is concomitant with changes in the expression of key transcription factors and cytokines. H3K27me3 and H3K4me3 levels in Jmjd3-deficient cells are correlated with altered gene expression through interactions with specific transcription factors. Our results identify Jmjd3 as an epigenetic factor in T-cell differentiation via changes in histone methylation and target gene expression.

Show MeSH
Related in: MedlinePlus