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A negative feedback loop mediated by the Bcl6-cullin 3 complex limits Tfh cell differentiation.

Mathew R, Mao AP, Chiang AH, Bertozzi-Villa C, Bunker JJ, Scanlon ST, McDonald BD, Constantinides MG, Hollister K, Singer JD, Dent AL, Dinner AR, Bendelac A - J. Exp. Med. (2014)

Bottom Line: Intriguingly, we found that Bcl6 was also highly and transiently expressed during the CD4(+)CD8(+) (double positive [DP]) stage of T cell development, in association with the E3 ligase cullin 3 (Cul3), a novel binding partner of Bcl6 which ubiquitinates histone proteins.Although they maintained an apparently normal phenotype after emigration, they expressed increased amounts of Batf and Bcl6 at basal state and produced explosive and prolonged Tfh responses upon subsequent antigen encounter.Ablation of Cul3 in mature CD4(+) splenocytes also resulted in dramatically exaggerated Tfh responses.

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Affiliation: Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637.

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The Bcl6–Cul3 complex directly regulates Batf and Bcl6. (a) ChIP-qPCR analysis of Bcl6 binding to the indicated genes in fresh thymocytes. Data are representative of two independent experiments (mean ± SEM). (b) qRT-PCR analysis of Batf and of Bcl6 (exons 2–3) in exon 7–9–deleted Bcl6cKO versus WT CD4+ SP thymocytes. Bar graphs represent mean ± SEM from five pairs of KO and controls from two independent experiments. (c) Transfection of Bcl6 together with luciferase-based reporters of Batf in HeLa cells. Mutated intronic regulatory sequences (m1 and m2) are as indicated. Bar graphs represent mean ± SEM from two independent experiments. (d) ChIP-qPCR analysis of H3K4me3 and acetyl-H3 along the Batf gene (indicated at bottom) in Cul3cKO versus littermate (LM) control CD4+ SP thymocytes. Bcl6-binding sequences are at 0.58 and 0.7 kb in the first intron. Data are representative of two independent experiments (mean ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig7: The Bcl6–Cul3 complex directly regulates Batf and Bcl6. (a) ChIP-qPCR analysis of Bcl6 binding to the indicated genes in fresh thymocytes. Data are representative of two independent experiments (mean ± SEM). (b) qRT-PCR analysis of Batf and of Bcl6 (exons 2–3) in exon 7–9–deleted Bcl6cKO versus WT CD4+ SP thymocytes. Bar graphs represent mean ± SEM from five pairs of KO and controls from two independent experiments. (c) Transfection of Bcl6 together with luciferase-based reporters of Batf in HeLa cells. Mutated intronic regulatory sequences (m1 and m2) are as indicated. Bar graphs represent mean ± SEM from two independent experiments. (d) ChIP-qPCR analysis of H3K4me3 and acetyl-H3 along the Batf gene (indicated at bottom) in Cul3cKO versus littermate (LM) control CD4+ SP thymocytes. Bcl6-binding sequences are at 0.58 and 0.7 kb in the first intron. Data are representative of two independent experiments (mean ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: As Batf was recently shown to be a key inducer of several Tfh genes, including Bcl6, cMaf, and Il21 (Ise et al., 2011), the simplest hypothesis explaining the above observations was that Bcl6–Cul3 complexes exerted a negative feedback on the Tfh program by repressing Batf and Bcl6. Recent chromatin immunoprecipitation (ChIP)–Seq studies of Bcl6-expressing macrophages identified Bcl6 binding to the first intron of the Batf gene, which contains a putative Bcl6-binding sequence motif (Barish et al., 2010, 2012). Using ChIP-qPCR, we demonstrated direct binding of Bcl6 to this intronic region of Batf in fresh thymocytes (Fig. 7 a). We also detected binding of Bcl6 to the Bcl6 proximal promoter, which includes a binding site previously identified in B cell lymphoma cells (Wang et al., 2002; Pasqualucci et al., 2003), as well as to Prdm1 and Bcl2 (Parekh et al., 2007). Thus, these findings demonstrated that, despite its transient expression, Bcl6 protein did bind key regulatory elements of multiple genes in fresh DP thymocytes.


A negative feedback loop mediated by the Bcl6-cullin 3 complex limits Tfh cell differentiation.

Mathew R, Mao AP, Chiang AH, Bertozzi-Villa C, Bunker JJ, Scanlon ST, McDonald BD, Constantinides MG, Hollister K, Singer JD, Dent AL, Dinner AR, Bendelac A - J. Exp. Med. (2014)

The Bcl6–Cul3 complex directly regulates Batf and Bcl6. (a) ChIP-qPCR analysis of Bcl6 binding to the indicated genes in fresh thymocytes. Data are representative of two independent experiments (mean ± SEM). (b) qRT-PCR analysis of Batf and of Bcl6 (exons 2–3) in exon 7–9–deleted Bcl6cKO versus WT CD4+ SP thymocytes. Bar graphs represent mean ± SEM from five pairs of KO and controls from two independent experiments. (c) Transfection of Bcl6 together with luciferase-based reporters of Batf in HeLa cells. Mutated intronic regulatory sequences (m1 and m2) are as indicated. Bar graphs represent mean ± SEM from two independent experiments. (d) ChIP-qPCR analysis of H3K4me3 and acetyl-H3 along the Batf gene (indicated at bottom) in Cul3cKO versus littermate (LM) control CD4+ SP thymocytes. Bcl6-binding sequences are at 0.58 and 0.7 kb in the first intron. Data are representative of two independent experiments (mean ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig7: The Bcl6–Cul3 complex directly regulates Batf and Bcl6. (a) ChIP-qPCR analysis of Bcl6 binding to the indicated genes in fresh thymocytes. Data are representative of two independent experiments (mean ± SEM). (b) qRT-PCR analysis of Batf and of Bcl6 (exons 2–3) in exon 7–9–deleted Bcl6cKO versus WT CD4+ SP thymocytes. Bar graphs represent mean ± SEM from five pairs of KO and controls from two independent experiments. (c) Transfection of Bcl6 together with luciferase-based reporters of Batf in HeLa cells. Mutated intronic regulatory sequences (m1 and m2) are as indicated. Bar graphs represent mean ± SEM from two independent experiments. (d) ChIP-qPCR analysis of H3K4me3 and acetyl-H3 along the Batf gene (indicated at bottom) in Cul3cKO versus littermate (LM) control CD4+ SP thymocytes. Bcl6-binding sequences are at 0.58 and 0.7 kb in the first intron. Data are representative of two independent experiments (mean ± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: As Batf was recently shown to be a key inducer of several Tfh genes, including Bcl6, cMaf, and Il21 (Ise et al., 2011), the simplest hypothesis explaining the above observations was that Bcl6–Cul3 complexes exerted a negative feedback on the Tfh program by repressing Batf and Bcl6. Recent chromatin immunoprecipitation (ChIP)–Seq studies of Bcl6-expressing macrophages identified Bcl6 binding to the first intron of the Batf gene, which contains a putative Bcl6-binding sequence motif (Barish et al., 2010, 2012). Using ChIP-qPCR, we demonstrated direct binding of Bcl6 to this intronic region of Batf in fresh thymocytes (Fig. 7 a). We also detected binding of Bcl6 to the Bcl6 proximal promoter, which includes a binding site previously identified in B cell lymphoma cells (Wang et al., 2002; Pasqualucci et al., 2003), as well as to Prdm1 and Bcl2 (Parekh et al., 2007). Thus, these findings demonstrated that, despite its transient expression, Bcl6 protein did bind key regulatory elements of multiple genes in fresh DP thymocytes.

Bottom Line: Intriguingly, we found that Bcl6 was also highly and transiently expressed during the CD4(+)CD8(+) (double positive [DP]) stage of T cell development, in association with the E3 ligase cullin 3 (Cul3), a novel binding partner of Bcl6 which ubiquitinates histone proteins.Although they maintained an apparently normal phenotype after emigration, they expressed increased amounts of Batf and Bcl6 at basal state and produced explosive and prolonged Tfh responses upon subsequent antigen encounter.Ablation of Cul3 in mature CD4(+) splenocytes also resulted in dramatically exaggerated Tfh responses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637.

Show MeSH
Related in: MedlinePlus