Abstract
The cytokine transcription profiles of developing T helper 1 and T helper 2 cells are imprinted and induced appropriately following stimulation of differentiated cells. Epigenetic regulation combines several mechanisms to ensure the inheritance of transcriptional programs. We found that the expression of the polycomb group proteins, whose role in maintaining gene silencing is well documented, was induced during development in both T helper lineages. Nevertheless, the polycomb proteins, YY1, Mel-18, Ring1A, Ezh2, and Eed, bound to the Il4 and Ifng loci in a differential pattern. In contrast to the prevailing dogma, the binding activity of the polycomb proteins in differentiated T helper cells was associated with cytokine transcription. The polycomb proteins bound to the cytokine genes under resting conditions, and their binding was induced dynamically following stimulation. The recruitment of the polycomb proteins Mel-18 and Ezh2 to the cytokine promoters was inhibited in the presence of cyclosporine A, suggesting the involvement of NFAT. Considering their binding pattern at the cytokine genes and their known function in higher order folding of regulatory elements, we propose a model whereby the polycomb proteins, in some contexts, positively regulate gene expression by mediating long-distance chromosomal interactions.
Highlights
Can be achieved in vitro by manipulating the cytokine milieu: IL-12 and IL-4 strongly potentiate Th1 and Th2 differentiation, respectively, via the transcription factors STAT4 and STAT6
The hyperacetylated status of the cytokine genes correlates with the selective binding of NFAT1, and we suggested that the differentiation process of Th cells reinforces chromatin structural changes, that facilitate differential accessibility to acute transcription factors (5)
We suggest that the known function of the Polycomb group (PcG) proteins in pairing DNA elements (19, 22, 23, 25–27, 47), is not necessarily associated with gene repression, but rather, under some circumstances, it may support active gene expression or maintain transcriptional status by mediating long-distance chromosomal interactions (2)
Summary
For Th1 differentiation, the cells were stimulated in the presence of 10 ng/ml recombinant mouse IL-12 (R&D systems) and 10 g/ml purified anti-IL-4 antibodies (11B11). For Th2 differentiation, cells were stimulated in the presence of 1000 units/ml mouse IL-4 (added as a supernatant of the 13L6 cell line), 5 g/ml purified anti-IFN␥ antibodies (XMG1.2), and 3 g/ml purified antiIL-12 antibodies (C178). Cells (10 –24 ϫ 107) were cross-linked on ice for 20 min, by adding a one-tenth volume of 11% formaldehyde solution (0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM HEPES, pH 8.0) directly to the media. PCR using Ifng promoter or VA primers was performed directly on input DNA purified from chromatin before immunoprecipitation. RT-PCR—Total RNA was extracted, reverse-transcribed, and amplified with the following primer sets: Il4, 5Ј-CATCGGCATTTTGAACGAGGTCA-3Ј and 5Ј-CTTATCGATGAATCCAGGCATCG-3Ј (Genomic: 4,610-bp product, cDNA: 240-bp product); Ifng, 5Ј-CATTGAAAGCCTAGAAAGTCTG-3Ј and 5Ј-CTCATGAATGCATCCTTTTTCG-3Ј (Genomic: 1,548-bp product, cDNA: 267-bp product); Gata3, 5Ј-GAACACTGAGCTGCCTGGCGCCGT-3Ј and 5Ј-CTTTGCGGGATAGTTTAGCAA-3Ј (Genomic: 812-bp product, cDNA: 391-bp product); T-bet 5ЈGCTACCCGCCCGTGGATGG-3Ј and 5Ј-CCGGTGTTGGGGGAGTCTGG-3Ј (Genomic: 13,279-bp product, cDNA: 384-bp product)
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