Jacob Eyal, Hod-Dvorai Reut, Ben-Mordechai Or Lea, Boyko Yulia, Avni Orly
Department of Immunology, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
J Mol Signal. 2011 May 30;6:5. doi: 10.1186/1750-2187-6-5.
Following antigen recognition, naive T helper (Th; CD4+) cells can differentiate toward one of several effector lineages such as Th1 and Th2; each expressing distinctive transcriptional profiles of cytokine genes. These cytokines eventually instruct the strategy of the immune response. In our search for factors that propagate the transcriptional programs of differentiated Th cells, we previously found that Polycomb group (PcG) proteins, which are known as epigenetic regulators that maintain repressive chromatin states, bind differentially the signature cytokine genes. Unexpectedly, their binding to the Ifng (Interferon-g) in Th1 cells and Il4 (Interleukin-4) in Th2 cells, was correlated with transcriptional activation. Therefore, in this study we aimed to determine the functional role of PcG proteins in the regulation of the expression of the signature cytokine genes.
PcG proteins were knocked down in primary and established murine Th cells using transduction of lentiviruses encoding short hairpin RNAs (shRNAs) directed to Mel-18, Ezh2, Eed and Ring1A, representative of two different PcG complexes. The chromatin structure and the binding activity of PcG proteins and transcription factors at the Ifng promoter were assessed by chromatin immunoprecipitation (ChIP) assays.
Downregulation of PcG proteins was consistent with their function as positive regulators of the signature cytokine genes in primary and established Th1 and Th2 cells. Moreover, the PcG protein Mel-18 was necessary to recruit the Th1-lineage specifying transcription factor T-bet, and the T cell receptor (TCR)-inducible transcription factor NFAT1 to the Ifng promoter in Th1 cells. Nevertheless, our results suggest that PcG proteins can function also as conventional transcriptional repressors in Th cells of their known target the Hoxa7 gene.
Our data support a model whereby the non-differentially expressed PcG proteins are recruited in a Th-lineage specific manner to their target genes to enforce the maintenance of specific transcriptional programs as transcriptional repressors or activators. Although our results suggest a direct effect of PcG proteins in the regulation of cytokine gene expression, indirect functions cannot be excluded.
在抗原识别后，初始辅助性T（Th；CD4+）细胞可向几种效应细胞谱系之一分化，如Th1和Th2；每种谱系都表达细胞因子基因独特的转录谱。这些细胞因子最终指导免疫反应的策略。在我们寻找传播分化的Th细胞转录程序的因子的过程中，我们先前发现多梳蛋白家族（PcG）蛋白，作为维持抑制性染色质状态的表观遗传调节因子，与标志性细胞因子基因的结合存在差异。出乎意料的是，它们在Th1细胞中与Ifng（干扰素-γ）以及在Th2细胞中与Il4（白细胞介素-4）的结合与转录激活相关。因此，在本研究中，我们旨在确定PcG蛋白在标志性细胞因子基因表达调控中的功能作用。
使用编码针对Mel-18、Ezh2、Eed和Ring1A的短发夹RNA（shRNA）的慢病毒转导，在原代和已建立的小鼠Th细胞中敲低PcG蛋白，Mel-18、Ezh2、Eed和Ring1A代表两种不同的PcG复合物。通过染色质免疫沉淀（ChIP）分析评估Ifng启动子处PcG蛋白和转录因子的染色质结构及结合活性。
PcG蛋白的下调与其作为原代和已建立的Th1和Th2细胞中标志性细胞因子基因的正调节因子的功能一致。此外，PcG蛋白Mel-18是在Th1细胞中招募Th1谱系特异性转录因子T-bet以及T细胞受体（TCR）诱导性转录因子NFAT1至Ifng启动子所必需的。然而，我们的结果表明，PcG蛋白在其已知靶基因Hoxa7的Th细胞中也可作为传统的转录抑制因子发挥作用。
我们的数据支持一种模型，即非差异表达的PcG蛋白以Th谱系特异性方式被招募至其靶基因，以作为转录抑制因子或激活因子来维持特定转录程序。尽管我们的结果表明PcG蛋白在细胞因子基因表达调控中具有直接作用，但间接功能也不能排除。
