The CCR4-NOT complex contributes to repression of Major Histocompatibility Complex class II transcription

The multi-subunit CCR4 (carbon catabolite repressor 4)-NOT (Negative on TATA) complex serves as a central coordinator of all different steps of eukaryotic gene expression. Accordingly, members of the CCR4-NOT complex have been implicated in a variety of biological functions. Here we performed a systematic and comparative analysis of cells where the CCR4-NOT subunits CNOT1, CNOT2 or CNOT3 were individually downregulated using doxycycline-inducible shRNAs. Microarray experiments showed that downregulation of either CNOT subunit resulted in elevated expression of major histocompatibility complex class II (MHC II) target genes which are found in a gene cluster on chromosome 6. Increased expression of MHC II genes after knock-down or knock-out of CNOT subunits was seen in a variety of cell systems and also in naïve macrophages from CNOT3 conditional knock-out mice. CNOT2-mediated repression of MHC II genes occurred independent from the master regulator class II transactivator (CIITA) and detectable changes of the chromatin structure at the chromosomal MHC II locus. CNOT2 downregulation resulted in an increased de novo transcription of mRNAs and tethering of CNOT2 to a regulatory region governing MHC II expression resulted in dimished transcription. These results expand the known repertoire of CCR4-NOT members for immune regulation and identify CNOT proteins as a novel group of corepressors serving to restrict inappropriate or exaggerated class II expression, which can be causative for various diseases. Cell culture experiment with HEK 293 cells, transfected either with Luciferase reporter only (control) or with DOX-inducible shRNAs against CCR4-NOT subunits. 1-2 technical replicates.

多亚基CCR4（carbon catabolite repressor 4，碳分解代谢物阻遏物4）-NOT（Negative on TATA，TATA结合抑制因子）复合物是真核基因表达所有不同步骤的核心协调因子。据此，CCR4-NOT复合物的成员已被证实参与多种生物学功能。本研究对采用强力霉素（doxycycline）诱导型短发卡RNA（shRNA）分别下调CCR4-NOT亚基CNOT1、CNOT2或CNOT3的细胞开展了系统性比较分析。微阵列实验结果显示，任一CNOT亚基的下调均可导致主要组织相容性复合体II类（major histocompatibility complex class II，MHC II）靶基因的表达上调，这类靶基因位于6号染色体的基因簇中。在多种细胞系统以及来自CNOT3条件性敲除小鼠的未成熟巨噬细胞中，均观察到CNOT亚基敲低或敲除后MHC II基因表达升高的现象。CNOT2介导的MHC II基因抑制作用不依赖于核心调控因子II类反式激活因子（class II transactivator，CIITA），且未在染色体MHC II位点检测到染色质结构的显著变化。CNOT2的下调会导致mRNA的新生转录增加，而将CNOT2锚定在调控MHC II表达的调控区域则会降低转录水平。本研究拓展了CCR4-NOT复合物成员在免疫调控中的已知功能谱系，并鉴定出CNOT蛋白作为一类新型核心抑制因子，可限制不当或过度的II类基因表达——此类异常表达可引发多种疾病。本研究采用仅转染荧光素酶报告基因（对照组）或转染靶向CCR4-NOT亚基的DOX诱导型shRNA的HEK 293细胞开展细胞培养实验，设置1-2次技术重复。