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

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. Overall design: 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.