Abstract
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. 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) genes which are found in a gene cluster on chromosome 6. Increased expression of MHC II genes after knock-down or knock-out of either CNOT subunit 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 also in the absence of the master regulator class II transactivator (CIITA) and did not cause detectable changes of the chromatin structure at the chromosomal MHC II locus. CNOT2 downregulation resulted in an increased de novo transcription of mRNAs whereas tethering of CNOT2 to a regulatory region governing MHC II expression resulted in diminished transcription. These results expand the known repertoire of CCR4-NOT members for immune regulation and identify CNOT proteins as a novel group of corepressors restricting class II expression.
Highlights
Eukaryotic gene expression is a multi-step process that is regulated at all levels from chromatin accessibility to the various steps of transcription, mRNA splicing, nuclear export, mRNA decay and protein translation[1]
In order to compare the cellular functions of CNOT1, CNOT2 and CNOT3 in a systematic manner, Human embryonic kidney (HEK)-293T cells allowing the individual knock-down of each subunit were produced
HEK-293T cells were transfected with pINDUCER plasmids encoding the individual shRNAs with specificity for CNOT1, CNOT2, CNOT3 or a target sequence against firefly luciferase (Luci) as a control
Summary
Eukaryotic gene expression is a multi-step process that is regulated at all levels from chromatin accessibility to the various steps of transcription, mRNA splicing, nuclear export, mRNA decay and protein translation[1]. One important regulator controlling all different levels of gene expression is the multi-subunit CCR4-NOT complex. This protein complex is evolutionary conserved in eukaryotes and consists of enzymatically active subunits as well as scaffolding proteins[2]. An example for a transcriptionally repressed gene cluster is provided by the MHC II genes These genes are typically only expressed in thymic epithelial cells (TECs) and professional antigen-presenting cells (APCs) of the immune system such as dendritic cells, B cells and activated macrophages[24]. Genetic and biochemical studies allowed the identification of four key trans-acting factors that regulate MHC II gene transcription by interacting with the SXY module: Regulatory Factor X 5 (RFX5), RFX-associated protein (RFXAP), RFX-associated ankyrin-containing protein (RFXANK) and, most importantly, CIITA31–34. Relevant downregulation of MHC II expression is mediated by the Epstein-Barr virus encoded BDLF3 protein to disable the immune system of the infected host[37]
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