Repression of Promoter Activity by CNOT2, a Subunit of the Transcription Regulatory Ccr4-Not Complex

Carin G.M Zwartjes,Sandrine Jayne,Debbie L.C Van Den Berg,H.T Marc Timmers

doi:10.1074/jbc.m311747200

Carin G.M Zwartjes, Sandrine Jayne + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m311747200

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Abstract

The evolutionary conserved Ccr4-Not complex controls mRNA metabolism at multiple levels in eukaryotic cells. Genetic analysis of not mutants in yeast identifies a negative role in transcription, which is dependent on core promoter structure. To obtain direct support for this we targeted individual core subunits of the human Ccr4-Not complex to promoters in transient transfections of human cells. In this experimental setup we found that the CNOT2 and CNOT9(hRcd1/hCaf40) subunits act as repressors of reporter gene activity. Interestingly, recruitment of other Ccr4-Not subunits did not affect the reporter gene. The major repression function of CNOT2 is localized in a specialized protein motif, the Not-Box. This conserved motif is present in all CNOT2 orthologs and surprisingly also in CNOT3 orthologs. Repression by the Not-Box was sensitive to treatment with the histone deacetylase inhibitor trichostatin A. In addition, mutation of a canonical TATA-box enhanced repression. Our experiments show for the first time direct regulation of promoter activity by components of the Ccr4-Not complex.

Highlights

Regulation of transcription by RNA polymerase II1 requires the interplay of many transcription factors at different levels [1]
The Gal4 expression plasmids were cotransfected into human osteosarcoma U2OS cells with the firefly luciferase reporter plasmid 7xGalTKLuc, which contains seven Gal4 binding sites fused to the herpes simplex virus thymidine kinase (TK) promoter, or with the control reporter plasmid TKLuc lacking any Gal4 binding sites
Not-Box-mediated Repression Is Sensitive to the Histone Deacetylase Inhibitor trichostatin A (TSA)—Our experiments suggest that the Not-Box motif as present in CNOT2 and CNOT3 orthologs can actively repress transcription

Summary

Introduction

Regulation of transcription by RNA polymerase II (pol II)1 requires the interplay of many transcription factors at different levels [1]. The Ccr4-Not Subunits CNOT2 and CNOT9(hRcd1/hCAF40) Repress Reporter Gene Activity—To investigate the ability of the human Ccr4-Not complex to regulate transcription the individual subunits (CNOT2, CNOT3, CNOT4, CNOT8, CNOT9(hRcd1/hCAF40), and the C-terminal 1,290 residues of CNOT1) were targeted to a pol II promoter by fusing each subunit to the Gal4(1–147) DBD. We observed that promoter targeting of the CNOT2 and CNOT9 subunits of the Ccr4-Not complex resulted in a strong repression of reporter gene activity.

Results

Conclusion