Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Sep 15;398(3):461-7.
doi: 10.1042/BJ20060406.

Involvement of the SMRT/NCoR-HDAC3 complex in transcriptional repression by the CNOT2 subunit of the human Ccr4-Not complex

Sandrine Jayne  1 , Carin G M Zwartjes, Frederik M A van Schaik, H Th Marc Timmers
Affiliations

Involvement of the SMRT/NCoR-HDAC3 complex in transcriptional repression by the CNOT2 subunit of the human Ccr4-Not complex

Sandrine Jayne et al. Biochem J. .

Abstract

In eukaryotic cells, the Ccr4-Not complex can regulate mRNA metabolism at various levels. Previously, we showed that promoter targeting of the CNOT2 subunit resulted in strong repression of RNA polymerase II transcription, which was sensitive to the HDAC (histone deacetylase) inhibitor, trichostatin A [Zwartjes, Jayne, van den Berg and Timmers (2004) J. Biol. Chem. 279, 10848-10854]. In the present study, the cofactor requirement for CNOT2-mediated repression was investigated. We found that coexpression of SMRT (silencing mediator for retinoic acid receptor and thyroid-hormone receptor) or NCoR (nuclear hormone receptor co-repressor) in combination with HDAC3 (or HDAC5 and HDAC6) augmented the repression by CNOT2. This repressive effect is mediated by the conserved Not-Box, which resides at the C-terminus of CNOT2 proteins. We observed physical interactions of CNOT2 with several subunits of the SMRT/NCoR-HDAC3 complex. Our results show that the SMRT/NCoR-HDAC3 complex is a cofactor of CNOT2-mediated repression and suggest that transcriptional regulation by the Ccr4-Not complex involves regulation of chromatin modification.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Overexpression of SMRT, NCoR and HDAC3 (or HDAC5 and HDAC6) augments transcriptional repression activities of CNOT2
(A) Effect of overexpression of class I HDACs, SMRT, NCoR and Sin3A on the repression activities of CNOT2. HEK-293T cells were transiently co-transfected with 2 μg of p7xGalTKLuc reporter plasmid, 1 μg of pCMV expression plasmid for Gal4DBD (white bars) or Gal4–CNOT2 fusion protein (black bars) and 3 μg of pCMV expression plasmid for co-repressor proteins. For the purpose of clarity, for each cofactor combination, the value obtained for Gal4–CNOT2 repression was divided by the value obtained with the Gal4DBD protein. As a consequence, on the graph, the fold repression of Gal4DBD is 1 for each cofactor. The effect of the repressors on the activities of Gal4DBD alone is less than 2-fold, except for SMRT/HDAC3, HDAC4 combinations, Sin3A, HDAC1 and Sin3A/HDAC1. The experiments shown are representative of at least three independent assays, performed in duplicate. (B) Effect of overexpression of class II HDACs, SMRT and NCoR on CNOT2-mediated repression. The assays were performed and the results are displayed as in (A).
Figure 2
Figure 2. Enhancement of repression by SMRT and NCoR requires the Not-Box
U2OS cells were transiently transfected as described in the legend of Figure 1. The effects of SMRT and NCoR on Gal4DBD alone were 0.97- and 1.2-fold respectively. The assays were performed in duplicate. The experiment shown is representative of at least three independent assays.
Figure 3
Figure 3. The Not-Box of CNOT2 interacts with HDAC3 in vivo
HEK-293T cells were transiently transfected with plasmids expressing FLAG-tagged HDAC3 and different Gal4 fusion proteins: Gal4DBD (lane 1), Gal4–CNOT2 (lane 2), Gal4–CNOT2-(334–540) (lane 3), Gal4–CNOT2-(437–540) (lane 4), Gal4–CNOT2-(256–540) (lane 5) or Gal4–CNOT2-(1–255) (lane 6). FLAG-tagged HDAC3 was precipitated using anti-FLAG beads in the presence of 100 mM KCl and the interacting proteins were analysed by SDS/PAGE and immunoblotting using the RK5C1 antibody (right panel). Proteins present in 10% of the input were analysed in the same way (left panel). Asterisks indicate positions of IgG proteins (heavy and light chains) and the arrow indicates the position of Gal4DBD protein. Positions of co-migrating marker proteins are indicated by their molecular mass in kDa to the left of the Figure.
Figure 4
Figure 4. The Not-Box of CNOT2 interacts with different subunits of the SMRT/NCoR–HDAC3 complex in a yeast two-hybrid assay
(A) Schematic drawing showing the known functional domains of SMRT and NCoR proteins. (B) The Not-Box of CNOT2 interacts with RD4 and with the RID of SMRT and NCoR. EGY48 cells were transformed with LexA and B42 expression plasmids together with a reporter plasmid. Galactosidase activity was detected by X-Gal (5-bromo-4-chloroindol-3-yl β-D-galactopyranoside) staining of yeast cells. The LexA fusion proteins are indicated to the left and the B42 fusion proteins are indicated above the panel. Full-length CNOT2 could not be tested as the LexA fusion protein behaves as a strong transcriptional activator in this assay. (C) Quantification of β-galactosidase activity. Lysates of yeast cells were prepared as described in the Experimental section. The indicated fold induction is relative to the activity of yeast cells expressing LexA and B42 control. (D) The Not-Box of CNOT2 interacts with GPS2 but not HDAC3 in the yeast two-hybrid assay. The assay was performed and the interactions were quantified as in (B, C).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Martens J. A., Winston F. Recent advances in understanding chromatin remodeling by Swi/Snf complexes. Curr. Opin. Genet. Dev. 2003;13:136–142. - PubMed
    1. Berger S. L. Histone modifications in transcriptional regulation. Curr. Opin. Genet. Dev. 2002;12:142–148. - PubMed
    1. Collart M. A., Timmers H. T. The eukaryotic Ccr4–not complex: a regulatory platform integrating mRNA metabolism with cellular signaling pathways? Prog. Nucleic Acid Res. Mol. Biol. 2004;77:289–322. - PubMed
    1. Denis C. L., Chen J. The CCR4–NOT complex plays diverse roles in mRNA metabolism. Prog. Nucleic Acid Res. Mol. Biol. 2003;73:221–250. - PubMed
    1. Yamashita A., Chang T. C., Yamashita Y., Zhu W., Zhong Z., Chen C. Y., Shyu A. B. Concerted action of poly(A) nucleases and decapping enzyme in mammalian mRNA turnover. Nat. Struct. Mol. Biol. 2005;12:1054–1063. - PubMed

Publication types