The Deacetylase HDAC1 Negatively Regulates the Cardiovascular Transcription Factor Krüppel-like Factor 5 through Direct Interaction

Takayoshi Matsumura,Toru Suzuki,Kenichi Aizawa,Yoshiko Munemasa,Shinsuke Muto,Masami Horikoshi,Ryozo Nagai

doi:10.1074/jbc.m410578200

Abstract

Transcription is regulated by a network of transcription factors and related cofactors that act in concert with the general transcription machinery. Elucidating their underlying interactions is important for understanding the mechanisms regulating transcription. Recently, we have shown that Krüppel-like factor KLF5, a member of the Sp/KLF family of zinc finger factors and a key regulator of cardiovascular remodeling, is regulated positively by the acetylase p300 and negatively by the oncogenic regulator SET through coupled interaction and regulation of acetylation. Here, we have shown that the deacetylase HDAC1 can negatively regulate KLF5 through direct interaction. KLF5 interacts with HDAC1 in the cell and in vitro. Gel shift DNA binding assay showed that their interaction inhibits the DNA binding activity of KLF5, suggesting a property of HDAC1 to directly affect the DNA binding affinity of a transcription factor. Reporter assay also revealed that HDAC1 suppresses KLF5-dependent promoter activation. Additionally, overexpression of HDAC1 suppressed KLF5-dependent activation of its endogenous downstream gene, platelet-derived growth factor-A chain gene, when activated by phorbol ester. Further, HDAC1 binds to the first zinc finger of KLF5, which is the same region where p300 interacts with KLF5 and, intriguingly, HDAC1 inhibits binding of p300 to KLF5. Direct competitive interaction between acetylase and deacetylase has been hitherto unknown. Collectively, the transcription factor KLF5 is negatively regulated by the deacetylase HDAC1 through direct effects on its activities (DNA binding activity, promoter activation) and further through inhibition of interaction with p300. These findings suggest a novel role and mechanism for regulation of transcription by deacetylase.

Highlights

Transcription is regulated by a network of transcription factors and related cofactors that act in concert with the general transcription machinery
In the process of dissecting the regulatory mechanisms of this family, we further showed that the histone chaperone TAF-I/ SET through its interaction with KLF5, a transcription factor of the Sp1 and Kruppel-like factor (Sp/KLF) family of zinc finger factors and a key regulator of cardiovascular remodeling as shown by null mutation in mice [20], represses the DNA binding, promoter activation, and growth stimulation activities of KLF5 by interaction as well as by non-catalytically “masking” it from acetylation by p300
KLF5 and HDAC1 Can Interact in the Cell and Directly Bind in Vitro—First, to see whether KLF5 and HDAC1 can interact in the cell, immunoprecipitation was done with HeLa cells overexpressing FLAG-tagged KLF5 and HDAC1 (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Preparation of Plasmid Constructs, Recombinant Epitope-tagged Protein, and Recombinant Adenovirus—The KLF5 expression vector pCAG-KLF5 was previously described [20]. After four washes with immunoprecipitation wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 100 ␮M ZnSO4, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 ␮g/ml leupeptin, and 1 ␮g/ml pepstatin), immunoprecipitate was subjected to SDS-PAGE analysis and immunoblotted with anti-HDAC1 mouse monoclonal antibody (Upstate Biotechnology). The cells were transfected with 90 ng of the reporter plasmid, 240 ng of pCAG-empty vector and/or pCAG-KLF5, and 90 ng of pcDNA3-empty vector and/or pcDNA3-HDAC1 in combination, as described in the figure legends, by liposome-mediated transfer (Tfx-20; Promega) according to the manufacturer’s instructions. Phorbol Ester-induced Expression of KLF5 and PDGF-A—After 48 h of growth arrest with serum-free medium, subconfluent rat smooth muscle cells [26] were transfected with 4 ␮g/6-well plate of either pcDNA3-empty vector or pcDNA3-HDAC1 expression vector using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The relative intensity in reference to internal 18 S rRNA was calculated by National Institutes of Health Image software

RESULTS

DISCUSSION

Additions and Corrections