Abstract
Accessibility of the genome to DNA-binding transcription factors is regulated by proteins that control the acetylation of amino-terminal lysine residues on nucleosomal histones. Specifically, histone deacetylase (HDAC) proteins repress transcription by deacetylating histones. To date, the only known regulatory mechanism of HDAC1 function is via interaction with associated proteins. Although the control of HDAC1 function by protein interaction and recruitment is well precedented, we were interested in exploring HDAC1 regulation by post-translational modification. Human HDAC1 protein was analyzed by ion trap mass spectrometry, and two phosphorylated serine residues, Ser(421) and Ser(423), were unambiguously identified. Loss of phosphorylation at Ser(421) and Ser(423) due to mutation to alanine or disruption of the casein kinase 2 consensus sequence directing phosphorylation reduced the enzymatic activity and complex formation of HDAC1. Deletion of the highly charged carboxyl-terminal region of HDAC1 also decreased its deacetylase activity and protein associations, revealing its requirement in maintaining HDAC1 function. Our results reinforce the importance of protein associations in modulating HDAC1 function and provide the first step toward characterizing the role of post-translational modifications in regulating HDAC activity in vivo.
Highlights
30,000 – 40,000 genes exist in a human cell, only a fraction of those genes are transcribed into mRNA and translated into the encoding protein in any given cell type [1, 2]
histone deacetylase (HDAC) proteins interact with cellular proteins implicated in cancer development, including the retinoblastoma tumor suppressor (Rb), metastasis-associated protein 2 (MTA2), and nuclear hormone receptors like the retinoic acid receptor [17]
To verify that HDAC1 is phosphorylated, T-Ag Jurkat cells were labeled in vivo with [32P]orthophosphate, and HDAC1 was immunoprecipitated with an HDAC1-specific antibody
Summary
30,000 – 40,000 genes exist in a human cell, only a fraction of those genes are transcribed into mRNA and translated into the encoding protein in any given cell type [1, 2]. Our data support a model where the carboxyl-terminal region of HDAC1 is essential for maintaining the protein association and enzymatic activity of HDAC1. Immunoprecipitated HDAC1, which migrated by SDS-PAGE identically to HDAC1 from lysates (data not shown), was labeled with 32P, indicating that the protein is phosphorylated in vivo (Fig. 1B).
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