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.Although 30,000 -40,000 genes exist in a human cell, only a fraction of those genes are transcribed into mRNA and ultimately translated into the encoding protein in any given cell type (1, 2). Transcription is regulated at many levels to selectively express only those proteins necessary for proper cell function. Although the role of DNA-binding transcription factors in controlling gene expression is well established, an additional level of regulation has been recently elucidated, the accessibility of genomic DNA to transcription factors (3).The genome is tightly packed into chromosomes through nucleosomal protein-DNA complexes. Each nucleosome contains ϳ200 base pairs of double-stranded DNA wrapped twice around a histone protein core (4). Although the carboxyl-terminal region of the histones are involved in forming the nucleosome core, the amino-terminal, lysine-rich tails are located outside of the nucleosome core (5). Various studies revealed a correlation between the acetylation of amino-terminal lysine residues on histones and transcriptional activity in chromatin (3,6). Therefore, the current model of transcriptional control includes DNA accessibility due to acetylation of histones.The acetylation state of histones is dependent on the activity of two proteins, histone acetyltransferase and histone deacetylase (HDAC), 1 which acetylate and deacetylate histones, respectively. Loss of HDAC activity using small molecule inhibitors correlates with the hyperacetylation of nucleosomal histones (7,8). Importantly, HDAC proteins in yeast and humans are necessary to achieve the full transcriptional repression and induction of diverse genes (9, 10).Not surprisingly, HDAC proteins are critica...

Histone deacetylase (HDAC) proteins are transcription regulators linked to cancer. As a result, multiple small molecule HDAC inhibitors are in various phases of clinical trials as anti-cancer drugs. The majority of HDAC inhibitors non-selectively influence the activities of eleven human HDAC isoforms, which are divided into distinct classes. This tutorial review focuses on the recent progress toward the identification of class-selective and isoform-selective HDAC inhibitors. The emerging trends suggest that subtle differences in the active sites of the HDAC isoforms can be exploited to dictate selectivity.

Protein phosphorylation plays a critical role in a variety of cellular functions. As a result, the monitoring of phosphoproteins in cells represents an important goal for proteomics research. To facilitate phosphoprotein detection, the first enzymatic phosphorylation-dependent biotinylation reaction of proteins is described. Specifically, kinase enzymes were coupled with an ATP-biotin conjugate to efficiently biotinylate substrate peptides and proteins after phosphate transfer. The kinase-mediated biotinylation reaction enables efficient detection of phosphoproteins in cell lysates or phosphopeptides after trypsin proteolysis, demonstrating its utility for proteomics research. Importantly, the studies reveal the cosubstrate promiscuity of kinase enzymes, laying the foundation for development of new chemical tools targeting the phosphoproteome.

Histone deacetylase 1 (HDAC1) has been linked to cell growth and cell cycle regulation, which makes it a widely recognized target for anticancer drugs. Whereas variations of the metal-binding and capping groups of HDAC inhibitors have been studied extensively, the role of the linker region is less well known, despite the potency of inhibitors with diverse linkers, such as MS-275. To facilitate a drug design that targets HDAC1, we assessed the influence of residues in the 11 Å channel of the HDAC1 active site on activity by using an alanine scan. The mutation of eight channel residues to alanine resulted in a substantial reduction in deacetylase activity. Molecular dynamics simulations indicated that alanine mutation results in significant movement of the active-site channel, which suggests that channel residues promote HDAC1 activity by influencing substrate interactions. With little characterization of HDAC1 available, the combined experimental and computational results define the active-site residues of HDAC1 that are critical for substrate/inhibitor binding and provide important insight into drug design.