Purification and enzymatic assay of class I histone deacetylase enzymes.

The reversible acetylation of histones has a profound influence on transcriptional status. Histone acetyltransferases catalyze the addition of these chemical modifications to histone lysine residues. Conversely, histone deacetylases (HDACs) catalyze the removal of these acetyl groups from histone lysine residues. As modulators of transcription, HDACs have found themselves as targets of several FDA-approved chemotherapeutic compounds which aim to inhibit enzyme activity. The ongoing efforts to develop targeted and isoform-specific HDAC inhibitors necessitates tools to study these modifications and the enzymes that maintain an equilibrium of these modifications. In this chapter, we present an optimized workflow for the isolation of recombinant protein and subsequent assay of class I HDAC activity. We demonstrate the application of this assay by assessing the activities of recombinant HDAC1, HDAC2, and SIN3B. This assay system utilizes readily available reagents and can be used to assess the activity and responsiveness of class I HDAC complexes to HDAC inhibitors.

Purification of active recombinant human histone deacetylase 1 (HDAC1) overexpressed in Escherichia coli.

OBJECTIVE: We attempted to overexpress Human Histone Deacetylase 1 (HDAC1) in Escherichia coli. RESULTS: A synthetic gene coding for HDAC1, and optimised for E. coli codon usage, was cloned into pBADHisB, generating pBAD-rHDAC1. This construct was used to transform E. coli TOP10, and the target protein was overexpressed and partially purified. According to its elution volume from a Superdex 200 column, the partially purified rHDAC1 was obtained in aggregated form, i.e., as an octamer. The dissociation of octameric HDAC1 was tested using several agents, among which sodium dodecyl sulfate was competent in partially dissociating rHDAC1 aggregates. When the enzyme activity was tested in vitro using 3H-acetyl-labelled histones both protein samples, aggregated and dissociated, were active. Hence, our results suggest that E. coli represents an alternative system for the production of the recombinant HDAC1. CONCLUSIONS: We described a procedure for the overexpression in E. coli of recombinant HDAC1, the purification of which in active form can be successfully performed, although yielding an octameric aggregate.

Discovering protein interactions and characterizing protein function using HaloTag technology.

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast. In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1. These examples demonstrate the power of this technology in enabling the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics.

PHF6 interacts with the nucleosome remodeling and deacetylation (NuRD) complex.

Mutations in PHF6 are the cause of Börjeson-Forssman-Lehman syndrome (BFLS), an X-linked intellectual disability (XLID) disorder, and both T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). The PHF6 gene encodes a protein with two plant homeodomain (PHD)-like zinc finger domains. As many PHD-like domains function to target chromatin remodelers to post-translationally modified histones, this suggests a role for PHF6 in chromatin regulation. However, PHD domains are usually found in association with a catalytic domain, a feature that is lacking in PHF6. This distinct domain structure and the minimal information on its cellular function prompted us to perform a proteomic screen to identify PHF6 binding partners. We expressed recombinant Flag-tagged PHF6 in HEK 293T cells for coimmunoprecipitation, and analyzed the purified products by mass spectrometry. We identified proteins involved in ribosome biogenesis, RNA splicing, and chromatin regulation, consistent with PHF6 localization to both the nucleoplasm and nucleolus. Notably, PHF6 copurified with multiple constituents of the nucleosome remodeling and deacetylation (NuRD) complex, including CHD4, HDAC1, and RBBP4. We demonstrate that this PHF6-NuRD complex is not present in the nucleolus but is restricted to the nucleoplasm. The association with NuRD represents the first known interaction for PHF6 and implicates it in chromatin regulation.

Histone deacetylase 1 expression in gastric cancer.

The aim of this study was to identify and evaluate novel prognostic markers for gastric cancer. Differential mRNA displays comparing paired tumor/normal stomach samples were assessed. Several differentially expressed cDNA fragments of candidate genes were identified, and one of these was further studied using quantitative reverse transcription-PCR in 140 human gastric carcinomas. To evaluate protein expression, immunohistochemical staining was performed in selected cases. One of the genes abundantly expressed in tumor tissue on the differential mRNA displays was identified as histone deacetylase 1 (HDAC). HDAC was overexpressed in the tumor tissue in 77% of the cases as determined by quantitative reverse transcription-PCR. Immunohistochemical staining revealed analogous results, showing strong expression in cancer cells. Patients were then classified into high (n=78) and low (n=62) expression groups according to the mean value of HDAC expression. High frequencies of lymph vessel and vascular vessel permeations, and advanced stage of the disease were recognized in the high expression group compared to the low expression group (p<0.05). Prognosis was significantly worse for the high expression group than for the low expression group (p<0.05), and multi-variate analysis demonstrated that HDAC expression was an independent prognostic factor. Although not significantly different, lymph node metastasis was recognized more frequently in the high expression group (p=0.07). In conclusion, the findings show that HDAC expression is associated with aggressive behavior of primary gastric cancer, and imply that determination of the HDAC expression status is useful for predicting prognosis in patients with gastric cancer.