George R. Green

H2B- and H3-Specific Histone Deacetylases Are Required for DNA Methylation in Neurospora crassa

Neurospora crassa utilizes DNA methylation to inhibit transcription of heterochromatin. DNA methylation is controlled by the histone methyltransferase DIM-5, which trimethylates histone H3 lysine 9, leading to recruitment of the DNA methyltransferase DIM-2. Previous work demonstrated that the histone deacetylase (HDAC) inhibitor trichostatin A caused a reduction in DNA methylation, suggesting involvement of histone deacetylation in DNA methylation. We therefore created mutants of each of the four classical N. crassa HDAC genes and tested their effect on histone acetylation levels and DNA methylation. Global increases in H3 and H4 acetylation levels were observed in both the hda-3 and the hda-4 mutants. Mutation of two of the genes, hda-1 and hda-3, caused partial loss of DNA methylation. The site-specific loss of DNA methylation in hda-1 correlated with loss of H3 lysine 9 trimethylation and increased H3 acetylation. In addition, an increase in H2B acetylation was observed by two-dimensional gel electrophoresis of histones of the hda-1 mutant. We found a similar increase in the Schizosaccharomyces pombe Clr3 mutant, suggesting that this HDAC has a previously unrecognized substrate and raising the possibility that the acetylation state of H2B may play a role in the regulation of DNA methylation and heterochromatin formation.

Extensive and Varied Modifications in Histone H2B of Wild-Type and Histone Deacetylase 1 Mutant Neurospora crassa

DNA methylation is deficient in a histone deacetylase 1 (HDA1) mutant (hda-1) strain of Neurospora crassa with inactivated histone deacetylase 1. Difference two-dimensional (2D) gels identified the primary histone deacetylase 1 target as histone H2B. Acetylation was identified by LC-MS/MS at five different lysines in wild-type H2B and at 11 lysines in hda-1 H2B, suggesting Neurospora H2B is a complex combination of different acetylated species. Individual 2D gel spots were shifted by single lysine acetylations. FTICR MS-observed methylation ladders identify an ensemble of 20-25 or more modified forms for each 2D gel spot. Twelve different lysines or arginines were methylated in H2B from the wild type or hda-1; only two were in the N-terminal tail. Arginines were modified by monomethylation, dimethylation, or deimination. H2B from wild-type and hda-1 ensembles may thus differ by acetylation at multiple sites, and by additional modifications. Combined with asymmetry-generated diversity in H2B structural states in nucleosome core particles, the extensive modifications identified here can create substantial histone-generated structural diversity in nucleosome core particles.

Purification and analysis of variant and modified histones using 2D PAGE.

Two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) systems employing combinations of acetic acid/urea (AU), acetic acid/urea/Triton X-100 (AUT) and sodium dodecyl sulfate (SDS) gel formulations are uniquely effective for resolution of histone variants and their modified derivatives. Coupled with Western transfer methods using modification-specific antibodies and recent advances in mass spectrometry, 2D PAGE emerges as a versatile tool for histone purification and analysis. This chapter describes 2D PAGE gel systems appropriate for histone proteins, including detailed procedures for designing, running, and staining gels. Methods for electrophoretic transfer of histones from AUTxSDS and AUTxAU 2D gels are described and evaluated. Alternatively, methods are provided for obtaining highly purified protein samples from fixed and stained gels via electroelution of proteins from specific gel spots.

Development of albumin microspheres containing Sp H1-DNA complexes: A novel gene delivery system

This study investigated the stability and transfection efficiency of plasmid DNA (pDNA) and sea urchin sperm histone H1 (Sp H1) complexes embedded in albumin microsphere formulations. Sp H1 increased the stability and transfection efficiency of pDNA, while providing a favourable sustained pDNA release profile. Encapsulating Sp H1-complexed pDNA into albumin microspheres further protected the pDNA from physical stress and heparin treatment. When compared with free pDNA encapsulated in albumin microspheres, the Sp H1-pDNA microsphere formulations exhibited decreased hydrophilicity, slower pDNA release profiles, protection against heparin-induced degradation of embedded pDNA and increased stability against physical stress. These results indicate that complex formation of pDNA with Sp H1 facilitates intracellular DNA transfer and that albumin microspheres-Sp H1-pDNA gene delivery formulations are suitable for controlled-release delivery of pDNA while offering protection of the pDNA from degradation and maintaining pDNA biological activity.

Amidation inhibitors 4-phenyl-3-butenoic acid and 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester are novel HDAC inhibitors with anti-tumorigenic properties

Summary4-Phenyl-3-butenoic acid (PBA) is an inhibitor of peptidylglycine alpha-amidating monooxygenase with anti-inflammatory properties that has been shown to inhibit the growth of ras-mutated epithelial and human lung carcinoma cells. In this report, we show that PBA also increases the acetylation levels of selected histone subtypes in a dose and time dependent manner, an effect that is attributable to the inhibition of histone deacetylase (HDAC) enzymes. Comparison studies with the known HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) using high resolution two-dimensional polyacrylamide gels and Western analysis provide evidence that PBA acts as an HDAC inhibitor within cells. PBA and a more potent amidation inhibitor, 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me), inhibit HDAC enzymes in vitro at micromolar concentrations, with IC50 values approximately 30 fold lower for AOPHA-Me than PBA for selected HDAC isoforms. Overall, these results indicate that PBA and AOPHA-Me are novel anti-tumorigenic HDAC inhibitors.