Craig A. Mizzen

The TAFII250 Subunit of TFIID Has Histone Acetyltransferase Activity

The transcription initiation factor TFIID is a multimeric protein complex composed of TATA box-binding protein (TBP) and many TBP-associated factors (TAF(II)s). TAF(II)s are important cofactors that mediate activated transcription by providing interaction sites for distinct activators. Here, we present evidence that human TAF(II)250 and its homologs in Drosophila and yeast have histone acetyltransferase (HAT) activity in vitro. HAT activity maps to the central, most conserved portion of dTAF(II)230 and yTAF(II)130. The HAT activity of dTAF(II)230 resembles that of yeast and human GCN5 in that it is specific for histones H3 and H4 in vitro. Our findings suggest that targeted histone acetylation at specific promoters by TAF(II)250 may be involved in mechanisms by which TFIID gains access to transcriptionally repressed chromatin.

Apoptotic Phosphorylation of Histone H2B Is Mediated by Mammalian Sterile Twenty Kinase

DNA in eukaryotic cells is associated with histone proteins; hence, hallmark properties of apoptosis, such as chromatin condensation, may be regulated by posttranslational histone modifications. Here we report that phosphorylation of histone H2B at serine 14 (S14) correlates with cells undergoing programmed cell death in vertebrates. We identify a 34 kDa apoptosis-induced H2B kinase as caspase-cleaved Mst1 (mammalian sterile twenty) kinase. Mst1 can phosphorylate H2B at S14 in vitro and in vivo, and the onset of H2B S14 phosphorylation is dependent upon cleavage of Mst1 by caspase-3. These data reveal a histone modification that is uniquely associated with apoptotic chromatin in species ranging from frogs to humans and provide insights into a previously unrecognized physiological substrate for Mst1 kinase. Our data provide evidence for a potential apoptotic histone code.

Overlapping but Distinct Patterns of Histone Acetylation by the Human Coactivators p300 and PCAF within Nucleosomal Substrates

A number of transcriptional coactivators possess intrinsic histone acetylase activity, providing a direct link between hyperacetylated chromatin and transcriptional activation. We have determined the core histone residues acetylated in vitro by recombinant p300 and PCAF within mononucleosomes. p300 specifically acetylates all sites of histones H2A and H2B known to be acetylated in bulk chromatin in vivo but preferentially acetylates lysines 14 and 18 of histone H3 and lysines 5 and 8 of histone H4. PCAF primarily acetylates lysine 14 of H3 but also less efficiently acetylates lysine 8 of H4. PCAF in its native form, which is present in a stable multimeric protein complex lacking p300/CBP, primarily acetylates H3 to a monoacetylated form, suggesting that PCAF-associated polypeptides do not alter the substrate specificity. These distinct patterns of acetylation by the p300 and PCAF may contribute to their differential roles in transcriptional regulation.

Phosphorylation of Linker Histone H1 Regulates Gene Expression In Vivo by Mimicking H1 Removal

Two Tetrahymena strains were created by gene replacement. One contained H1 with all phosphorylation sites mutated to alanine, preventing phosphorylation. The other had these sites changed to glutamic acid, mimicking the fully phosphorylated state. Global gene expression was not detectably changed in either strain. Instead, H1 phosphorylation activated or repressed specific genes in a manner that was remarkably similar to the effects of knocking out the gene encoding H1. These studies demonstrate a role for H1 phosphorylation in the regulation of transcription in vivo and suggest that it acts by mimicking the partial removal of H1.

Ligand specific effects on aluminum incorporation and toxicity in neurons and astrocytes.

Aluminum is present in many manufactured foods and medicines and is added to drinking water for purification purposes. It has been proposed that aluminum is a contributing factors to several neurodegenerative disorders such as Alzheimers disease. However, this remains controversial primarily due to the unusual properties of aluminum and a lack of information on its cellular sites of action. To resolve some of these questions, we have examined aluminum uptake in both neuronal and astroglial cells as well as the role of metal speciation. The relative accumulation of four aluminum salts, aluminum maltolate, aluminum lactate, aluminum chloride and aluminum fluoride, was investigated and correlated with cell viability and intracellular distribution as determined by morin staining. Significant differences in aluminum incorporation and toxicity were observed in both neuronal and glia cells with the largest effects exhibited by the maltol species. This was accompanied by a nuclear accumulation in the neuronal cell line that was contrasted by the perinuclear, vesicular distribution in astrocytes that partially co-localized with cathepsin D, a lysosomal marker. These findings demonstrate differences in aluminum species and highlights the importance of these factors in modulating the toxic effect of aluminum.

Histone acetyltransferase Hbo1: Catalytic activity, cellular abundance, and links to primary cancers

In addition to the well-characterized proteins that comprise the pre-replicative complex, recent studies suggest that chromatin structure plays an important role in DNA replication initiation. One of these chromatin factors is the histone acetyltransferase (HAT) Hbo1 which is unique among HAT enzymes in that it serves as a positive regulator of DNA replication. However, several of the basic properties of Hbo1 have not been previously examined, including its intrinsic catalytic activity, its molecular abundance in cells, and its pattern of expression in primary cancer cells. Here we show that recombinant Hbo1 can acetylate nucleosomal histone H4 in vitro, with a preference for lysines 5 and 12. Using semi-quantitative western blot analysis, we find that Hbo1 is approximately equimolar with the number of active replication origins in normal human fibroblasts but is an order of magnitude more abundant in both MCF7 and Saos-2 established cancer cell lines. Immunohistochemistry for Hbo1 in 11 primary human tumor types revealed strong Hbo1 protein expression in carcinomas of the testis, ovary, breast, stomach/esophagus, and bladder.

A novel H2A/H4 nucleosomal histone acetyltransferase in Tetrahymena thermophila.

ABSTRACT Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymenamacronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be ∼80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena.

Identification and mutation of phosphorylation sites in a linker histone. Phosphorylation of macronuclear H1 is not essential for viability in Tetrahymena

Linker histone phosphorylation has been suggested to play roles in both chromosome condensation and transcriptional regulation. In the ciliated protozoan Tetrahymena, in contrast to many eukaryotes, histone H1 of macronuclei is highly phosphorylated during interphase. Macronuclei divide amitotically without overt chromosome condensation in this organism, suggesting that requirements for phosphorylation of macronuclear H1 may be limited to transcriptional regulation. Here we report the major sites of phosphorylation of macronuclear H1 in Tetrahymena thermophila. Five phosphorylation sites, present in a single cluster, were identified by sequencing 32P-labeled peptides isolated from tryptic peptide maps. Phosphothreonine was detected within two TPVK motifs and one TPTK motif that resemble established p34 cdc2 kinase consensus sequences. Phosphoserine was detected at two non-proline-directed sites that do not resemble known kinase consensus sequences. Phosphorylation at the two noncanonical sites appears to be hierarchical because it was observed only when a nearby p34 cdc2 site was also phosphorylated. Cells expressing macronuclear H1 containing alanine substitutions at all five of these phosphorylation sites were viable even though macronuclear H1 phosphorylation was abolished. These data suggest that the five sites identified comprise the entire collection of sites utilized by Tetrahymena and demonstrate that phosphorylation of macronuclear H1, like the protein itself, is not essential for viability in Tetrahymena.

Capillary electrophoresis of histone H1 variants at neutral pH in dynamically modified fused-silica tubing

Existing methods for the analysis of histone H1 by capillary electrophoresis (CE) employ acidic buffers (pH <3.0) to suppress silanol ionization and minimize the loss of these extremely basic proteins by adsorption to capillary walls. Here we describe the use of Polybrene (PB) as a dynamic modification reagent in a simple procedure that facilitates the analysis of chicken H1 at neutral pH. PB is adsorbed to the inner surfaces of capillaries to render them cationic prior to use and a low concentration of PB is included in the electrolyte to replenish the coating during use. Inclusion of ethylenediaminetetraacetic acid (EDTA) in the electrolyte results in the assembly of a dynamic cation‐exchange layer upon the immobilized PB that influences the relative mobilities of H1 variants. The six nonallelic variants of H1 known in this species as well as certain allelic variants are resolved. Because the procedure is effective in preventing the adsorption of proteins as basic as H1 at neutral pH, this strategy should facilitate CE analyses of many basic proteins under conditions that maintain their native conformation.

ISOLATION AND CHARACTERIZATION OF IN VIVO MODIFIED HISTONES AND AN ACTIVITY GEL ASSAY FOR IDENTIFICATION OF HISTONE ACETYLTRANSFERASES

Publisher Summary Chromatin in vivo is intrinsically a dynamic structure, undergoing pronounced conformational changes in response to specific biological stimuli. Histones themselves are subject to multiple posttranslational modifications such as phosphorylation and acetylation. Histone acetylation is brought about and maintained by a balance of two classes of competing enzymes: the on reaction is catalyzed by enzymes called histone acetyltransferases (HATS); the opposing off reaction (that is, the removal of acetyl groups from histones) is catalyzed by deacetylase (HDACs) enzymes. This chapter discusses the techniques used for isolating and analyzing Tetrahymena histones modified in vivo. Histones are easily extracted from isolated nuclei by virtue of their acid solubility. Following extraction, individual histones can be isolated by reverse phase high-performance liquid chromatography (RP-HPLC) or be analyzed by gel electrophoresis. Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) effectively resolves all Tetrahymena histones except H3 and H2A, better resolution is achieved on acid-urea (or triton-acid-urea) gels that separate proteins according to their size, charge, and, in the case of triton-containing acid-urea gels, detergent-binding properties.