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Craig A Mizzen

Publications and source records attributed to Craig A Mizzen.

10 recordsLinked to original sources
PubMed2006

Functional CpG methylation system in a social insect.

DNA methylation systems are well characterized in vertebrates, but methylation in Drosophila melanogaster and other invertebrates remains controversial. Using the recently sequenced honey bee genome, we present a bioinformatic, molecular, and biochemical characterization of a functional DNA methylation system in an insect. We report on catalytically active orthologs of the vertebrate DNA methyltransferases Dnmt1 and Dnmt3a and b, two isoforms that contain a methyl-DNA binding domain, genomic 5-methyl-deoxycytosine, and CpG-methylated genes. The honey bee provides an opportunity to study the roles of methylation in social contexts.

Amino Acid Sequence↗
PubMed2006

Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila.

Linker histone H1 is highly phosphorylated in normal growing Tetrahymena thermophila but becomes noticeably dephosphorylated in response to certain conditions such as prolonged starvation. Because phosphorylation of H1 has been associated with the regulation of gene expression, DNA repair, and other critical processes, we sought to use mass spectrometry-based approaches to obtain an in depth phosphorylation "signature" for this linker histone. Histone H1 from both growing and starved Tetrahymena was analyzed by nanoflow reversed-phase HPLC MS/MS following enzymatic digestions, propionic anhydride derivatization, and phosphopeptide enrichment via IMAC. We confirmed five phosphorylation sites identified previously and detected two novel sites of phosphorylation and two novel minor sites of acetylation. The sequential order of phosphorylation on H1 was deduced by using mass spectrometry to define the modified sites on phosphorylated H1 isoforms separated by cation-exchange chromatography. Relative levels of site-specific phosphorylation on H1 isolated from growing and starved Tetrahymena were obtained using a combination of stable isotopic labeling, IMAC, and tandem mass spectrometry.

Amino Acid Sequence↗
PubMed2006

Quantitative analysis of modified proteins and their positional isomers by tandem mass spectrometry: human histone H4.

Here we show that fragment ion abundances from dissociation of ions created from mixtures of multiply modified histone H4 (11 kDa) or of N-terminal synthetic peptides (2 kDa) correspond to their respective intact ion abundances measured by Fourier transform mass spectrometry. Isomeric mixtures of modified forms of the same protein are resolved and quantitated with a precision of </=5% using the relative ratios of their fragment ions, with intact protein ions created by electrospray greatly easing many of the systematic biases that more strongly affect small peptides (e.g., differences in ionization efficiency and ion m/z values). The ion fragmentation methods validated here are directly extensible to intact human proteins to derive quantitative information on the highly related and often isomeric protein forms created by combinatorial arrays of posttranslational modifications.

Chromatography, High Pressure Liquid↗
PubMed2006

Modifications of H3 and H4 during chromatin replication, nucleosome assembly, and histone exchange.

Histone posttranslational modifications that accompany DNA replication, nucleosome assembly, and H2A/H2B exchange were examined in human tissue culture cells. Through microsequencing analysis and chromatin immunoprecipitation, it was found that a subset of newly synthesized H3.2/H3.3 is modified by acetylation and methylation at sites that correlate with transcriptional competence. Immunoprecipitation experiments suggest that cytosolic predeposition complexes purified from cells expressing FLAG-H4 contain H3/H4 dimers, not tetramers. Studies of the deposition of newly synthesized H2A/H2B onto replicating and nonreplicating chromatin demonstrated that H2A/H2B exchange takes place in chromatin regions that contain acetylated H4; however, there is no single pattern of H4 acetylation that accompanies exchange. H2A/H2B exchange is also largely independent of the deposition of replacement histone variant, H3.3. Finally, immunoprecipitation of nucleosomes replicated in the absence of de novo nucleosome assembly showed that histone modifications do not prevent the transfer of parental histones to newly replicated DNA and thus have the potential to serve as means of epigenetic inheritance. Our experiments provide an in-depth analysis of the "histone code" associated with chromatin replication and dynamic histone exchange in human cells.

Acetylation↗
PubMed2006

Mass spectrometric characterization of human histone H3: a bird's eye view.

The modification of H3 in asynchronous HeLa cells was profiled using Top Down Mass Spectrometry. A broad distribution of species differing by 14 Da and containing less than 3% unmodified protein was observed for all three variants. Species of up to +168 Da were observed for H3.1, and fragmentation of all species by Electron Capture Dissociation (ECD) revealed approximately 5% methylation of K4 and approximately 50% dimethylation of K9. K14 and K23 were major sites of acetylation. H3.3 was slightly hypermodified with the apex of the distribution shifted by approximately +14 Da compared to H3.1. H3.1 (50% and 15%) from colchicine-treated cells was monophosphorylated and diphosphorylated, respectively, with equivalent modification of S10 and S28.

Acetylation↗
PubMed2006

Gene-specific characterization of human histone H2B by electron capture dissociation.

The basis set of protein forms expressed by human cells from the H2B gene family was determined by Top Down Mass Spectrometry. Using Electron Capture Dissociation for MS/MS of H2B isoforms, direct evidence for the expression of unmodified H2B.Q, H2B.A, H2B.K/T, H2B.J, H2B.E, H2B.B, H2B.F, and monoacetylated H2B.A was obtained from asynchronous HeLa cells. H2B.A was the most abundant form, with the overall expression profile not changing significantly in cells arrested in mitosis by colchicine or during mid-S, mid-G2, G2/M, and mid-G1 phases of the cell cycle. Modest hyperacetylation of H2B family members was observed after sodium butyrate treatment.

Acetylation↗
PubMed2006

Precise characterization of human histones in the H2A gene family by top down mass spectrometry.

Top Down analysis revealed that at least fourteen genes encoding histone H2A are coexpressed in HeLa cells. Characterization of these species revealed that all except H2A.Z and H2A.F/Z were alpha-N-acetylated, H2A.O and H2A.C,D,I,N,P were the most abundant, and those exceeding approximately 10% abundance lacked post-translational modifications. This unequivocal identification of H2A forms illustrates the advantages of Top Down Mass Spectrometry and provides a global perspective of H2A regulation through the cell cycle.

Acetylation↗
PubMed2003

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."

Apoptosis↗
PubMed2003

Modulation of histone acetylation by [4-(acetylamino)-N-(2-amino-phenyl) benzamide] in HCT-8 colon carcinoma.

CI-994 or N-acetyldinaline [4-(acetylamino)-N-(2-amino-phenyl) benzamide] is an antitumor cytostatic agent currently undergoing clinical trial. Although several changes in cellular metabolism induced by the drug have been characterized, the primary molecular mechanism of its antitumor activity has been previously unknown. Here, we show that CI-994 is a histone deacetylase (HDAC) inhibitor that causes histone hyperacetylation in living cells. In assays of isolated enzymes, CI-994 inhibited HDAC-1 and HDAC-2 in a concentration-dependent fashion but had no effect on the activity of the prototypical histone acetyltransferase GCN5. Acetylated histone H3-specific Western blots were used to monitor histone acetylation in HCT-8 colon carcinoma cells treated with CI-994 in vitro. CI-994 induced hyperacetylation of H3 in a time- and dose-dependent fashion. H3 hyperacetylation was detectable as early as 30 min after the addition of CI-994 to cells. These data demonstrate that inhibition of HDAC is an early event in cells treated with CI-994 and suggest that this inhibition is mechanistically related to the antitumor activity of this compound.

Acetylation↗