High mobility group (HMG) A1 proteins are subject to a number of post-translational modifications, which may regulate their function in gene transcription and other cellular processes. We examined, by using mass spectrometry, the acetylation of HMGA1a and HMGA1b proteins induced by histone acetyltransferases p300 and PCAF in vitro and in PC-3 human prostate cancer cells in vivo. It turned out that five lysine residues in HMGA1a, i.e., Lys-14, Lys-64, Lys-66, Lys-70, and Lys-73, could be acetylated by both p300 and PCAF. We further quantified the level of acetylation by analyzing, with LC-MS/MS, the proteolytic peptides of the in vitro or in vivo acetylated HMGA1 proteins where the unmodified lysine residues were chemically derivatized with a perdeuterated acetyl group. Quantification results revealed that p300 and PCAF exhibited different site preferences for the acetylation; the preference of p300 acetylation followed the order of Lys-64
Lys-70Lys-66Lys-14
Lys73, whereas the selectivity of PCAF acetylation followed the sequence of Lys-70
Lys-73  Lys-64
Lys-66  Lys-14. HMGA1b was acetylated in a very similar fashion as HMGA1a. We also demonstrated that C-terminal phosphorylation of HMGA1 proteins did not affect the in vitro acetylation of the two proteins by either p300 or PCAF. Moreover, we examined the acetylation of lysine residues in HMGA1a and HMGA1b isolated from PC-3 human prostate cancer cells. Our results showed that all the above five lysine residues were also acetylated in vivo, with Lys-64, Lys-66 and Lys-70 in HMGA1a exhibiting higher levels of acetylation than Lys-14 and Lys-73.

Epigenetic regulation involves reversible changes in DNAmethylation and/or histone modification patterns1?7. Short interfering RNAs (siRNAs) can direct DNA methylation and heterochromatic histone modifications, causing sequence-specific transcriptional gene silencing1,4,8,9. In animals and yeast, histone H2B is known to be monoubiquitinated, and this regulates the methylation of histone H3 (refs 10, 11).However, the relationship between histone ubiquitination and DNA methylation has not been investigated. Here we show that mutations in an Arabidopsis deubiquitination enzyme, SUP32/UBP26, decrease the dimethylation on lysine 9 of H3, suppress siRNA-directed methylation of DNA and release heterochromatic silencing of transgenes as well as transposons. We found that Arabidopsis histone H2B is monoubiquitinated at lysine 143 and that the levels of ubiquitinated H2B and trimethyl H3 at lysine 4 increase in sup32 mutant plants. SUP32/UBP26 can deubiquitinate H2B, and chromatin immunoprecipitation assays suggest an association between H2B ubiquitination and release of silencing. These data suggest that H2B deubiquitination by SUP32/UBP26 is required for heterochromatic histone H3 methylation and DNA methylation.

Max is a ubiquitous transcription factor with a bHLHZip [basic HLH (helix?loop?helix) leucine zipper] DNA-binding/dimerization domain and the central component of the Myc/Max/Mad transcription factor network that controls cell growth, proliferation, differentiation and apoptotic cell death in metazoans. Max is the obligatory DNA-binding and dimerization partner for all the bHLHZip regulators of the Myc/Max/Mad network,including the Myc family of oncoproteins and the Mad family of Myc antagonists, which recognize E-box DNA elements in the regulatory regions of target genes. Max lacks a transcription regulatory domain and is the only member of the network that efficiently homodimerizes. Binding of Max homodimers to E-box elements suppresses the transcription regulatory functions of its network partners and of other non-network E-box-binding regulators. In contrast with its highly regulated partners, Max is a constitutively expressed and phosphorylated protein. Phosphorylation is, however, the only Max post-translational modification identified so far. In the present study, we have analysed Max posttranslational modifications by MS. We have found that Max is acetylated at several lysine residues (Lys-57, Lys-144 and Lys-145) in mammalian cells. Max acetylation is stimulated by inhibitors of histone deacetylases and by overexpression of the p300 co-activator/HAT (histone acetyltransferase). The p300HAT also directly acetylates Max in vitro at these three residues. Interestingly, the three Max residues acetylated in vivo and in vitro by p300 are important for Max nuclear localization and Max-mediated suppression of Myc transactivation. These results uncover novel post-translational modifications of Max and suggest the potential regulation of specific Max complexes by p300 and reversible acetylation.

Histone variants and their post-translational modifications help regulate chromosomal functions. Htz1 is an evolutionarily conserved H2A variant found at several promoters in the yeast Saccharomyces cerevisiae. In this study, we undertook a genome-wide analysis of Htz1 and its modifications in yeast. Using mass spectrometric analysis, we determined that Htz1 is acetylated at Lys 3, Lys 8, Lys 10, and Lys 14 within its N-terminal tail, with K14 being the most abundant acetylated site. ChIP and microarray analysis were then used to compare the location of Htz1-K14 acetylation to that of Htz1 genome-wide. The data presented here demonstrate that while Htz1 is associated preferentially with the promoters of repressed genes, K14 acetylation is enriched at the promoters of active genes, and requires two known histone acetyltransferases, Gcn5 and Esa1. In support of our genome-wide analysis, we found that the acetylatable lysines of Htz1 are required for its full deposition during nucleosome reassembly upon repression of PHO5. Since the majority of Htz1 acetylation is seen at active promoters, where nucleosomes are known to be disassembled, our data argue for a dynamic process in which reassembly of Htz1 is regulated by its acetylation at promoters during transcription.

The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases (HATs) GCN5 and Tip60. Here, we report a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. We show that p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1 to 110 and acetylates Myc in a TAD-dependent manner in vivo at several lysine residues located between the TAD and DNA-binding domain. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. Whereas p300 and CBP can stabilize Myc independently of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene, and p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. Our results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation.

MS/MS of electrosprayed ions is shown to have the capacity to discriminate between peptides that differ by configuration about their r-carbons. It is not necessary for the peptides to possess tertiary structures that are affected by stereochemistry, since five epimers of the pentapeptide, H2N-Gly-Leu-Ser-Phe-Ala-OH (GLSFA) all display different collisionally activated dissociation (CAD)patterns of their protonated parent ions. The figure of merit, r, is a ratio of ratios of fragment ion abundances between stereoisomers, where r ) 1 corresponds to no stereochemical effect. Values of r as high as 3.8 are seen for diastereomer pairs. Stereochemical effects are also seen for the diprotonated dodecapeptide H2N-Leu-Val- Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-OH (LVFFAEDVGSNK), a tryptic fragment from the amyloid â-protein. Triply charged complexes of the protonated dodecapeptide with cobalt(II) ions undergo CAD at lower collision energies than do doubly protonated LVFFAEDVGSNK ions. Statistically significant (p < 0.01) differences between the all-L-dodecapeptide and the ones containing a D-serine or a D-aspartic acid are observed.

The c-Myc oncoprotein (Myc) functions as a transcription regulator in association with an obligatory partner, Max, to control cell growth and differentiation. The Myc:Max complex regulates specific genes by recognizing ??E-box?? DNA sequences and promoter-bound factors such as Miz-1. Myc recruits histone acetyltransferases (HATs) to modify chromatin and is, itself, acetylated in mammalian cells by several of these HATs including p300/CBP, GCN5, and Tip60. The Myc residues that are directly modified by these different HATs remain unknown. Here, we have analyzed the acetylation of recombinant Myc:Max complexes by purified p300 HAT in vitro by using MALDI-TOF and LC-ESI-MS/MS mass spectrometry. These analyses identify six lysine residues in human Myc (K143, K157, K275, K317, K323, and K371) as direct substrates for p300. Our results further indicate that p300 can acetylate DNA-bound Myc:Max complexes and that acetylated Myc:Max heterodimers efficiently interact with Miz-1.

Methylation is a relatively stable histone modification,yet regulation of the transition between mono-, di-, and trimethylation of lysine (K) residues may control dynamic processes such as transcription and DNA repair. Identifying factors that regulate the ability of methyltransferases to perform successive rounds of methylation on the same lysine residue is important for understanding the functions of histone methylation. Previous reports have indicated that ubiquitylation of histone H2B K123 is required for methylation of lysines 4 and 79 of histone H3 by the methyl-transferases Set1 and Dot1, respectively. In contrast, by using chromatin immunoprecipitation and mass spectrometry, we find that ubiquitylation of H2B-K123 is dispensable for monomethylation of H3-K4 and H3-K79 but is required for the transition from monomethylation to subsequent methylation states. Dot1 binding to chromatin occurs normally in the absence of H2B-K123 ubiquitylation, suggesting that ubiquitylation does not regulate enzyme recruitment but does regulate the processive activity of the histone methyltransferase

In Saccharomyces cerevisiae, known histone acetylation sites regulating gene activity are located in the N-terminal tails protruding from the nucleosome core. We report lysine 56 in histone H3 as a novel acetylation site that is located in the globular domain, where it extends toward the DNA major groove at the entry-exit points of the DNA superhelix as it wraps nucleosome. We show that K56 acetylation is enriched preferentially at certain active genes, such as those coding for histones. SPT10, a putative acetyltransferase, is required for cell cycle-specific K56 acetylation at histone genes. This allows recruitment of the nucleosome remodeling factor Snf5 and subsequent transcription. These findings indicate that histone H3 K56 acetylation at the entry-exit gate enables recruitment of the SWI/SNF nucleosome remodeling complex and so regulates gene activity.

Histone acetylation, methylation, and phosphorylation occur predominantly in the unstructured N-terminal domains or histone ?tails?. These modifications and others comprise a ?histone code? that directly facilitates or antagonizes association of regulatory proteins with nucleosomes to mediate changes in chromatin structure and activity. Methylation of histone H3 outside of the tail region at lysine 79 has been reported for a variety of species ranging from yeast to humans and in some gene-specific cases appears to be associated with active chromatin and transcription. Whether methylation of lysine 79 is associated with other posttranslational modifications of the H3 tail is unknown. Using mass spectrometric relative quantitation, a mass spectrometric ?Western blot?, we compare methylation at lysines 4, 9, and 79 with acetylation of human histone H3. We find that the total levels of lysine 4 and 79 methylation (combined mono-, di-, and trimethylation) in the H3 population increase with the degree of H3 tail acetylation. The total amount of lysine 4 methylation increases progressively from less than 10% in the nonacetylated H3 to greater than 90% in the penta-acetylated H3. In addition, significant levels of lysine 4 trimethylation also occur in combination with the pentaacetylated H3 species. In contrast, the level of H3 lysine 9 trimethylation is greatest for the monoacetylated species while H3 lysine 9 acetylation occurs predominantly in hyperacetylated (tetra- and penta-acetylated) H3 isoforms. Together, these results indicate that methylation of lysine 4 and 79 as well as the switch from lysine 9 methylation to acetylation are coordinated synchronously with H3 hyperacetylation as marks of active chromatin.

Testis-specific histones are synthesized and accumulated at specific stages of mammalian spermatogenesis. Their proposed functions range from facilitation of the replacement of somatic histones by protamines to epigenetic control of gene transcription. Several testis histone variants were characterized in mouse and rat; however, few are known in humans. Here we report the identification and characterization of a novel human histone 2B gene (H2BFWT) located at Xq22.2, which encodes a highly divergent H2B variant. The H2BFWT gene contains two introns and is transcribed exclusively in testis, where the spliced polyadenylated mRNA was detected. Genomic PCR, Southern blot analysis, and BLAST-based searches indicate that H2BFWT evolved in the primate lineage or has been lost in rodents. In transfected Chinese hamster cells, GFP-tagged H2BFWT targeted to large fluorescent bodies that partially colocalize with the interstitial telomeric blocks. Therefore, H2BFWT may have telomere-associated functions and participate in the telomere-binding complex in the human sperm

Protonated threonine and its allo diastereomer exhibit different proportions of collisionally activated dissociation (CAD) product ions. N-Methylation attenuates these differences. Water loss from protonated allo-threonine gives protonated trans-3-methylaziridinecarboxylic acid via an internal SN2 pathway, rather than protonated vinylglycine.

Histone acetylation and methylation play a critical role in transcription and gene regulation. Identification of sites of lysine acetylation and methylation in histones or other proteins by mass spectrometry (MS) is of increasing interest. In this paper, we report the use of MS to differentiate between peptides containing acetylated or tri-methylated lysines. High accuracy matrix-assisted laser desorption/ionization-time of flight MS gives better than five parts per million measurement accuracy, which is sufficient to verify acetylation and/or methylation. Electrospray ionization tandem mass spectrometry was used to assign modification sites and to differentiate acetylation from methylation. Typically, an immonium ion at m/z 98 corresponds to a mono-methylated lysine and an immonium ion at m/z 126 corresponds to an acetylated lysine. The neutral loss ion (MH1-59) is unique for a tri-methylated lysine. For a peptide with two or more modification sites of acetylation or tri-methylation or one site containing partial acetylation and tri-methylation, the a2-, b2-type ion is the characteristic index for an acetylated lysine whereas the b2-59 ion is indicative of a tri-methylated lysine in the N-terminus. The y-type ions and y-59 ions are charateristic of an acetylated lysine and a tri-methylated lysine at the C-terminus, respectively. We demonstrated that a lysine in a peptide modified by methylation or acetylation can be differentiated by MS using our method. Even if more then one lysine is present in a peptide and different modifications of this amino acid occur, they can be distinguished. This method was successful for the determination of the acetylation and methylation status of lysine 9 of histone H3 in chicken erythrocytes and human HeLa cell lines.

In plant reproduction, pollination is an essential process that delivers the sperm through specialized extracellular matrices (ECM) of the pistil to the ovule. Although specific mechanisms of guidance for pollen tubes through the pistil are not known, the female tissues play a critical role in this event. Many studies have documented the existence of diffusible chemotropic factors in the lily stigma that can induce pollen tube chemotropism in vitro, but no molecules have been isolated to date. In this study, we identified a chemotropic compound from the stigma by use of biochemical methods. We purified a lily stigma protein that is active in an in vitro chemotropism assay by using cation exchange, gel filtration, and HPLC. Tryptic digestion of the protein yielded peptides that identified the protein as a plantacyanin (basic blue protein), and this was confirmed by cloning the cDNA from the lily stigma. Plantacyanins are small cell wall proteins of unknown function. The measured molecular mass by electrospray ionization ion source MS is 9,898 Da, and the molecular mass of the mature protein (calculated from the cDNA) is 9,900.2 Da. Activity of the lily plantacyanin (named chemocyanin) is enhanced in the presence of stigma/stylar cysteine-rich adhesin, previously identified as a pollen tube adhesin in the lily style.

MH+ ions from sec-alkyl m-dimethylamino ethers have been studied by MS/MS using chemical ionization (CI) and electrospray ionization (ESI). Collisionally activated dissociation (CAD) of MH+ from the vicinally perdeuterated 3-hexyl ether, CH3CD2CHOArCD2CH2CH3, shows that alkene expulsion is >96% regioselective, yielding m/z 139 and m/z 124 fragment ions. The fact that alkene elimination is not 100% regioselective (but gives about 3% m/z 138) is attributed to a small amount of ion?neutral complex formation. CAD of MH+ ions from vicinally monodeuterated ethers (the 3-hexyl-4-d1 ether (1) and the sec-butyl-3-d1 ether (2)) demonstrates that the erythro and threo diastereomers give significantly different m/z 139:m/z 138 fragment ion intensity ratios. These intensity ratios are sufficiently reproducible that they can be used to quantitate the proportions of erythro and threo in mixtures. DFT calculations showsubstantial differences between the 4-center transition states for charge-remote elimination vs. alkene elimination from a neutral precursor.

Abstract DNA methylation and histone modification are two major epigenetic pathways that interplay to regulate transcriptional activity and other genome functions. Dnmt3L is a regulatory factor for the de novo DNA methyltransferases Dnmt3a and Dnmt3b. Though recent biochemical studies have revealed that Dnmt3L binds to the tail of histone H3 with unmethylated lysine 4 in vitro, the requirement of chromatin components for DNA methylation has not been examined, and functional evidence for the connection of modified histone tails to DNA methylation is still lacking. Here we used the budding yeast Saccharomyces cerevisiae as a model system to investigate the chromatin determinants of DNA methylation through ectopic expression of murine Dnmt3a and Dnmt3L. We found that the N-terminus of histone H3 tail is required for de novo methylation, while the central part encompassing lysines 9 and 27 as well as the H4 tail are dispensable. DNA methylation occurs predominantly in heterochromatin regions lacking H3K4 methylation. In mutant strains depleted of H3K4 methylation, the DNA methylation level increased five-fold. The methylation activity of Dnmt3a largely depends on the Dnmt3L''''''''s PHD domain recognizing the histone H3 tail with unmethylated lysine 4. Functional analysis of Dnmt3L in mouse ES cells confirmed that the chromatin-recognition ability of Dnmt3L''''''''s PHD domain is indeed required for efficient methylation at the promoter of the endogenous Dnmt3L gene. These findings establish the histone H3 tail N-terminus with an unmethylated lysine 4 as a chromatin determinant for DNA methylation.