Agus Darwanto

Identification and Characterization of Propionylation at Histone H3 Lysine 23 in Mammalian Cells

Propionylation has been identified recently as a new type of protein post-translational modification. Bacterial propionyl-CoA synthetase and human histone H4 are propionylated at specific lysine residues that have been known previously to be acetylated. However, other proteins subject to this modification remain to be identified, and the modifying enzymes involved need to be characterized. In this work, we report the discovery of histone H3 propionylation in mammalian cells. Propionylation at H3 lysine Lys23 was detected in the leukemia cell line U937 by mass spectrometry and Western analysis using a specific antibody. In this cell line, the propionylated form of Lys23 accounted for 7%, a level at least 6-fold higher than in other leukemia cell lines (HL-60 and THP-1) or non-leukemia cell lines (HeLa and IMR-90). The propionylation level in U937 cells decreased remarkably during monocytic differentiation, indicating that this modification is dynamically regulated. Moreover, in vitro assays demonstrated that histone acetyltransferase p300 can catalyze H3 Lys23 propionylation, whereas histone deacetylase Sir2 can remove this modification in the presence of NAD+. These results suggest that histone propionylation might be generated by the same set of enzymes as for histone acetylation and that selection of donor molecules (propionyl-CoA versus acetyl-CoA) may determine the difference of modifications. Because like acetyl-CoA, propionyl-CoA is an important intermediate in biosynthesis and energy production, histone H3 Lys23 propionylation may provide a novel epigenetic regulatory mark for cell metabolism.

A Modified “Cross-talk” between Histone H2B Lys-120 Ubiquitination and H3 Lys-79 Methylation

Western blot analysis is currently the major method utilized for quantitatively assessing histone global modifications. However, there is a growing need to develop a highly specific, accurate, and multisite quantitative method. Herein, we report a liquid chromatography-tandem mass spectrometry-multiple reaction monitoring method to simultaneously quantify multisite modifications with unmatched specificity, sensitivity, and throughput. With one set of purification of histones by high pressure liquid chromatography or SDS-PAGE, nearly 20 modification sites including acetylation, propionylation, methylation, and ubiquitination were quantified within 2 h for two samples to be compared. Using this method, the relative levels of H2B ubiquitination and H3 Lys-79 methylation were quantified in the U937 human leukemia cell line, U937 derivative cell lines overexpressing anti-secretory factor 10 (AF10) and mutant AF10 with the deletion of the hDot1 binding domain OM-LZ. We found that H2B ubiquitination is inversely correlated with H3 Lys-79 methylation. Therefore, we propose that a catalytic and inhibitory loop mechanism may better describe the cross-talk relationship between H2B ubiquitination and H3 Lys-79 methylation.

Maternal cocaine administration causes an epigenetic modification of protein kinase Cε gene expression in fetal rat heart

Protein kinase Cϵ (PKCϵ) plays a pivotal role in cardioprotection during cardiac ischemia and reperfusion injury. Recent studies demonstrated that prenatal cocaine exposure caused a decrease in PKCϵ expression and increased heart susceptibility to ischemic injury in adult offspring, suggesting an in utero programming of PKCϵ gene expression pattern in the heart. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for cocaine-mediated repression of the PKCϵ gene in the heart. Pregnant rats were administered either saline or cocaine intraperitoneally (15 mg/kg) twice daily from days 15 to 20 of gestational age, and term fetal hearts were studied. Cocaine treatment significantly decreased PKCϵ mRNA and protein levels in the heart. CpG dinucleotides found in cAMP response element-binding protein (CREB), CREB/c-Jun1, and CREB/c-Jun2 binding sites at the proximal promoter region of the PKCϵ gene were densely methylated and were not affected by cocaine. In contrast, methylation of CpGs in the activator protein 1 (AP-1) binding sites was low but was significantly increased by cocaine. Reporter gene assays showed that the AP-1 binding site played a strong stimulatory role of PKCϵ gene transcription. Methylation of the AP-1 binding sites significantly decreased AP-1 binding to the PKCϵ promoter. Supershift analyses implicated c-Jun homodimers binding to the AP-1 binding sites. Cocaine did not affect nuclear c-Jun levels or the binding of c-Jun to the unmethylated AP-1 binding sites. The results indicate a role for DNA methylation in cocaine-mediated PKCϵ gene repression in the developing heart and suggest an epigenetic mechanism affecting this gene linked with vulnerability of ischemic injury in the heart of adult offspring.

Mass Spectrometric Studies on Epigenetic Interaction Networks in Cell Differentiation

Arrest of cell differentiation is one of the leading causes of leukemia and other cancers. Induction of cell differentiation using pharmaceutical agents has been clinically attempted for the treatment of these cancers. Epigenetic regulation may be one of the underlying molecular mechanisms controlling cell proliferation or differentiation. Here, we report on the use of proteomics-based differential protein expression analysis in conjunction with quantification of histone modifications to decipher the interconnections among epigenetic modifications, their modifying enzymes or mediators, and changes in the associated pathways/networks that occur during cell differentiation. During phorbol-12-myristate 13-acetate-induced differentiation of U937 cells, fatty acid synthesis and its metabolic processing, the clathrin-coated pit endocytosis pathway, and the ubiquitin/26 S proteasome degradation pathways were up-regulated. In addition, global histone H3/H4 acetylation and H2B ubiquitination were down-regulated concomitantly with impaired chromatin remodeling machinery, RNA polymerase II complexes, and DNA replication. Differential protein expression analysis established the networks linking histone hypoacetylation to the down-regulated expression/activity of p300 and linking histone H2B ubiquitination to the RNA polymerase II-associated FACT-RTF1-PAF1 complex. Collectively, our approach has provided an unprecedentedly systemic set of insights into the role of epigenetic regulation in leukemia cell differentiation.

Mechanisms of base selection by human single-stranded selective monofunctional uracil-DNA glycosylase

hSMUG1 (human single-stranded selective monofunctional uracil-DNA glyscosylase) is one of three glycosylases encoded within a small region of human chromosome 12. Those three glycosylases, UNG (uracil-DNA glycosylase), TDG (thymine-DNA glyscosylase), and hSMUG1, have in common the capacity to remove uracil from DNA. However, these glycosylases also repair other lesions and have distinct substrate preferences, indicating that they have potentially redundant but not overlapping physiological roles. The mechanisms by which these glycosylases locate and selectively remove target lesions are not well understood. In addition to uracil, hSMUG1 has been shown to remove some oxidized pyrimidines, suggesting a role in the repair of DNA oxidation damage. In this paper, we describe experiments in which a series of oligonucleotides containing purine and pyrimidine analogs have been used to probe mechanisms by which hSMUG1 distinguishes potential substrates. Our results indicate that the preference of hSMUG1 for mispaired uracil over uracil paired with adenine is best explained by the reduced stability of a duplex containing a mispair, consistent with previous reports with Escherichia coli mispaired uracil-DNA glycosylase. We have also extended the substrate range of hSMUG1 to include 5-carboxyuracil, the last in the series of damage products from thymine methyl group oxidation. The properties used by hSMUG1 to select damaged pyrimidines include the size and free energy of solvation of the 5-substituent but not electronic inductive properties. The observed distinct mechanisms of base selection demonstrated for members of the uracil glycosylase family help explain how considerable diversity in chemical lesion repair can be achieved.

Impact of Base Analogues within a CpG Dinucleotide on the Binding of DNA by the Methyl-Binding Domain of MeCP2 and Methylation by DNMT1

The epigenetic control of transcription requires the selective recognition of methylated CpG dinucleotides by methylation-sensitive sequence-specific DNA binding proteins. In order to probe the mechanism of selective interaction of the methyl-binding protein with methylated DNA, we have prepared a series of oligonucleotides containing modified purines and pyrimidines at the recognition site, and we have examined the binding of these oligonucleotides to the methyl-binding domain (MBD) of the methyl-CpG-binding protein 2 (MeCP2). Our results suggest that pyrimidine 5-substituents similar in size to a methyl group facilitate protein binding; however, binding affinity does not correlate with the hydrophobicity of the substituent, and neither the 4-amino group of 5-methylcytosine (mC) nor Watson-Crick base pair geometry is essential for MBD binding. However, 5-substituted uracil analogues in one strand do not direct human DNA methyltransferase 1 (DNMT1) methylation of the opposing strand, as does mC. Important recognition elements do include the guanine O6 and N7 atoms present in the major groove. Unexpectedly, removal of the guanine 2-amino group from the minor groove substantially enhances MBD binding, likely resulting from DNA bending at the substitution site. The enhanced binding of the MBD to oligonucleotides containing several cytosine analogues observed here is better explained by a DNA-protein interface mediated by structured water as opposed to hydrophobic interactions.

Cloning and Characterization of Rhodotorula glutinis Thymine Hydroxylase

Thymine hydroxylase (TH) is a member of the alpha-ketoglutarate-dependent nonheme iron dioxygenase family that includes a series of DNA repair proteins including alkB. Substantial interest in this family of enzymes derives from their capacity to modify DNA bases and precursors by oxidation. Previously, a sequence has been published for cloned Rhodotorula glutinis TH. However, the minimal reported activity of this enzyme, coupled with inconsistencies with previously published mass spectrometry data, compelled us to reexamine TH. The sequence reported here differs from the previously reported sequence at two amino acid positions and is consistent with previously reported mass spectrometry data. The cloned enzyme characterized in this report displayed substantial activity, indicating that the sequence differences are critical for activity. The substrate selectivity of TH against a series of pyrimidine analogues is consistent with that reported for the wild-type enzyme and, in part, explains the mode of selection of uracil analogues. A preliminary model of the active site has been constructed for the purposes of comparing TH with other members of this family. TH and alkB share in common the capacity to oxidize N-methyl groups. However, TH has the added capacity to oxidize the 5-methyl group of thymine, a property that is potentially important for enzymes that could act on DNA and modify DNA-protein interactions.

Mechanisms of Base Selection by the Escherichia coli Mispaired Uracil Glycosylase

The repair of the multitude of single-base lesions formed daily in cells of all living organisms is accomplished primarily by the base excision repair pathway that initiates repair through a series of lesion-selective glycosylases. In this article, single-turnover kinetics have been measured on a series of oligonucleotide substrates containing both uracil and purine analogs for the Escherichia coli mispaired uracil glycosylase (MUG). The relative rates of glycosylase cleavage have been correlated with the free energy of helix formation and with the size and electronic inductive properties of a series of uracil 5-substituents. Data are presented that MUG can exploit the reduced thermodynamic stability of mispairs to distinguish U:A from U:G pairs. Discrimination against the removal of thymine results primarily from the electron-donating property of the thymine 5-methyl substituent, whereas the size of the methyl group relative to a hydrogen atom is a secondary factor. A series of parameters have been obtained that allow prediction of relative MUG cleavage rates that correlate well with observed relative rates that vary over 5 orders of magnitude for the series of base analogs examined. We propose that these parameters may be common among DNA glycosylases; however, specific glycosylases may focus more or less on each of the parameters identified. The presence of a series of glycosylases that focus on different lesion properties, all coexisting within the same cell, would provide a robust and partially redundant repair system necessary for the maintenance of the genome.

Aryl Hydrocarbon Receptor Ligand 5F 203 Induces Oxidative Stress That Triggers DNA Damage in Human Breast Cancer Cells.

Breast tumors often show profound sensitivity to exogenous oxidative stress. Investigational agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) induces aryl hydrocarbon receptor (AhR)-mediated DNA damage in certain breast cancer cells. Since AhR agonists often elevate intracellular oxidative stress, we hypothesize that 5F 203 increases reactive oxygen species (ROS) to induce DNA damage, which thwarts breast cancer cell growth. We found that 5F 203 induced single-strand break formation. 5F 203 enhanced oxidative DNA damage that was specific to breast cancer cells sensitive to its cytotoxic actions, as it did not increase oxidative DNA damage or ROS formation in nontumorigenic MCF-10A breast epithelial cells. In contrast, AhR agonist and procarcinogen benzo[a]pyrene and its metabolite, 1,6-benzo[a]pyrene quinone, induced oxidative DNA damage and ROS formation, respectively, in MCF-10A cells. In sensitive breast cancer cells, 5F 203 activated ROS-responsive kinases: c-Jun-N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38). AhR antagonists (alpha-naphthoflavone, CH223191) or antioxidants (N-acetyl-l-cysteine, EUK-134) attenuated 5F 203-mediated JNK and p38 activation, depending on the cell type. Pharmacological inhibition of AhR, JNK, or p38 attenuated 5F 203-mediated increases in intracellular ROS, apoptosis, and single-strand break formation. 5F 203 induced the expression of cytoglobin, an oxidative stress-responsive gene and a putative tumor suppressor, which was diminished with AhR, JNK, or p38 inhibition. Additionally, 5F 203-mediated increases in ROS production and cytoglobin were suppressed in AHR100 cells (AhR ligand-unresponsive MCF-7 breast cancer cells). Our data demonstrate 5F 203 induces ROS-mediated DNA damage at least in part via AhR, JNK, or p38 activation and modulates the expression of oxidative stress-responsive genes such as cytoglobin to confer its anticancer action.

pH-Dependent configurations of a 5-chlorouracil-guanine base pair.

Hypochlorous acid (HOCl) from activated neutrophils at sites of inflammation can react with and damage biological molecules, including nucleic acids. The reaction of HOCl with cytosine analogues can generate multiple products, including 5-chlorouracil (ClU). In this paper, we have constructed oligonucleotides containing ClU paired opposite guanine (ClU-G). Melting studies indicate that oligonucleotide duplexes containing the ClU-G mispair are substantially less stable than those containing a ClU-A base pair. The melting temperature of the ClU-G mispair is not experimentally distinguishable from that of a T-G pair. NMR studies indicate that the ClU-G base pair adopts a wobble geometry at neutral pH, similar to a T-G mispair. The exchangeable protons of the ClU-G mispair broaden rapidly with an increase in temperature, indicating that the ClU-G mispair is less stable and opens more easily than the surrounding adjacent base pairs. Unlike the ClU-A base pair studied previously [Theruvathu, J. A., et al. (2009) Biochemistry 48, 7539-7546], the ClU-G mispair undergoes a pH-dependent structural change, assuming an ionized base pair configuration that approximates a Watson-Crick base pair at higher pH. Ionization of ClU in a DNA template could promote mispair formation and mutation, in accord with previous studies on other 5-halouracil analogues. The electron-withdrawing 5-chloro substituent facilitates ionization of the ClU N3 proton, promoting mispair formation, but it also renders the glycosidic bond susceptible to base cleavage by DNA repair glycosylases.