Butyrate-induced histone hyperacetylation in human and mouse cells: estimation of putative sites of histone acetylation in vivo

Human and mouse cells in culture were treated with various concentrations of sodium butyrate. Acid-extracted histones of control and butyrate-treated cells were analyzed by two-dimensional gel electrophoresis. All core histones of the control cells contained modified forms. All core histones of the butyrate-treated cells were hyperacetylated. Depending on the number of acetylation sites per molecule, each histone or histone variant exhibited a characteristic number of acetylated forms. This number was the same for each histone common in human and mouse cells treated with butyrate. Histones 2A.1, 2A.2, and 2A.X have two sites of inner acetylation; 2A.Z has 3; 2B's have 5; and each one of the H3 variants as well as H4 have 4.     

A cooperative activation loop among SWI/SNF, γ‐H2AX and H3 acetylation for DNA double‐strand break repair

Although recent studies highlight the importance of histone modifications and ATP‐dependent chromatin remodelling in DNA double‐strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser‐139 (S139ph) after DNA damage, binds to γ‐H2AX (the phosphorylated form of H2AX)‐containing nucleosomes in S139ph‐dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to γ‐H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with γ‐H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to γ‐H2AX nucleosomes. S139ph stimulates the H3 acetylation on γ‐H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on γ‐H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, γ‐H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.     

Persistent histone modifications at the BDNF and Cdk‐5 promoters following extinction of nicotine‐seeking in rats

Drugs of addiction lead to a wide range of epigenetic changes at the promoter regions of genes directly implicated in learning and memory processes. We have previously shown that the histone deactylase inhibitor, sodium butyrate (NaB), accelerates the extinction of nicotine‐seeking and provides resistance to relapse. Here, we explore the potential molecular mechanisms underlying this effect. Rats received intravenous nicotine or saline self‐administration, followed by 6 days of extinction training, with each extinction session followed immediately by treatment with NaB or vehicle. On the last day of extinction, rats were killed and the medial ventral prefrontal cortex retained for chromatin immunoprecipitation and quantitative polymerase chain reaction (qPCR). A history of nicotine exposure significantly decreased H3K14 acetylation at the brain‐derived neurotrophic factor (BDNF) exon IV promoter, and this effect was abolished with NaB treatment. In contrast, nicotine self‐administration alone, resulted in a significant decrease in histone methylation at the H3K27me3 and H3K9me2 marks in the promoter regions of BDNF exon IV and cyclin‐dependent kinase 5 (Cdk‐5). Quantitative PCR‐identified changes in several genes associated with NaB treatment that were independent of nicotine exposure; however, an interaction of nicotine history and NaB treatment was detected only in the expression of BDNF IV and BDNF IX. Together these results suggest that nicotine self‐administration leads to a number of epigenetic changes at both the BDNF and Cdk‐5 promoters, and that these changes may contribute to the enhanced extinction of nicotine‐seeking by NaB.     

Titration_DB: Storage and analysis of NMR‐monitored protein pH titration curves

NMR‐monitored pH titration experiments are routinely used to measure site‐specific protein pKa values. Accurate experimental pKa values are essential in dissecting enzyme catalysis, in studying the pH‐dependence of protein stability and ligand binding, in benchmarking pKa prediction algorithms, and ultimately in understanding electrostatic effects in proteins. However, due to the complex ways in which pH‐dependent electrostatic and structural changes manifest themselves in NMR spectra, reported apparent pKa values are often dependent on the way that NMR pH‐titration curves are analyzed. It is therefore important to retain the raw NMR spectroscopic data to allow for documentation and possible re‐interpretation. We have constructed a database of primary NMR pH‐titration data, which is accessible via a web interface. Here, we report statistics of the database contents and analyze the data with a global perspective to provide guidelines on best practice for fitting NMR titration curves. Titration_DB is available at http://enzyme.ucd.ie/Titration_DB . Proteins 2010. © 2009 Wiley‐Liss, Inc.     

CLONING,EXPRESSION, AND CHARACTERIZATION OF A HISTONE‐LIKE PROTEIN FROM THE MARINE DINOFLAGELLATE LINGULODINIUM POLYEDRUM (DINOPHYCEAE)1

Although nucleosomes and histones are lacking in dinoflagellate nuclei, small basic histone‐like proteins have been reported, but their function(s) is unknown. In this study we cloned and sequenced a gene for a histone‐like protein from the dinoflagellate Lingulodinium polyedrum (Stein) Dodge (HLp) (formerly Gonyaulax polyedra Stein) and investigated its post‐translational modification and DNA‐binding activities. HLp appears to be acetylated in L. polyedrum, and we identified several L. polyedrum proteins that possess histone acetyltransferase activity and may be responsible for this modification. HLp binds weakly to L. polyedrum DNA but to certain specific sequences with higher affinity, consistent with its having a regulatory function.     

NMR‐based characterization of a refolding intermediate of β2‐microglobulin labeled using a wheat germ cell‐free system

In patients with dialysis‐related amyloidosis, β2‐microglobulin (β2‐m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of β2‐m is a prerequisite to the formation of amyloid fibrils, and that the folding intermediate trapped by the non‐native trans‐Pro32 isomer leads to the formation of amyloid fibrils. Although clarifying the structure of this refolding intermediate by high resolution NMR spectroscopy is important, this has been made difficult by the limited lifetime of the intermediate. Here, we studied the structure of the refolding intermediate using a combination of amino acid selective labeling with wheat germ cell‐free protein synthesis and NMR techniques. The HSQC spectra of β2‐ms labeled selectively at either phenylalanine, leucine, or valine enabled us to monitor the structures of the refolding intermediate. The results suggested that the refolding intermediate has an overall fold and cores similar to the native structure, but contains disordered structures around Pro32. The fluctuation of the β‐sheet regions especially the last half of the βB strand and the first half of the βE strand, both suggested to be important for amyloidogenicity, may transform β2‐m into an amyloidogenic structure.     

Effects of histone acetylation on chromatin structure

The effect of histone acetylation was monitored on CHO chromatin structure, following the addition of 7 mM Na-butyrate to the cell culture medium. The properties of both control and hyperacetylated chromatins and nuclei were investigated by circular dichroism, ethidium bromide intercalation, differential scanning calorimetry, and affinity chromatography. Our results are compatible with modest but significant alterations in the various levels of chromatin organization, as a result of the charge neutralization of some lysine residues within the N-terminal region of the histonic octamer. Namely, large statistically significant differences do exist in the heat capacity thermograms of native nuclei, where unfolding into single nucleofilament of the highly packed native chromatin superfiber appears associated with acetylation; at the same time CD, EB, and affinity chromatography point to modest but consistent differences in the compactness of isolated nucleosomes and polynucleosomes. J. Cell. Biochem. 64:466–475. © 1997 Wiley-Liss, Inc.     

Design of high‐affinity S100‐target hybrid proteins

S100B and S100A10 are dimeric, EF‐hand proteins. S100B undergoes a calcium‐dependant conformational change allowing it to interact with a short contiguous sequence from the actin‐capping protein CapZ (TRTK12). S100A10 does not bind calcium but is able to recruit the N‐terminus of annexin A2 important for membrane fusion events, and to form larger multiprotein complexes such as that with the cation channel proteins TRPV5/6. In this work, we have designed, expressed, purified, and characterized two S100‐target peptide hybrid proteins comprised of S100A10 and S100B linked in tandem to annexin A2 (residues 1–15) and CapZ (TRTK12), respectively. Different protease cleavage sites (tobacco etch virus, PreScission) were incorporated into the linkers of the hybrid proteins. In situ proteolytic cleavage monitored by ¹H‐¹⁵N HSQC spectra showed the linker did not perturb the structures of the S100A10‐annexin A2 or S100B‐TRTK12 complexes. Furthermore, the analysis of the chemical shift assignments (¹H, ¹⁵N, and ¹³C) showed that residues T102‐S108 of annexin A2 formed a well‐defined α‐helix in the S100A10 hybrid while the TRTK12 region was unstructured at the N‐terminus with a single turn of α‐helix from D108‐K111 in the S100B hybrid protein. The two S100 hybrid proteins provide a simple yet extremely efficient method for obtaining high yields of intact S100 target peptides. Since cleavage of the S100 hybrid protein is not necessary for structural characterization, this approach may be useful as a scaffold for larger S100 complexes.     

Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX–DNMT3–DNMT3L domain

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX–DNMT3–DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino‐terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1α to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.     

Role of histone acetylation in the development of diabetic retinopathy and the metabolic memory phenomenon

Hyperglycemia is considered as one of the major determinants in the development of diabetic retinopathy, but the progression of retinopathy resists arrest after hyperglycemia is terminated, suggesting a metabolic memory phenomenon. Diabetes alters the expression of retinal genes, and this continues even after good glycemic control is re‐instituted. Since the expression of genes is affected by chromatin structure that is modulated by post‐translational modifications of histones, our objective is to investigate the role of histone acetylation in the development of diabetic retinopathy, and in the metabolic memory phenomenon. Streptozotocin‐induced rats were maintained either in poor glycemic control (PC, glycated hemoglobin, GHb >11%) or good glycemic control (GC, GHb <6%) for 12 months, or allowed to be in PC for 6 months followed by in GC for 6 months (PC‐GC). On a cellular level, retinal endothelial cells, the target of histopathology of diabetic retinopathy, were incubated in 5 or 20 mM glucose for 4 days. Activities of histone deacetylase (HDAC) and histone acetyltransferase (HAT), and histone acetylation were quantified. Hyperglycemia activated HDAC and increased HDAC1, 2, and 8 gene expressions in the retina and its capillary cells. The activity HAT was compromised and the acetylation of histone H3 was decreased. Termination of hyperglycemia failed to provide any benefits to diabetes‐induced changes in retinal HDAC and HAT, and histone H3 remained subnormal. This suggests “in principle” the role of global acetylation of retinal histone H3 in the development of diabetic retinopathy and in the metabolic memory phenomenon associated with its continued progression. J. Cell. Biochem. 110: 1306–1313, 2010. © 2010 Wiley‐Liss, Inc.