Histone Type Research Articles

Insight the C-Site Pocket Conformational Changes Responsible for Sirtuin 2 Activity Using Molecular Dynamics Simulations

Sirtuin belongs to a family of typical histone deacetylase which regulates the fundamental cellular biological processes including gene expression, genome stability, mitosis, nutrient metabolism, aging, mitochondrial function, and cell motility. Michael et. al. reported that B-site mutation (Q167A and H187A) decreased the SIRT2 activity but still the structural changes were not reported. Hence, we performed 5 ns molecular dynamics (MD) simulation on SIRT2 Apo-form and complexes with substrate/NAD+ and inhibitor of wild type (WT), Q167A, and H187A. The results revealed that the assembly and disassembly of C-site induced by presence of substrate/NAD+ and inhibitor, respectively. This assembly and disassembly was mainly due to the interaction between the substrate/NAD+ and inhibitor and F96 and the distance between F96 and H187 which are present at the neck of the C-site. MD simulations suggest that the conformational change of L3 plays a major role in assembly and disassembly of C-site. Our current results strongly suggest that the distinct conformational change of L3 as well as the assembly and disassembly of C-site plays an important role in SIRT2 deacetylation function. Our study unveiled the structural changes of SIRT2 in presence of NAD+ and inhibitor which should be helpful to improve the inhibitory potency of SIRT2.

Transcriptional signatures mediated by acetylation overlap with early-stage Alzheimer’s disease

The mechanisms by which environmental influences lead to the development of complex neurodegenerative diseases are largely unknown. It is known, however, that epigenetic mechanisms can mediate alterations in transcription due to environmental influences. In order to identify genes susceptible to regulation in the adult cortex by one type of epigenetic mechanism, histone, and protein acetylation, we treated mice with the histone deacetylase inhibitor Trichostatin A (TSA). After 1week of treatment with TSA, RNA was extracted from the brain cortices of mice and gene expression differences were analyzed by microarray profiling. The altered genes were then compared with genes differentially expressed in microarray studies of disease by database and literature searches. Genes regulated by TSA were found to significantly overlap with differentially expressed genes in the Alzheimer's disease (AD) brain. Several TSA-regulated genes involved in chromatin remodeling and epigenetic reprogramming including histone cluster 1, H4h (Hist1H4h), methionine adenosyltransferase II, alpha (Mat2a), and 5-methyltetrahydrofolate homocysteine reductase (Mtrr) overlapped with genes altered in early-stage AD in gray matter. We also show that the expression of hemoglobin, which has been shown to be altered in neurons in the AD brain, is regulated by TSA treatment. This analysis suggests involvement of epigenetic mechanisms in neurons in early stages of AD.

Acetyl-CoA Carboxylase Regulates Global Histone Acetylation

Histone acetylation depends on intermediary metabolism for supplying acetyl-CoA in the nucleocytosolic compartment. However, because nucleocytosolic acetyl-CoA is also used for de novo synthesis of fatty acids, histone acetylation and synthesis of fatty acids compete for the same acetyl-CoA pool. The first and rate-limiting reaction in de novo synthesis of fatty acids is carboxylation of acetyl-CoA to form malonyl-CoA, catalyzed by acetyl-CoA carboxylase. In yeast Saccharomyces cerevisiae, acetyl-CoA carboxylase is encoded by the ACC1 gene. In this study, we show that attenuated expression of ACC1 results in increased acetylation of bulk histones, globally increased acetylation of chromatin histones, and altered transcriptional regulation. Together, our data indicate that Acc1p activity regulates the availability of acetyl-CoA for histone acetyltransferases, thus representing a link between intermediary metabolism and epigenetic mechanisms of transcriptional regulation.

Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules

The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.

Saccharomyces Cerevisiae MHF Complex Structurally Resembles the Histones (H3-H4)2 Heterotetramer and Functions as a Heterotetramer

Fanconi anemia (FA) is a chromosomal instability disorder associated with deficiencies in the Fanconi anemia complementation group (FANC) network. A complex consisting of FANCM-associated histone-fold proteins 1 and 2 (MHF1 and MHF2) has been shown to act cooperatively with FANCM in DNA damage repair in the FA pathway. Here we report the structure of Saccharomyces cerevisiae MHF complex in which MHF1 and MHF2 assume a typical histone fold, and the complex has a heterotetrameric architecture similar to that of the histones (H3-H4)₂ heterotetramer. Loop L2 of MHF1 is probably involved in DNA binding, and loop L3 and helices α2 and α3 of one MHF1 subunit interact with those of the other to form two heterotetramer interfaces. Further genetic data demonstrate that the heterotetramer assembly is essential for the function of the complex in DNA repair. These results provide, to the best of our knowledge, new mechanistic insights into the function of the MHF complex.

Micrococcal Nuclease Does Not Substantially Bias Nucleosome Mapping

We have mapped sequence-directed nucleosome positioning on genomic DNA molecules using high-throughput sequencing. Chromatins, prepared by reconstitution with either chicken or frog histones, were separately digested to mononucleosomes using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both enzymes preferentially cleave internucleosomal (linker) DNA, although they do so by markedly different mechanisms. MNase has hitherto been very widely used to map nucleosomes, although concerns have been raised over its potential to introduce bias. Having identified the locations and quantified the strength of both the chicken or frog histone octamer binding sites on each DNA, the results obtained with the two enzymes were compared using a variety of criteria. Both enzymes displayed sequence specificity in their preferred cleavage sites, although the nature of this selectivity was distinct for the two enzymes. In addition, nucleosomes produced by CAD nuclease are 8–10 bp longer than those produced with MNase, with the CAD cleavage sites tending to be 4–5 bp further out from the nucleosomal dyad than the corresponding MNase cleavage sites. Despite these notable differences in cleavage behaviour, the two nucleases identified essentially equivalent patterns of nucleosome positioning sites on each of the DNAs tested, an observation that was independent of the histone type. These results indicate that biases in nucleosome positioning data collected using MNase are, under our conditions, not significant.

HIstome--a relational knowledgebase of human histone proteins and histone modifying enzymes

Histones are abundant nuclear proteins that are essential for the packaging of eukaryotic DNA into chromosomes. Different histone variants, in combination with their modification ‘code’, control regulation of gene expression in diverse cellular processes. Several enzymes that catalyze the addition and removal of multiple histone modifications have been discovered in the past decade, enabling investigations of their role(s) in normal cellular processes and diverse pathological conditions. This sudden influx of data, however, has resulted in need of an updated knowledgebase that compiles, organizes and presents curated scientific information to the user in an easily accessible format. Here, we present HIstome, a browsable, manually curated, relational database that provides information about human histone proteins, their sites of modifications, variants and modifying enzymes. HIstome is a knowledgebase of 55 human histone proteins, 106 distinct sites of their post-translational modifications (PTMs) and 152 histone-modifying enzymes. Entries have been grouped into 5 types of histones, 8 types of post-translational modifications and 14 types of enzymes that catalyze addition and removal of these modifications. The resource will be useful for epigeneticists, pharmacologists and clinicians. HIstome: The Histone Infobase is available online at http://www.iiserpune.ac.in/∼coee/histome/ and http://www.actrec.gov.in/histome/.

A Peek into the Complex Realm of Histone Phosphorylation

Although discovered long ago, posttranslational phosphorylation of histones has been in the spotlight only recently. Information is accumulating almost daily on phosphorylation of histones and their roles in cellular physiology and human diseases. An extensive cross talk exists between phosphorylation and other posttranslational modifications, which together regulate various biological processes, including gene transcription, DNA repair, and cell cycle progression. Recent research on histone phosphorylation has demonstrated that nearly all histone types are phosphorylated at specific residues and that these modifications act as a critical intermediate step in chromosome condensation during cell division, transcriptional regulation, and DNA damage repair. As with all young fields, apparently conflicting and sometimes controversial observations about histone phosphorylations and their true functions in different species are found in the literature. Accumulating evidence suggests that instead of functioning strictly as part of a general code, histone phosphorylation probably functions by establishing cross talk with other histone modifications and serving as a platform for recruitment or release of effector proteins, leading to a downstream cascade of events. Here we extensively review published information on the complexities of histone phosphorylation, the roles of proteins recognizing these modifications and the resuting physiological outcome, and, importantly, future challenges and opportunities in this fast-moving field.

Phylogenetic analysis and classification of the Brassica rapa SET-domain protein family

BackgroundThe SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain is an evolutionarily conserved sequence of approximately 130-150 amino acids, and constitutes the catalytic site of lysine methyltransferases (KMTs). KMTs perform many crucial biological functions via histone methylation of chromatin. Histone methylation marks are interpreted differently depending on the histone type (i.e. H3 or H4), the lysine position (e.g. H3K4, H3K9, H3K27, H3K36 or H4K20) and the number of added methyl groups (i.e. me1, me2 or me3). For example, H3K4me3 and H3K36me3 are associated with transcriptional activation, but H3K9me2 and H3K27me3 are associated with gene silencing. The substrate specificity and activity of KMTs are determined by sequences within the SET domain and other regions of the protein.ResultsHere we identified 49 SET-domain proteins from the recently sequenced Brassica rapa genome. We performed sequence similarity and protein domain organization analysis of these proteins, along with the SET-domain proteins from the dicot Arabidopsis thaliana, the monocots Oryza sativa and Brachypodium distachyon, and the green alga Ostreococcus tauri. We showed that plant SET-domain proteins can be grouped into 6 distinct classes, namely KMT1, KMT2, KMT3, KMT6, KMT7 and S-ET. Apart from the S-ET class, which has an interrupted SET domain and may be involved in methylation of nonhistone proteins, the other classes have characteristics of histone methyltransferases exhibiting different substrate specificities: KMT1 for H3K9, KMT2 for H3K4, KMT3 for H3K36, KMT6 for H3K27 and KMT7 also for H3K4. We also propose a coherent and rational nomenclature for plant SET-domain proteins. Comparisons of sequence similarity and synteny of B. rapa and A. thaliana SET-domain proteins revealed recent gene duplication events for some KMTs.ConclusionThis study provides the first characterization of the SET-domain KMT proteins of B. rapa. Phylogenetic analysis data allowed the development of a coherent and rational nomenclature of this important family of proteins in plants, as in animals. The results obtained in this study will provide a base for nomenclature of KMTs in other plant species and facilitate the functional characterization of these important epigenetic regulatory genes in Brassica crops.

The Nuclear Transport Machinery Recognizes Nucleoplasmin–Histone Complexes

The nuclear transport of the chromatin remodeling protein nucleoplasmin and chromatin building histones is mediated by importins. Nucleoplasmin (NP) contains a classical bipartite nuclear localization signal (NLS) that is recognized by the importin α/β heterodimer, while histones present multiple NLS-like motifs that are recognized by importin β family members for nuclear targeting. To explore the possibility of a cotransport of histones and their chaperone NP to the nucleus, we have analyzed the assembly of complexes of NP/histones with importins by means of fluorescence anisotropy, centrifugation in sucrose gradients, and isothermal titration calorimetry. Data show that importin α ΔIBB (a truncated form of importin α lacking the autoinhibitory N-terminal domain) and histones (linker, H5, and nucleosomal core, H2AH2B) can simultaneously bind to NP. Analysis of the binding energetics reveals an enthalpy-driven formation of high affinity ternary, NP/Δα/H5 and NP/Δα/H2AH2B, complexes. We find that different amount of importin α molecules can be loaded on NP/histone complexes dependent on the histone type, linker or core, and the amount of bound histones. We further demonstrate that NP/H5 complexes can also incorporate importin α/β, thus forming quaternary NP/histones/α/β complexes that might represent a putative coimport pathway for nuclear import of histones and their chaperone protein NP, enhancing the histone import efficiency.

Visualization of neuropeptides in paraffin-embedded tissue sections of the central nervous system in the decapod crustacean, Penaeus monodon, by imaging mass spectrometry

The distributions of neuropeptides in paraffin-embedded tissue sections (PETS) of the eyestalk, brain, and thoracic ganglia of the shrimp Penaeus monodon were visualized by imaging mass spectrometry (IMS). Peptide signals were obtained from PETS without affecting morphological features. Twenty-nine neuropeptides comprising members of FMRFamide, SIFamides, crustacean hyperglycaemic hormone, orcokinin-related peptides, tachykinin-related peptides, and allatostatin A were detected and visualized. Among these findings we first identified tachykinin-related peptide as a novel neuropeptide in this shrimp species. We found that these neuropeptides were distributed at specific areas in the three neural organs. In addition, 28 peptide sequences derived from 4 types of constitutive proteins, including actin, histones, arginine kinase, and cyclophilin A were also detected. All peptide sequences were verified by liquid chromatography–tandem mass spectrometry. The use of IMS on acetic acid-treated PETS enabled us to identify peptides and obtain their specific localizations in correlation with the undisturbed histological structure of the tissue samples.

Synergistic effects between natural histone mixtures and polyethylenimine in non-viral gene delivery in vitro

Nanoparticles made of plasmid DNA (pDNA) and cationic polymers are promising strategies for non-viral gene delivery. However, many cationic polymers are toxic to cells when used in higher concentrations. Positively charged proteins, such as histones, are biodegradable and a good alternative, especially for potential in vivo applications. It has previously been shown that histones are able to complex DNA and mediate transfection of cells. To investigate possible synergistic effects between the different histone types and to avoid the use of recombinant proteins, we analysed whether natural histone mixtures would be functional as gene carriers. Core and linker histones from calf thymus and from chicken erythrocytes were used to transfect different cell lines. The protein mixtures efficiently complexed the pDNA, and the resulting particles entered the cells. However, only marginal expression of the gene encoded by the pDNA was observed. Transfection rates increased drastically when minimal amounts of the basic polymer polyethylenimine (PEI) were added to the particles. Neither PEI nor histones alone mediated any transfection under the conditions where a combination of both worked efficiently, and the combined particles were well tolerated by the cells. These results demonstrate that histone mixtures from natural sources in combination with minimal amounts of PEI can be used as gene carriers. This might have consequences for the development of novel gene delivery strategies, such as DNA vaccines, with minimal side-effects.

Genetics Talks to Epigenetics? The Interplay Between Sequence Variants and Chromatin Structure

Transcription is regulated by two major mechanisms. On the one hand, changes in DNA sequence are responsible for genetic gene regulation. On the other hand, chromatin structure regulates gene activity at the epigenetic level. Given the fundamental participation of these mechanisms in transcriptional regulation of virtually any gene, they are likely to co-regulate a significant proportion of the genome. The simple concept behind this idea is that a mutation may have a significant impact on local chromatin structure by modifying DNA methylation patterns or histone type recruitment. Yet, the relevance of these interactions is poorly understood. Elucidating how genetic and epigenetic mechanisms co-participate in regulating transcription may assist in some of the unresolved cases of genetic variant-phenotype association. One example is loci that have biologically predictable functions but genotypes that fail to correlate with phenotype, particularly disease outcome. Conversely, a crosstalk between genetics and epigenetics may provide a mechanistic explanation for cases in which a convincing association between phenotype and a genetic variant has been established, but the latter does not lie in a promoter or protein coding sequence. Here, we review recently published data in the field and discuss their implications for genetic variant-phenotype association studies.

Data Processing Algorithms for Analysis of High Resolution MSMS Spectra of Peptides with Complex Patterns of Posttranslational Modifications

The emergence of efficient fragmentation methods such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) provides the opportunity for detailed structural characterization of heavily covalently modified large peptides and small proteins such as intact histones. Even with effective gas phase ion isolation so that a single molecular precursor ion is selected, the MSMS spectrum of a heavily modified peptide may reveal the presence of a mixture of peptides with the same amino acid sequence and the same total number of posttranslational modification (PTM) moieties (same PTM composition) but with different PTM configurations or site-specific occupancy isoforms. Currently available data analysis methods depend on a deisotoping procedure, which becomes less effective when spectra (fragmentation patterns) contain many overlapping isotopic distributions. Peptide database search engines can only identify the most abundant PTM configuration (PTM arrangement on different residues) in such mixtures. To identify all the PTM configurations present in these mixtures and to estimate their relative abundances, we extended our fragment assignment by visual assistance program to search for ions representing all possible configurations, subjected to the total PTM composition constraint. This resulted in the identification of PTM configurations supported by unique fragment ions, and their relative abundances were estimated by use of a non-negative least squares procedure.

The evolutionary history of histone H3 suggests a deep eukaryotic root of chromatin modifying mechanisms

BackgroundThe phenotype of an organism is an outcome of both its genotype, encoding the primary sequence of proteins, and the developmental orchestration of gene expression. The substrate of gene expression in eukaryotes is the chromatin, whose fundamental units are nucleosomes composed of DNA wrapped around each two of the core histone types H2A, H2B, H3 and H4. Key regulatory steps involved in the determination of chromatin conformations are posttranslational modifications (PTM) at histone tails as well as the assembly of histone variants into nucleosomal arrays. Although the mechanistic background is fragmentary understood, it appears that the chromatin signature of metazoan cell types is inheritable over generations. Even less understood is the conservation of epigenetic mechanisms among eukaryotes and their origins.ResultsIn the light of recent progress in understanding the tree of eukaryotic life we discovered the origin of histone H3 by phylogenetic analyses of variants from all supergroups, which allowed the reconstruction of ancestral states. We found that H3 variants evolved frequently but independently within related species of almost all eukaryotic supergroups. Interestingly, we found all core histone types encoded in the genome of a basal dinoflagellate and H3 variants in two other species, although is was reported that dinoflagellate chromatin is not organized into nucleosomes.Most probably one or more animal/nuclearid H3.3-like variants gave rise to H3 variants of all opisthokonts (animals, choanozoa, fungi, nuclearids, Amoebozoa). H3.2 and H3.1 as well as H3.1t are derivatives of H3.3, whereas H3.2 evolved already in early branching animals, such as Trichoplax. H3.1 and H3.1t are probably restricted to mammals.We deduced a model for protoH3 of the last eukaryotic common ancestor (LECA) confirming a remarkable degree of sequence conservation in comparison to canonical human H3.1. We found evidence that multiple PTMs are conserved even in putatively early branching eukaryotic taxa (Euglenozoa/Excavata).ConclusionsAt least a basal repertoire of chromatin modifying mechanisms appears to share old common ancestry and may thus be inherent to all eukaryotes. We speculate that epigenetic principles responsive to environmental triggers may have had influenced phenotypic variation and concomitantly may potentially have had impact on eukaryotic diversification.

Butyrate and propionate induced activated or non-activated neutrophil apoptosis via HDAC inhibitor activity but without activating GPR-41/GPR-43 pathways

Objective Decreased neutrophil apoptosis is implicated in persistent inflammation resulting in systemic inflammatory response syndrome and multiple organ dysfunctions syndromes. Short-chain fatty acids (SCFAs) may be a candidate to control neutrophil apoptosis because SCFAs are normally produced in the gut and related products have been approved for human use. We investigated the effects of SCFAs on apoptosis of activated and non-activated neutrophils and their mechanisms. Methods Purified neutrophils obtained from healthy volunteers were preincubated for 1 h with or without the G-protein receptor (GPR) inhibitor pertussis toxin (100 ng/mL) or U-73122 (50 ng/mL), extracellular signal-related protein kinase inhibitor PD98059 (10 μM), mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 (25 μM), Jun kinase inhibitor-I (2 μM), caspase-3 and -7 inhibitor Z-VAD-FMK (100 μM), caspase-8 inhibitor Z-IETD-FMK (50 μM), or caspase-9 inhibitor Z-LEHD-FMK (50 μM). The cells were then cultured with or without SCFAs or trichostatin A, a typical histone deacetylase inhibitor, in the presence or absence of lipopolysaccharide (1 μg/mL) or tumor necrosis factor-α (100 ng/mL). Neutrophil apoptosis was assessed by annexin V staining using flow cytometry. The GPR-41 and -43 and apoptosis-related proteins (bax, mcl-1, a1) mRNA were measured by quantitative real-time polymerase chain reaction and the expression of acetylated histone H3 was determined by western blot. Results The caspase inhibitors inhibited butyrate- and propionate-induced neutrophil apoptosis treated or untreated with lipopolysaccharide or tumor necrosis factor-α, whereas GPR and MAPK inhibitors had no effect. The mRNA expressions of GPR-43 and a1 protein were reduced by butyrate and propionate. The expressions of acetylated histone H3 were induced by butyrate and propionate. Conclusion These results suggest that butyrate and propionate increase apoptosis of neutrophils irrespective of their activation state, by factors other than GPRs and MAPKs, and their mechanisms likely relate to their histone deacetylase inhibition activity, which may control a1 mRNA expression.

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A–H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A–H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A–H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A–H2B dimers onto DNA.

Cytokeratin 8/18 overexpression and complex formation as an indicator of GST-P positive foci transformation into hepatocellular carcinomas

Screening of the proteome of microdissected glutathione S-transferase placental form (GST-P) positive foci and normal-appearing liver on anionic (Q10), and cationic (CM10) surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip arrays demonstrated significant overexpression of cytokeratin 8 (CK8; m/z 54,020), cytokeratin 18 (CK18; m/z 47,760), microsomal cytochrome 5A (m/z 15,224) and histone type 2 H2aa3 (m/z 15,964) in the livers of rats initiated with diethylnitrosamine (DEN) followed by 10 weeks on phenobarbital (PB) at a dose of 500 ppm. Furthermore, formation of CK8 and CK18 complexes due to CK8 phosphorylation at Ser73 and Ser431 was found to be strongly associated with promotion of hepatocarcinogenesis by PB and the development of hepatocellular carcinomas. The data were confirmed by immunohistochemistry and real-time Q-PCR and profound overexpression of CK8 and CK18 (CK8/18) proteins and mRNAs were detected in several large size GST-P positive foci and liver tumors. A strong correlation between CK8/18 positive foci development and multiplicity of hepatocellular carcinomas was further observed. Moreover, elevation of CK8/18 was strongly associated with induction of cell proliferation in GST-P positive foci and tumors. In conclusion, our data imply that CK8/18 overexpression, those two cytokeratins complex formation associated with histone type 2 H2aa3 up-regulation and intermediate filament reorganization may drive neoplastic transformation of GST-P positive foci during rat hepatocarcinogenesis leading to the formation of hepatocellular carcinomas.

Effect of Histone Acetylation on Nucleosome Dynamics Revealed by spFRET Microscopy

Nucleosomes form the basic unit of DNA compaction in eukaryotes. Besides condensing the DNA, nucleosomes play a crucial role in gene regulation by modulating access to the nucleosomal DNA for DNA-processing proteins. Accessibility of DNA within the nucleosome can be realized by spontaneous unwrapping (DNA breathing), and by ATP-dependent remodeling enzymes. Both mechanisms are regulated by specific post-translational modifications to the nucleosome histones. We applied single-pair FRET measurements to characterize the effect of specific histone modifications on DNA breathing dynamics in individual nucleosomes. DNA labeled with a FRET pair was used to reconstitute nucleosomes with three types of histones: native chicken erythrocyte histones, recombinant unmodified histones, and recombinant histones acetylated at H3K56. Using alternating laser excitation to select for the correct label stoichiometry, in combination with both widefield TIRF microscopy on immobilized nucleosomes and FCS on freely diffusing nucleosomes, we were able to quantify DNA breathing dynamics at timescales ranging from milliseconds to minutes. Uncovering the effect of histone modifications on the dynamic behavior of single nucleosomes provides insight in the physical mechanisms underlying gene regulation.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Neprilysin gene expression requires binding of the amyloid precursor protein intracellular domain to its promoter: implications for Alzheimer disease

Amyloid beta-peptide (Abeta) accumulation leads to neurodegeneration and Alzheimer disease; however, amyloid metabolism is a dynamic process and enzymic mechanisms exist for Abeta removal. Considerable controversy surrounds whether the intracellular domain of the amyloid precursor protein (AICD) regulates expression of the Abeta-degrading metalloprotease, neprilysin (NEP). By comparing two neuroblastoma cell lines differing substantially in NEP expression, we show by chromatin immunoprecipitation (ChIP) that AICD is bound directly to the NEP promoter in high NEP-expresser (NB7) cells but not in low-expresser (SH-SY5Y) cells. The methylation status of the NEP promoter does not regulate expression in these cells, whereas the histone deacetylase inhibitors trichostatin A and valproate partly restore NEP expression and activity in SH-SY5Y cells. ChIP analysis also reveals AICD binding to the NEP promoter in rat primary neurons but not in HUVEC cells. Chromatin remodelling of crucial Alzheimer disease-related genes by valproate could provide a new therapeutic strategy.