Histone Sequences Research Articles

Identification of 987P Protein Receptors for Enterotoxigenic Escherichia coli

The 987P fimbriae of enterotoxigenic Escherichia coli (ETEC) mediate adhesive interactions with brush border vesicle (BBV) of the intestinal epithelial cells from the neonatal piglets. By adhering to intestinal epithelial cells, producing localized multiplication, the 987P ETEC can progress to mucosal surface colonization and concomitant effective enterotoxin delivery. To identify the receptors for the 987P, BBV proteins from piglet intestinal villous epithelial cells were separated by SDS-PAGE and analyzed by ligand blot, protein bands with a set of 32–35 kD recognized by the 987P fimbriae were subjected to in gel proteolysis with trypsin. The tryptic fragments were separated by microbore reversed phase HPLC (RP-HPLC), samples shown to contain one major peak by MALDI-MS were submitted to Edman sequencing, three peptides were sequenced successfully and the all of three peptides matched the sequence of human or porcine histone H 1 proteins. Porcine histone H 1 proteins isolated from both piglet intestinal epithelial cells and BBV demonstrated the same SDS-PAGE migration pattern and 987P-binding properties as the 987P-specific protein receptors from piglet intestinal brush border did. The results indicated that the 987P protein receptors are piglet BBV-derived Histone H 1 proteins.

Asymmetric histone modifications between the original and derived loci of human segmental duplications

A systematic analysis of histone modifications between human segmental duplications shows that two seemingly identical genomic copies have distinct epigenomic properties.

Distinct binding modes specify the recognition of methylated histones H3K4 and H4K20 by JMJD2A-tudor

The lysine demethylase JMJD2A has the unique property of binding trimethylated peptides from two different histone sequences (H3K4me3 and H4K20me3) through its tudor domains. Here we show using X-ray crystallography and calorimetry that H3K4me3 and H4K20me3, which are recognized with similar affinities by JMJD2A, adopt radically different binding modes, to the extent that we were able to design single point mutations in JMJD2A that inhibited the recognition of H3K4me3 but not H4K20me3 and vice versa.

Structural Basis for Lower Lysine Methylation State-Specific Readout by MBT Repeats of L3MBTL1 and an Engineered PHD Finger

Human L3MBTL1, which contains three malignant brain tumor (MBT) repeats, binds monomethylated and dimethylated lysines, but not trimethylated lysines, in several histone sequence contexts. In crystal structures of L3MBTL1 complexes, the monomethyl- and dimethyllysines insert into a narrow and deep cavity of aromatic residue-lined pocket 2, while a proline ring inserts into shallower pocket 1. We have also engineered a single Y to E substitution within the aromatic cage of the BPTF PHD finger, resulting in a reversal of binding preference from trimethyl- to dimethyllysine in an H3K4 sequence context. In both the "cavity insertion" (L3MBTL1) and "surface groove" (PHD finger) modes of methyllysine recognition, a carboxylate group both hydrogen bonds and ion pairs to the methylammonium proton. Our structural and binding studies of these two modules provide insights into the molecular principles governing the decoding of lysine methylation states, thereby highlighting a methylation state-specific layer of histone mark readout impacting on epigenetic regulation.

Characterization of antimicrobial histone sequences and posttranslational modifications by mass spectrometry

Histones typically play a role in DNA packaging and transcription regulation. These proteins are heavily modified by acetylation, methylation, phosphorylation and/or ubiquitination, and various combinations of these modifications alter histone functions and form the basis of the histone code. Furthermore, histones, including those found in shrimp, have recently been found to possess antimicrobial properties; however, the sequences and posttranslational modifications of shrimp histones are largely unknown. In this study mass spectrometry was used to characterize the primary structure of the shrimp antimicrobial histone. A combination of in-solution digestion and in-gel propionylation/digestion followed by LC-MS-MS and MALDI-TOF-TOF analysis was used. Over 80% of each histone sequence was obtained by in-solution digestion; however, none of the N-terminal domains was sequenced with this method. An in-gel propionylation method was optimized to recover and sequence the extremely hydrophilic histone N-termini. This method was then applied to shrimp hemocyte lysates separated on a 1-D SDS-PAGE gel. Overall, 95% coverage was obtained for the histone sequences as well as the identification of posttranslational sites such as acetylation, methylation and phosphorylation.

Control of Splicing Efficiency by the Mouse Histone H2a Element in a Murine Leukemia Virus–based Retroviral Vector

While using various human complementary DNA (cDNA) sequences in the context of the murine leukemia virus (MLV)-based retroviral vector, it was found that a retroviral vector containing some human cDNA sequences produces unusually low viral titer. One of those sequences is that for the human IL-1 receptor antagonist protein (IL1RN). The RNA analysis showed that a cryptic splice acceptor sequence is present in the middle of its coding region, resulting in the deletion of the packaging signal sequence and the removal of some coding sequences that lead to low viral titer and a low level of the transgene product. We tested whether the mouse Hist2h2aa1 element (mH2aE), previously shown to suppress the splicing function, could inhibit the cryptic splicing in the context of MLV-based retroviral vectors. It was found that the mH2aE could efficiently suppress such unwanted splicing event, thus increasing the amount of unspliced transcript, which eventually led to the increase in the level of IL1RN expression and viral titer. The mH2aE could also be used to control unusually high splicing activity. Our data suggested that the mH2aE could be used for the fine-tuning of the splicing process, thus improving the level of gene expression and viral titer in the context of retroviral vectors.

PTMs on H3 Variants before Chromatin Assembly Potentiate Their Final Epigenetic State

Histone posttranslational modifications (PTMs) and sequence variants regulate genome function. Although accumulating evidence links particular PTM patterns with specific genomic loci, our knowledge concerning where and when these PTMs are imposed remains limited. Here, we find that lysine methylation is absent prior to histone incorporation into chromatin, except at H3K9. Nonnucleosomal H3.1 and H3.3 show distinct enrichments in H3K9me, such that H3.1 contains more K9me1 than H3.3. In addition, H3.3 presents other modifications, including K9/K14 diacetylated and K9me2. Importantly, H3K9me3 was undetectable in both nonnucleosomal variants. Notably, initial modifications on H3 variants can potentiate the action of enzymes as exemplified with Suv39HMTase to produce H3K9me3 as found in pericentric heterochromatin. Although the set of initial modifications present on H3.1 is permissive for further modifications, in H3.3 a subset cannot be K9me3. Thus, initial modifications impact final PTMs within chromatin.

Remembering the cell fate during cellular differentiation

Higher eukaryote contains several hundreds of different cell types, each with a distinctive set of property defined by a unique gene expression pattern, even though every cell (with minor exception) shares the common genome. During cellular differentiation, the committed gene expression pattern is set up and propagated through numerous cell divisions. Therefore, cells must have evolved some elegant and inherent mechanisms to remember their expression states for the requirement of the stability of differentiation and development. Here we speculate a hypothetically cellular memory mechanism. In this hypothesis, the cell-cell variation during cellular differentiation may result from the inherent stochastic gene expression. The evolution of histone and distant regulatory sequences change the parameters of expression stochasticity. S-phase-dependent gene activation and epigenetic marks on chromatin provide means to discriminate transcriptionally active and repressive states. Eventually, mitotic memory mechanisms have been developed through which these expression states are transmitted through numerous cell divisions.

Trypanosomatid histones.

The histones are responsible for packaging and regulating access to eukaryotic genomes. Trypanosomatids are flagellated protists that diverged early from the eukaryotic lineage and include parasites that cause disease in humans and other mammals. Here, we review the properties of histones in parasitic trypanosomatids, from gene organization and sequence to expression, post-translational modification and function within chromatin. Phylogenetic and experimental analysis indicates that certain specifically conserved histone sequence motifs, particularly within the N-terminal 'tail' domains, possibly represent functionally important modification substrates conserved throughout the eukaryotic lineage. For example, histone H3 contains a highly conserved methylation substrate. Trypanosomatids also possess at least three variant histones. Among these is an orthologue of H2A.Z, a histone involved in protecting 'active' chromatin from silencing in yeast. Histones provide docking platforms for a variety of regulatory factors. The presence of histone modification and variant histones in trypanosomatids therefore represents evidence for a network that provides the discrimination required to regulate transcription, recombination, repair and chromosome replication and segregation.

Sin mutations alter inherent nucleosome mobility.

Previous studies have identified sin mutations that alleviate the requirement for the yeast SWI/SNF chromatin remodelling complex, which include point changes in the yeast genes encoding core histones. Here we characterise the biochemical properties of nucleosomes bearing these mutations. We find that sin mutant nucleosomes have a high inherent thermal mobility. As the SWI/SNF complex can alter nucleosome positioning, the higher mobility of sin mutant nucleosomes provides a means by which sin mutations may substitute for SWI/SNF function. The location of sin mutations also provides a new opportunity for insights into the mechanism for nucleosome mobilisation. We find that both mutations altering histone DNA contacts at the nucleosome dyad and mutations in the dimer-tetramer interface influence nucleosome mobility. Furthermore, incorporation of H2A.Z into nucleosomes, which also alters dimer-tetramer interactions, affects nucleosome mobility. Thus, variation of histone sequence or subtype provides a means by which eukaryotes may regulate access to chromatin through alterations to nucleosome mobility.

Mining Core Histone Sequences from Public Protein Databases

Publisher Summary Constructing an online database of histones and histone fold-containing proteins has allowed analyzing histone sequence variation in some detail. This chapter describes how one has inventoried core histone protein sequences. The issues involved in such an undertaking are for the most part not unique to histone sequences. The initial goal was to collect as many reported histone sequences as one could find. There are many variations on the basic Basic Local Alignment Search Tool (BLAST) search protocol. For text-based searches of sequence annotation the Entrez search engine at the NCBI Web site is used. For sequence-based searching, several varieties of the popular BLAST are used for pair-wise alignment algorithm. To abnegate sequence redundancy all the unique sequence identifiers are extracted from the BLAST outputs into a file, and use this file to generate a corresponding library of FASTA records. Several parameters are investigated in this chapter for their effects in the identification of histone H3 sequences.

Histone gene complement, variant expression, and mRNA processing in a urochordate Oikopleura dioica that undergoes extensive polyploidization.

Considerable data exist on coding sequences of histones in a wide variety of organisms. Much more restricted information is available on total histone gene complement, gene organization, transcriptional regulation, and histone mRNA processing. In particular, there is a significant phylogenetic gap in information for the urochordates, a subphylum near the invertebrate-vertebrate transition. In this study, we show that the appendicularian Oikopleura dioica has a histone gene complement that is similar to that of humans, though its genome size is 40- to 50-fold smaller. At a total length of 3.5 kb, the H3, H4, H1, H2A, and H2B quintet cluster is the most compact described thus far, but despite very rapid early developmental cleavage cycles, no extensive tandem repeats of the cluster were present. The high degree of variation within each of the complements of O. dioica H2A and H2B subtypes resembled that found in plants as opposed to more closely related vertebrate and invertebrate species, and developmental stage-specific expression of different subtypes was observed. The linker histone H1 was present in relatively few copies per haploid genome and contained short N- and C-terminal tails, a feature similar to that of copepods but different from many standard model organisms. The 3'UTRs of the histone genes contained both the consensus stem-loop sequence and the polyadenylation signals but lacked the consensus histone downstream element that is involved in the processing of histone mRNAs in echinoderms and vertebrates. Two types of transcripts were found, i.e., those containing both the stem-loop and a polyA tail as well as those cleaved at the normal site just 3' of the stem-loop. The O. dioica data are an important addition to the limited number of eukaryotes for which sufficiently extensive information on histone gene complements is available. Increasingly, it appears that understanding the evolution of histone gene organization, transcriptional regulation, and mRNA processing will depend at least as much on comparative analysis of constraints imposed by certain life history features and cell biological characteristics as on projections based on simple phylogenetic relationships.

Diseño y evaluación de tres oligonucleótidos para la detección de Leishmania por PCR

Leishmaniasis affects public health in 88 countries of the world and represents an important obstacle for their socioeconomic development. The methods used to diagnose this disease take time and are frequently traumatic to the patient. Materials and methods Genomic DNA from different Leishmania species were extracted. The carboxi-end terminal of a partial sequence of the H2B histone of Leishmania (V.) peruviana was used to design three oligonucleotides (LEISH 1, LEISH 2, LEISH3) to design a PCR technique as an alternative method to diagnose this disease rapidly. Results: A PCR was performed with these primers, which amplified succesfully 123bp (LEISH 1/ LEISH 2) and 139bp (LEISH1/ LEISH3) of the partial sequence studied. The sensitivity of direct PCR was appropiate even when 1fg of purified DNA of L. (V.) peruviana and 2 parasites was used. The specificity was 100% in all Leishmania species analyzed and did not recognized neither Trypanosoma nor human DNA. Conclusions: The oligonucleotides designed could be used in the detection of parasite from different kind of samples like fresh blood with anticoagulant, biopsy and dermal scrapping. PCR has become an alternative method to diagnose leishmaniasis because of its speed, specificity and sensitivity.

Characterization of Seiridium spp. Associated with Cypress Canker Based on ß-Tubulin and Histone Sequences.

Cypress canker is a serious disease that has devastated Cupressus spp. in many parts of the world. In Mediterranean Europe it has caused the deaths of millions of trees. Three species of Seiridium, S. cardinale, S. cupressi, and S. unicorne, are associated with cypress canker. Considerable debate surrounds the taxonomic status of these fungi. They have been viewed as a single morphologically variable species, three distinct taxa; or two species based on the presence or absence of conidial appendages. Studies based on ribosomal DNA (ITS1, ITS2, and 5.8S gene) sequence failed to separate the cypress canker fungi. In an attempt to distinguish between the species associated with cypress canker we used histone and partial ß-tubulin sequences of fourteen isolates of Seiridium spp. from cypress. Analysis of sequence data showed Seiridium isolates from Cupressus spp., residing in two major clades. One clade accommodated S. unicorne isolates from Portugal and South Africa. The other major clade consisted of two subclades containing non-appendaged S. cardinale isolates. We believe the larger second clade, represents the cypress canker pathogens while the other clade contains the less pathogenic S. unicorne, which has a host range beyond Cupressus. This study thus provides strong evidence to support previous morphological data suggesting three distinct species are associated with cypress canker.

Crystal structures of recombinant histones HMfA and HMfB from the hyperthermophilic archaeon Methanothermus fervidus

The hyperthermophilic archaeon Methanothermus fervidus contains two small basic proteins, HMfA (68 amino acid residues) and HMfB (69 residues) that share a common ancestry with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. HMfA and HMfB have sequences that differ at 11 locations, they have different structural stabilities, and the complexes that they form with DNA have different electrophoretic mobilities. Here, crystal structures are documented for recombinant (r) HMfA at a resolution of 1.55 Å refined to a crystallographic R-value of 19.8 % (tetragonal form) and at 1.48 Å refined to a R-value of 18.8 % (orthorhombic form), and for rHMfB at 1.9 Å refined to a R-value of 18.0 %. The rHMfA and rHMfB monomers have structures that are just histone folds in which a long central α-helix (α2; 29 residues) is separated from shorter N-terminal (α1; 11 residues) and C-terminal (α3; 10 residues) α-helices by two loops (L1 and L2; both 6 residues). Within L1 and L2, three adjacent residues are in extended (β) conformation. rHMfA and rHMfB assemble into homodimers, with the α2 helices anti-parallel aligned and crossing at an angle of close to 35°, and with hydrogen bonds formed between the extended, parallel regions of L1 and L2 resulting in short β-ladders. Dimerization creates a novel N-terminal structure that contains four proline residues, two from each monomer. As prolines are present at these positions in all archaeal histone sequences, this proline-tetrad structure is likely to be a common feature of all archaeal histone dimers. Almost all residues that participate in monomer-monomer interactions are conserved in HMfA and HMfB, consistent with the ability of these monomers to form both homodimers and (HMfA+HMfB) heterodimers. Differences in side-chain interactions that result from non-conservative residue differences in HMfA and HMfB are identified, and the structure of a (rHMfA) 2-DNA complex is presented based on the structures documented here and modeled by homology to histone-DNA interactions in the eukaryal nucleosome.

Molecular characterization of Fusarium subglutinans associated with mango malformation

Abstract Mango malformation is a serious disease of Mangifera indica in tropical and subtropical regions of the world. This disease is caused by Fusarium subglutinans, which is also associated with diseases on many other hosts, such as pineapple, pine, maize and sugarcane. The F. subglutinans strains associated with different hosts are virtually indistinguishable using morphological characters, but can be easily differentiated using histone and beta-tubulin gene sequencing, and some have subsequently been assigned to distinct species. The aim of this study was to characterize F. subglutinans isolates associated with mango malformation using histone H3 gene sequencing and to compare them with other isolates in the Gibberella fujikuroi complex. Analysis of histone sequence data revealed the presence of two phylogenetically distinct groups of F. subglutinans isolates associated with mango malformation. We also considered the identity of the two groups of isolates associated with mango malformation and determined their relatedness to other Fusarium spp. For this purpose, portions of the beta-tubulin gene were sequenced and compared with the beta-tubulin sequences deposited in the National Center for Biotechnology Information (NCBI) nucleotide database. This comparison to the NCBI database indicated that one group of F. subglutinans strains isolated from mango constitutes a unique lineage in the G. fujikuroi complex. Based on beta-tubulin and histone H3 sequencing, the second group of isolates is conspecific with the F. subglutinans strains previously reported to be the causal agent of mango malformation.

DNA Supercoiling and Temperature Adaptation: A Clue to Early Diversification of Life?

Cellular systems to control an appropriate DNA geometry for function probably evolved simultaneously with DNA genomes. Such systems are basically DNA topoisomerases and DNA-binding proteins. Therefore, their distribution in extant organisms may be a source of information on early evolution and the nature of the last common ancestor (cenancestor). Most living beings need the strand-opening potential of negative DNA supercoiling to allow transcription and other DNA-dependent processes. Mesophiles have global negatively supercoiled DNA, essentially due to gyrase (introducing negative supercoils) in bacteria and to DNA wrapping around histone cores in eukaryotes. Mesophilic archaea, halophilic methanogens, and halophiles might use a gyrase, whereas some methanogens might use histone wrapping. The existence of these two distinct mechanisms suggests that mesophily appeared at least twice in evolution. On the other hand, only one system which is based on reverse gyrase (introducing positive supercoils) appears to be required for hyperthermophilic life. Archaeal hyperthermophiles lacking gyrase have relaxed to positively supercoiled DNA, but hyperthermophilic bacteria of the genus Thermotoga, which have both gyrase and reverse gyrase, have negative supercoiling. This suggests that reverse gyrase is necessary at least locally, but whereas these hyperthermophilic bacteria favor general melting potential and stability at critical active regions, hyperthermophilic archaea favor general linking excess and local melting. In this context, the existence of a thermophilic (60-80 degrees C) ancestor endowed with only relaxing topoisomerases is hypothesized. Such temperatures allow a compromise between melting potential and stability, i.e., an appropriate DNA geometry for function. Subsequent duplication and functional specialization of existing DNA topoisomerases would then have facilitated adaptation to hyperthermophily and mesophily in archaea and bacteria, respectively. If reverse gyrase is an ancient character in hyperthermophilic bacteria, the cenancestor would have already been a hyperthermophile. Histone sequence homology and similarities of nucleosome structural dynamics suggest that eukaryotes inherited this system for DNA structural homeostasis from methanogenic euryarchaea. Some mesophilic archaea would have improved their adaptability to mesophily by importing gyrase from bacteria.

Development of a Scintillation Proximity Assay for Histone Deacetylase Using a Biotinylated Peptide Derived from Histone-H4

Measurement of histone deacetylase activity is usually accomplished by incubation of the enzyme(s) with acetate-radiolabeled histones or synthetic peptides based on histone sequences, followed by extraction and quantification of released radiolabeled acetic acid. Consequently, this assay is both time consuming and extremely limiting when large numbers of samples are involved. We have now developed a simple, two-step histone deacetylase assay that is based on the scintillation proximity assay (SPA) principle. A biotinylated [3H]acetyl histone H4 peptide substrate was synthesized and shown to generate a radioactive signal upon binding to streptavidin-coated SPA beads. Incubation of biotinylated [3H]acetyl peptide with HeLa nuclear extract (source of histone deacetylase) resulted in a time- and protein-dependent decrease in the SPA signal, providing a measure of enzyme activity. The histone deacetylase-mediated decrease in SPA counts was accompanied by a proportional appearance in free3H-labeled acetate in the assay mixture. Histone deacetylase activity measured by SPA was concordant with that determined via the traditional ethyl acetate extraction procedure. Furthermore, a broad range of histone deacetylase inhibitors was demonstrated to have comparable effects on the catalytic activity of the HeLa nuclei enzyme using both assays. The histone deacetylase SPA system described here should be readily applicable for automated high-throughput screening and therefore facilitate the discovery of new inhibitors of histone deacetylases.

Histone Sequence Database: sequences, structures, post-translational modifications and genetic loci.

The Histone Sequence Database is an annotated and searchable collection of all available histone and histone fold sequences and structures. Particular emphasis has been placed on documenting conflicts between similar sequence entries from a number of source databases, conflicts that are not necessarily documented in the source databases themselves. New additions to the database include compilations of post-translational modifications for each of the core and linker histones, as well as genomic information in the form of map loci for the human histone gene complement, with the genetic loci linked to Online Mendelian Inheritance in Man (OMIM). The database is freely accessible through the World Wide Web at either http://genome.nhgri.nih.gov/histones/ or http://www.ncbi.nlm.nih. gov/Baxevani/HISTONES

Histone Sequence Database: new histone fold family members.

Searches of the major public protein databases with core and linker chicken and human histone sequences have resulted in the compilation of an annotated set of histone protein sequences. In addition, new database searches with two distinct motif search algorithms have identified several members of the histone fold family, including human DRAP1 and yeast CSE4. Database resources include information on conflicts between similar sequence entries in different source databases, multiple sequence alignments, links to the Entrez integrated information retrieval system, structures for histone and histone fold proteins, and the ability to visualize structural data through Cn3D. The database currently contains >1000 protein sequences, which are searchable by protein type, accession number, organism name, or any other free text appearing in the definition line of the entry. All sequences and alignments in this database are available through the World Wide Web at http://www.nhgri.nih. gov/DIR/GTB/HISTONES or http://www.ncbi.nlm.nih. gov/Baxevani/HISTONES