Trypanosoma brucei histones are heavily modified with combinatorial post-translational modifications and mark Pol II transcription start regions with hyperacetylated H2A.

Trypanosomes diverged from the main eukaryotic lineage about 600 million years ago, and display some unusual genomic and epigenetic properties that provide valuable insight into the early processes employed by eukaryotic ancestors to regulate chromatin-mediated functions. We analysed Trypanosoma brucei core histones by high mass accuracy middle-down mass spectrometry to map core histone post-translational modifications (PTMs) and elucidate cis-histone combinatorial PTMs (cPTMs). T. brucei histones are heavily modified and display intricate cPTMs patterns, with numerous hypermodified cPTMs that could contribute to the formation of non-repressive euchromatic states. The Trypanosoma brucei H2A C-terminal tail is hyperacetylated, containing up to five acetylated lysine residues. MNase-ChIP-seq revealed a striking enrichment of hyperacetylated H2A at Pol II transcription start regions, and showed that H2A histones that are hyperacetylated in different combinations localised to different genomic regions, suggesting distinct epigenetic functions. Our genomics and proteomics data provide insight into the complex epigenetic mechanisms used by this parasite to regulate a genome that lacks the transcriptional control mechanisms found in later-branched eukaryotes. The findings further demonstrate the complexity of epigenetic mechanisms that were probably shared with the last eukaryotic common ancestor.

SeqPredNN: a neural network that generates protein sequences that fold into specified tertiary structures.

BACKGROUND: The relationship between the sequence of a protein, its structure, and the resulting connection between its structure and function, is a foundational principle in biological science. Only recently has the computational prediction of protein structure based only on protein sequence been addressed effectively by AlphaFold, a neural network approach that can predict the majority of protein structures with X-ray crystallographic accuracy. A question that is now of acute relevance is the "inverse protein folding problem": predicting the sequence of a protein that folds into a specified structure. This will be of immense value in protein engineering and biotechnology, and will allow the design and expression of recombinant proteins that can, for instance, fold into specified structures as a scaffold for the attachment of recombinant antigens, or enzymes with modified or novel catalytic activities. Here we describe the development of SeqPredNN, a feed-forward neural network trained with X-ray crystallographic structures from the RCSB Protein Data Bank to predict the identity of amino acids in a protein structure using only the relative positions, orientations, and backbone dihedral angles of nearby residues. RESULTS: We predict the sequence of a protein expected to fold into a specified structure and assess the accuracy of the prediction using both AlphaFold and RoseTTAFold to computationally generate the fold of the derived sequence. We show that the sequences predicted by SeqPredNN fold into a structure with a median TM-score of 0.638 when compared to the crystal structure according to AlphaFold predictions, yet these sequences are unique and only 28.4% identical to the sequence of the crystallized protein. CONCLUSIONS: We propose that SeqPredNN will be a valuable tool to generate proteins of defined structure for the design of novel biomaterials, pharmaceuticals, catalysts, and reporter systems. The low sequence identity of its predictions compared to the native sequence could prove useful for developing proteins with modified physical properties, such as water solubility and thermal stability. The speed and ease of use of SeqPredNN offers a significant advantage over physics-based protein design methods.

Bioinformatics education--perspectives and challenges out of Africa.

The discipline of bioinformatics has developed rapidly since the complete sequencing of the first genomes in the 1990s. The development of many high-throughput techniques during the last decades has ensured that bioinformatics has grown into a discipline that overlaps with, and is required for, the modern practice of virtually every field in the life sciences. This has placed a scientific premium on the availability of skilled bioinformaticians, a qualification that is extremely scarce on the African continent. The reasons for this are numerous, although the absence of a skilled bioinformatician at academic institutions to initiate a training process and build sustained capacity seems to be a common African shortcoming. This dearth of bioinformatics expertise has had a knock-on effect on the establishment of many modern high-throughput projects at African institutes, including the comprehensive and systematic analysis of genomes from African populations, which are among the most genetically diverse anywhere on the planet. Recent funding initiatives from the National Institutes of Health and the Wellcome Trust are aimed at ameliorating this shortcoming. In this paper, we discuss the problems that have limited the establishment of the bioinformatics field in Africa, as well as propose specific actions that will help with the education and training of bioinformaticians on the continent. This is an absolute requirement in anticipation of a boom in high-throughput approaches to human health issues unique to data from African populations.

The epigenome of Trypanosoma brucei: a regulatory interface to an unconventional transcriptional machine.

The epigenome represents a major regulatory interface to the eukaryotic genome. Nucleosome positions, histone variants, histone modifications and chromatin associated proteins all play a role in the epigenetic regulation of DNA function. Trypanosomes, an ancient branch of the eukaryotic evolutionary lineage, exhibit some highly unusual transcriptional features, including the arrangement of functionally unrelated genes in large, polymerase II transcribed polycistronic transcription units, often exceeding hundreds of kilobases in size. It is generally believed that transcription initiation plays a minor role in regulating the transcript level of genes in trypanosomes, which are mainly regulated post-transcriptionally. Recent advances have revealed that epigenetic mechanisms play an essential role in the transcriptional regulation of Trypanosoma brucei. This suggested that the modulation of gene activity, particularly that of pol I transcribed genes, is, indeed, an important control mechanism, and that the epigenome is critical in regulating gene expression programs that allow the successful migration of this parasite between hosts, as well as the continuous evasion of the immune system in mammalian hosts. A wide range of epigenetic signals, readers, writers and erasers have been identified in trypanosomes, some of which have been mapped to essential genetic functions. Some epigenetic mechanisms have also been observed to be unique to trypanosomes. We review recent advances in our understanding of epigenetic control mechanisms in T. brucei, the causative agent of African sleeping sickness, and highlight the utility of epigenetic targets in the possible development of new therapies for human African trypanosomiasis.

AnchorMS: a bioinformatics tool to derive structural information from the mass spectra of cross-linked protein complexes.

SUMMARY: Mass spectrometry is being increasingly used in the structural elucidation of mega-Dalton protein complexes in an approach termed MS3D, referring to the application of MS to the study of macromolecular structures. This involves the identification of cross-linked residues in the constituent proteins of chemically cross-linked multi-subunit complexes. AnchorMS was developed to simplify MS3D studies by identifying cross-linked peptides in complex peptide mixtures, and to determine the specific residues involved in each cross-link. When identifying cross-linked peptide pairs (CLPP), AnchorMS implements a mathematical model to exclude false positives by using a dynamic score threshold to estimate the number of false-positive peak matches expected in an MS/MS spectrum. This model was derived from CLPPs with randomly generated sequences. AnchorMS does not require specific sample labeling or pre-treatment, and AnchorMS is especially suited for discriminating between CLPPs that differ only in the cross-linked residue pairs. AVAILABILITY: AnchorMS was coded in Python, and is available as a free web service at cbio.ufs.ac.za/AnchorMS.

Secondary structures of the core histone N-terminal tails: their role in regulating chromatin structure.

The core histone N-terminal tails dissociate from their binding positions in nucleosomes at moderate salt concentrations, and appear unstructured in the crystal. This suggested that the tails contributed minimally to chromatin structure. However, in vitro studies have shown that the tails were involved in a range of intra- and inter-nucleosomal as well as inter-fibre contacts. The H4 tail, which is essential for chromatin compaction, was shown to contact an adjacent nucleosome in the crystal. Acetylation of H4K16 was shown to abolish the ability of a nucleosome array to fold into a 30 nm fibre. The application of secondary structure prediction software has suggested the presence of extended structured regions in the histone tails. Molecular Dynamics studies have further shown that sections of the H3 and H4 tails assumed α-helical and ß-strand content that was enhanced by the presence of DNA, and that post-translational modifications of the tails had a major impact on these structures. Circular dichroism and NMR showed that the H3 and H4 tails exhibited significant α-helical content, that was increased by acetylation of the tail. There is thus strong evidence, both from biophysical and from computational approaches, that the core histones tails, particularly that of H3 and H4, are structured, and that these structures are influenced by post-translational modifications. This chapter reviews studies on the position, binding sites and secondary structures of the core histone tails, and discusses the possible role of the histone tail structures in the regulation of chromatin organization, and its impact on human disease.

Physical cell-cell contact elicits specific transcriptomic responses in wine yeast species.

Fermenting grape juice provides a habitat for a well-mapped and evolutionarily relevant microbial ecosystem consisting of many natural or inoculated strains of yeasts and bacteria. The molecular nature of many of the ecological interactions within this ecosystem remains poorly understood, with the partial exception of interactions of a metabolic nature such as competition for nutrients and production of toxic metabolites/peptides. Data suggest that physical contact between species plays a significant role in the phenotypic outcome of interspecies interactions. However, the molecular nature of the mechanisms regulating these phenotypes remains unknown. Here, we present a transcriptomic analysis of physical versus metabolic contact between two wine relevant yeast species, Saccharomyces cerevisiae and Lachancea thermotolerans. The data show that these species respond to the physical presence of the other species. In S. cerevisiae, physical contact results in the upregulation of genes involved in maintaining cell wall integrity, cell wall structural components, and genes involved in the production of H2S. In L. thermotolerans, HSP stress response genes were the most significantly upregulated gene family. Both yeasts downregulated genes belonging to the FLO family, some of which play prominent roles in cellular adhesion. qPCR analysis indicates that the expression of some of these genes is regulated in a species-specific manner, suggesting that yeasts adjust gene expression to specific biotic challenges or interspecies interactions. These findings provide fundamental insights into yeast interactions and evolutionary adaptations of these species to the wine ecosystem.IMPORTANCEWithin the wine ecosystem, yeasts are the most relevant contributors to alcoholic fermentation and wine organoleptic characteristics. While some studies have described yeast-yeast interactions during alcoholic fermentation, such interactions remain ill-defined, and little is understood regarding the molecular mechanisms behind many of the phenotypes observed when two or more species are co-cultured. In particular, no study has investigated transcriptional regulation in response to physical interspecies cell-cell contact, as opposed to the generally better understood/characterized metabolic interactions. These data are of direct relevance to our understanding of microbial ecological interactions in general while also creating opportunities to improve ecosystem-based biotechnological applications such as wine fermentation. Furthermore, the presence of competitor species has rarely been considered an evolutionary biotic selection pressure. In this context, the data reveal novel gene functions. This, and further such analysis, is likely to significantly enlarge the genome annotation space.

Nano-electrospray tandem mass spectrometric analysis of the acetylation state of histones H3 and H4 in stationary phase in Saccharomyces cerevisiae.

BACKGROUND: The involvement of histone acetylation in facilitating gene expression is well-established, particularly in the case of histones H3 and H4. It was previously shown in Saccharomyces cerevisiae that gene expression was significantly down-regulated and chromatin more condensed in stationary phase compared to exponential phase. We were therefore interested in establishing the acetylation state of histone H3 and H4 in stationary and in exponential phase, since the regulation of this modification could contribute to transcriptional shut-down and chromatin compaction during semi-quiescence. RESULTS: We made use of nano-spray tandem mass spectrometry to perform a precursor ion scan to detect an m/z 126 immonium ion, diagnostic of an Nε-acetylated lysine residue that allowed unambiguous identification of acetylated as opposed to tri-methylated lysine. The fragmentation spectra of peptides thus identified were searched with Mascot against the Swiss-Prot database, and the y-ion and b-ion fragmentation series subsequently analyzed for mass shifts compatible with acetylated lysine residues. We found that K9, K14 and K36 of histone H3 and K12 and K16 of histone H4 were acetylated in exponential phase (bulk histones), but could not detect these modifications in histones isolated from stationary phase cells at the sensitivity level of the mass spectrometer. The corresponding un-acetylated peptides were, however, observed. A significantly higher level of acetylation of these residues in exponential phase was confirmed by immuno-blotting. CONCLUSION: H4K16 acetylation was previously shown to disrupt formation of condensed chromatin in vitro. We propose that de-acetylation of H4K16 allowed formation of condensed chromatin in stationary phase, and that acetylation of H3K9, H3K14, H3K36, and H4K12 reflected the active transcriptional state of the yeast genome in exponential phase.

The Saccharomyces cerevisiae linker histone Hho1p is essential for chromatin compaction in stationary phase and is displaced by transcription.

The importance of core histones in the regulation of DNA function by chromatin is clear. However, little is known about the role of the linker histone. We investigated the role of H1 in Saccharomyces cerevisiae during extensive transcriptional reprogramming in stationary phase. Although the levels of linker histone Hho1p remained constant during growth to semiquiescence, there was a genome-wide increase in binding to chromatin. Hho1p was essential for compaction of chromatin in stationary phase, but not for general transcriptional repression. A clear, genome-wide anticorrelation was seen between the level of bound Hho1p and gene expression. Surprisingly, the rank order of gene activity was maintained even in the absence of Hho1p. Based on these findings, we suggest that linker histone Hho1p has a limited role in transcriptional regulation and that the dynamically exchanging linker histone may be evicted from chromatin by transcriptional activity.

Histone octamer helical tubes suggest that an internucleosomal four-helix bundle stabilizes the chromatin fiber.

A major question in chromatin involves the exact organization of nucleosomes within the 30-nm chromatin fiber and its structural determinants of assembly. Here we investigate the structure of histone octamer helical tubes via the method of iterative helical real-space reconstruction. Accurate placement of the x-ray structure of the histone octamer within the reconstructed density yields a pseudoatomic model for the entire helix, and allows precise identification of molecular interactions between neighboring octamers. One such interaction that would not be obscured by DNA in the nucleosome consists of a twofold symmetric four-helix bundle formed between pairs of H2B-alpha3 and H2B-alphaC helices of neighboring octamers. We believe that this interface can act as an internucleosomal four-helix bundle within the context of the chromatin fiber. The potential relevance of this interface in the folding of the 30-nm chromatin fiber is discussed.

The effect of epigenetic modifications on the secondary structures and possible binding positions of the N-terminal tail of histone H3 in the nucleosome: a computational study.

The roles of histone tails as substrates for reversible chemical modifications and dynamic cognate surfaces for the binding of regulatory proteins are well established. Despite these crucial roles, experimentally derived knowledge of the structure and possible binding sites of histone tails in chromatin is limited. In this study, we utilized molecular dynamics of isolated histone H3 N-terminal peptides to investigate its structure as a function of post-translational modifications that are known to be associated with defined chromatin states. We observed a structural preference for α-helices in isoforms associated with an inactive chromatin state, while isoforms associated with active chromatin states lacked α-helical content. The physicochemical effect of the post-translational modifications was highlighted by the interaction of arginine side-chains with the phosphorylated serine residues in the inactive isoform. We also showed that the isoforms exhibit different tail lengths, and, using molecular docking of the first 15 N-terminal residues of an H3 isoform, identified potential binding sites between the superhelical gyres on the octamer surface, close to the site of DNA entry/exit in the nucleosome. We discuss the possible functional role of the binding of the H3 tail within the nucleosome on both nucleosome and chromatin structure and stability.

Non-random clustering of stress-related genes during evolution of the S. cerevisiae genome.

BACKGROUND: Coordinately regulated genes often physically cluster in eukaryotic genomes, for reasons that remain unclear. RESULTS: Here we provide evidence that many S. cerevisiae genes induced by starvation and other stresses reside in non-random clusters, where transcription of these genes is repressed in the absence of stress. Most genes essential for growth or for rapid, post-transcriptional responses to stress in cycling cells map between these gene clusters. Genes that are transcriptionally induced by stresses include a large fraction of rapidly evolving paralogues of duplicated genes that arose during an ancient whole genome duplication event. Many of these rapidly evolving paralogues have acquired new or more specialized functions that are less essential for growth. The slowly evolving paralogues of these genes are less likely to be transcriptionally repressed in the absence of stress, and are frequently essential for growth or for rapid stress responses that may require constitutive expression of these genes in cycling cells. CONCLUSION: Our findings suggest that a fundamental organizing principle during evolution of the S. cerevisiae genome has been clustering of starvation and other stress-induced genes in chromosome regions that are transcriptionally repressed in the absence of stress, from which most genes essential for growth or rapid stress responses have been excluded. Chromatin-mediated repression of many stress-induced genes may have evolved since the whole genome duplication in parallel with functions for proteins encoded by these genes that are incompatible with growth. These functions likely provide fitness effects that escape detection in assays of reproductive capacity routinely employed to assess evolutionary fitness, or to identify genes that confer stress-resistance in cycling cells.

Calculating the statistical significance of physical clusters of co-regulated genes in the genome: the role of chromatin in domain-wide gene regulation.

Physical clusters of co-regulated, but apparently functionally unrelated, genes are present in many genomes. Despite the important implication that the genomic environment contributes appreciably to the regulation of gene expression, no simple statistical method has been described to identify physical clusters of co-regulated genes. Here we report the development of a model that allows the direct calculation of the significance of such clusters. We have implemented the derived statistical relation in a software program, Pyxis, and have analyzed a selection of Saccharomyces cerevisiae gene expression microarray data sets. We have identified many gene clusters where constituent genes exhibited a regulatory dependence on proteins previously implicated in chromatin structure. Specifically, we found that Tup1p-dependent gene domains were enriched close to telomeres, which suggested a new role for Tup1p in telomere silencing. In addition, we identified Sir2p-, Sir3p- and Sir4p-dependent clusters, which suggested the presence of Sir-mediated heterochromatin in previously unidentified regions of the yeast genome. We also showed the presence of Sir4p-dependent gene clusters bordering the HMRa heterothallic locus, which suggested leaky termination of the heterochromatin by the boundary elements. These results demonstrate the utility of Pyxis in identifying possible higher order genomic features that may contribute to gene regulation in extended domains.

The Saccharomyces cerevisiae linker histone Hho1p, with two globular domains, can simultaneously bind to two four-way junction DNA molecules.

Saccharomyces cerevisiae encodes a single linker histone, Hho1p, with two globular domains. This raised the possibility that Hho1p could bind to two nucleosome cores simultaneously. To evaluate this idea, we studied the ability of a four-way junction, immobilized on the surface of a magnetic bead, to pull down a radiolabeled four-way junction in the presence of different Hho1 proteins. Four-way junctions are known to bind to H1, presumably due to structure similarities to the DNA at the nucleosomal entry/exit point. We found a significant increase in the ability of full-length Hho1p to pull down radiolabeled four-way junction DNA under ionic conditions where both globular domains could bind. The binding was structure specific, since the use of double-stranded DNA, or a mutant Hho1p in which the second DNA binding site of globular domain 1 was abolished, resulted in a significant decrease in bridged binding. Additionally, bridged binding required a covalent attachment between the two globular domains, since factor Xa protease treatment of the complex formed by a modified Hho1p that contained a factor Xa cleavage site between the two globular domains resulted in a significant release of radiolabeled four-way junction. These findings demonstrated that the two globular domains independently associated with two different four-way junction molecules in a manner that required amino acid residues implicated in structure-specific binding in the nucleosome. We discuss the implication of these findings on the chromatin structure of yeast and propose a model where a single Hho1 protein binds to two serially adjacent nucleosomes.

Saccharomyces cerevisiae Set1p is a methyltransferase specific for lysine 4 of histone H3 and is required for efficient gene expression.

Several homologues of the Drosophila Su(var)3-9 protein were recently reported to methylate lysine 9 of histone H3. Whereas this methylation signal served to recruit heterochromatin-associated proteins to transcriptionally silenced regions, histone H3 methylated at lysine 4 was associated with transcriptionally active areas of the genome. These findings suggested that the interplay between lysine 4 and 9 methylation is crucial in eukaryotic gene regulation. Here we provide evidence that Saccharomyces cerevisiae Set1p is a methyltransferase specific for lysine 4 of histone H3. In addition, we show that the absence of Set1p and lysine 4 methylation result in decreased transcription of approximately 80% of the genes in S. cerevisiae. Hierarchical clustering analysis of the set1(-) expression profile revealed a correspondence to that of a mad2(-) strain, suggesting that the transcriptional defect in the set1(-) strain may be due to changes in chromatin structure. These findings establish a central role for methylation of histone H3 lysine 4 in transcriptional regulation.