Adaptive Evolution in TRIF Leads to Discordance between Human and Mouse Innate Immune Signaling.

The TIR domain-containing adapter inducing IFN-ß (TRIF) protein is an innate immune system protein that mediates the MyD88-independent toll-like receptor response pathway in mice and humans. Previously, we identified positive selection at seven distinct residues in mouse TRIF (mTRIF), as compared with human and other mammalian orthologs, thus predicting protein functional shift in mTRIF. We reconstructed TRIF for the most recent common ancestor of mouse and human, and mutated this at the seven sites to their extant mouse/human states. We overexpressed these TRIF mutants in immortalized human and mouse cell lines and monitored TRIF-dependent cytokine production and gene expression induction. We show that optimal TRIF function in human and mouse is dependent on the identity of the positively selected sites. These data provide us with molecular data relating observed differences in response between mouse and human MyD88-independent signaling in the innate immune system with protein functional change.

Characterization of the substrate binding site of an iron detoxifying membrane transporter from Plasmodium falciparum.

BACKGROUND: Plasmodium species are entirely dependent upon their host as a source of essential iron. Although it is an indispensable micronutrient, oxidation of excess ferrous iron to the ferric state in the cell cytoplasm can produce reactive oxygen species that are cytotoxic. The malaria parasite must therefore carefully regulate the processes involved in iron acquisition and storage. A 273 amino acid membrane transporter that is a member of the vacuolar iron transporter (VIT) family and an orthologue of the yeast Ca2+-sensitive cross complementer (CCC1) protein plays a major role in cytosolic iron detoxification of Plasmodium species and functions in transport of ferrous iron ions into the endoplasmic reticulum for storage. While this transporter, termed PfVIT, is not critical for viability of the parasite evidence from studies of mice infected with VIT-deficient Plasmodium suggests it could still provide an efficient target for chemoprophylactic treatment of malaria. Individual amino acid residues that constitute the Fe2+ binding site of the protein were identified to better understand the structural basis of substrate recognition and binding by PfVIT. METHODS: Using the crystal structure of a recently published plant VIT as a template, a high-quality homology model of PfVIT was constructed to identify the amino acid composition of the transporter's substrate binding site and to act as a guide for subsequent mutagenesis studies. To test the effect of mutation of the substrate binding-site residues on PfVIT function a yeast complementation assay assessed the ability of overexpressed, recombinant wild type and mutant PfVIT to rescue an iron-sensitive deletion strain (ccc1∆) of Saccharomyces cerevisiae yeast from the toxic effects of a high concentration of extracellular iron. RESULTS: The combined in silico and mutagenesis approach identified a methionine residue located within the cytoplasmic metal binding domain of the transporter as essential for PfVIT function and provided insight into the structural basis for the Fe2+-selectivity of the protein. CONCLUSION: The structural model of the metal binding site of PfVIT opens the door for rational design of therapeutics to interfere with iron homeostasis within the malaria parasite.

Gene Fusions Derived by Transcriptional Readthrough are Driven by Segmental Duplication in Human.

Gene fusion occurs when two or more individual genes with independent open reading frames becoming juxtaposed under the same open reading frame creating a new fused gene. A small number of gene fusions described in detail have been associated with novel functions, for example, the hominid-specific PIPSL gene, TNFSF12, and the TWE-PRIL gene family. We use Sequence Similarity Networks and species level comparisons of great ape genomes to identify 45 new genes that have emerged by transcriptional readthrough, that is, transcription-derived gene fusion. For 35 of these putative gene fusions, we have been able to assess available RNAseq data to determine whether there are reads that map to each breakpoint. A total of 29 of the putative gene fusions had annotated transcripts (9/29 of which are human-specific). We carried out RT-qPCR in a range of human tissues (placenta, lung, liver, brain, and testes) and found that 23 of the putative gene fusion events were expressed in at least one tissue. Examining the available ribosome foot-printing data, we find evidence for translation of three of the fused genes in human. Finally, we find enrichment for transcription-derived gene fusions in regions of known segmental duplication in human. Together, our results implicate chromosomal structural variation brought about by segmental duplication with the emergence of novel transcripts and translated protein products.

Yeast epigenetics: the inheritance of histone modification states.

Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) are two of the most recognised and well-studied model systems for epigenetic regulation and the inheritance of chromatin states. Their silent loci serve as a proxy for heterochromatic chromatin in higher eukaryotes, and as such both species have provided a wealth of information on the mechanisms behind the establishment and maintenance of epigenetic states, not only in yeast, but in higher eukaryotes. This review focuses specifically on the role of histone modifications in governing telomeric silencing in S. cerevisiae and centromeric silencing in S. pombe as examples of genetic loci that exemplify epigenetic inheritance. We discuss the recent advancements that for the first time provide a mechanistic understanding of how heterochromatin, dictated by histone modifications specifically, is preserved during S-phase. We also discuss the current state of our understanding of yeast nucleosome dynamics during DNA replication, an essential component in delineating the contribution of histone modifications to epigenetic inheritance.

Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins.

Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are suppressed by deletion of either SIR2 or SIR3. While these and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using MCM ChIP-Seq, we show that a SIR2 deletion rescued MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner in wild type (i.e. CDC6) cells. In addition, we present evidence that both Sir2 and Sir3 bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin-SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 -correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly modify the local chromatin environment of euchromatic DNA replication origins.

A model for the evolution of biological specificity: a cross-reacting DNA-binding protein causes plasmid incompatibility.

Few biological systems permit rigorous testing of how changes in DNA sequence give rise to adaptive phenotypes. In this study, we sought a simplified experimental system with a detailed understanding of the genotype-to-phenotype relationship that could be altered by environmental perturbations. We focused on plasmid fitness, i.e., the ability of plasmids to be stably maintained in a bacterial population, which is dictated by the plasmid's replication and segregation machinery. Although plasmid replication depends on host proteins, the type II plasmid partitioning (Par) machinery is entirely plasmid encoded and relies solely on three components: parC, a centromere-like DNA sequence, ParR, a DNA-binding protein that interacts with parC, and ParM, which forms actin-like filaments that push two plasmids away from each other at cell division. Interactions between the Par operons of two related plasmids can cause incompatibility and the reduced transmission of one or both plasmids. We have identified segregation-dependent plasmid incompatibility between the highly divergent Par operons of plasmids pB171 and pCP301. Genetic and biochemical studies revealed that the incompatibility is due to the functional promiscuity of the DNA-binding protein ParRpB171, which interacts with both parC DNA sequences to direct plasmid segregation, indicating that the lack of DNA binding specificity is detrimental to plasmid fitness in this environment. This study therefore successfully utilized plasmid segregation to dissect the molecular interactions between genotype, phenotype, and fitness.

An evolutionarily 'young' lysine residue in histone H3 attenuates transcriptional output in Saccharomyces cerevisiae.

The DNA entry and exit points on the nucleosome core regulate the initial invasion of the nucleosome by factors requiring access to the underlying DNA. Here we describe in vivo consequences of eliminating a single protein-DNA interaction at this position through mutagenesis of histone H3 Lys 42 to alanine. This substitution has a dramatic effect on the Saccharomyces cerevisiae transcriptome in both the transcriptional output and landscape of mRNA species produced. We attribute this in part to decreased histone H3 occupancy at transcriptionally active loci, leading to enhanced elongation. Additionally we show that this lysine is methylated in vivo, and genetic studies of methyl-lysine mimics suggest that this modification may be crucial in attenuating gene expression. Interestingly, this site of methylation is unique to Ascomycota, suggesting a recent evolutionary innovation that highlights the evolvability of post-translational modifications of chromatin.

Yin and Yang of histone H2B roles in silencing and longevity: a tale of two arginines.

In budding yeast, silent chromatin is defined at the region of telomeres, rDNA loci, and silent mating loci. Although the silent chromatin at different loci shows structural similarity, the underlying mechanism to establish, maintain, and inherit these structures may be fundamentally different. In this study, we found two arginine residues within histone H2B, which are specifically required to maintain either the telomeric or the rDNA silenct chromatin. Arginine 95 (R95) plays a specific role at telomeres, whereas arginine 102 (R102) is required to maintain the silent chromatin at rDNA and to ensure the integrity of rDNA loci by suppressing recombination between rDNA repeats. R95 mutants show enhanced rDNA silencing but a paradoxically low Sir2 protein abundance. Furthermore weakened silencing at telomeres in R95 mutants can be suppressed by a specific SIR3 allele, SIR3-D205N, which increases the affinity of Sir proteins to telomeres, suggesting H2B-R95 may directly mediate telomeric Sir protein-nucleosome interactions. Double mutations of R95 and R102 lead to desilencing of both rDNA and telomeres, indicating both arginines are necessary to ensure integrity of silent chromatin at these loci. Furthermore, mutations of R102 cause accumulation of extrachromosomal rDNA circles and reduce life span, suggesting that histone H2B contributes to longevity.

Histone h3 exerts a key function in mitotic checkpoint control.

It has been firmly established that many interphase nuclear functions, including transcriptional regulation, are regulated by chromatin and histones. How mitotic progression and quality control might be influenced by histones is less well characterized. We show that histone H3 plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis. Prior to anaphase, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. Lack of tension due to erroneous attachment activates the spindle assembly checkpoint, which corrects the mistakes and ensures segregation fidelity. A histone H3 mutation impairs the ability of yeast cells to activate the checkpoint in a tensionless crisis, leading to missegregation and aneuploidy. The defects in tension sensing result directly from an attenuated H3-Sgo1p interaction essential for pericentric recruitment of Sgo1p. Reinstating the pericentric enrichment of Sgo1p alleviates the mitotic defects. Histone H3, and hence the chromatin, is thus a key factor transmitting the tension status to the spindle assembly checkpoint.

HistoneHits: a database for histone mutations and their phenotypes.

Histones are the basic protein components of nucleosomes. They are among the most conserved proteins and are subject to a plethora of post-translational modifications. Specific histone residues are important in establishing chromatin structure, regulating gene expression and silencing, and responding to DNA damage. Here we present HistoneHits, a database of phenotypes for systematic collections of histone mutants. This database combines assay results (phenotypes) with information about sequences, structures, post-translational modifications, and evolutionary conservation. The web interface presents the information through dynamic tables and figures. It calculates the availability of data for specific mutants and for nucleosome surfaces. The database currently includes 42 assays on 677 mutants multiply covering 405 of the 498 residues across yeast histones H3, H4, H2A, and H2B. We also provide an interface with an extensible controlled vocabulary for research groups to submit new data. Preliminary analyses confirm that mutations at highly conserved residues and modifiable residues are more likely to generate phenotypes. Buried residues and residues on the lateral surface tend to generate more phenotypes, while tail residues generate significantly fewer phenotypes than other residues. Yeast mutants are cross referenced with known human histone variants, identifying a position where a yeast mutant causes loss of ribosomal silencing and a human variant increases breast cancer susceptibility. All data sets are freely available for download.

Probing nucleosome function: a highly versatile library of synthetic histone H3 and H4 mutants.

Nucleosome structural integrity underlies the regulation of DNA metabolism and transcription. Using a synthetic approach, a versatile library of 486 systematic histone H3 and H4 substitution and deletion mutants that probes the contribution of each residue to nucleosome function was generated in Saccharomyces cerevisiae. We probed fitness contributions of each residue to perturbations of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for transcriptional silencing onto the nucleosome surface. Each histone mutant was tagged with unique molecular barcodes, facilitating identification of histone mutant pools through barcode amplification, labeling, and TAG microarray hybridization. Barcodes were used to score complex phenotypes such as competitive fitness in a chemostat, DNA repair proficiency, and synthetic genetic interactions, revealing new functions for distinct histone residues and new interdependencies among nucleosome components and their modifiers.

Insights into the role of histone H3 and histone H4 core modifiable residues in Saccharomyces cerevisiae.

The biological significance of recently described modifiable residues in the globular core of the bovine nucleosome remains elusive. We have mapped these modification sites onto the Saccharomyces cerevisiae histones and used a genetic approach to probe their potential roles both in heterochromatic regions of the genome and in the DNA repair response. By mutating these residues to mimic their modified and unmodified states, we have generated a total of 39 alleles affecting 14 residues in histones H3 and H4. Remarkably, despite the apparent evolutionary pressure to conserve these near-invariant histone amino acid sequences, the vast majority of mutant alleles are viable. However, a subset of these variant proteins elicit an effect on transcriptional silencing both at the ribosomal DNA locus and at telomeres, suggesting that posttranslational modification(s) at these sites regulates formation and/or maintenance of heterochromatin. Furthermore, we provide direct mass spectrometry evidence for the existence of histone H3 K56 acetylation in yeast. We also show that substitutions at histone H4 K91, K59, S47, and R92 and histone H3 K56 and K115 lead to hypersensitivity to DNA-damaging agents, linking the significance of the chemical identity of these modifiable residues to DNA metabolism. Finally, we allude to the possible molecular mechanisms underlying the effects of these modifications.

The DNA binding domain of the gene 2.5 single-stranded DNA-binding protein of bacteriophage T7.

Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA-binding protein that is essential for viral survival. Its crystal structure reveals a conserved oligosaccharide/oligonucleotide binding fold predicted to interact with single-stranded DNA. However, there is no experimental evidence to support this hypothesis. Recently, we reported a genetic screen for lethal mutations in gene 2.5 that we are using to identify functional domains of the gene 2.5 protein. This screen uncovered a number of mutations that led to amino acid substitutions in the proposed DNA binding domain. Three variant proteins, gp2.5-Y158C, gp2.5-K152E, and gp2.5-Y111C/Y158C, exhibit a decrease in binding affinity for oligonucleotides. A fourth, gp2.5-K109I, exhibits an altered mode of binding single-stranded DNA. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta26C, binds single-stranded DNA 10-fold more tightly than the wild-type protein. The three altered proteins defective in single-stranded DNA binding cannot mediate the annealing of homologous DNA, whereas gp2.5-Delta26C mediates the reaction more effectively than does wild-type. Gp2.5-K109I retains this annealing ability, albeit slightly less efficiently. With the exception of gp2.5-Delta26C, all variant proteins form dimers in solution and physically interact with T7 DNA polymerase.