rapid-identification-chemical-genetic-interactions-saccharomyces

Rapid Identification of Chemical Genetic Interactions in <em>Saccharomyces cerevisiae</em>

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

University of Victoria

The cis-trans isomerization of proline serves as a regulatory switch in signaling pathways. We identify the proline isomerase Fpr4, a member of the FK506 binding protein family in Saccharomyces cerevisiae, as an enzyme which binds the amino-terminal tail of histones H3 and H4 and catalyses the isomerization of H3 proline P30 and P38 in vitro. We show that P38 is necessary for methylation of K36 and that isomerization by Fpr4 inhibits the ability of Set2 to methylate H3 K36 in vitro. These results suggest that the conformational state of P38, controlled by Fpr4, is important for methylation of H3K36 by Set2. Consistent with such an antagonistic role, abrogation of Fpr4 catalytic activity in vivo results in increased levels of H3K36 methylation and delayed transcriptional induction kinetics of specific genes in yeast. These results identify proline isomerization as a novel noncovalent histone modification that regulates transcription and provides evidence for crosstalk between histone lysine methylation and proline isomerization.

Proline isomerization of histone H3 regulates lysine methylation and gene expression.

Several recent studies from the field of epigenetics have combined chromatin-immunoprecipitation (ChIP) with next-generation high-throughput sequencing technologies to describe the locations of histone post-translational modifications (PTM) and DNA methylation genome-wide. While these reports begin to quench the chromatin biologists thirst for visualizing where in the genome epigenetic marks are placed, they also illustrate several advantages of sequencing based genomics compared to microarray analysis. Accordingly, next-generation sequencing (NGS) technologies are now challenging microarrays as the tool of choice for genome analysis. The increased affordability of comprehensive sequence-based genomic analysis will enable new questions to be addressed in many areas of biology. It is inevitable that massively-parallel sequencing platforms will supercede the microarray for many applications, however, there are niches for microarrays to fill and interestingly we may very well witness a symbiotic relationship between microarrays and high-throughput sequencing in the future.

Advantages of next-generation sequencing versus the microarray in epigenetic research.

Yeast Fpr4p belongs to the FK506-binding protein (FKBP) class of peptidyl proline isomerases (PPIases), and has been implicated in regulating the cis-trans conversion of proline residues within histone tails. Here we report the (1)H, (13)C and (15)N chemical shift assignments for the bacterially expressed C-terminal PPIase catalytic domain of Fpr4p. Prediction of secondary structure reveals similarity to domains from other members of the FKBP proline isomerases, including yeast Fpr1p and the prototypic PPIase region from human FKBP12.

Chemical shift assignments of the catalytic domain from the yeast proline isomerase Fpr4p.

The post-translational modification of proteins and enzymes provides a dynamic and reversible means to control protein function and transmit biological signals. While covalent modifications such as phosphorylation and acetylation have drawn much attention, in the past decade the involvement of peptidyl-proline isomerases (PPIs) in signaling and post-translational modification of protein function has become increasingly apparent. Three distinct families of PPI enzymes (parvulins, cyclophilins, and FK506-binding proteins (FKBPs)) each have the capacity to catalyze cis-trans proline isomerization in substrate proteins, and this modification can regulate both structure and function. In eukaryotic cells, a subset of these enzymes is localized to the nucleus, where they regulate gene expression at multiple control points. Here we summarize this body of work that together establishes a clear role of these enzymes as evolutionarily conserved players in the control of both transcription of mRNAs and the assembly of chromatin.

The roles of peptidyl-proline isomerases in gene regulation.

The 55th Annual Canadian Society for Molecular Biosciences Conference on Epigenetics and Genomic Stability in Whistler, Canada, 14-18 March 2012, brought together 31 speakers from different nationalities. The organizing committee, led by Jim Davie (Chair) at the University of Manitoba (Manitoba, Canada), consisted of several established researchers in the fields of chromatin and epigenetics from across Canada. The meeting was centered on the contribution of epigenetics to gene expression, DNA damage and repair, and the role of environmental factors. A few interesting talks on replication added some insightful information on the controversial issue of histone post-translational modifications as genuine epigenetic marks that are inherited through cell division.

55th Annual Canadian Society for Molecular Biosciences Conference on Epigenetics and Genomic Stability. Whistler, British Columbia, Canada, 14–18 March 2012.

Alkylated guanidinium compounds exhibit microbiocidal activity in marine environments, yet the mode of action of these compounds has not been defined. A comprehensive chemical-genetic approach in budding yeast was used to define the biological processes affected by these compounds. N-Butyl-N'-decylguanidinium and N-hexyl-N'-(3-hydroxypropyl)-N''-octylguanidinium chlorides were shown to prevent yeast growth in a dose-dependent manner. All non-essential genes required for tolerance of sub-lethal amounts of these biocides were identified. These unbiased and systematic screens reveal the two related guanidinium compounds have a non-overlapping spectrum of targets in vivo. A functional tryptophan biosynthetic pathway is essential for tolerance of both biocides, which identifies tryptophan amino acid import as one process affected by these compounds. Further analysis of hypersensitive gene lists demonstrates that the substitutions on alkylated guanidiums confer important functional differences in vivo: one derivative renders the ability to generate acidic vacuoles essential, while the other is synthetically lethal with mutants in the transcriptional response to chemical stress. Altogether the results define the common and distinct biological processes affected by biocidal alkylated guanidinium salts.

Chemical-genetic identification of the biochemical targets of polyalkyl guanidinium biocides.

Peptidylprolyl isomerases have been implicated in chromatin regulation through their association with histones, chromatin-modifying enzymes and DNA-binding transcription factors. As with other post-translational modifications to proteins, a mechanistic understanding of the regulation of biological processes is fostered by loss-of-function studies both in vitro and in vivo. For peptidylprolyl isomerases, this can be accomplished with small-molecule inhibitors with high affinity for the isomerase active site or by mutation of amino acid residues that contribute to catalysis. In the present article, we review caveats to each of these approaches, and place emphasis on the thorough characterization of loss-of-function mutations in FKBPs (FK506-binding proteins). Using a case study of mutagenesis of the nuclear FKBP25 peptidylprolyl isomerase enzyme, we demonstrate that certain mutations generate a loss-of-function phenotype because they induce a complete loss of the FKBP domain fold, whereas other mutations are 'surgical' in that they ablate catalytic isomerase activity, while maintaining domain structure. Peptidylprolyl isomerases are thought to have both catalytic and non-catalytic functions, but differentiating between these mechanisms has proved to be challenging. The domain-destabilizing and surgical mutants described will facilitate the characterization of these two reported functions of peptidylprolyl isomerases.

Resolving the functions of peptidylprolyl isomerases: insights from the mutagenesis of the nuclear FKBP25 enzyme.

The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro(16) and Pro(30) of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro(38). This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond.

Structure and activity of the peptidyl-prolyl isomerase domain from the histone chaperone Fpr4 toward histone H3 proline isomerization.

Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification.

Many proteins in the immune system are also expressed in the brain. One such class of immune proteins are T-cell receptors (TCRs), whose functions in T lymphocytes in adaptive immunity are well characterized. In the brain, TCRs are confined to neocortical neurons, but their functional role has not been determined. In mouse layer 1 neocortical neurons, TCR activation inhibited α7 nicotinic currents. TCRs modulated α7 currents via tyrosine phosphorylation of α7 nicotinic receptors (nAChRs) through src tyrosine kinases because eliminating lck kinase expression, coexpressing fyn kinase dead, or mutating tyrosine to alanine in α7 blocked the effect of TCR activation. We found that TCR stimulation decreased surface α7 nAChRs and reduced single-channel conductance. These results reveal that TCRs play a major role in the modulation of cholinergic neurotransmission in the brain mediated by α7 nAChRs and that this has a profound effect on regulating neuronal excitability.

T-cell receptor activation decreases excitability of cortical interneurons by inhibiting α7 nicotinic receptors.

Peptidyl-proline isomerases of the FK506-binding protein (FKBP) family belong to a class of enzymes that catalyze the cis-trans isomerization of prolyl-peptide bonds in proteins. A handful of FKBPs are found in the nucleus, implying that the isomerization of proline in nuclear proteins is enzymatically controlled. FKBP25 is a nuclear protein that has been shown to associate with chromatin modifiers and transcription factors. In this study, we performed the first proteomic characterization of FKBP25 and found that it interacts with numerous ribosomal proteins, ribosomal processing factors, and a small selection of chromatin modifiers. In agreement with previous reports, we found that nucleolin is a major FKBP25-interacting protein and demonstrated that this interaction is dependent on rRNA. FKBP25 interacts with the immature large ribosomal subunit in nuclear extract but does not associate with mature ribosomes, implicating this FKBP's action in ribosome biogenesis. Despite engaging nascent 60S ribosomes, FKBP25 does not affect steady-state levels of rRNAs or its pre-rRNA intermediates. We conclude that FKBP25 is likely recruited to preribosomes to chaperone one of the protein components of the ribosome large subunit.

Article	Year
Proline isomerization of histone H3 regulates lysine methylation and gene expression. Cell\| PubMed ID: 16959570	2006
Advantages of next-generation sequencing versus the microarray in epigenetic research. Briefings in functional genomics & proteomics\| PubMed ID: 19535508	2009
Chemical shift assignments of the catalytic domain from the yeast proline isomerase Fpr4p. Biomolecular NMR assignments\| PubMed ID: 21898050	2012
The roles of peptidyl-proline isomerases in gene regulation. Biochemistry and cell biology = Biochimie et biologie cellulaire\| PubMed ID: 21999350	2012
55th Annual Canadian Society for Molecular Biosciences Conference on Epigenetics and Genomic Stability. Whistler, British Columbia, Canada, 14–18 March 2012. Epigenomics\| PubMed ID: 22690661	2012
Chemical-genetic identification of the biochemical targets of polyalkyl guanidinium biocides. Organic & biomolecular chemistry\| PubMed ID: 23689276	2013
Resolving the functions of peptidylprolyl isomerases: insights from the mutagenesis of the nuclear FKBP25 enzyme. Biochemical Society transactions\| PubMed ID: 23697935	2013
Structure and activity of the peptidyl-prolyl isomerase domain from the histone chaperone Fpr4 toward histone H3 proline isomerization. The Journal of biological chemistry\| PubMed ID: 23888048	2013
Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification. Nature\| PubMed ID: 24352239	2014
T-cell receptor activation decreases excitability of cortical interneurons by inhibiting α7 nicotinic receptors. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 24381265	2014
The prolyl isomerase, FKBP25, interacts with RNA-engaged nucleolin and the pre-60S ribosomal subunit. RNA (New York, N.Y.)\| PubMed ID: 24840943	2014

Christopher J. Nelson

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