Predicting genes associated with RNA methylation pathways using machine learning.

RNA methylation plays an important role in functional regulation of RNAs, and has thus attracted an increasing interest in biology and drug discovery. Here, we collected and collated transcriptomic, proteomic, structural and physical interaction data from the Harmonizome database, and applied supervised machine learning to predict novel genes associated with RNA methylation pathways in human. We selected five types of classifiers, which we trained and evaluated using cross-validation on multiple training sets. The best models reached 88% accuracy based on cross-validation, and an average 91% accuracy on the test set. Using protein-protein interaction data, we propose six molecular sub-networks linking model predictions to previously known RNA methylation genes, with roles in mRNA methylation, tRNA processing, rRNA processing, but also protein and chromatin modifications. Our study exemplifies how access to large omics datasets joined by machine learning methods can be used to predict gene function.

Methylation of histone H3 at lysine 37 by Set1 and Set2 prevents spurious DNA replication.

DNA replication initiates at genomic locations known as origins of replication, which, in S. cerevisiae, share a common DNA consensus motif. Despite being virtually nucleosome-free, origins of replication are greatly influenced by the surrounding chromatin state. Here, we show that histone H3 lysine 37 mono-methylation (H3K37me1) is catalyzed by Set1p and Set2p and that it regulates replication origin licensing. H3K37me1 is uniformly distributed throughout most of the genome, but it is scarce at replication origins, where it increases according to the timing of their firing. We find that H3K37me1 hinders Mcm2 interaction with chromatin, maintaining low levels of MCM outside of conventional replication origins. Lack of H3K37me1 results in defective DNA replication from canonical origins while promoting replication events at inefficient and non-canonical sites. Collectively, our results indicate that H3K37me1 ensures correct execution of the DNA replication program by protecting the genome from inappropriate origin licensing and spurious DNA replication.

A computational platform for high-throughput analysis of RNA sequences and modifications by mass spectrometry.

The field of epitranscriptomics continues to reveal how post-transcriptional modification of RNA affects a wide variety of biological phenomena. A pivotal challenge in this area is the identification of modified RNA residues within their sequence contexts. Mass spectrometry (MS) offers a comprehensive solution by using analogous approaches to shotgun proteomics. However, software support for the analysis of RNA MS data is inadequate at present and does not allow high-throughput processing. Existing software solutions lack the raw performance and statistical grounding to efficiently handle the numerous modifications found on RNA. We present a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), that addresses these shortcomings. We demonstrate the capability of NASE to reliably identify a wide range of modified RNA sequences in four original datasets of varying complexity. In human tRNA, we characterize over 20 different modification types simultaneously and find many cases of incomplete modification.

Phosphorylation of Histone H4T80 Triggers DNA Damage Checkpoint Recovery.

In response to genotoxic stress, cells activate a signaling cascade known as the DNA damage checkpoint (DDC) that leads to a temporary cell cycle arrest and activation of DNA repair mechanisms. Because persistent DDC activation compromises cell viability, this process must be tightly regulated. However, despite its importance, the mechanisms regulating DDC recovery are not completely understood. Here, we identify a DNA-damage-regulated histone modification in Saccharomyces cerevisiae, phosphorylation of H4 threonine 80 (H4T80ph), and show that it triggers checkpoint inactivation. H4T80ph is critical for cell survival to DNA damage, and its absence causes impaired DDC recovery and persistent cell cycle arrest. We show that, in response to genotoxic stress, p21-activated kinase Cla4 phosphorylates H4T80 to recruit Rtt107 to sites of DNA damage. Rtt107 displaces the checkpoint adaptor Rad9, thereby interrupting the checkpoint-signaling cascade. Collectively, our results indicate that H4T80ph regulates DDC recovery.

RNA Binding by Histone Methyltransferases Set1 and Set2.

Histone methylation at H3K4 and H3K36 is commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) and is catalyzed by Saccharomyces cerevisiae Set1 and Set2, respectively. Here we report that both methyltransferases can be UV cross-linked to RNA in vivo High-throughput sequencing of the bound RNAs revealed strong Set1 enrichment near the transcription start site, whereas Set2 was distributed along pre-mRNAs. A subset of transcripts showed notably high enrichment for Set1 or Set2 binding relative to RNAPII, suggesting functional posttranscriptional interactions. In particular, Set1 was strongly bound to the SET1 mRNA, Ty1 retrotransposons, and noncoding RNAs from the ribosomal DNA (rDNA) intergenic spacers, consistent with its previously reported silencing roles. Set1 lacking RNA recognition motif 2 (RRM2) showed reduced in vivo cross-linking to RNA and reduced chromatin occupancy. In addition, levels of H3K4 trimethylation were decreased, whereas levels of dimethylation were increased. We conclude that RNA binding by Set1 contributes to both chromatin association and methyltransferase activity.

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

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.

Distinct transcriptional outputs associated with mono- and dimethylated histone H3 arginine 2.

Dimethylation of histone H3 Arg2 (H3R2me2) maintains transcriptional silencing by inhibiting Set1 mediated trimethylation of H3K4. Here we demonstrate that Arg2 is also monomethylated (H3R2me1) in yeast but that its functional characteristics are distinct from H3R2me2: (i) H3R2me1 does not inhibit histone H3 Lys4 (H3K4) methylation; (ii) it is present throughout the coding region of genes; and (iii) it correlates with active transcription. Collectively, these results indicate that different H3R2 methylation states have defined roles in gene expression.

Histone H3 tail clipping regulates gene expression.

Induction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters. Here we identify a histone H3 endopeptidase activity in Saccharomyces cerevisiae that may regulate these events. The endopeptidase cleaves H3 after Ala21, generating a histone that lacks the first 21 residues and shows a preference for H3 tails carrying repressive modifications. In vivo, the H3 N terminus is clipped, specifically within the promoters of genes following the induction of transcription. H3 clipping precedes the process of histone eviction seen when genes become fully active. A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells. These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression.

Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation.

Modifications on histones control important biological processes through their effects on chromatin structure. Methylation at lysine 4 on histone H3 (H3K4) is found at the 5' end of active genes and contributes to transcriptional activation by recruiting chromatin-remodelling enzymes. An adjacent arginine residue (H3R2) is also known to be asymmetrically dimethylated (H3R2me2a) in mammalian cells, but its location within genes and its function in transcription are unknown. Here we show that H3R2 is also methylated in budding yeast (Saccharomyces cerevisiae), and by using an antibody specific for H3R2me2a in a chromatin immunoprecipitation-on-chip analysis we determine the distribution of this modification on the entire yeast genome. We find that H3R2me2a is enriched throughout all heterochromatic loci and inactive euchromatic genes and is present at the 3' end of moderately transcribed genes. In all cases the pattern of H3R2 methylation is mutually exclusive with the trimethyl form of H3K4 (H3K4me3). We show that methylation at H3R2 abrogates the trimethylation of H3K4 by the Set1 methyltransferase. The specific effect on H3K4me3 results from the occlusion of Spp1, a Set1 methyltransferase subunit necessary for trimethylation. Thus, the inability of Spp1 to recognize H3 methylated at R2 prevents Set1 from trimethylating H3K4. These results provide the first mechanistic insight into the function of arginine methylation on chromatin.

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

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.

Human but not yeast CHD1 binds directly and selectively to histone H3 methylated at lysine 4 via its tandem chromodomains.

Defining the protein factors that directly recognize post-translational, covalent histone modifications is essential toward understanding the impact of these chromatin "marks" on gene regulation. In the current study, we identify human CHD1, an ATP-dependent chromatin remodeling protein, as a factor that directly and selectively recognizes histone H3 methylated on lysine 4. In vitro binding studies identified that CHD1 recognizes di- and trimethyl H3K4 with a dissociation constant (Kd) of approximately 5 microm, whereas monomethyl H3K4 binds CHD1 with a 3-fold lower affinity. Surprisingly, human CHD1 binds to methylated H3K4 in a manner that requires both of its tandem chromodomains. In vitro analyses demonstrate that unlike human CHD1, yeast Chd1 does not bind methylated H3K4. Our findings indicate that yeast and human CHD1 have diverged in their ability to discriminate covalently modified histones and link histone modification-recognition and non-covalent chromatin remodeling activities within a single human protein.

Chromatin modifier enzymes, the histone code and cancer.

In all organisms, cell proliferation is orchestrated by coordinated patterns of gene expression. Transcription results from the activity of the RNA polymerase machinery and depends on the ability of transcription activators and repressors to access chromatin at specific promoters. During the last decades, increasing evidence supports aberrant transcription regulation as contributing to the development of human cancers. In fact, transcription regulatory proteins are often identified in oncogenic chromosomal rearrangements and are overexpressed in a variety of malignancies. Most transcription regulators are large proteins, containing multiple structural and functional domains some with enzymatic activity. These activities modify the structure of the chromatin, occluding certain DNA regions and exposing others for interaction with the transcription machinery. Thus, chromatin modifiers represent an additional level of transcription regulation. In this review we focus on several families of transcription activators and repressors that catalyse histone post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and SUMOylation); and how these enzymatic activities might alter the correct cell proliferation program, leading to cancer.

The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth.

Remodelling of the nuclear membrane is essential for the dynamic changes of nuclear architecture at different stages of the cell cycle and during cell differentiation. The molecular mechanism underlying the regulation of nuclear membrane biogenesis is not known. Here we show that Smp2, the yeast homologue of mammalian lipin, is a key regulator of nuclear membrane growth during the cell cycle. Smp2 is phosphorylated by Cdc28/Cdk1 and dephosphorylated by a nuclear/endoplasmic reticulum (ER) membrane-localized CPD phosphatase complex consisting of Nem1 and Spo7. Loss of either SMP2 or its dephosphorylated form causes transcriptional upregulation of key enzymes involved in lipid biosynthesis concurrent with a massive expansion of the nucleus. Conversely, constitutive dephosphorylation of Smp2 inhibits cell division. We show that Smp2 associates with the promoters of phospholipid biosynthetic enzymes in a Nem1-Spo7-dependent manner. Our data suggest that Smp2 is a critical factor in coordinating phospholipid biosynthesis at the nuclear/ER membrane with nuclear growth during the cell cycle.

Methylation of H3 lysine 4 at euchromatin promotes Sir3p association with heterochromatin.

Set1p methylates lysine 4 of histone H3 and can activate transcription by recruiting the chromatin-remodeling factor Isw1p. In addition, Lys-4-methylated H3 is required for maintenance of silencing at the telomeres, rDNA, and HML locus in Saccharomyces cerevisiae. The molecular mechanism underlying the role of Set1p in silencing is not known. Here we report that euchromatic methylation of H3 Lys-4 is necessary to maintain silencing at specific heterochromatic sites. Inactivation of Set1p catalytic activity or mutation of H3 Lys-4 leads to decreased binding of the silent information regulator Sir3p at heterochromatic sites. Concomitantly, there is an increase in the amount of Sir3p bound to genes located in subtelomeric regions. Consistent with this result is the finding that in vitro, Sir3p preferentially binds histone H3 tails when methylation is absent at H3 Lys-4, a situation found in heterochromatin. The inability of Sir3p to bind methylated H3 Lys-4 tails suggests a model whereby H3 Lys-4 methylation prevents Sir3p association at euchromatic sites and therefore concentrates Sir3p at unmodified, heterochromatic regions of the genome.

Methylation of histone H3 K4 mediates association of the Isw1p ATPase with chromatin.

Set1p methylates lysine 4 (K4) of histone H3 and regulates the expression of many genes in yeast. Here we use a biochemical approach to identify a protein, Isw1p, which recognizes chromatin preferentially when it is di- and trimethylated at K4 H3. We show that on certain actively transcribed genes, the Isw1p chromatin remodeling ATPase requires K4 H3 methylation to associate with chromatin in vivo. Analysis of one such gene, MET16, shows that the enzymatic activities of Set1p and Isw1p are functionally connected: Set1p methylation and Isw1p ATPase generate specific chromatin changes at the 5' end of the gene, are necessary for the correct distribution of RNA polymerase II over the coding region, and are required for the recruitment of the cleavage and polyadenylation factor Rna15p. These results indicate that K4 H3 methylation and Isw1p ATPase activity are intimately linked in regulating transcription of certain genes in yeast.

Mechanisms of P/CAF auto-acetylation.

P/CAF is a histone acetyltransferase enzyme which was originally identified as a CBP/p300-binding protein. In this manuscript we report that human P/CAF is acetylated in vivo. We find that P/CAF is acetylated by itself and by p300 but not by CBP. P/CAF acetylation can be an intra- or intermolecular event. The intermolecular acetylation requires the N-terminal domain of P/CAF. The intramolecular acetylation targets five lysines (416-442) at the P/CAF C-terminus, which are in the nuclear localisation signal (NLS). Finally, we show that acetylation of P/CAF leads to an increment of its histone acetyltransferase (HAT) activity. These findings identify a new post-translation modification on P/CAF which may regulate its function.

Active genes are tri-methylated at K4 of histone H3.

Lysine methylation of histones in vivo occurs in three states: mono-, di- and tri-methyl. Histone H3 has been found to be di-methylated at lysine 4 (K4) in active euchromatic regions but not in silent heterochromatic sites. Here we show that the Saccharomyces cerevisiae Set1 protein can catalyse di- and tri-methylation of K4 and stimulate the activity of many genes. Using antibodies that discriminate between the di- and tri-methylated state of K4 we show that di-methylation occurs at both inactive and active euchromatic genes, whereas tri-methylation is present exclusively at active genes. It is therefore the presence of a tri-methylated K4 that defines an active state of gene expression. These findings establish the concept of methyl status as a determinant for gene activity and thus extend considerably the complexity of histone modifications.