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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., The Kouzarides laboratory is supported by Cancer Research UK (grants RG72100 and RG96894) and core support from the Wellcome Trust (core grant WT203144) and Cancer Research UK (grant C6946/A24843). For the purpose of open access, the authors have applied a CC BY public copyright license to any author accepted manuscript version arising from this submission. G.M.-Z. was funded by a European Molecular Biology Organization (EMBO) long-term fellowship (ALTF907-2014) and Asociación Española Contra el Cáncer (POSTD18021MILL). K.T. was supported by a Sir Henry Wellcome Fellowship (grant RG94424). T.B. was supported by the European Research Council (ERC; StG 309952) and the Helmholtz Association.

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.

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.

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.

8 pages, 7 figures.-- PMID: 12888487 [PubMed].-- PMCID: PMC169960., 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., This study was funded by an EU grant to H.S.R., a Cancer Research UK grant to T.K. and by grants (PB-98-0468) and (SAF 2002-00741) from the Spanish Ministerio de Ciencia y Tecnología to M.M.B.

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.

A description of a new histone modification, methylation of glutamine, on histone H2A in yeast and human cells. Post-translational modifications of histones are important for many DNA-templated processes. Here, Tony Kouzarides and colleagues identify a novel histone modification, methylation of glutamine, on histone H2A in yeast and human cells. The modification is exclusive to the nucleolus, being enriched over rDNA, and the enzyme responsible is a previously described rRNA methyltransferase. Glutamine methylation facilitates rDNA transcription by regulating binding of the histone chaperone complex FACT to nucleosomes, and it seems to have evolved as a modification dedicated to ribosomal biosynthesis. The finding that a protein can catalyse the methylation of both proteins and RNA raises the possibility that many other enzymes may have such dual specificity. Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes1. 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) complex2. 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.

SummaryThe 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.

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.

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.

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.

The Nup84p complex consists of five nucleoporins (Nup84p, Nup85p, Nup120p, Nup145p-C, and Seh1p) and Sec13p, a bona fide subunit of the COPII coat complex. We show that a pool of green fluorescent protein–tagged Sec13p localizes to the nuclear pores in vivo, and identify sec13 mutant alleles that are synthetically lethal with nup85Δ and affect the localization of a green fluorescent protein–Nup49p reporter protein. In the electron microscope, sec13 mutants exhibit structural defects in nuclear pore complex (NPC) and nuclear envelope organization. For the assembly of the complex, Nup85p, Nup120p, and Nup145p-C are essential. A highly purified Nup84p complex was isolated from yeast under native conditions and its molecular mass was determined to be 375 kD by quantitative scanning transmission electron microscopy and analytical ultracentrifugation, consistent with a monomeric complex. Furthermore, the Nup84p complex exhibits a Y-shaped, triskelion-like morphology 25 nm in diameter in the transmission electron microscope. Thus, the Nup84p complex constitutes a paradigm of an NPC structural module with distinct composition, structure, and a role in nuclear mRNA export and NPC bio- genesis.

Two membrane proteins were identified through their genetic interaction with the nucleoporin Nup84p and shown to participate in nuclear envelope morphogenesis in yeast. One component is a known sporulation factor Spo7p, and the other, Nem1p, a novel protein whose C-terminal domain is conserved during eukaryotic evolution. Spo7p and Nem1p localize to the nuclear/ER membrane and behave biochemically as integral membrane proteins. Nem1p binds to Spo7p via its conserved C-terminal domain. Although cells without Spo7p or Nem1p are viable, they exhibit a drastically altered nuclear morphology with long, pore-containing double nuclear membrane extensions. These protrusions emanate from a core nucleus which contains the DNA, and penetrate deeply into the cytoplasm. Interestingly, not only Spo7(-) and Nem1(-), but also several nucleoporin mutants are defective in sporulation. Thus, Spo7p and Nem1p, which exhibit a strong genetic link to nucleoporins of the Nup84p complex, fulfil an essential role in formation of a spherical nucleus and meiotic division.

The hrs1-1 mutation was isolated as an extragenic suppressor of the hyperrecombination phenotype of hpr1Δ cells. We have cloned, sequenced and deleted from the genome the HRS1 gene. The DNA sequence of the HRS1 gene reveals that it is identical to PGD1, a gene with no reported function, and that the Hrs1p protein contains polyglutamine stretches typically found in transcription factors. We have purified a His(6) tagged version of Hrs1p protein from E. coli and have obtained specific anti-Hrs1p polyclonal antibodies. We show that Hrs1p is a 49-kD nuclear protein, as determined by indirect immunofluorescence microscopy and Western blot analysis. The hrs1Δ null mutation reduces the frequency of deletions in wild-type and hpr1Δ backgrounds sevenfold below wild-type and rad52 levels. Furthermore, hrs1Δ cells show reduced induction of the GAL1,10 promoter relative to wild-type cells. Our results suggest that Hrs1p is required for the formation of deletions between direct repeats and that it may function in gene expression. This suggests a connection between gene expression and direct repeat recombination. In this context, we discuss the possible roles of Hrs1p and Hpr1p in initiation of direct-repeat recombination.

The HPR1 gene of Saccharomyces cerevisiae is involved in maintaining low levels of deletions between DNA repeats. To understand how deletions initiate in the absence of the Hpr1 protein and the mechanisms of recombination leading to deletions in S. cerevisiae, we have isolated mutations as suppressors of the hyper-deletion phenotype of the hpr1 delta mutation. The mutations defined five different genes called HRS for hyper-recombination suppression. They suppress the hyper-deletion phenotype of hpr1 delta strains for three direct repeat systems tested. The mutations eliminated the hyper-deletion phenotype of hpr1 delta strains either completely (hrs1-1 and hrs2-1) or significantly (hrs3-1, hrs4-1 and hrs5-1). None of the mutations has a clear effect on the levels of spontaneous and double-strand break-induced deletions. Among other characteristics we have found are the following: (1) one mutation, hrs1-1, reduces the frequency of deletions in rad52-1 strains 20-fold, suggesting that the HRS1 gene is involved in the formation of RAD52-independent deletions; (2) the hrs2-1 hpr1 delta mutant is sensitive to methyl-methane-sulfonate and the single mutants hpr1 delta and hrs2-1 are resistant, which suggests that the HPR1 and HRS2 proteins may have redundant DNA repair functions; (3) the hrs4-1 mutation confers a hyper-mutator phenotype and (4) the phenotype of lack of activation of gene expression observed in hpr1 delta strains is only partially suppressed by the hrs2-1 mutation, which suggests that the possible functions of the Hpr1 protein in gene expression and recombination repair can be separated. We discuss the possible relationship between the HPR1 and the HRS genes and their involvement in initiation of the events responsible for deletion formation.

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.