Philip Zegerman

Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain

Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing. The chromo domain of HP1 is necessary for both targeting and transcriptional repression. In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homologue of the mammalian SUV39H1 histone methylase. Both Clr4 and SUV39H1 methylate specifically lysine 9 of histone H3 (ref. 6). Here we show that HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4. The chromo domain of HP1 is identified as its methyl-lysine-binding domain. A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing. These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a ‘methyl marker’ on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state.

Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast.

Cyclin-dependent kinases (CDKs) drive major cell cycle events including the initiation of chromosomal DNA replication. We identified two S phase CDK (S-CDK) phosphorylation sites in the budding yeast Sld3 protein that, together, are essential for DNA replication. Here we show that, when phosphorylated, these sites bind to the amino-terminal BRCT repeats of Dpb11. An Sld3–Dpb11 fusion construct bypasses the requirement for both Sld3 phosphorylation and the N-terminal BRCT repeats of Dpb11. Co-expression of this fusion with a phospho-mimicking mutant in a second essential CDK substrate, Sld2, promotes DNA replication in the absence of S-CDK. Therefore, Sld2 and Sld3 are the minimal set of S-CDK targets required for DNA replication. DNA replication in cells lacking G1 phase CDK (G1-CDK) required expression of the Cdc7 kinase regulatory subunit, Dbf4, as well as Sld2 and Sld3 bypass. Our results help to explain how G1- and S-CDKs promote DNA replication in yeast.

Histone H3 Lysine 4 Methylation Disrupts Binding of Nucleosome Remodeling and Deacetylase (NuRD) Repressor Complex

Histone N-terminal tails are post-translationally modified in many ways. At lysine residues, histones can be either acetylated or methylated. Both modifications lead to the binding of specific proteins; bromodomain proteins, such as GCN5, bind acetyl lysines and the chromodomain protein, HP1, binds methyl lysine 9 of histone H3. Here we show that the previously characterized transcriptional repressor complex NuRD (nucleosomeremodeling and deacetylase) binds to the histone H3 N-terminal tail and that methylation at lysine 4, but not lysine 9, prevents binding. Given that lysine 4 methylation is found at sites of active transcription, these results suggest that a function of lysine 4 methylation is to disrupt the association of histones with a repressor complex.

Checkpoint Dependent Inhibition of DNA Replication Initiation by Sld3 and Dbf4 Phosphorylation

The initiation of eukaryotic DNA replication is regulated by three protein kinase classes: cyclin-dependent kinases (CDK), Dbf4-dependent kinase (DDK) and the DNA damage checkpoint kinases. CDK phosphorylation of two key initiation factors, Sld2 and Sld3, promotes essential interactions with Dpb11 (refs 2–4), whereas DDK acts by phosphorylating subunits of the Mcm2-7 helicase. CDK has an additional role in replication by preventing the re-loading of Mcm2-7 during the S, G2 and M phases, thus preventing origin re-firing and re-replication. During the G1 phase, both CDK and DDK are downregulated, which allows origin licensing and prevents premature replication initiation. Origin firing is also inhibited during the S phase when DNA damage or replication fork stalling activates the checkpoint kinases. Here we show that, analogous to the situation in the G1 phase, the Saccharomyces cerevisiae checkpoint kinase Rad53 inhibits both CDK- and DDK-dependent pathways, which acts redundantly to block further origin firing. Rad53 acts on DDK directly by phosphorylating Dbf4, whereas the CDK pathway is blocked by Rad53-mediated phosphorylation of the downstream CDK substrate, Sld3. This allows CDK to remain active during the S phase in the presence of DNA damage, which is crucial to prevent re-loading of Mcm2-7 onto origins that have already fired. Our results explain how checkpoints regulate origin firing and demonstrate that the slowing of S phase by the ‘intra-S checkpoint’ is primarily due to the inhibition of origin firing.

Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast

Eukaryotic chromosomes are replicated from multiple origins that initiate throughout the S‐phase of the cell cycle. Why all origins do not fire simultaneously at the beginning of S‐phase is not known, but two kinase activities, cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK), are continually required throughout the S‐phase for all replication initiation events. Here, we show that the two CDK substrates Sld3 and Sld2 and their binding partner Dpb11, together with the DDK subunit Dbf4 are in low abundance in the budding yeast, Saccharomyces cerevisiae. Over‐expression of these factors is sufficient to allow late firing origins of replication to initiate early and together with deletion of the histone deacetylase RPD3, promotes the firing of heterochromatic, dormant origins. We demonstrate that the normal programme of origin firing prevents inappropriate checkpoint activation and controls S‐phase length in budding yeast. These results explain how the competition for limiting DDK kinase and CDK targets at origins regulates replication initiation kinetics during S‐phase and establishes a unique system with which to investigate the biological roles of the temporal programme of origin firing.

E1A directly binds and regulates the P/CAF acetyltransferase

The P/CAF protein has intrinsic histone acetyltransferase (HAT) activity and is capable of binding the transcriptional co‐activator CBP. Here we show that P/CAF can regulate transcription and that this function is independent of its binding to CBP. The HAT domain of P/CAF has transcriptional activation potential in yeast. In mammalian cells P/CAF can stimulate transcription of the RSV promoter, using the activity of its HAT domain. We show that the adenovirus protein E1A targets P/CAF and sequesters its transcriptional activity. Binding of E1A to P/CAF is direct, independent of CBP and requires residues within E1A conserved region 1. We find that the P/CAF binding residues in E1A are within a motif shown to be essential for efficient disruption of myogenesis by E1A. The fact that E1A can directly bind and regulate the activity of P/CAF, independently of its regulation of CBP, highlights an important role for P/CAF in the process of cell differentiation.

DNA replication as a target of the DNA damage checkpoint

Faithful inheritance of the genome from mother to daughter cell requires that it is replicated accurately, in its entirety, exactly once. DNA replication not only has to have high fidelity, but also has to cope with exogenous and endogenous agents that damage DNA during the life cycle of a cell. The DNA damage checkpoint, which monitors and responds to defects in the genome, is critical for the completion of replication. The focus of this review is how DNA replication is regulated by the checkpoint response in the presence of DNA damage and fork stalling agents.

Titration of four replication factors is essential for the Xenopus laevis midblastula transition.

Regulating the MBT It has been known for more than 30 years that a defined number of cell divisions in the frog embryo precede a crucial developmental event called the midblastula transition (MBT). Collart et al. (p. 893, published online 1 August) now elucidate a mechanism involved in the control of the MBT. DNA replication initiation factors are titrated out during early cell divisions, which controls the elongation of the cell cycle and the onset of zygotic transcription during the MBT. Increasing numbers of nuclei compared with the cytoplasmic volume promotes a key developmental step in frog embryos. The rapid, reductive early divisions of many metazoan embryos are followed by the midblastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the MBT. We show that four DNA replication factors—Cut5, RecQ4, Treslin, and Drf1—are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis. The levels of these factors regulate multiple events of the MBT, including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.

Quantitative Proteomics Reveals the Basis for the Biochemical Specificity of the Cell Cycle Machinery

Cyclin-dependent kinases comprise the conserved machinery that drives progress through the cell cycle, but how they do this in mammalian cells is still unclear. To identify the mechanisms by which cyclin-cdks control the cell cycle, we performed a time-resolved analysis of the in vivo interactors of cyclins E1, A2, and B1 by quantitative mass spectrometry. This global analysis of context-dependent protein interactions reveals the temporal dynamics of cyclin function in which networks of cyclin-cdk interactions vary according to the type of cyclin and cell-cycle stage. Our results explain the temporal specificity of the cell-cycle machinery, thereby providing a biochemical mechanism for the genetic requirement for multiple cyclins in vivo and reveal how the actions of specific cyclins are coordinated to control the cell cycle. Furthermore, we identify key substrates (Wee1 and c15orf42/Sld3) that reveal how cyclin A is able to promote both DNA replication and mitosis.

A novel human Ada2 homologue functions with Gcn5 or Brg1 to coactivate transcription.

ABSTRACT In yeast, the transcriptional adaptor yeast Ada2 (yAda2) is a part of the multicomponent SAGA complex, which possesses histone acetyltransferase activity through action of the yGcn5 catalytic enzyme. yAda2, among several SAGA proteins, serves to recruit SAGA to genes via interactions with promoter-bound transcription factors. Here we report identification of a new human Ada2 homologue, hAda2β. Ada2β differs both biochemically and functionally from the previously characterized hAda2α, which is a stable component of the human PCAF (human Gcn5 homologue) acetylase complex. Ada2β, relative to Ada2α, interacted selectively, although not stably, with the Gcn5-containing histone acetylation complex TFTC/STAGA. In addition, Ada2β interacted with Baf57 (a component of the human Swi/Snf complex) in a yeast two-hybrid screen and associated with human Swi/Snf in vitro. In functional assays, hAda2β (but not Ada2α), working in concert with Gcn5 (but not PCAF) or Brg1 (the catalytic component of hSwi/Snf complex), increased transcription via the B-cell-specific transcription factor Pax5/BSAP. These findings support the view that Gcn5 and PCAF have distinct roles in vivo and suggest a new mechanism of coactivator function, in which a single adaptor protein (Ada2β) can coordinate targeting of both histone acetylation and chromatin remodeling activities.