Tatyana I. Kalashnikova

Histone H2A.Z Regulates Transcription and Is Partially Redundant with Nucleosome Remodeling Complexes

Nucleosomes impose a block to transcription that can be overcome in vivo by remodeling complexes such as SNF/SWI and histone modification complexes such as SAGA. Mutations in the major core histones relieve transcriptional repression and bypass the requirement for SNF/SWI and SAGA. We have found that the variant histone H2A.Z regulates gene transcription, and deletion of the gene encoding H2A.Z strongly increases the requirement for SNF/SWI and SAGA. This synthetic genetic interaction is seen at the level of single genes and acts downstream of promoter nucleosome reorganization. H2A.Z is preferentially crosslinked in vivo to intergenic DNA at the PH05 and GAL1 loci, and this association changes with transcriptional activation. These results describe a novel pathway for regulating transcription using variant histones to modulate chromatin structure.

Cln3 activates G1-specific transcription via phosphorylation of the SBF bound repressor Whi5.

G1-specific transcriptional activation by Cln3/CDK initiates the budding yeast cell cycle. To identify targets of Cln3/CDK, we analyzed the SBF and MBF transcription factor complexes by multidimensional protein interaction technology (MudPIT). Whi5 was identified as a stably bound component of SBF but not MBF. Inactivation of Whi5 leads to premature expression of G1-specific genes and budding, whereas overexpression retards those processes. Whi5 inactivation bypasses the requirement for Cln3 both for transcriptional activation and cell cycle initiation. Whi5 associates with G1-specific promoters via SBF during early G1 phase, then dissociates coincident with transcriptional activation. Dissociation of Whi5 is promoted by Cln3 in vivo. Cln/CDK phosphorylation of Whi5 in vitro promotes its dissociation from SBF complexes. Mutation of putative CDK phosphorylation sites, at least five of which are phosphorylated in vivo, strongly reduces SBF-dependent transcription and delays cell cycle initiation. Like mammalian Rb, Whi5 is a G1-specific transcriptional repressor antagonized by CDK.

Regulation and recognition of SCFGrr1 targets in the glucose and amino acid signaling pathways.

ABSTRACT SCFGrr1, one of several members of the SCF family of E3 ubiquitin ligases in budding Saccharomyces cerevisiae, is required for both regulation of the cell cycle and nutritionally controlled transcription. In addition to its role in degradation of Gic2 and the CDK targets Cln1 and Cln2, Grr1 is also required for induction of glucose- and amino acid-regulated genes. Induction of HXT genes by glucose requires the Grr1-dependent degradation of Mth1. We show that Mth1 is ubiquitinated in vivo and degraded via the proteasome. Furthermore, phosphorylated Mth1, targeted by the casein kinases Yck1/2, binds to Grr1. That binding depends upon the Grr1 leucine-rich repeat (LRR) domain but not upon the F-box or basic residues within the LRR that are required for recognition of Cln2 and Gic2. Those observations extend to a large number of Grr1-dependent genes, some targets of the amino acid-regulated SPS signaling system, which are properly regulated in the absence of those basic LRR residues. Finally, we show that regulation of the SPS targets requires the Yck1/2 casein kinases. We propose that casein kinase I plays a similar role in both nutritional signaling pathways by phosphorylating pathway components and targeting them for ubiquitination by SCFGrr1.

DNA replication checkpoint promotes G1-S transcription by inactivating the MBF repressor Nrm1

The cell cycle transcriptional program imposes order on events of the cell-cycle and is a target for signals that regulate cell-cycle progression, including checkpoints required to maintain genome integrity. Neither the mechanism nor functional significance of checkpoint regulation of the cell-cycle transcription program are established. We show that Nrm1, an MBF-specific transcriptional repressor acting at the transition from G1 to S phase of the cell cycle, is at the nexus between the cell cycle transcriptional program and the DNA replication checkpoint in fission yeast. Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. This leads to the expression of genes encoding components that function in DNA replication and repair pathways important for cell survival in response to arrested DNA replication.

Histone-Histone Interactions and Centromere Function

ABSTRACT Cse4p is a structural component of the core centromere ofSaccharomyces cerevisiae and is a member of the conserved CENP-A family of specialized histone H3 variants. The histone H4 allelehhf1-20 confers defects in core centromere chromatin structure and mitotic chromosome transmission. We have proposed that Cse4p and histone H4 interact through their respective histone fold domains to assemble a nucleosome-like structure at centromeric DNA. To test this model, we targeted random mutations to the Cse4p histone fold domain and isolated three temperature-sensitive cse4alleles in an unbiased genetic screen. Two of the cse4alleles contain mutations at the Cse4p-H4 interface. One of these requires two widely separated mutations demonstrating long-range cooperative interactions in the structure. The third cse4allele is mutated at its helix 2-helix 3 interface, a region required for homotypic H3 fold dimerization. Overexpression of wild-type Cse4p and histone H4 confer reciprocal allele-specific suppression ofcse4 and hhf1 mutations, providing strong evidence for Cse4p-H4 protein interaction. Overexpression of histone H3 is dosage lethal in cse4 mutants, suggesting that histone H3 competes with Cse4p for histone H4 binding. However, the relative resistance of the Cse4p-H4 pathway to H3 interference argues that centromere chromatin assembly must be highly regulated.

DNA replication stress differentially regulates G1/S genes via Rad53‐dependent inactivation of Nrm1

MBF and SBF transcription factors regulate a large family of coordinately expressed G1/S genes required for early cell‐cycle functions including DNA replication and repair. SBF is inactivated upon S‐phase entry by Clb/CDK whereas MBF targets are repressed by the co‐repressor, Nrm1. Using genome‐wide expression analysis of cells treated with methyl methane sulfonate (MMS), hydroxyurea (HU) or camptothecin (CPT), we show that genotoxic stress during S phase specifically induces MBF‐regulated genes. This occurs via direct phosphorylation of Nrm1 by Rad53, the effector checkpoint kinase, which prevents its binding to MBF target promoters. We conclude that MBF‐regulated genes are distinguished from SBF‐regulated genes by their sensitivity to activation by the S‐phase checkpoint, thereby, providing an effective mechanism for enhancing DNA replication and repair and promoting genome stability.

Stb1 Collaborates with Other Regulators To Modulate the G1-Specific Transcriptional Circuit

ABSTRACT G1-specific transcription in the budding yeast Saccharomyces cerevisiae depends upon SBF and MBF. Whereas inactivation of SBF-regulated genes during the G1/S transition depends upon mitotic B-type cyclins, inactivation of MBF has been reported to involve multiple regulators, Nrm1 and Stb1. Nrm1 is a transcriptional corepressor that inactivates MBF-regulated transcription via negative feedback as cells exit G1 phase. Cln/cyclin-dependent kinase (CDK)-dependent inactivation of Stb1, identified via its interaction with the histone deacetylase (HDAC) component Sin3, has also been reported to inactivate MBF-regulated transcription. This report shows that Stb1 is a stable component of both SBF and MBF that binds G1-specific promoters via Swi6 during G1 phase. It is important for the growth of cells in which SBF or MBF is inactive. Although dissociation of Stb1 from promoters as cells exit G1 correlates with Stb1 phosphorylation, phosphorylation is only partially dependent upon Cln1/2 and is not involved in transcription inactivation. Inactivation depends upon Nrm1 and Clb/CDK activity. Stb1 inactivation dampens maximal transcriptional induction during late G1 phase and also derepresses gene expression in G1-phase cells prior to Cln3-dependent transcriptional activation. The repression during G1 also depends upon Sin3. We speculate that the interaction between Stb1 and Sin3 regulates the Sin3/HDAC complex at G1-specific promoters.

THE SBF AND MBF-ASSOCIATED PROTEIN MSA1 IS REQUIRED FOR PROPER TIMING OF G1-SPECIFIC TRANSCRIPTION IN SACCHAROMYCES CEREVISIAE

In the budding yeast Saccharomyces cerevisiae, cell cycle initiation is prompted during G1 phase by Cln3/cyclin-dependent protein kinase-mediated transcriptional activation of G1-specific genes. A recent screening performed to reveal novel interactors of SCB-binding factor (SBF) and MCB-binding factor (MBF) identified, in addition to the SBF-specific repressor Whi5 and the MBF-specific corepressor Nrm1, a pair of homologous proteins, Msa1 and Msa2 (encoded by YOR066w and YKR077w), as interactors of SBF and MBF, respectively. MSA1 is expressed periodically during the cell cycle with peak mRNA levels occurring at the late M/early G1 phase and peak protein levels occurring in early G1. Msa1 associates with SBF- and MBF-regulated target promoters consistent with a role in G1-specific transcriptional regulation. Msa1 affects cell cycle initiation by advancing the timing of transcription of G1-specific genes. Msa1 binds to SBF- and MBF-regulated promoters and binding is maximal during the G1 phase. Binding depends upon the cognate transcription factor. Msa1 overexpression advances the timing of SBF-dependent transcription and budding, whereas depletion delays both indicators of cell cycle initiation. Similar effects on MBF-regulated transcription are observed. Based upon these results, we conclude that Msa1 acts to advance the timing of G1-specific transcription and cell cycle initiation.

Repression of G1/S transcription is mediated via interaction of the GTB motifs of Nrm1 and Whi5 with Swi6.

ABSTRACT In Saccharomyces cerevisiae, G1/S transcription factors MBF and SBF regulate a large family of genes important for entry to the cell cycle and DNA replication and repair. Their regulation is crucial for cell viability, and it is conserved throughout evolution. MBF and SBF consist of a common component, Swi6, and a DNA-specific binding protein, Mbp1 and Swi4, respectively. Transcriptional repressors bind to and regulate the activity of both transcription factors. Whi5 binds to SBF and represses its activity at the beginning of the G1 phase to prevent early activation. Nrm1 binds to MBF to repress transcription as cells progress through S phase. Here, we describe a protein motif, the GTB motif (for G1/S transcription factor binding), in Nrm1 and Whi5 that is required to bind to the transcription factors. We also identify a region of the carboxy terminus of Swi6 that is required for Nrm1 and Whi5 binding to their target transcription factors and show that mutation of this region overrides the repression of MBF- and SBF-regulated genes by Nrm1 and Whi5. Finally, we show that the GTB motif is the core of a functional module that is necessary and sufficient for targeting of the transcription factors by their cognate repressors.