RAP, RAP, open up! New wrinkles for RAP1 in yeast

doi:10.1016/S0168-9525(99)01936-8

Trends in Genetics

Volume 16, Issue 2, 1 February 2000, Pages 51-53

https://doi.org/10.1016/S0168-9525(99)01936-8 Get rights and content

Abstract

RAP1 (repressor/activator protein 1) from budding yeast is well known for its involvement in gene activation and repression, telomere structure and function, and replication. Recent studies have examined additional roles for RAP1 in heterochromatin boundary-element formation, creation of hotspots for meiotic recombination, and chromatin opening. These studies provide new insight into the ability of this abundant DNA-binding protein to participate in a diverse array of functions taking place in a chromatin environment.

Section snippets

RAP1 and boundary elements

Boundary elements are DNA sequences that can delimit the spread of heterochromatin and/or euchromatin, thus defining a discrete border between the two. Bi and Broach⁴ sought to identify such elements by looking for sequences that would, when strategically placed, prevent silencing of reporter genes placed into the homothallic mating loci, HML and HMR, of the yeast S. cerevisiae. Although one could quibble about equating HML and HMR with metazoan heterochromatin, they share important

RAP1 and chromatin opening

One way in which RAP1 could contribute to creating a boundary element is by preventing the formation of one or more nucleosomes in the vicinity of the RAP1-binding sites, thus creating a gap in the local chromatin that might inhibit the spread of heterochromatin⁴. A similar role for RAP1 in opening chromatin has been proposed in the context of transcription⁷. The yeast HIS4 gene can be activated independently by GCN4 or by BAS1 and BAS2; in either case, a RAP1-binding site in the promoter is

RAP1 and meiotic recombination

Domains of RAP1 have been assigned to some of its functions1, 2 (Fig. 1). RAP1 binding induces a 50–60° bend in DNA in vitro, and the N-terminal third of the protein is needed for this property. The central third of the protein is needed for DNA binding, and adjacent to this region is a short (∼66 bp) segment that can activate transcription in the context of GAL4 fusions. Also in the C-terminal third of the protein are regions that are needed for telomeric silencing, and for interaction with

Chromatin opening: a common theme?

Chromatin opening by RAP1 was shown to assist GCN4 binding at the HIS4 promoter⁶, and was suggested to be important in boundary-element formation by TEF promoter sequences (Ref. 4). Similarly, it seems likely that chromatin opening by RAP1 is an important determinant of meiotic hotspot activity at the HIS4 promoter⁵. Meiotic hotspots are associated with high levels of meiosis-specific double-strand breaks in DNA, and these breaks are found in regions of open chromatin, as defined by

Acknowledgements

Thanks to T. Petes for critically reading the manuscript. Work in the author’s laboratory is supported by grant GM51993 from the NIH.

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Cited by (101)

Fundamentals of G-quadruplex biology
2020, Annual Reports in Medicinal Chemistry
Several decades elapsed between the first descriptions of G-quadruplex nucleic acid structures (G4s) assembled in vitro and the emergence of experimental findings indicating that such structures can form and function in living systems. A large body of evidence now supports roles for G4s in many aspects of nucleic acid biology, spanning processes from transcription and chromatin structure, mRNA processing, protein translation, DNA replication and genome stability, and telomere and mitochondrial function. Nonetheless, it must be acknowledged that some of this evidence is tentative, which is not surprising given the technical challenges associated with demonstrating G4s in biology. Here I provide an overview of evidence for G4 biology, focusing particularly on the many potential pitfalls that can be encountered in its investigation, and briefly discuss some of broader biological processes that may be impacted by G4s including cancer.
Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant
2017, Journal of Biological Chemistry
Repressor activator protein 1 (Rap1) performs multiple vital cellular functions in the budding yeast Saccharomyces cerevisiae. These include regulation of telomere length, transcriptional repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of mRNA-encoding genes, including the highly transcribed ribosomal protein- and glycolytic enzyme-encoding genes. Studies of the contributions of Rap1 to telomere length regulation and transcriptional repression have yielded significant mechanistic insights. However, the mechanism of Rap1 transcriptional activation remains poorly understood because Rap1 is encoded by a single copy essential gene and is involved in many disparate and essential cellular functions, preventing easy interpretation of attempts to directly dissect Rap1 structure-function relationships. Moreover, conflicting reports on the ability of Rap1-heterologous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenge use of this approach to study Rap1. Described here is the development of an altered DNA-binding specificity variant of Rap1 (Rap1^AS). We used Rap1^AS to map and characterize a 41-amino acid activation domain (AD) within the Rap1 C terminus. We found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. In summary, we show here that Rap1 contains an AD required for Rap1-dependent gene transcription. The Rap1^AS variant will likely also be useful for studies of the functions of Rap1 in other biological pathways.
Role of CK2-dependent phosphorylation of Ifh1 and Crf1 in transcriptional regulation of ribosomal protein genes in Saccharomyces cerevisiae
2016, Biochimica et Biophysica Acta - Gene Regulatory Mechanisms
In Saccharomyces cerevisiae, Fhl1 is involved in the regulation of ribosomal protein (RP) genes through interaction with either its coactivator Ifh1 or corepressor Crf1, depending on nutrient conditions. Interaction of Fhl1 with Ifh1 or Crf1 is achieved through a forkhead-associated (FHA) domain of Fhl1, which binds to forkhead-binding (FHB) domains of Ifh1 and Crf1. Here, we demonstrate that CK2-dependent phosphorylation of T681 and T348 residues, located in the FHB domains of Ifh1 and Crf1, respectively, provides binding sites for the FHA domain of Fhl1. Cells expressing Ifh1^T681A mutant showed reduced association of Ifh1 at the RP gene promoters and decreased levels of RP gene transcripts, thereby reducing the growth rate. On the other hand, cells expressing Crf1^T348A showed a defect in repressing RP gene transcription upon inhibition of target of rapamycin complex 1 (TORC1) by rapamycin treatment. Taken together, these findings suggest the mechanisms by which CK2-dependent recruitment of Ifh1 and Crf1 at the RP gene promoters governs the transcription of RP genes.
The wrapping loop and Rap1 C-terminal (RCT) domain of yeast Rap1 modulate access to different DNA binding modes
2015, Journal of Biological Chemistry
Budding yeast Rap1 is a specific double-stranded DNA-binding protein involved in repression and activation of gene transcription and in the establishment of the nucleoprotein complex formed at telomeres. The DNA-binding domain (DBD) of Rap1 forms a high affinity complex with DNA where both Myb-like domains bind to the recognition site. However, we recently showed that the DBD can also access an alternative, lower affinity DNA-binding mode where a single Myb-like domain binds. This results in Rap1-DNA complexes with stoichiometry higher than previously anticipated. In this work, we show that the ability of the DBD to form higher stoichiometry complexes on DNA is maintained also in larger Rap1 constructs. This indicates that transition between at least two DNA-binding modes is a general property of the protein and not a specific feature of the DBD in isolation. The transition between binding modes is modulated by the C-terminal wrapping loop within the DBD, consistent with the proposed model in which the transient opening of this region allows a switch between binding modes. Finally, we provide evidence that the Rap1 C terminus interacts with the DNA-binding domain, suggesting a complex network of interactions that couples changes in conformation of the protein to the binding of its DNA recognition sequence.
Background: Rap1 is a general regulator of transcription and a component of the telomere.
Results: Rap1 binds DNA in at least two binding modes.
Conclusion: Access to binding modes in vitro is affected by different regions of the protein.
Significance: The ability of Rap1 to access different binding modes may impact its functions.
Adaptive response and tolerance to sugar and salt stress in the food yeast Zygosaccharomyces rouxii
2014, International Journal of Food Microbiology
Citation Excerpt :
Under osmotic stimuli, the transcriptional repressor activator protein Rap1 binds the GPD1 promoter and induced the GPD1 transcription. On the contrary, the specific inactivation of all Rap1 binding sites completely abolishes the osmostress-induced transcription of GPD1 (Eriksson et al., 2000; Morse, 2000). A third evidence for the epigenetic regulation of cellular osmo-adaptiation involves the HOG pathway.
The osmotolerant and halotolerant food yeast Zygosaccharomyces rouxii is known for its ability to grow and survive in the face of stress caused by high concentrations of non-ionic (sugars and polyols) and ionic (mainly Na⁺ cations) solutes. This ability determines the success of fermentation on high osmolarity food matrices and leads to spoilage of high sugar and high salt foods. The knowledge about the genes, the metabolic pathways, and the regulatory circuits shaping the Z. rouxii sugar and salt-tolerance, is a prerequisite to develop effective strategies for fermentation control, optimization of food starter culture, and prevention of food spoilage. This review summarizes recent insights on the mechanisms used by Z. rouxii and other osmo and halotolerant food yeasts to endure salts and sugars stresses. Using the information gathered from S. cerevisiae as guide, we highlight how these non-conventional yeasts integrate general and osmoticum-specific adaptive responses under sugar and salts stresses, including regulation of Na⁺ and K⁺-fluxes across the plasma membrane, modulation of cell wall properties, compatible osmolyte production and accumulation, and stress signalling pathways. We suggest how an integrated and system-based knowledge on these mechanisms may impact food and biotechnological industries, by improving the yeast spoilage control in food, enhancing the yeast-based bioprocess yields, and engineering the osmotolerance in other organisms.
Nuclear pore interactions with the genome
2014, Current Opinion in Genetics and Development
Citation Excerpt :
Consistent with a role for the NPC in regulating endogenous boundaries, loss of Nup2 leads to the spread of silencing from telomeres [50,51]. Furthermore, Nup-bound regions in yeast are enriched for the binding of the transcription factor Rap1, which also has boundary activity [52]. In a Drosophila embryonic cell line, NPC binding overlaps with the insulator protein, Suppressor of Hairy-wing [53].
Within the nucleus, chromatin is functionally organized into distinct nuclear compartments. The nuclear periphery, containing Nuclear Pore Complexes (NPCs), plays an important role in the spatial organization of chromatin and in transcriptional regulation. The role of Nuclear Pore Proteins (Nups) in transcription and their involvement in leukemia and viral integration has renewed interest in understanding their mechanism of action. Nups bind to both repressed and active genes, often in a regulated fashion. Nups can associate with chromatin both at the NPC and inside the nucleoplasm. These interactions are guided by evolutionarily conserved mechanisms that involve promoter DNA elements and trans-acting factors. These interactions can also lead to interchromosomal clustering of co-regulated genes. Nups affect gene expression by promoting stronger transcription, by limiting the spread of repressed chromatin or by altering chromatin structure. Nups can promote epigenetic regulation by establishing boundary elements and poising recently repressed genes for faster reactivation.

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Trends in Genetics

Abstract

Section snippets

RAP1 and boundary elements

RAP1 and chromatin opening

RAP1 and meiotic recombination

Chromatin opening: a common theme?

Acknowledgements

References (22)

RAP1: a protean regulator in yeast

Trends Genet.

Chromatin remodeling by transcriptional activation domains in a yeast episome

J. Biol. Chem.

The protein CTCF is required for the enhancer-blocking activity of vertebrate insulators

Cell

Repressor Activator Protein 1 and its ligands: organising chromatin domains

Nucleic Acids Mol. Biol.

A protein-counting mechanism for telomere length regulation in yeast

Science

UAS_rpg can function as a heterochromatin boundary element in yeast

Genes Dev.

Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain

Genetics

Chromatin opening and transactivator potentiation by RAP1 in Saccharomyces cerevisiae

Mol. Cell. Biol.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

Mol. Cell. Biol.

GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5′ TGACTC 3′ sequences

Proc. Natl. Acad. Sci. U. S. A.

In vivo analysis of functional regions within yeast Rap1p

Mol. Cell. Biol.

Cited by (101)

Fundamentals of G-quadruplex biology

Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant

Role of CK2-dependent phosphorylation of Ifh1 and Crf1 in transcriptional regulation of ribosomal protein genes in Saccharomyces cerevisiae

The wrapping loop and Rap1 C-terminal (RCT) domain of yeast Rap1 modulate access to different DNA binding modes

Adaptive response and tolerance to sugar and salt stress in the food yeast Zygosaccharomyces rouxii

Nuclear pore interactions with the genome

Trends in Genetics

OutlookRAP, RAP, open up! New wrinkles for RAP1 in yeast

Abstract

Section snippets

RAP1 and boundary elements

RAP1 and chromatin opening

RAP1 and meiotic recombination

Chromatin opening: a common theme?

Acknowledgements

Trends Genet.

J. Biol. Chem.

Cell

Repressor Activator Protein 1 and its ligands: organising chromatin domains

Nucleic Acids Mol. Biol.

A protein-counting mechanism for telomere length regulation in yeast

Science

UASrpg can function as a heterochromatin boundary element in yeast

Genes Dev.

Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain

Genetics

Chromatin opening and transactivator potentiation by RAP1 in Saccharomyces cerevisiae

Mol. Cell. Biol.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

Mol. Cell. Biol.

GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5′ TGACTC 3′ sequences

Proc. Natl. Acad. Sci. U. S. A.

In vivo analysis of functional regions within yeast Rap1p

Mol. Cell. Biol.

Outlook
RAP, RAP, open up! New wrinkles for RAP1 in yeast

UAS_rpg can function as a heterochromatin boundary element in yeast