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1.
bioRxiv ; 2024 May 13.
Article in English | MEDLINE | ID: mdl-38798450

ABSTRACT

Nuclear pore proteins (Nups) in yeast, flies and mammals physically interact with hundreds or thousands of chromosomal sites, which impacts transcriptional regulation. In budding yeast, transcription factors mediate interaction of Nups with enhancers of highly active genes. To define the molecular basis of this mechanism, we exploited a separation-of-function mutation in the Gcn4 transcription factor that blocks its interaction with the nuclear pore complex (NPC) without altering its DNA binding or activation domains. SILAC mass spectrometry revealed that this mutation reduces the interaction of Gcn4 with the highly conserved nuclear export factor Crm1/Xpo1. Crm1 both interacts with the same sites as Nups genome-wide and is required for Nup2 to interact with the yeast genome. In vivo, Crm1 undergoes extensive and stable interactions with the NPC. In vitro, Crm1 binds to Gcn4 and these proteins form a complex with the nuclear pore protein Nup2. Importantly, the interaction between Crm1 and Gcn4 does not require Ran-GTP, suggesting that it is not through the nuclear export sequence binding site. Finally, Crm1 stimulates DNA binding by Gcn4, supporting a model in which allosteric coupling between Crm1 binding and DNA binding permits docking of transcription factor-bound enhancers at the NPC.

2.
NAR Genom Bioinform ; 6(1): lqae012, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38327869

ABSTRACT

Defining the in vivo DNA binding specificity of transcription factors (TFs) has relied nearly exclusively on chromatin immunoprecipitation (ChIP). While ChIP reveals TF binding patterns, its resolution is low. Higher resolution methods employing nucleases such as ChIP-exo, chromatin endogenous cleavage (ChEC-seq) and CUT&RUN resolve both TF occupancy and binding site protection. ChEC-seq, in which an endogenous TF is fused to micrococcal nuclease, requires neither fixation nor antibodies. However, the specificity of DNA cleavage during ChEC has been suggested to be lower than the specificity of the peaks identified by ChIP or ChIP-exo, perhaps reflecting non-specific binding of transcription factors to DNA. We have simplified the ChEC-seq protocol to minimize nuclease digestion while increasing the yield of cleaved DNA. ChEC-seq2 cleavage patterns were highly reproducible between replicates and with published ChEC-seq data. Combined with DoubleChEC, a new bioinformatic pipeline that removes non-specific cleavage sites, ChEC-seq2 identified high-confidence cleavage sites for three different yeast TFs that are strongly enriched for their known binding sites and adjacent to known target genes.

3.
bioRxiv ; 2023 Oct 18.
Article in English | MEDLINE | ID: mdl-37905156

ABSTRACT

Defining the in vivo DNA binding specificity of transcription factors (TFs) has relied nearly exclusively on chromatin immunoprecipitation (ChIP). While ChIP reveals TF binding patterns, its resolution is low. Higher resolution methods employing nucleases such as ChIP-exo, chromatin endogenous cleavage (ChEC-seq) and CUT&RUN resolve both TF occupancy and binding site protection. ChEC-seq, in which an endogenous TF is fused to micrococcal nuclease, requires neither fixation nor antibodies. However, the specificity of DNA cleavage during ChEC has been suggested to be lower than the specificity of the peaks identified by ChIP or ChIP-exo, perhaps reflecting non-specific binding of transcription factors to DNA. We have simplified the ChEC-seq protocol to minimize nuclease digestion while increasing the yield of cleaved DNA. ChEC-seq2 cleavage patterns were highly reproducible between replicates and with published ChEC-seq data. Combined with DoubleChEC, a new bioinformatic pipeline that removes non-specific cleavage sites, ChEC-seq2 identified high-confidence cleavage sites for three different yeast TFs that are strongly enriched for their known binding sites and adjacent to known target genes.

4.
Dev Cell ; 49(6): 936-947.e4, 2019 06 17.
Article in English | MEDLINE | ID: mdl-31211995

ABSTRACT

Loss of nuclear pore complex (NPC) proteins, transcription factors (TFs), histone modification enzymes, Mediator, and factors involved in mRNA export disrupts the physical interaction of chromosomal sites with NPCs. Conditional inactivation and ectopic tethering experiments support a direct role for the TFs Gcn4 and Nup2 in mediating interaction with the NPC but suggest an indirect role for factors involved in mRNA export or transcription. A conserved "positioning domain" within Gcn4 controls interaction with the NPC and inter-chromosomal clustering and promotes transcription of target genes. Such a function may be quite common; a comprehensive screen reveals that tethering of most yeast TFs is sufficient to promote targeting to the NPC. While some TFs require Nup100, others do not, suggesting two distinct targeting mechanisms. These results highlight an important and underappreciated function of TFs in controlling the spatial organization of the yeast genome through interaction with the NPC.


Subject(s)
Basic-Leucine Zipper Transcription Factors/metabolism , Chromatin/metabolism , Genome, Fungal , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Active Transport, Cell Nucleus , Basic-Leucine Zipper Transcription Factors/genetics , Chromatin/genetics , Nuclear Pore/genetics , Nuclear Pore Complex Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
5.
Mol Biol Cell ; 27(19): 2980-93, 2016 10 01.
Article in English | MEDLINE | ID: mdl-27489341

ABSTRACT

On activation, the GAL genes in yeast are targeted to the nuclear periphery through interaction with the nuclear pore complex. Here we identify two cis-acting "DNA zip codes" from the GAL1-10 promoter that are necessary and sufficient to induce repositioning to the nuclear periphery. One of these zip codes, GRS4, is also necessary and sufficient to promote clustering of GAL1-10 alleles. GRS4, and to a lesser extent GRS5, contribute to stronger expression of GAL1 and GAL10 by increasing the fraction of cells that respond to the inducer. The molecular mechanism controlling targeting to the NPC is distinct from the molecular mechanism controlling interchromosomal clustering. Targeting to the nuclear periphery and interaction with the nuclear pore complex are prerequisites for gene clustering. However, once formed, clustering can be maintained in the nucleoplasm, requires distinct nuclear pore proteins, and is regulated differently through the cell cycle. In addition, whereas targeting of genes to the NPC is independent of transcription, interchromosomal clustering requires transcription. These results argue that zip code-dependent gene positioning at the nuclear periphery and interchromosomal clustering represent interdependent phenomena with distinct molecular mechanisms.


Subject(s)
Galactokinase/genetics , Galactokinase/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Trans-Activators/metabolism , Cell Nucleus/metabolism , Gene Expression Regulation, Fungal/genetics , Multigene Family , Nuclear Pore/metabolism , Nuclear Pore Complex Proteins/metabolism , Promoter Regions, Genetic/genetics , Protein Transport/genetics , Protein Transport/physiology , Saccharomyces cerevisiae/metabolism , Trans-Activators/genetics , Transcription Factors/metabolism , Transcription, Genetic
6.
Microb Cell ; 2(12): 481-490, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26688804

ABSTRACT

Many genes localize at the nuclear periphery through physical interaction with the nuclear pore complex (NPC). We have found that the yeast INO1 gene is targeted to the NPC both upon activation and for several generations after repression, a phenomenon called epigenetic transcriptional memory. Targeting of INO1 to the NPC requires distinct cis-acting promoter DNA zip codes under activating conditions and under memory conditions. When at the nuclear periphery, active INO1 clusters with itself and with other genes that share the GRS I zip code. Here, we show that during memory, the two alleles of INO1 cluster in diploids and endogenous INO1 clusters with an ectopic INO1 in haploids. After repression, INO1 does not cluster with GRS I - containing genes. Furthermore, clustering during memory requires Nup100 and two sets of DNA zip codes, those that target INO1 to the periphery when active and those that target it to the periphery after repression. Therefore, the interchromosomal clustering of INO1 that occurs during transcriptional memory is dependent upon, but mechanistically distinct from, the clustering of active INO1. Finally, while localization to the nuclear periphery is not regulated through the cell cycle during memory, clustering of INO1 during memory is regulated through the cell cycle.

7.
Methods Cell Biol ; 122: 463-85, 2014.
Article in English | MEDLINE | ID: mdl-24857743

ABSTRACT

Many genes in budding yeast Saccharomyces cerevisiae associate with the nuclear pore complex (NPC), which impacts their location within the nucleus and their transcriptional regulation. To understand how eukaryotic genomes are spatially organized, we have used multiple approaches for analyzing the localization and transcription of genes. We have used these approaches to study the role of DNA elements in targeting genomic loci to the NPC and how these interactions regulate transcription, chromatin structure and the spatial organization of the yeast genome. These studies combine yeast molecular genetics with live-cell microscopy and biochemistry. Here, we present detailed protocols for these cytological and molecular approaches.


Subject(s)
DNA, Fungal/genetics , Genome, Fungal/genetics , Lac Operon/genetics , Nuclear Pore/genetics , Cell Line, Tumor , Chromatin Immunoprecipitation/methods , Genetic Variation , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Microscopy, Confocal/methods , Plasmids/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription, Genetic
8.
Genetics ; 193(3): 829-51, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23335340

ABSTRACT

There is increasing evidence that certain Vacuolar protein sorting (Vps) proteins, factors that mediate vesicular protein trafficking, have additional roles in regulating transcription factors at the endosome. We found that yeast mutants lacking the phosphatidylinositol 3-phosphate [PI(3)P] kinase Vps34 or its associated protein kinase Vps15 display multiple phenotypes indicating impaired transcription elongation. These phenotypes include reduced mRNA production from long or G+C-rich coding sequences (CDS) without affecting the associated GAL1 promoter activity, and a reduced rate of RNA polymerase II (Pol II) progression through lacZ CDS in vivo. Consistent with reported genetic interactions with mutations affecting the histone acetyltransferase complex NuA4, vps15Δ and vps34Δ mutations reduce NuA4 occupancy in certain transcribed CDS. vps15Δ and vps34Δ mutants also exhibit impaired localization of the induced GAL1 gene to the nuclear periphery. We found unexpectedly that, similar to known transcription elongation factors, these and several other Vps factors can be cross-linked to the CDS of genes induced by Gcn4 or Gal4 in a manner dependent on transcriptional induction and stimulated by Cdk7/Kin28-dependent phosphorylation of the Pol II C-terminal domain (CTD). We also observed colocalization of a fraction of Vps15-GFP and Vps34-GFP with nuclear pores at nucleus-vacuole (NV) junctions in live cells. These findings suggest that Vps factors enhance the efficiency of transcription elongation in a manner involving their physical proximity to nuclear pores and transcribed chromatin.


Subject(s)
Class III Phosphatidylinositol 3-Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Transcription Elongation, Genetic , Vacuolar Sorting Protein VPS15/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Cell Nucleus/metabolism , Class III Phosphatidylinositol 3-Kinases/genetics , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , GC Rich Sequence , Galactokinase/genetics , Galactokinase/metabolism , Gene Deletion , Histone Acetyltransferases/metabolism , Nuclear Pore/metabolism , Phenotype , Phosphorylation , Promoter Regions, Genetic , Protein Transport , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Vacuolar Sorting Protein VPS15/genetics , Vacuoles/metabolism
9.
Dev Cell ; 22(6): 1234-46, 2012 Jun 12.
Article in English | MEDLINE | ID: mdl-22579222

ABSTRACT

Active genes in yeast can be targeted to the nuclear periphery through interaction of cis-acting "DNA zip codes" with the nuclear pore complex. We find that genes with identical zip codes cluster together. This clustering was specific; pairs of genes that were targeted to the nuclear periphery by different zip codes did not cluster together. Insertion of two different zip codes (GRS I or GRS III) at an ectopic site induced clustering with endogenous genes that have that zip code. Targeting to the nuclear periphery and interaction with the nuclear pore is a prerequisite for gene clustering, but clustering can be maintained in the nucleoplasm. Finally, we find that the Put3 transcription factor recognizes the GRS I zip code to mediate both targeting to the NPC and interchromosomal clustering. These results suggest that zip-code-mediated clustering of genes at the nuclear periphery influences the three-dimensional arrangement of the yeast genome.


Subject(s)
Chromosomes, Fungal/metabolism , DNA/genetics , Gene Expression Regulation, Fungal , Glycine-tRNA Ligase/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism , Chromosomes, Fungal/genetics , Glycine-tRNA Ligase/genetics , Multigene Family , Nuclear Pore/metabolism , Promoter Regions, Genetic , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
10.
Cell Cycle ; 10(3): 392-5, 2011 Feb 01.
Article in English | MEDLINE | ID: mdl-21228627

ABSTRACT

In yeast, many genes are targeted to the nuclear periphery through interaction with the Nuclear Pore Complex upon activation. Targeting requires nucleoporin proteins and DNA elements in the promoters of these genes. We have recently found that targeting is regulated through the cell cycle. Immediately following the initiation of DNA replication, active genes lose peripheral localization for ~30 minutes. This regulation is mediated by cyclic phosphorylation of a nucleoporin by Cdk1. Some genes that are targeted to the nuclear periphery upon activation remain at the nuclear periphery after repression, a phenomenon called transcriptional memory. Curiously, the mechanism that regulates localization of active genes to the nuclear periphery does not regulate the localization of the same genes after repression, suggesting that these genes are targeted by two distinct mechanisms. Finally, the localization of other parts of the genome that localize at the nuclear periphery seem to be regulated by a distinct mechanism, suggesting that the spatial organization of the genome is tightly coupled to the progression of the cell cycle.


Subject(s)
CDC2 Protein Kinase/physiology , Models, Genetic , Nuclear Pore/metabolism , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/genetics , Biomarkers/analysis , Cell Cycle , Nuclear Envelope/ultrastructure , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore Complex Proteins/physiology , Phosphorylation , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Telomere/genetics , Telomere/metabolism , Transcriptional Activation
11.
Methods Enzymol ; 470: 569-80, 2010.
Article in English | MEDLINE | ID: mdl-20946825

ABSTRACT

DNA within the yeast nucleus is spatially organized. Yeast telomeres cluster together at the nuclear periphery, centromeres cluster together near the spindle pole body, and both the rDNA repeats and tRNA genes cluster within the nucleolus. Furthermore, the localization of individual genes to subnuclear compartments can change with changes in transcriptional status. As such, yeast researchers interested in understanding nuclear events may need to determine the subnuclear localization of parts of the genome. This chapter describes a straightforward quantitative approach using immunofluorescence and confocal microscopy to localize chromosomal loci with respect to well characterized nuclear landmarks.


Subject(s)
Chromosomes, Fungal/genetics , Fluorescent Antibody Technique/methods , Yeasts/genetics , Cell Nucleolus/genetics , Cell Nucleus/genetics , Centromere/genetics , Microscopy, Confocal , Microscopy, Fluorescence , Telomere/genetics
12.
Mol Biol Cell ; 21(19): 3421-32, 2010 Oct 01.
Article in English | MEDLINE | ID: mdl-20702586

ABSTRACT

Many inducible genes in yeast are targeted to the nuclear pore complex when active. We find that the peripheral localization of the INO1 and GAL1 genes is regulated through the cell cycle. Active INO1 and GAL1 localized at the nuclear periphery during G1, became nucleoplasmic during S-phase, and then returned to the nuclear periphery during G2/M. Loss of peripheral targeting followed the initiation of DNA replication and was lost in cells lacking a cyclin-dependent kinase (Cdk) inhibitor. Furthermore, the Cdk1 kinase and two Cdk phosphorylation sites in the nucleoporin Nup1 were required for peripheral targeting of INO1 and GAL1. Introduction of aspartic acid residues in place of either of these two sites in Nup1 bypassed the requirement for Cdk1 and resulted in targeting of INO1 and GAL1 to the nuclear periphery during S-phase. Thus, phosphorylation of a nuclear pore component by cyclin dependent kinase controls the localization of active genes to the nuclear periphery through the cell cycle.


Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle/genetics , Galactokinase/genetics , Genes, Fungal/genetics , Myo-Inositol-1-Phosphate Synthase/genetics , Nuclear Pore Complex Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/cytology , Cell Cycle/drug effects , DNA Replication/drug effects , Galactokinase/metabolism , Mutation/genetics , Myo-Inositol-1-Phosphate Synthase/metabolism , Nuclear Envelope/drug effects , Nuclear Envelope/enzymology , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Repressor Proteins/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism
13.
PLoS Biol ; 5(4): e81, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17373856

ABSTRACT

Many genes are recruited to the nuclear periphery upon transcriptional activation. The mechanism and functional significance of this recruitment is unclear. We find that recruitment of the yeast INO1 and GAL1 genes to the nuclear periphery is rapid and independent of transcription. Surprisingly, these genes remain at the periphery for generations after they are repressed. Localization at the nuclear periphery serves as a form of memory of recent transcriptional activation, promoting reactivation. Previously expressed GAL1 at the nuclear periphery is activated much more rapidly than long-term repressed GAL1 in the nucleoplasm, even after six generations of repression. Localization of INO1 at the nuclear periphery is necessary and sufficient to promote more rapid activation. This form of transcriptional memory is chromatin based; the histone variant H2A.Z is incorporated into nucleosomes within the recently repressed INO1 promoter and is specifically required for rapid reactivation of both INO1 and GAL1. Furthermore, H2A.Z is required to retain INO1 at the nuclear periphery after repression. Therefore, H2A.Z-mediated localization of recently repressed genes at the nuclear periphery represents an epigenetic state that confers memory of transcriptional activation and promotes reactivation.


Subject(s)
Cell Nucleus/genetics , Epigenesis, Genetic , Histones/physiology , Transcription, Genetic , Base Sequence , Chromatin Immunoprecipitation , DNA Primers , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae Proteins/genetics
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