https://www.cell.com/cell/pdf/S0092-8674(04)00448-9.pdf

Genome-wide localization of the nuclear transport machinery couples transcriptional status and nuclear organization.

Cells have evolved sophisticated multi-protein complexes that can regulate gene activity at various steps of the transcription process. Recent advances highlight the role of nuclear positioning in the control of gene expression and have put nuclear envelope components at centre stage. On the inner face of the nuclear envelope, active genes localize to nuclear-pore structures whereas silent chromatin localizes to non-pore sites. Nuclear-pore components seem to not only recruit the RNA-processing and RNA-export machinery, but contribute a level of regulation that might enhance gene expression in a heritable manner.

/pdf/the-nuclear-envelope-and-transcriptional-control-1zqaumd7cb.pdf

The nuclear envelope and transcriptional control.

The organization of the genome in the nucleus of a eukaryotic cell is fairly complex and dynamic. Various features of the nuclear architecture, including compartmentalization of molecular machines and the spatial arrangement of genomic sequences, help to carry out and regulate nuclear processes, such as DNA replication, DNA repair, gene transcription, RNA processing, and mRNA transport. Compartmentalized multiprotein complexes undergo extensive modifications or exchange of protein subunits, allowing for an exquisite dynamics of structural components and functional processes of the nucleus. The architecture of the interphase nucleus is linked to the spatial arrangement of genes and gene clusters, the structure of chromatin, and the accessibility of regulatory DNA elements. In this review, we discuss recent studies that have provided exciting insight into the interplay between nuclear architecture, genome organization, and gene expression.

/pdf/dynamics-and-interplay-of-nuclear-architecture-genome-1twnrsk2nf.pdf

Dynamics and interplay of nuclear architecture, genome organization, and gene expression

The spatial arrangement of chromatin within the nucleus can affect reactions that occur on the DNA and is likely to be regulated. Here we show that activation of INO1 occurs at the nuclear membrane and requires the integral membrane protein Scs2. Scs2 antagonizes the action of the transcriptional repressor Opi1 under conditions that induce the unfolded protein response (UPR) and, in turn, activate INO1. Whereas repressed INO1 localizes throughout the nucleoplasm, the gene is recruited to the nuclear periphery upon transcriptional activation. Recruitment requires the transcriptional activator Hac1, which is produced upon induction of the UPR, and is constitutive in a strain lacking Opi1. Artificial recruitment of INO1 to the nuclear membrane permits activation in the absence of Scs2, indicating that the intranuclear localization of a gene can profoundly influence its mechanism of activation. Gene recruitment to the nuclear periphery, therefore, is a dynamic process and appears to play an important regulatory role.

https://escholarship.org/content/qt8rg9620z/qt8rg9620z.pdf?t=qafv3j

Gene recruitment of the activated INO1 locus to the nuclear membrane.

Nuclear Retention of Unspliced mRNAs in Yeast Is Mediated by Perinuclear Mlp1

The silent mating-type loci (HML and HMR) in the yeast Saccharomyces cerevisiae are maintained in a transcriptionally inactive state due to the formation of a specialized chromatin structure analogous to heterochromatin of higher eukaryotes. Silencing of the mating-type loci requires flanking sequence elements, termed silencers, which bind the transcription factors Rap1 and Abf1, as well as the multisubunit origin recognition complex (ORC) (reviewed in references 11, 15, and 27). Together, these proteins recruit the silencing proteins, Sir1, Sir2, Sir3, and Sir4, that participate in the formation of heterochromatin.

Genes placed near telomeres are also silenced. Telomeric silencing depends on Rap1, which binds to telomeric TG1-3 repeats and recruits Sir3 and Sir4 (28, 29). A Sir complex consisting of Sir2, Sir3, and Sir4 then spreads from the telomeres to nearby nucleosomes to form silent chromatin (16, 17). Although RAP1 is an essential gene, mutations that delete the 3′ end of the gene (rap1ΔC) are viable but lead to a complete loss of telomeric silencing (22, 26, 28). This is due to the inability of Rap1ΔC to recruit Sir3 and Sir4 to the telomeres.

Expressing Sir proteins as GAL4 DNA-binding domain (GBD) hybrids and tethering them to defective silencers in which binding sites for the ORC, Rap1, and/or Abf1 have been replaced by Gal4 binding sites can lead to silencing. This so-called targeted silencing was first demonstrated with GBD-Sir1 but has since been shown with the other Sir proteins as well as with Rap1 and Orc1 (5, 6, 38). Targeted silencing also has been achieved by forming GBD hybrids with endoplasmic reticulum or Golgi proteins and tethering them to a partially defective HMR E silencer (1). Overexpression of such membrane protein hybrids causes them to accumulate in the endoplasmic reticulum (which is contiguous with the nuclear envelope). As a consequence, the DNA-binding domain of Gal4 is in the nucleus but is anchored to the nuclear membrane. In this case it is thought that silencing occurs because the HMR locus, with Gal4 sites at the E silencer, is drawn to the periphery of the nucleus where there is a high concentration of Sir proteins (1).

Circular autonomously replicating sequence (ARS) plasmids that lack a mechanism for mitotic segregation are preferentially retained in mother cells, resulting in the generation of plasmid-free daughters. In the absence of selection, these plasmids are lost from logarithmically growing cultures (30). In contrast, ARS plasmids that contain embedded telomeric sequences or the HMR E silencer are segregated efficiently between dividing cells and are stably propagated (8, 19, 23, 24). Plasmid segregation mediated by the HMR E silencer requires the Sir proteins, and segregation mediated by telomeric sequences is improved by the silencing factors. Previously, we showed that tethering a specific domain of Sir4, the so-called partitioning and anchoring domain (PAD4), directly to ARS plasmids also confers efficient mitotic segregation (2). Using a DNA-topology assay that measures axial rotation of intracellular DNA segments, it was shown that the tethered PAD4 domain of Sir4 also immobilizes the DNA to which it is bound (2). The partitioning and DNA immobilization data suggested a model in which the Sir4 domain attaches to a nuclear component, such as a chromosome or the nuclear membrane, that divides symmetrically between cells at mitosis.

Here we describe the results of a screen for factors that, when tethered to a telomere, can reestablish silencing at a telomere in a rap1Δc mutant defective in silencing. In this screen we identified three known Sir-interacting proteins, Rap1, Sir1, and Rad7, as well as a novel protein, Esc1, which we show interacts with the PAD4 domain of Sir4. We further show that Esc1 is located at the nuclear periphery and is essential for the partitioning and anchoring of plasmids by the PAD4 domain of Sir4. The results suggest that Esc1 helps recruit Sir4 to the nuclear periphery.

Catherine V. Laiosa

Papers

Esc1, a Nuclear Periphery Protein Required for Sir4-Based Plasmid Anchoring and Partitioning