Biomedical subjects

Marc R Gartenberg

Publications and source records attributed to Marc R Gartenberg.

4 recordsLinked to original sources

PubMed2005

Targeting of cohesin by transcriptionally silent chromatin.

Eukaryotic DNA replication produces sister chromatids that are linked together until anaphase by cohesin, a ring-shaped protein complex that is thought to act by embracing both chromatids. Cohesin is enriched at centromeres, as well as discrete sites along chromosome arms where transcription positions the complex between convergent gene pairs. A relationship between cohesin and Sir-mediated transcriptional silencing has also begun to emerge. Here we used fluorescence microscopy and site-specific recombination to characterize interactions between newly replicated copies of the silent HMR mating-type locus. HMR was tagged with lac-GFP and flanked by binding sites for an inducible site-specific recombinase. Excision of the locus in cells with sister chromatids produced two chromatin circles that remained associated with one another. Pairing of the circles required silent chromatin, cohesin, and the RSC chromatin-remodeling complex. Chromatin immunoprecipitation showed that targeting of cohesin to the locus is Sir-dependent, and functional tests showed that silent chromatin acts in a continuous fashion to maintain cohesion. Remarkably, loss of silencing led to loss of cohesin from linear chromosomal templates but not from excised chromatin circles. The results are consistent with a model in which cohesin binds silent chromatin via topological linkage to individual chromatids.

Cell Cycle Proteins↗

PubMed2005

Multiple pathways tether telomeres and silent chromatin at the nuclear periphery: functional implications for sir-mediated repression.

The positioning of chromosomal domains within interphase nuclei is thought to contribute to establishment and maintenance of epigenetic control. Using GFP-tagged chromosomal domains, LexA-fusion targeting and live microscopy, we investigated mechanisms through which chromatin can be anchored to the nuclear envelope (NE). We find that a subdomain of the silencing information regulator Sir4 (Sir4(PAD)) and yKu80 are sufficient to tether a chromatin region to the nuclear periphery, independently of their silencing function. Sir4(PAD) interacts with Esc1 (Establishes Silent Chromatin 1), a large acidic protein, localized at the nuclear periphery in the absence of Sir4 and yKu. Sir4 also binds to the periphery through yKu80, whose perinuclear ligand is unidentified. Both pathways are involved in the localization of natural telomeres. To show that silent chromatin can also mediate anchorage, we uncoupled the HMR silent mating-type locus from the chromosome using inducible site-specific recombination. Real-time cytological techniques reveal the position and dynamics of the excised locus. We show that the silent HMR ring associates with the NE in a SIR-dependent manner, while derepressed excised rings move without detectable constraint throughout the nucleoplasm. Dual anchoring pathways thus cooperate to generate high concentrations of SIR proteins in perinuclear foci, which in turn promote repression.

Cell Nucleus↗

PubMed2004

Sir-mediated repression can occur independently of chromosomal and subnuclear contexts.

Epigenetic mechanisms silence the HM mating-type loci in budding yeast. These loci are tightly linked to telomeres, which are also repressed and held together in clusters at the nuclear periphery, much like mammalian heterochromatin. Yeast telomere anchoring can occur in the absence of silent chromatin through the DNA end binding factor Ku. Here we examine whether silent chromatin binds the nuclear periphery independently of telomeres and whether silencing persists in the absence of anchorage. HMR was excised from the chromosome by inducible site-specific recombination and tracked by real-time fluorescence microscopy. Silent rings associate with the nuclear envelope, while nonsilent rings move freely throughout the nucleus. Silent chromatin anchorage requires the action of either Ku or Esc1. In the absence of both proteins, rings move throughout the nucleoplasm yet remain silent. Thus, transcriptional repression can be sustained without perinuclear anchoring.

Antigens, Nuclear↗

PubMed2002

Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning.

A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1. A previously uncharacterized protein, Esc1 (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7. Telomeric silencing was slightly decreased in Deltaesc1 mutants, but silencing of the HM loci was unaffected. On the other hand, targeted silencing by various tethered proteins was greatly weakened in Deltaesc1 mutants. Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262). When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo. Here, both phenomena were shown to require ESC1. Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1. Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing. GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes. Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery.

Basic Helix-Loop-Helix Proteins↗