Localization of Sir2p: the nucleolus as a compartment for silent information regulators

Abstract

In wild-type budding yeast strains, the proteins encoded by SIR3, SIR4 and RAP1 co-localize with telomeric DNA in a limited number of foci in interphase nuclei. Immunostaining of Sir2p shows that in addition to a punctate staining that coincides with Rap1 foci, Sir2p localizes to a subdomain of the nucleolus. The presence of Sir2p at both the spacer of the rDNA repeat and at telomeres is confirmed by formaldehyde cross-linking and immunoprecipitation with anti-Sir2p antibodies. In strains lacking Sir4p, Sir3p becomes concentrated in the nucleolus, by a pathway requiring SIR2 and UTH4, a gene that regulates life span in yeast. The unexpected nucleolar localization of Sir2p and Sir3p correlates with observed effects of sir mutations on rDNA stability and yeast longevity, defining a new site of action for silent information regulatory factors.

Distinct Roles for the Essential MYST Family HAT Esa1p in Transcriptional Silencing

“…Nop1p staining in wild-type cells at 28°C appeared as a characteristic discrete crescent shape apart from the bulk of the DAPIstaining chromatin ( Figure 6A, top). Most of Sir2p colocalized with the Nop1p staining in the nucleolar crescent and some Sir2p localized to telomeric foci with the bulk of the chromatin, consistent with previous results (Gotta et al, 1997).…”

“…After reversal of cross-linking, DNA was purified by phenol-chloroform extraction and recovered by ethanol precipitation. Precipitated DNA was analyzed by molecular amplification using the 25S-1, 25S-2, 5S, and NTS primers described in Gotta et al (1997) and the POL1 primers described in Papamichos-Chronakis et al (2002).…”

“…Indirect immunofluorescence was carried out as described (Gotta et al, 1997;Stone et al, 2000). Briefly, cells were logarithmically grown in YPD for 4 h at either 28 or 37°C and fixed by adding paraformaldehyde to a final concentration of 3.3% at 30°C for 10 min.…”

“…In addition to being found directly in the nucleolus and rDNA (Gotta et al, 1997;Huang and Moazed, 2003), Sir2p functions to suppress recombination within the highly repetitive rDNA array (Gottlieb and Esposito, 1989). Indeed, many rDNA silencing defective mutants are correlated with increases in rDNA recombination (Smith et al, 1999).…”

“…After reversal of cross-linking, DNA was purified by phenol-chloroform extraction and recovered by ethanol precipitation. Precipitated DNA was analyzed by molecular amplification using the 25S-1, 25S-2, 5S, and NTS primers described in Gotta et al (1997) and the POL1 primers described in Papamichos-Chronakis et al (2002).To generate the triple-HA-tagged ESA1 construct (ESA1-3HA), sequence immediately 3Ј to the ESA1 ORF was amplified using Deep-Vent polymerase (New England Biolabs) and oligos T3 and D1-ESA1 (5Ј-CGCGTTTAAACGC- A. S. Clarke et al …”