General Regulatory Factors (GRFs) as Genome Partitioners

Geneviève Fourel,Tsuyoshi Miyake,Pierre-Antoine Defossez,Rong Li,Éric Gilson

doi:10.1074/jbc.m202578200

Abstract

Insulators are sequences that uncouple adjacent chromosome domains. Here we have shown that Saccharomyces cerevisiae Rap1p and Abf1p proteins are endowed with a potent insulating capacity. Insulating domains in Rap1p coincide with previously described transcription activation domains, whereas four adjacent subdomains spanning the whole of the Abf1p C terminus (440-731) were found to display autonomous insulating capacity. That both Rap1p and Abf1p silencing domains either contain or largely overlap with an insulating domain suggests that insulation conveys some undefined chromosome organization capacity that also contributes a function in silencing. Together with Reb1p and Tbf1p, previously involved in the activity of Saccharomyces cerevisiae subtelomeric insulators, insulating potential emerges as a supplementary common property of General Regulatory Factors (GRFs). Thus GRFs, which bind to sites scattered throughout the genome within promoters, would not only play a key role in regulating gene expression but also partition the genome in functionally independent domains.

Highlights

Abf1p, Reb1p, and Rap1p are three yeast transcription factors known as General Regulatory Factors, or GRFs.1 They share a number of characteristics
This idea is further substantiated by the fact that the SWI/SNF chromatin remodeling complex appears to compensate for the effect of deleting a Reb1p site in the GAL1 promoter or an Abf1p site in ARS1, on transcription and replication initiation, respectively [10, 11]
URA3 OFF colonies are detected by virtue of their growth on medium containing 5-fluoro-orotic acid (FOA)

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains—Yeast strains described in this study are derivatives of W303–1a and were obtained following standard genetic manipulations as described in Ref. 20. Constructs were sequenced, and expression of the Gal4p chimeras was checked by immunoblotting using an anti-HA antibody coupled with peroxidase (Roche Molecular Biochemicals). Strains expressing roughly similar levels of fusion proteins were selected, and at least two independent transformants derived from distinct plasmid clones were analyzed for each construct in silencing/insulation assays. CEN-ARS pRS315- and pRS313-derived plasmids used to express Gal4-Abf1p chimeras are described in Ref. 24. A BglII-BamHI fragment spanning four LexA sites was obtained by PCR using plasmid psh1834 and inserted at the BglII site of pUASURTEL [19]. The LexAGal chimera was constructed in the pEG202 vector and contained Gal residues 799 –1081

RESULTS

Genome Partitioning

DISCUSSION