SFP1 primarily transcriptional targets and its interaction with other ribosomes biogenesis factors

Proper regulation of cell growth is a primary concern of all living organisms. The fundamental engine that drives cell growth is ribosomes biogenesis, a remarkably energy-intensive process that is highly regulated at the transcriptional level in the budding yeast S. cerevisiae. Sfp1p, a stress- and nutrient-sensitive transcription factor is involved in this control. It is localized in the nucleus of rapidly growing cells where it activates Ribosomal Protein (RP) and Ribosomal Biogenesis (Ribi) genes. All studies done so far to address the nuclear role of Sfp1p have used ChIP, and have identified RP, but not Ribi promoters as sites of Sfp1p binding. However, the ChIP method may fail to yield signals for proteins that very transiently [1] or indirectly bind with DNA. In addition, the formaldehyde crosslinking step of the ChIP protocol is sensed as a stress by the cell and results in rapid relocation of a fraction of Sfp1p from the nucleus to the cytoplasm. An interesting alternative to ChIP for the identification of transcription factor binding sites is the DamID method [2]. In this method, the E.coli DNA methyltransferase (Dam) is fused to the chromatin-binding protein of interest, targeting the methylase to the native binding site of this factor and resulting in local methylation of the DNA. We are currently adapting this strategy to analyze the methylation pattern resulting from the expression of a Sfp1-Dam fusion protein in yeast.

[1] Schmiedeberg, L., Skene, P., Deaton, A., and Bird, A. (2009). A temporal threshold for formaldehyde crosslinking and fixation. PLoS One 4, e4636.

[2] B. van Steensel et al. (2003). BioTechniques 35,346-357