A connection between the FACT complex and cell progression in Saccharomyces cerevisiae = Una conexión entre el complejo FACT y progresión de la célula Saccharomyces cerevisiae

Resumen

The FACT complex is an essential component of eukaryotic ce lls able to modify the chromatin structure allowing transcription by the RNA polymerase II. The FACT that this complex is essential in all organisms tested added to that it has been recently shown to be a target of curaxins, a potential novel anticancer compound, makes this complex an interesting subject of study. In Saccharomyces cerevisiae, this complex is essential, and mutants affected in Spt16, one of the two subunits of this complex, show a clear arrest in the G1/S transition of the cell cycle and a transcriptional defect that does not seem to affect equally all genes. Previous work in our lab has shown that this mutant presents a transcriptional repression of CLN3, a G1-cyclin that plays a key role during the G1/S transition controlling the expression of many genes required to enter DNA replication. In this work, we have focused on the role of this complex in cell cycle progression and in transcription in the yeast Saccharomyces cerevisiae. The first part of our work has focused on the cell cycle defect described for mutants affected in the Spt16 subunit of the FACT complex. Our results confirm that the absence of a functional FACT complex leads to a transcriptional down-regulation of the cyclin CLN3, which specifically operates through its promoter. Down-regulation is unrelated to regulatory elements previously described for this promoter, and seems to operate through the creation of a more closed chromatin structure that does not depend on the presence of this complex during its transcription. In addition to cell cycle defects, we show that FACT dysfunction leads to a defect in chromatin reassembly during transcription that generates a pool of free histones evicted from transcribed chromatin. This novel source of free histones is harmful in conditions where histone degradation is impaired and shows a positive correlation whit the cell cycle defects described for this mutant. Using a mutant version of Spt6, another complex involved in other complex involver in chromatin reassembly also generates a clear G1/S transition defect and a transcriptional repression of CLN3. Our results clearly suggest a link between chromatin reposition and the G1/S transition and we propose that free histones may be a signal in the call to sense the chromatin state before entering DNA replication. The second part of this work has focused on the role of FACT on RNA polymerase II transcription. We have performed two different genome wide approaches to measure the amount (ChIP on ChIP) and the activity (Genomic Run On) of RNA polymerase II in the absence of the FACT complex. Our results show that FACT plays an important global role in the amount of total RNA polymerase II that remains associated to the coding region of a gene, which seems to have an inverse correlation ribosomal protein genes are especially sensitive to the inactivation of FACT, which contributes to maintain the proportion of active (non-arrested) RNA polymerase II molecules on these genes during elongation.