FACT Prevents the Accumulation of Free Histones Evicted from Transcribed Chromatin and a Subsequent Cell Cycle Delay in G1

The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA–damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication

Excess histone levels mediate cytotoxicity via multiple mechanisms

The accumulation of excess histone proteins in cells has deleterious consequences such as genomic instability in the form of excessive chromosome loss, enhanced sensitivity to DNA damaging agents and cytotoxicity. Hence, the synthesis of histone proteins is tightly regulated at multiple steps and transcriptional as well as posttranscriptional regulation of histone proteins is well established. Additionally, we have recently demonstrated that histone protein levels are regulated posttranslationally by the DNA damage checkpoint kinase Rad53 and ubiquitin-proteasome dependent proteolysis in the budding yeast. However, the underlying mechanism(s) via which excess histones exert their deleterious effects in vivo are not clear. Here we have investigated the mechanistic basis for the deleterious effects of excess histones in budding yeast. We find that the presence of excess histones saturates certain histone modifying enzymes, potentially interfering with their activities. Additionally, excess histones appear to bind non-specifically to DNA as well as RNA, which can adversely affect their metabolism. Microarray analysis revealed that upon overexpression of histone gene pairs, about 240 genes were either up or downregulated by two-fold or more. Overall, we present evidence that excess histones are likely to mediate their cytotoxic effects via multiple mechanisms that are primarily dependent on inappropriate electrostatic interactions between the positively charged histones and diverse negatively charged molecules in the cell. Our findings help explain the basis for the existence of multiple distinct mechanisms that contribute to the tight control of histone protein levels in cells and highlight their importance in maintaining genomic stability and cell viability