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

Abstract

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.

FACT Proteins, SUPT16H and SSRP1, Are Transcriptional Suppressors of HIV-1 and HTLV-1 That Facilitate Viral Latency

“…Further study of FACT proteins in human retroviral transcription, as well as more profound analysis of FACT-mediated transcriptional regulation at the genome-wide scale in retrovirus-infected cells, may help to unravel the complicated functions of FACT that could be modulated by viral components. In addition, FACT is also a multifunctional protein complex that regulates other cellular processes, such as DNA replication (62)(63)(64), DNA damage response (65,66), and cell cycle progression (67,68). Whether FACT-mediated nontranscriptional functions may affect HIV-1/HTLV-1 replication is unclear and requires further investigation.…”

Free Histones and the Cell Cycle

“…This model agrees with a recent publication which reports how Spt16 promotes the redeposition of the original H3 and H4 histones evicted by elongating Pol II (Jamai et al, 2009). The protective role against evicted histones is probably not an exclusive function of FACT, but a function of the other factors that cooperate during chromatin reassembly, like Spt6, for which we have also provided some evidence (Morillo-Huesca et al, 2010).…”

“…In addition to this, we have found an interesting connection between free histone levels and cell cycle defects in the G1/S transition. We postulate that free or non-chromatin-bound histones can trigger the down-regulation of CLN3, thereby arresting cells at G1 (START) and contributing to control free histone levels before starting DNA replication (Morillo-Huesca et al, 2010). Our results indicate a so far unknown connection between chromatin dynamics and cell cycle regulation.…”

“…This result, in combination with the lack of phosphorylation of Rad53 after FACT inactivation, indicates that excess histones are involved in this phenomenon. This conclusion is strengthened by the results of the experiments that manipulate in vivo histone levels: (i) deletion of one of the two loci encoding H2A and H2B partially suppressed the ts phenotype and the accumulation of G1 cells of the spt16 mutant, indicating that a reduction in histone levels can alleviate the cdc phenotype due to a FACT dysfunction; (ii) overproduction of histone levels in wild-type cells leads to a clear accumulation of the cells in G1 (asynchronous culture) and a more marked delay in the entry of synchronised cells in the S-phase (Morillo-Huesca et al, 2010). This delay in the G1/S transition also correlates with CLN3 down-regulation.…”

“…According to this model, we observed that deleting the HTA2-HTB2 locus can partially suppress the ts phenotype of spt16-197. Using co-immunoprecipitation assays to detect free non-chromatin-bound histones, we demonstrated that Pol II-dependent transcription in the absence of active FACT causes an accumulation of evicted histones, which could become toxic for the cell if not targeted for degradation by Rad53 (Morillo-Huesca et al, 2010). …”