Essential histone chaperones collaborate to regulate transcription and chromatin integrity

SUMMARYHistone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. We have discovered that the physical interaction between two essential histone chaperones, Spt6 and Spn1/Iws1, is required for transcriptional accuracy and nucleosome organization. To understand this requirement, we have isolated suppressors of an spt6 mutation that disrupts the Spt6-Spn1 interaction. Several suppressors are in a third essential histone chaperone, FACT, while another suppressor is in the transcription elongation factor Spt5/DSIF. The FACT suppressors weaken FACT-nucleosome interactions and bypass the requirement for Spn1, possibly by restoring a necessary balance between Spt6 and FACT on chromatin. In contrast, the Spt5 suppressor modulates Spt6 function in a Spn1-dependent manner. Despite these distinct mechanisms, both suppressors alleviate the nucleosome organization defects caused by disruption of the Spt6-Spn1 interaction. Taken together, we have uncovered a network in which histone chaperones and other elongation factors coordinate transcriptional integrity and chromatin structure.

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The role of FACT in managing chromatin: disruption, assembly, or repair?

Nucleic Acids Research ◽

10.1093/nar/gkaa912 ◽

2020 ◽

Vol 48 (21) ◽

pp. 11929-11941

Author(s):

Tim Formosa ◽

Fred Winston

Keyword(s):

Dna Repair ◽

Dna Replication ◽

Normal State ◽

Elongation Factor ◽

Histone Chaperone ◽

The Core ◽

Transcription Elongation Factor ◽

Differentiated Cells ◽

Chromatin Integrity

Abstract FACT (FAcilitates Chromatin Transcription) has long been considered to be a transcription elongation factor whose ability to destabilize nucleosomes promotes RNAPII progression on chromatin templates. However, this is just one function of this histone chaperone, as FACT also functions in DNA replication. While broadly conserved among eukaryotes and essential for viability in many organisms, dependence on FACT varies widely, with some differentiated cells proliferating normally in its absence. It is therefore unclear what the core functions of FACT are, whether they differ in different circumstances, and what makes FACT essential in some situations but not others. Here, we review recent advances and propose a unifying model for FACT activity. By analogy to DNA repair, we propose that the ability of FACT to both destabilize and assemble nucleosomes allows it to monitor and restore nucleosome integrity as part of a system of chromatin repair, in which disruptions in the packaging of DNA are sensed and returned to their normal state. The requirement for FACT then depends on the level of chromatin disruption occurring in the cell, and the cell's ability to tolerate packaging defects. The role of FACT in transcription would then be just one facet of a broader system for maintaining chromatin integrity.

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Ribavirin-Induced Intracellular GTP Depletion Upregulates Factor VII Expression

Blood ◽

10.1182/blood.v120.21.1113.1113 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1113-1113

Author(s):

Atsuo Suzuki ◽

Yuhri Miyawaki ◽

Eriko Okuyama ◽

Moe Murata ◽

Ando Yumi ◽

...

Keyword(s):

Gene Transcription ◽

Transcription Initiation ◽

Transcription Elongation ◽

Elongation Factor ◽

Elongation Rate ◽

The Other ◽

Factor Vii ◽

Dependent Manner ◽

Transcription Elongation Factor ◽

Impdh Inhibitors

Abstract Abstract 1113 In this study, we investigated the molecular basis of upregulation of factor VII (FVII) gene expression by ribavirin, and found that intracellular GTP depletion induced by ribavirin activated FVII gene transcription and modulated transcription elongation. In 2006, Yamamoto et al. reported that anti-hepatitis C virus (HCV) agent ribavirin elevated the activity of FVII in HCV-infected hemophilia patients; however, the precise mechanisms were still unknown. In addition, the anti-HCV mechanisms of ribavirin were not yet fully elucidated, although the extended studies have been done. We investigated the effects of ribavirin in vitro and confirmed the approximately 4-fold upregulation of FVII mRNA by ribavirin treatment in HepG2 cells. FVII mRNA was increased in a dose-dependent manner up to 100μg/mL of ribavirin at a lower concentration than therapeutic concentration of 150μg/mL. FVII mRNA induction by ribavirin was also observed in a time-dependent manner from 24 h to 72 h after treatment. Ribavirin metabolite ribavirin 5'-monophosphate is one of the IMP dehydrogenase (IMPDH) inhibitors, and the other IMPDH inhibitors mycophenolic acid (MPA) and 6-mercaptupurine (6-MP) also induced FVII upregulation. It is well known that inhibition of IMPDH causes intracellular GTP depletion, and guanosine supplementation to salvage GTP could reverse FVII mRNA increase in ribavirin-treated cells. These results indicated that cellular GTP reduction associated with FVII gene upregulation. The mechanisms of gene upregulation by GTP depletion were not elucidated. The promoter activities and mRNA stability of FVII were analyzed under ribavirin treatment. The FVII gene promoter activity was enhanced up to 1.5-fold by ribavirin treatment; however the activation did not reach 4-fold induction of FVII mRNA increase. There was no significant change of FVII mRNA half-life in ribavirin-treated cells. Since the promoter activation might display transcription initiation capacity, the contribution of transcription elongation stage was further investigated. Transcription elongation was regulated by phosphorylation of carbo-terminal domain (CTD) of RNA polymerase II (PolII). Transcription elongation factor P-TEFb (positive-transcription elongation factor b), which consists as a complex of CDK9 and cyclin T, phosphorylates Ser of PolII CTD. The kinase activity of P-TEFb could be inhibited by 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB). In FVII gene upregulation, DRB completely canceled ribavirin-induced FVII mRNA increase. We also performed nuclear run-on assay to verify the potential transcription elongation capacity of paused PolII, and observed a dramatic increase of FVII mRNA in ribavirin-treated cells. These results suggested that ribavirin-induced FVII gene upregulation was caused not only by transcription initiation but also by accelerated transcription elongation rate. There are various transcription factor associated with transcription elongation in addition to P-TEFb, such as elongin, ELL (eleven nineteen-lysine rich leukemia). We found that ELL3, a member of ELL family protein, was upregulated by ribavirin treatment. A ELL3 mRNA increase occurred prior to FVII mRNA upregulation, and the ELL3 upregulation was also canceled by guanosine supplementation. These results indicated ELL3 induction by ribavirin was also a response to cellular GTP depletion. To confirm the contribution of ELL3 protein to FVII gene transcription elongation, we used siRNAs specific to ELL3 and as expected, knockdown of ELL3 resulted in diminished FVII upregulation. A chromatin immunoprecipitation (ChIP) revealed ELL3 recruitment to the FVII gene, and the recruitments of PolII and CDK9 were also enhanced by ribavirin treatment. Taken together, FVII gene upregulation by ribavirin was associated with intracellular GTP depletion. The GTP reduction mainly modulates transcription elongation rate rather than transcription initiation, though the relationships between cellular GTP depletion and enhanced transcription elongation must be investigated. This study uncovered candidate mechanisms of ribavirin and the other IMPDH inhibitors and highlights a development of novel pharmaceutical therapies for hemophilia. Disclosures: No relevant conflicts of interest to declare.

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Xeroderma pigmentosum C is involved in Epstein–Barr virus DNA replication

Journal of General Virology ◽

10.1099/vir.0.83212-0 ◽

2007 ◽

Vol 88 (12) ◽

pp. 3234-3243 ◽

Cited By ~ 11

Author(s):

Chih-Chung Lu ◽

Yi-Chun Chen ◽

Jiin-Tarng Wang ◽

Pei-Wen Yang ◽

Mei-Ru Chen

Keyword(s):

Dna Repair ◽

Dna Replication ◽

Xeroderma Pigmentosum ◽

Epstein Barr Virus ◽

Excision Repair ◽

Mutant Cell ◽

Dependent Manner ◽

Barr Virus ◽

Epstein Barr ◽

Lytic Dna Replication

Cellular mismatch and base-excision repair machineries have been shown to be involved in Epstein–Barr Virus (EBV) lytic DNA replication. We report here that nucleotide-excision repair (NER) may also play an important role in EBV lytic DNA replication. Firstly, the EBV BGLF4 kinase interacts with xeroderma pigmentosum C (XPC), the critical DNA damage-recognition factor of NER, in yeast and in vitro, as demonstrated by yeast two-hybrid and glutathione S-transferase pull-down assays. Simultaneously, XPC was shown, by indirect immunofluorescence and co-immunoprecipitation assays, to interact and colocalize with BGLF4 in EBV-positive NA cells undergoing lytic viral replication. In addition, the efficiency of EBV DNA replication was reduced about 30–40 % by an XPC small interfering RNA. Expression of BGLF4 enhances cellular DNA-repair activity in p53-defective H1299/bcl2 cells in a host-cell reactivation assay. This enhancement was not observed in the XPC-mutant cell line XP4PA-SV unless complemented by ectopic XPC, suggesting that BGLF4 may stimulate DNA repair in an XPC-dependent manner. Overall, we suggest that the interaction of BGLF4 and XPC may be involved in DNA replication and repair and thereby enhance the efficiency of viral DNA replication.

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Key histone chaperones have distinct roles in replisome progression and genomic stability

10.1101/2020.02.25.964221 ◽

2020 ◽

Author(s):

Ioannis Tsirkas ◽

Daniel Dovrat ◽

Yang Lei ◽

Angeliki Kalyva ◽

Diana Lotysh ◽

...

Keyword(s):

Dna Replication ◽

Chromatin Structure ◽

Replication Fork ◽

Genome Stability ◽

Spontaneous Mutation ◽

Genomic Stability ◽

Yeast Cells ◽

Eukaryotic Cells ◽

Histone Chaperones ◽

Replication Fork Progression

AbstractReplication-coupled (RC) nucleosome assembly is an essential process in eukaryotic cells in order to maintain chromatin structure during DNA replication. The deposition of newly synthesized H3/H4 histones during DNA replication is facilitated by specialized histone chaperones. Although the contribution of these histone chaperones to genomic stability has been thoroughly investigated, their effect on replisome progression is much less understood. By exploiting a time-lapse microscopy system for monitoring DNA replication in individual live cells, we examined how mutations in key histone chaperones including CAC1, RTT106, RTT109 and ASF1, affect replication fork progression. Our experiments revealed that mutations in CAC1 or RTT106 that directly deposit histones on the DNA, slowdown replication fork progression. In contrast, analysis of cells mutated in the intermediary ASF1 or RTT109 histone chaperones revealed that replisome progression is not affected. We found that mutations in histone chaperones including ASF1 and RTT109 lead to extended G2/M duration, elevated number of RPA foci and in some cases, increased spontaneous mutation rate. Our research suggests that histone chaperones have distinct roles in enabling high replisome progression and maintaining genome stability during cell cycle progression.Author SummaryHistone chaperones (HC) play key roles in maintaining the chromatin structure during DNA replication in eukaryotic cells. Despite extensive studies on HCs, little is known regarding their importance for replication fork progression during S-phase. Here, we utilized a live-cell imaging approach to measure the progression rates of single replication forks in individual yeast cells mutated in key histone chaperones. Using this approach, we show that mutations in CAC1 or RTT106 HCs that directly deposit histones on the DNA lead to slowdown of replication fork progression. In contrast, mutations in ASF1 or RTT109 HCs that transfers H3/H4 to CAC1 or RTT106, do not affect replisome progression but lead to post replication defects. Our results reveal distinct functions of HCs in replication fork progression and maintaining genome stability.

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A Novel Function of the DNA Repair Gene rhp6 in Mating-Type Silencing by Chromatin Remodeling in Fission Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5511 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5511-5522 ◽

Cited By ~ 26

Author(s):

Jagmohan Singh ◽

Vintoo Goel ◽

Amar J. S. Klar

Keyword(s):

Dna Repair ◽

Gene Regulation ◽

Dna Replication ◽

Mating Type ◽

Chromatin Structure ◽

Temperature Sensitive ◽

Repair Gene ◽

Daughter Cells ◽

Dna Repair Gene

ABSTRACT Recent studies have indicated that the DNA replication machinery is coupled to silencing of mating-type loci in the budding yeastSaccharomyces cerevisiae, and a similar silencing mechanism may operate in the distantly related yeast Schizosaccharomyces pombe. Regarding gene regulation, an important function of DNA replication may be in coupling of faithful chromatin assembly to reestablishment of the parental states of gene expression in daughter cells. We have been interested in isolating mutants that are defective in this hypothesized coupling. An S. pombe mutant fortuitously isolated from a screen for temperature-sensitive growth and silencing phenotype exhibited a novel defect in silencing that was dependent on the switching competence of the mating-type loci, a property that differentiates this mutant from other silencing mutants of S. pombe as well as of S. cerevisiae. This unique mutant phenotype defined a locus which we named sng1 (for silencing not governed). Chromatin analysis revealed a switching-dependent unfolding of the donor locimat2P and mat3M in thesng1 − mutant, as indicated by increased accessibility to the in vivo-expressed Escherichia coli dammethylase. Unexpectedly, cloning and sequencing identified the gene as the previously isolated DNA repair gene rhp6. RAD6, an rhp6 homolog in S. cerevisiae, is required for postreplication DNA repair and ubiquitination of histones H2A and H2B. This study implicates the Rad6/rhp6 protein in gene regulation and, more importantly, suggests that a transient window of opportunity exists to ensure the remodeling of chromatin structure during chromosome replication and recombination. We propose that the effects of thesng1−/rhp6 − mutation on silencing are indirect consequences of changes in chromatin structure.

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