Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing

BACKGROUND: Position-dependent gene silencing in yeast involves many factors, including the four HIR genes and nucleosome assembly proteins Asf1p and chromatin assembly factor I (CAF-I, encoded by the CAC1-3 genes). Both cac Delta asfl Delta and cac Delta hir Delta double mutants display synergistic reductions in heterochromatic gene silencing. However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously been explored. RESULTS: Our biochemical and genetic studies of yeast Asf1p revealed links to Hir protein function. In vitro, an active histone deposition complex was formed from recombinant yeast Asf1p and histones H3 and H4 that lack a newly synthesized acetylation pattern. This Asf1p/H3/H4 complex generated micrococcal nuclease--resistant DNA in the absence of DNA replication and stimulated nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis. Also, Asf1p bound to the Hir1p and Hir2p proteins in vitro and in cell extracts. In vivo, the HIR1 and ASF1 genes contributed to silencing the heterochromatic HML locus via the same genetic pathway. Deletion of either HIR1 or ASF1 eliminated telomeric gene silencing in combination with pol30--8, encoding an altered form of the DNA polymerase processivity factor PCNA that prevents CAF-I from contributing to silencing. Conversely, other pol30 alleles prevented Asf1/Hir proteins from contributing to silencing. CONCLUSIONS: Yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity.     

Interaction of the yeast Pso5/Rad16 and Sgs1 proteins: influences on DNA repair and aging.

The interaction trap method was used to isolate putative binding partners of Rad16/Pso5, a protein responsible for repair of silent DNA. One of the interactors found was Sgs1, a DNA helicase influencing the life span of Saccharomyces cerevisiae, with homology to the human BLM, WRN and RECQL4 proteins. Using the same fusion proteins from the two-hybrid screening, we show evidence that both proteins also interact in vitro. We tested isogenic strains, containing mutant alleles of the two genes in single and double mutant combination, for phenotypic similarity. Life span in sgs1Delta single and sgs1Delta rad16Delta double mutants is about 40% of that of WT, and the rad16/pso5Delta single mutant also had its life span reduced to 75%. Sensitivity to different mutagens, whose lesions are poorly repaired in rad16/pso5Delta mutants, was tested in sgs1Delta mutants. The sgs1Delta conferred sensitivity to MMS, H2O2 and was moderately sensitive to UV(254nm) (UVC) and 4-NQO. An epistatic interaction between rad16 and sgs1 mutations after UVC, 4-NQO and H2O2 was observed. Moreover, we found that in a top3 background, functional Sgs1p and Rad16p apparently channel MMS, 4-NQO and H2O2 induced lesions into aberrant DNA repair. Our results demonstrate that Sgs1 is not only involved in genome stability, somatic recombination and aging, but is also implicated, together with Rad16/Pso5, in the repair of specific DNA damage.     

The histone chaperone complex HIR maintains nucleosome occupancy and counterbalances impaired histone deposition in CAF‐1 complex mutants

Chromatin organization is essential for coordinated gene expression, genome stability, and inheritance of epigenetic information. The main components involved in chromatin assembly are specific complexes such as Chromatin Assembly Factor 1 (CAF‐1) and Histone Regulator (HIR), which deposit histones in a DNA synthesis‐dependent or ‐independent manner, respectively. Here, we characterize the role of the plant orthologs Histone Regulator A (HIRA), Ubinuclein (UBN) and Calcineurin Binding protein 1 (CABIN1), which constitute the HIR complex. Arabidopsis loss‐of‐function mutants for the various subunits of the complex are viable, but hira mutants show reduced fertility. We show that loss of HIRA reduces extractable histone H3 protein levels and decreases nucleosome occupancy at both actively transcribed genes and heterochromatic regions. Concomitantly, HIRA contributes to maintenance of silencing of pericentromeric repeats and certain transposons. A genetic analysis based on crosses between mutants deficient in subunits of the CAF‐1 and HIR complexes showed that simultaneous loss of both the CAF‐1 and HIR histone H3 chaperone complexes severely affects plant survival, growth and reproductive development. Our results suggest that HIRA partially rescues impaired histone deposition in fas mutants to preserve nucleosome occupancy, implying plasticity in histone variant interaction and deposition.     

Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin-producing Escherichia coli O157:H7

In general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid-rich conditions. We found that the Shiga Toxin-producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K-12 strain in Luria-Bertani (LB)-2-morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini-Tn5 mutagenesis and, from 50,000 colonies, five mutants that showed a clear acetic acid-sensitive phenotype were isolated. The insertion of mini-Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O-polysaccharide, loss of both O-polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O-polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O-polysaccharide and ECA, and normal growth was restored in organic acid-rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O-polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O-polysaccharide and ECA as well as an acid resistance mechanism.     

A role for histone H2B during repair of UV-induced DNA damage in Saccharomyces cerevisiae

To investigate the role of the nucleosome during repair of DNA damage in yeast, we screened for histone H2B mutants that were sensitive to UV irradiation. We have isolated a new mutant, htb1-3, that shows preferential sensitivity to UV-C. There is no detectable difference in bulk chromatin structure or in the number of UV-induced cis-syn cyclobutane pyrimidine dimers (CPD) between HTB1 and htb1-3 strains. These results suggest a specific effect of this histone H2B mutation in UV-induced DNA repair processes rather than a global effect on chromatin structure. We analyzed the UV sensitivity of double mutants that contained the htb1-3 mutation and mutations in genes from each of the three epistasis groups of RAD genes. The htb1-3 mutation enhanced UV-induced cell killing in rad1Delta and rad52Delta mutants but not in rad6Delta or rad18Delta mutants, which are defective in postreplicational DNA repair (PRR). When combined with other mutations that affect PRR, the histone mutation increased the UV sensitivity of strains with defects in either the error-prone (rev1Delta) or error-free (rad30Delta) branches of PRR, but did not enhance the UV sensitivity of a strain with a rad5Delta mutation. When combined with a ubc13Delta mutation, which is also epistatic with rad5Delta, the htb1-3 mutation enhanced UV-induced cell killing. These results suggest that histone H2B acts in a novel RAD5-dependent branch of PRR.     

Genetic ablation of the CDP/Cux protein C terminus results in hair cycle defects and reduced male fertility

Murine CDP/Cux, a homologue of the Drosophila Cut homeoprotein, modulates the promoter activity of cell cycle-related and cell-type-specific genes. CDP/Cux interacts with histone gene promoters as the DNA binding subunit of a large nuclear complex (HiNF-D). CDP/Cux is a ubiquitous protein containing four conserved DNA binding domains: three Cut repeats and a homeodomain. In this study, we analyzed genetically targeted mice (Cutl1(tm2Ejn), referred to as Delta C) that express a mutant CDP/Cux protein with a deletion of the C terminus, including the homeodomain. In comparison to the wild-type protein, indirect immunofluorescence showed that the mutant protein exhibited significantly reduced nuclear localization. Consistent with these data, DNA binding activity of HiNF-D was lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (Delta C(-/-)) mice, indicating the functional loss of CDP/Cux protein in the nucleus. No significant difference in growth characteristics or total histone H4 mRNA levels was observed between wild-type and Delta C(-/-) MEFs in culture. However, specific histone genes (H4.1 and H1) containing CDP/Cux binding sites have reduced expression levels in homozygous mutant MEFs. Stringent control of growth and differentiation appears to be compromised in vivo. Homozygous mutant mice have stunted growth (20 to 50% weight reduction), a high postnatal death rate of 60 to 70%, sparse abnormal coat hair, and severely reduced fertility. The deregulated hair cycle and severely diminished fertility in Cutl1(tm2Ejn/tm2Ejn) mice suggest that CDP/Cux is required for the developmental control of dermal and reproductive functions.     

Yaf9, a novel NuA4 histone acetyltransferase subunit,is required for the cellular response to spindle stress in yeast

Yaf9 is one of three proteins in budding yeast containing a YEATS domain. We show that Yaf9 is part of a large complex and that it coprecipitates with three known subunits of the NuA4 histone acetyltransferase. Although Esa1, the catalytic subunit of NuA4, is essential for viability, we found that yaf9 Delta mutants are viable but hypersensitive to microtubule depolymerizing agents and synthetically lethal with two different mutants of the mitotic apparatus. Microtubules depolymerized more readily in the yaf9Delta mutant compared to the wild type in the presence of nocodazole, and recovery of microtubule polymerization and cell division from limiting concentrations of nocodazole was inhibited. Two other NuA4 mutants (esa1-1851 and yng2 Delta) and nonacetylatable histone H4 mutants were also sensitive to benomyl. Furthermore, wild-type budding yeast were more resistant to benomyl when grown in the presence of trichostatin A, a histone deacetylase inhibitor. These results strongly suggest that acetylation of histone H4 by NuA4 is required for the cellular resistance to spindle stress.     

A novel high-throughput genetic screen for stress-responsive mutants of Arabidopsis thaliana reveals new loci involving stress responses

Activation sequence-1 (as-1) cognate promoter elements are widespread in the promoters of plant defense-related genes as well as in plant pathogen promoters, and may play important roles in the activation of defense-related genes. The as-1-type elements are highly responsive to multiple stress stimuli such as jasmonic acid (JA), salicylic acid (SA), H(2)O(2), xenobiotics and heavy metals, and therefore provide a unique opportunity for identifying additional signaling components and cross-talk points in the various signaling networks. A single as-1-type cis-element-driven GUS reporter Arabidopsis line responsive to JA, SA, H(2)O(2), xenobiotics and heavy metals was constructed for mutagenesis. A large-scale T-DNA mutagenesis has been conducted in the reporter background, and an efficient high-throughput mutant screen was established for isolating mutants with altered responses to the stress chemicals. A number of mutants with altered stress responses were obtained, some of which appear to identify new components in the as-1-based signal transduction pathways. We characterized a mutant (Delta8L4) with a T-DNA insertion in the coding sequence of the gene At4g24275. The as-1-regulated gene expression and GUS reporter gene expression were altered in the Delta8L4 mutant, but there was no change in the expression of genes lacking as-1 elements in their promoters. The phenotype observed with the Delta8L4 mutant was further verified using RNAi plants for At4g24275 (8L4-RNAi), suggesting the feasibility of use of this high-throughput mutant screening in isolating stress-signaling mutants.     

The RAD6/BRE1 histone modification pathway in Saccharomyces confers radiation resistance through a RAD51-dependent process that is independent of RAD18

We examine ionizing radiation (IR) sensitivity and epistasis relationships of several Saccharomyces mutants affecting post-translational modifications of histones H2B and H3. Mutants bre1Delta, lge1Delta, and rtf1Delta, defective in histone H2B lysine 123 ubiquitination, show IR sensitivity equivalent to that of the dot1Delta mutant that we reported on earlier, consistent with published findings that Dot1p requires H2B K123 ubiquitination to fully methylate histone H3 K79. This implicates progressive K79 methylation rather than mono-methylation in IR resistance. The set2Delta mutant, defective in H3 K36 methylation, shows mild IR sensitivity whereas mutants that abolish H3 K4 methylation resemble wild type. The dot1Delta, bre1Delta, and lge1Delta mutants show epistasis for IR sensitivity. The paf1Delta mutant, also reportedly defective in H2B K123 ubiquitination, confers no sensitivity. The rad6Delta, rad51null, rad50Delta, and rad9Delta mutations are epistatic to bre1Delta and dot1Delta, but rad18Delta and rad5Delta show additivity with bre1Delta, dot1Delta, and each other. The bre1Delta rad18Delta double mutant resembles rad6Delta in sensitivity; thus the role of Rad6p in ubiquitinating H2B accounts for its extra sensitivity compared to rad18Delta. We conclude that IR resistance conferred by BRE1 and DOT1 is mediated through homologous recombinational repair, not postreplication repair, and confirm findings of a G1 checkpoint role for the RAD6/BRE1/DOT1 pathway.     

Replication-independent histone deposition by the HIR complex and Asf1

The orderly deposition of histones onto DNA is mediated by conserved assembly complexes, including chromatin assembly factor-1 (CAF-1) and the Hir proteins . CAF-1 and the Hir proteins operate in distinct but functionally overlapping histone deposition pathways in vivo . The Hir proteins and CAF-1 share a common partner, the highly conserved histone H3/H4 binding protein Asf1, which binds the middle subunit of CAF-1 as well as to Hir proteins . Asf1 binds to newly synthesized histones H3/H4 , and this complex stimulates histone deposition by CAF-1 . In yeast, Asf1 is required for the contribution of the Hir proteins to gene silencing . Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 comprise the HIR complex, which copurifies with the histone deposition protein Asf1. Together, the HIR complex and Asf1 deposit histones onto DNA in a replication-independent manner. Histone deposition by the HIR complex and Asf1 is impaired by a mutation in Asf1 that inhibits HIR binding. These data indicate that the HIR complex and Asf1 proteins function together as a conserved eukaryotic pathway for histone replacement throughout the cell cycle.     

Histone dosage regulates DNA damage sensitivity in a checkpoint-independent manner by the homologous recombination pathway

In eukaryotes, multiple genes encode histone proteins that package genomic deoxyribonucleic acid (DNA) and regulate its accessibility. Because of their positive charge, ‘free’ (non-chromatin associated) histones can bind non-specifically to the negatively charged DNA and affect its metabolism, including DNA repair. We have investigated the effect of altering histone dosage on DNA repair in budding yeast. An increase in histone gene dosage resulted in enhanced DNA damage sensitivity, whereas deletion of a H3–H4 gene pair resulted in reduced levels of free H3 and H4 concomitant with resistance to DNA damaging agents, even in mutants defective in the DNA damage checkpoint. Studies involving the repair of a HO endonuclease-mediated DNA double-strand break (DSB) at the MAT locus show enhanced repair efficiency by the homologous recombination (HR) pathway on a reduction in histone dosage. Cells with reduced histone dosage experience greater histone loss around a DSB, whereas the recruitment of HR factors is concomitantly enhanced. Further, free histones compete with the HR machinery for binding to DNA and associate with certain HR factors, potentially interfering with HR-mediated repair. Our findings may have important implications for DNA repair, genomic stability, carcinogenesis and aging in human cells that have dozens of histone genes.