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Chromatin Disassembly Mediated by the Histone Chaperone Asf1 Is Essential for Transcriptional Activation of the Yeast PHO5 and PHO8 Genes
Abstract
Nucleosome loss from a promoter region has recently been described as a potential mechanism for transcriptional regulation. We investigated whether H3/H4 histone chaperones mediate the loss of nucleosomes from the promoter of the yeast PHO5 gene during transcriptional activation. We found that antisilencing function 1 (Asf1p) mediates nucleosome disassembly from the PHO5 promoter in vivo. We show that nucleosome disassembly also occurs at a second promoter, that of the PHO8 gene, during activation, and we demonstrate that this is also mediated by Asf1p. Furthermore, we show that nucleosome disassembly is essential for PHO5 and PHO8 activation. Contrary to the current dogma, we demonstrate that nucleosome disassembly is not required to enable binding of the Pho4p activator to its PHO5 UASp2 site in vivo. Finally, we show that nucleosomes are reassembled over the PHO5 promoter during repression. As such, nucleosome disassembly and reassembly are important mechanisms for transcriptional activation and repression, respectively.
... More recently, acetylation on the lateral surface of the nucleosome (H3 K64Ac, K122Ac) has also been implicated in transcription activation (Tropberger et al. 2013, Di Cerbo et al. 2014. Furthermore, the acetylation of H3 K56, which lies in the N-terminal α-helix of H3, is a hallmark of histones that are newly deposited during replication and transcription (Li et al. 2008), and this mark has been implicated in nucleosome assembly during replication and DNA repair (Masumoto et al. 2005;Recht et al. 2006) as well as in turnover of nucleosomes during transcription (Adkins, Howar and Tyler 2004;Schwabish and Struhl 2006). ...
... CAF-I deposits H3/H4 on newly replicated DNA (Kaufman et al. 1995), which it distinguishes from other cellular DNA by its interaction with PCNA (Shibahara and Stillman 1999). The histone chaperone Asf1 transfers H3/H4 to CAF-I during replication-coupled chromatin assembly (Tyler et al. 1999), but it also has replication-independent functions in that it is involved in the deposition of H3/H4 during transcription as well as in nucleosome disassembly at promoters during transcription activation (Adkins, Howar and Tyler 2004;Schwabish and Struhl 2006). The interaction of SAS-I with these chromatin assembly factors implies that SAS-I-mediated H4 K16 acetylation occurs in processes mediated by these factors, i.e. replication and transcription. ...
... We furthermore tested the effect of CAF-I and Asf1. While CAF-I function is restricted to replication-coupled chromatin assembly, Asf1 also has roles both in assembly and disassembly of H3/H4 during transcription (Adkins, Howar and Tyler 2004;Schwabish and Struhl 2006). cac1 did not decrease H4 K16Ac deposition (Fig. 3B), which may not be surprising, since the cells in this experiment were maintained in G1-phase. ...
The histone acetyltransferase Sas2 is part of the SAS-I complex and acetylates lysine 16 of histone H4 (H4 K16Ac) in the genome of Saccharomyces cerevisiae. Sas2-mediated H4 K16Ac is strongest over the coding region of genes with low expression. However, it is unclear how Sas2-mediated acetylation is incorporated into chromatin. Our previous work has shown physical interactions of SAS-I with the histone chaperones CAF-I and Asf1, suggesting a link between SAS-I mediated acetylation and chromatin assembly. Here, we find that Sas2-dependent H4 K16Ac in bulk histones requires passage of the cells through the S-phase of the cell cycle, and the rate of increase in H4 K16Ac depends on both CAF-I and Asf1, whereas steady-state levels and genome-wide distribution of H4 K16Ac show only mild changes in their absence. Furthermore, H4 K16Ac is deposited in chromatin at genes upon repression, and this deposition requires the histone chaperone Spt6, but not CAF-I, Asf1, HIR or Rtt106. Altogether, our data indicate that Spt6 controls H4 K16Ac levels by incorporating K16-unacetylated H4 in strongly transcribed genes. Upon repression, Spt6 association is decreased, resulting in less deposition of K16-unacetylated H4 and therefore in a concomitant increase of H4 K16Ac that is recycled during transcription.
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... Recruitment of transcription machinery to promoters is restricted by nucleosomes 19 . Histone chaperones and ATP-dependent chromatin-remodelling complexes are accordingly important for gene regulation given they regulate nucleosome dynamics at promoters, restricting or increasing promoter accessibility for recruitment of transcription factors and RNA polymerase II (Pol II) [19][20][21][22][23] . ...
... Do other histone chaperones have a role at the promoters? It has been reported that histone chaperone Asf1 mediates nucleosome disassembly from the PHO5 promoter for its activation in yeast 22,23 . In human, histone chaperones HIRA and ASF1a localise at active promoters where BRG1, a human SWI/ SNF complex subunit, also localises 52 . ...
The Pleiotropic Drug Resistance (PDR) network is central to the drug response in fungi, and its overactivation is associated with drug resistance. However, gene regulation of the PDR network is not well understood. Here, we show that the histone chaperone Rtt106 and the chromatin remodeller SWI/SNF control expression of the PDR network genes and confer drug resistance. In Saccharomyces cerevisiae, Rtt106 specifically localises to PDR network gene promoters dependent on transcription factor Pdr3, but not Pdr1, and is essential for Pdr3-mediated basal expression of the PDR network genes, while SWI/SNF is essential for both basal and drug-induced expression. Also in the pathogenic fungus Candida glabrata, Rtt106 and SWI/SNF regulate drug-induced PDR gene expression. Consistently, loss of Rtt106 or SWI/SNF sensitises drug-resistant S. cerevisiae mutants and C. glabrata to antifungal drugs. Since they cooperatively drive PDR network gene expression, Rtt106 and SWI/SNF represent potential therapeutic targets to combat antifungal resistance.
... For example, CAF-1 interacts with the replication processivity clamp PCNA and Asf1 interacts with RFC, which loads PCNA, and the MCM helicase (Groth et al. 2007;Franco et al. 2005). In addition to their role in nucleosome assembly, Asf1 and Rtt109 indirectly promote nucleosome disassembly through H3K56 acetylation (Adkins, Howar and Tyler 2004;Schwabish and Struhl 2006;Korber et al. 2006). The multifunctional roles of Asf1 and Rtt1109 (promoting nucleosome disassembly as well as binding of H3 to CAF-1 and Rtt106) thus make it difficult to predict what roles these factors might play in modulating heteroduplex rejection. ...
... This was surprising, given that Asf1 and Rtt109 act upstream of CAF-1 and Rtt106 in the nucleosome deposition pathway. Asf1 and Rtt109 have been implicated in nucleosome removal during DNA replication and transcriptional activation (Groth et al. 2007;Ransom, Dennehey, and Tyler 2010;Adkins, Howar and Tyler 2004;Schwabish and Struhl 2006;Korber et al. 2006). Such a nucleosome removal activity could also act during DNA recombination, and in its absence, lead to chromatin acting to stabilize strand invasion intermediates that are refractory to heteroduplex rejection. ...
Recombination between divergent DNA sequences is actively prevented by heteroduplex rejection mechanisms. In baker's yeast such anti-recombination mechanisms can be initiated by the recognition of DNA mismatches in heteroduplex DNA by MSH proteins, followed by recruitment of the Sgs1-Top3-Rmi1 helicase-topoisomerase complex to unwind the recombination intermediate. We previously showed that the repair/rejection decision during single-strand annealing recombination is temporally regulated by MSH protein levels and by factors that excise non-homologous single-stranded tails. These observations, coupled with recent studies indicating that mismatch repair factors interact with components of the histone chaperone machinery, encouraged us to explore roles for epigenetic factors and chromatin conformation in regulating the decision to reject vs. repair recombination between divergent DNA substrates. This work involved the use of an inverted repeat recombination assay thought to measure sister chromatid repair during DNA replication. Our observations are consistent with the histone chaperones CAF-1 and Rtt106 and the histone deacetylase Sir2 acting to suppress heteroduplex rejection and the Rpd3, Hst3 and Hst4 deacetylases acting to promote heteroduplex rejection. These observations and double mutant analysis have led to a model in which nucleosomes located at DNA lesions stabilize recombination intermediates and compete with mismatch repair factors that mediate heteroduplex rejection.
... Recent studies have provided insight into the roles of histone modifications and histone chaperones in regulating chromatin assembly (24)(25)(26)(27). Histone modifications affect chromatin assembly in various ways, including the regulation of histone folding and processing, histone nuclear import, and the interaction between histones and histone chaperones (28)(29)(30)(31). For example, H4K5&K12ac, a diacetylation catalyzed by histone acetyltransferase 1(HAT1)-RbAp46, is detected on newly synthesized histone H4 from yeast to humans as an early modification occurring on H3-H4 (32)(33)(34)(35)(36). ...
... Newly synthesized H3-H4 molecules associate with HAT1, which acetylates H4K5&K12 (31,32,34,35,44). After acetylation, the histones are bound to ASF1, which associates with importin 4. Thus, downregulation of H4K12ac may disrupt the association of H3/H4 with ASF1 and importin 4, inhibiting histone nuclear translocation (28,45,46). After nuclear import, the H3-H4 dimer of the ASF1 complex is transferred to the central histone chaperone CAF-1, which binds two H3-H4 dimers to form (H3-H4) 2 tetramers and deposits them onto replicating DNA through the CAF-1proliferating cell nuclear antigen interaction (47,48). ...
Acrolein is a major component of cigarette smoke and cooking fumes. Previously, we reported that acrolein compromises chromatin assembly; however, underlying mechanisms have not been defined. Here, we report that acrolein reacts with lysine residues including lysines 5 and 12 on histone H4 in vitro and in vivo, sites important for chromatin assembly. Acrolein-modified histones are resistant to acetylation, suggesting that the reduced H4K12 acetylation following acrolein exposure is likely due to the formation of acrolein-histone lysine adducts. Accordingly, the association of H3/H4 with the histone chaperone ASF1 and importin 4 is disrupted and the translocation of GFP-tagged H3 is inhibited in cells exposed to acrolein. Interestingly, in vitro plasmid supercoiling assays reveal that treatment of either histones or ASF1 with acrolein has no effect on formation of plasmid supercoiling, indicating that acrolein-protein adduct formation itself does not directly interfere with nucleosome assembly. Notably, exposure of histones to acrolein prior to histone acetylation leads to the inhibition of RSF (Remodeling and Spacing Factor) chromatin assembly, which requires acetylated histones for efficient assembly. These results suggest that acrolein compromises chromatin assembly via reacting with histone lysine residues at the sites critical for chromatin assembly and prevents these sites from physiological modifications.
... It was discovered that it caused a loss of position-dependent transcriptional silencing when overexpressed, making it an essential component of chromatin-related functions [6]. Asf1 facilitates multiple processes, including the assembly and disassembly of nucleosomes, the cellular response to DNA damage, and the coordination of DNA-templated activities, such as replication, transcription, and repair [7][8][9]. The deletion of ASF1 renders cells sensitive to agents that induce DNA damage and replicational stress [8]. ...
The problem of low-dose irradiation has been discussed in the scientific literature for several decades, but it is impossible to come to a generally accepted conclusion about the presence of any specific features of low-dose irradiation in contrast to acute irradiation. We were interested in the effect of low doses of UV radiation on the physiological processes, including repair processes in cells of the yeast Saccharomyces cerevisiae, in contrast to high doses of radiation. Cells utilize excision repair and DNA damage tolerance pathways without significant delay of the cell cycle to address low levels of DNA damage (such as spontaneous base lesions). For genotoxic agents, there is a dose threshold below which checkpoint activation is minimal despite the measurable activity of the DNA repair pathways. Here we report that at ultra-low levels of DNA damage, the role of the error-free branch of post-replicative repair in protection against induced mutagenesis is key. However, with an increase in the levels of DNA damage, the role of the error-free repair branch is rapidly decreasing. We demonstrate that with an increase in the amount of DNA damage from ultra-small to high, asf1Δ-specific mutagenesis decreases catastrophically. A similar dependence is observed for mutants of gene-encoding subunits of the NuB4 complex. Elevated levels of dNTPs caused by the inactivation of the SML1 gene are responsible for high spontaneous reparative mutagenesis. The Rad53 kinase plays a key role in reparative UV mutagenesis at high doses, as well as in spontaneous repair mutagenesis at ultra-low DNA damage levels.
... Histone chaperones mediate nucleosome assembly or disassembly in several DNA-dependent processes and regulate chromatin functions through the assistance of histone modification [9][10][11]. Anti-silencing function 1 (ASF1) is a highly conserved histone H3-H4 chaperone linked to the modulation of cell cycle, DNA damage repair, as well as transcription regulation [12]. ...
Anti-silencing function 1B (ASF1B) has been reported to be associated with the occurrence of many kinds of tumors. However, the biological effect and action mechanism of ASF1B in pancreatic cancer (PC) tumorigenesis remain unclear. The expression and prognosis value of ASF1B in PC were analyzed using GEPIA, GEO, and Kaplan–Meier plotter databases. The diagnostic value of ASF1B in PC was determined by receiver operating characteristic curve. The relationship between ASF1B expression and the clinical feathers in PC was investigated based on TCGA. qRT-PCR and western blot analyses were used to measure ASF1B expression in PC cells. Cell proliferation was evaluated by MTT and EdU assays, and apoptosis was examined by TUNEL and caspase-3 activity assays. Western blot analysis was utilized to detect the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, Bax, Bcl-2, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling proteins. ASF1B was overexpressed in several digestive cancers, including PC. Upregulated ASF1B was correlated with the poor prognosis and clinical features in PC patients. The area under the curve (AUC) value of ASF1B was 0.990. ASF1B was also overexpressed in PC cells. ASF1B silencing inhibited PC cell proliferation, promoted apoptosis, and increased caspase-3 activity, which were accompanied by the reduction of PCNA and cyclin D1 expression and increase of the ratio of Bax/Bcl-2 expression. Additionally, ASF1B silencing suppressed the PI3K/Akt pathway and 740Y-P treatment partially abolished the effects of ASF1B knockdown on PC cells. In conclusion, ASF1B silencing retarded proliferation and promoted apoptosis in PC cells by inactivation of the PI3K/Akt pathway.
... Depletion of ASF1 had a considerable effect on productive transcription. ASF1 is required for nucleosome disruption in yeast Adkins, Howar, and Tyler 2004), and for both assembly and disassembly of H3 histones during RNAPII elongation (Marc A. Schwabish and Struhl 2006). In mammalian cells, ASF1 is localised by ChIP at promoters of transcriptionally active genes (Pchelintsev et al. 2013). ...
The packaging of DNA into nucleosomes represents a challenge for transcription. Nucleosome disruption and histone eviction enables RNA Polymerase II progression through DNA, a process that compromises chromatin integrity and the maintenance of epigenetic information. Here, we used the imaging SNAP-tag system to distinguish new and old histones and monitor chromatin re-assembly coupled to transcription incells. First, we uncovered a loss of both old variants H3.1 and H3.3 that depends on transcriptional activity, with a major effect on H3.3. Focusing on transcriptionally active domains, we revealed a local enrichment in H3.3 with dynamics involving both new H3.3 incorporation and old H3.3 retention. Mechanistically, we demonstrate that the HIRA chaperone is critical to handle both new and old H3.3, and showed that this implicates different pathways. The de novo H3.3 deposition depends strictly on HIRA trimerization as well as its partner UBN1 while ASF1 interaction with HIRA can be bypassed. In contrast, the recycling of H3.3 requires HIRA but proceeds independently of UBN1 or HIRA trimerization and shows an absolute dependency on ASF1-HIRA interaction. Therefore, we propose a model where HIRA can coordinate these distinct pathways for old H3.3 recycling and new H3.3 deposition during transcription to finetune chromatin states.
... ASF1 functions by transferring H3-H4 heterodimers to the histone chaperone CAF-1 or HIRA for nucleosome assembly 23 and contributes to heterochromatin formation [24][25][26] . In addition to its role in nucleosome assembly, ASF1 also plays a role in nucleosome disassembly and histone exchange [27][28][29][30] . Here, we find that ASF1 forms a complex with RIF1 in response to DNA damage through a B-domain, which is also responsible for the interactions of CAF-1 and HIRA with ASF1. ...
The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identify a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair. The 53BP1-RIF1 pathway is important for DNA repair. Here, the authors identified the histone chaperone ASF1, which functions as a suppressor of DNA end resection through changing high-order chromatin structure, as a partner of RIF1. This finding links DNA repair and dynamic changes of high-order chromatin structure.
... Replication coupled chromatin assembly (Tyler et al., 1999) Replication independent chromatin assembly (Rufiange et al., 2007) Heterochromatic gene silencing (Sharp et al., 2001) Nucleosome disassembly during gene expression (Adkins et al., 2004;Erkina and Erkine, 2015) Somatic cellular reprogramming and maintenance of pluripotency (Gonzalez-Munoz et al., 2014) Cell cycle progression, chromatin assembly during endoreduplication (Zhu et al., 2011) Double strand break (DSB) pathway (Huang et al., 2018;Zhu et al., 2011) UV induced DNA damage repair (Lario et al., 2013) Tolerance against heat and oxidative stress (Weng et al., 2014) AtFKBP53 elements of the viruses that enter and persist in the nucleus also interact with the cellular chromatin machinery and are also subjected to the process of chromatin formation in some cases. Animal and plant DNA viruses generally use the cellular replication enzyme for replication and form chromatin structure similar to the cellular nucleosome. ...
Nucleosomes are assembled or disassembled with the aid of histone chaperones in a cell. Viruses can exist either as minichromosomes/episomes or can integrate into the host genome and in both the cases the viral proteins interact and manipulate the cellular nucleosome assembly machinery to ensure their survival and propagation. Recent studies have provided insight into the mechanism and role of histone chaperones in nucleosome assembly and disassembly on the virus genome. Further, the interactions between viral proteins and histone chaperones have been implicated in the integration of the virus genome into the host genome. This review highlights the recent progress and future challenges in understanding the role of histone chaperones in viruses with DNA or RNA genome and their role in governing viral pathogenesis.
... During replication it collaborates with the CAF-1 complex [4] while replication-independent histone deposition also requires the HIR proteins (composed of Hir1, Hir2, Hir3, and Hpc2) [5]. In addition, Asf1 is capable of mediating chromatin disassembly as well as chromatin reassembly during transcription [17,18]. The binding of Asf1 to a H3-H4 dimer prevents it from tetramerizing prematurely. ...
Acetylation on lysine 56 of histone H3 of the yeast Saccharomyces cerevisiae has been implicated in many cellular processes that affect genome stability. Despite being the object of much research, the complete scope of the roles played by K56 acetylation is not fully understood even today. The acetylation is put in place at the S-phase of the cell cycle, in order to flag newly synthesized histones that are incorporated during DNA replication. The signal is removed by two redundant deacetylases, Hst3 and Hst4, at the entry to G2/M phase. Its crucial location, at the entry and exit points of the DNA into and out of the nucleosome, makes this a central modification, and dictates that if acetylation and deacetylation are not well concerted and executed in a timely fashion, severe genomic instability arises. In this review, we explore the wealth of information available on the many roles played by H3K56 acetylation and the deacetylases Hst3 and Hst4 in DNA replication and repair.
... [37,38] Among these ASF1 (anti-silencing function 1) and CAF-1 (chromatin assembly factor 1), are key H3-H4 histone chaperone factors, which also play a crucial role in replicationcoupled nucleosome assembly and eviction at promoters during fork progression and transcriptional activation. [39][40][41] Another important Cancer heterogeneity F I G U R E 1 Biological processes in which linker histone H1 is involved. Linker histone H1 is implicated in the regulation of DNA metabolism and chromatin processes by facilitating or inhibiting the recruitment of protein complexes to chromatin. ...
During early embryonic development in several metazoans, accurate DNA replication is ensured by high number of replication origins. This guarantees rapid genome duplication coordinated with fast cell divisions. In Xenopus laevis embryos this program switches to one with a lower number of origins at a developmental stage known as mid-blastula transition (MBT) when cell cycle length increases and gene transcription starts. Consistent with this regulation, somatic nuclei replicate poorly when transferred to eggs, suggesting the existence of an epigenetic memory suppressing replication assembly origins at all available sites. Recently, it was shown that histone H1 imposes a non-permissive chromatin configuration preventing replication origin assembly on somatic nuclei. This somatic state can be erased by SSRP1, a subunit of the FACT complex. Here, we further develop the hypothesis that this novel form of epigenetic memory might impact on different areas of vertebrate biology going from nuclear reprogramming to cancer development.
... ASF1 is involved in both Replication-Coupled and Replication-Independent histone deposition pathways. During gene transcription in yeast and Arabidopsis, ASF1 association with chromatin results promotes H3K56 acetylation, in turn facilitating nucleosome disassembly, allowing RNA PolII recruitment to gene promoters 10,39,40 . ASF1-mediated chromatin remodeling is also required for DNA replication and gene silencing, and its role in nucleosome assembly and disassembly is notable in centromeric regions, where ASF1 displays overlapping function with the CenH3 chaperone Sim3 in fission yeast 41,42 . ...
Sexual reproduction in flowering plants is distinct from that in animals since gametogenesis requires production of haploid spores, which divide and differentiate into specialised gametophyte structures. Anti-Silencing Function 1 (ASF1) is a histone H3/H4 chaperone involved in chromatin remodeling during cell division, which we have found plays a critical role in gametophyte development in Arabidopsis thaliana. Using mutant alleles for the two ASF1 homologs, asf1a and asf1b, we show that ASF1 is required for successful development of gametophytes and acquisition of fertilisation competency. On the female side, reproductive failure is caused by aberrant development of ovules, leading to gamete degeneration. On the male side, we show both in vitro and in vivo that asf1 mutant pollen tube growth is stunted, limiting fertilisation to ovules nearest the stigma. Consistent with ASF1 importance in gametogenesis, we show that ASF1A and ASF1B are expressed throughout female and male gametogenesis. We show that the gametogenesis defects can be corrected by ASF1A and ASF1B transgenes, and that ASF1A and ASF1B act redundantly. Thus, in contrast to the role of ASF1 in sporophytic cell cycle progression, our data indicate that during reproduction, ASF1 is required for the precise nuclei differentiation necessary for gametophyte maturation and fertilisation.
... We therefore looked at histone turnover and occupancy of the histone chaperones Asf1 and HIR in spt16-197 and spt6-1004 cells. Asf1 is notoriously difficult to detect by ChIP (discussed in Adkins et al. [2004]), but one study suggested that it binds to the transcribed regions of highly transcribed genes (Dewari and Bhargava, 2014). These experiments, however, did not include controls with no-tag samples. ...
Genomic DNA is framed by additional layers of information, referred to as the epigenome. Epigenomic marks such as DNA methylation, histone modifications, and histone variants are concentrated on specific genomic sites, where they can both instruct and reflect gene expression. How this information is maintained, notably in the face of transcription, is not completely understood. Specifically, the extent to which modified histones themselves are retained through RNA polymerase II passage is unclear. Here, we show that several histone modifications are mislocalized when the transcription-coupled histone chaperones FACT or Spt6 are disrupted in Saccharomyces cerevisiae. In the absence of functional FACT or Spt6, transcription generates nucleosome loss, which is partially compensated for by the increased activity of non-transcription-coupled histone chaperones. The random incorporation of transcription-evicted modified histones scrambles epigenomic information. Our work highlights the importance of local recycling of modified histones by FACT and Spt6 during transcription in the maintenance of the epigenomic landscape.
... However, our MNase-seq analyses showed that the distribution of nucleosome pair distances as well as the positioning of nucleosomes around TSSs is not changed in the asf1 mutant compared to the wild type. By these analyses, we cannot exclude effects of asf1 on nucleosome turnover as was observed in at the PHO5 gene promoter in yeast [82][83][84][85], or short-lived nucleosome positioning effects, but it is also possible that the effect of asf1 on transcript levels depends on other chromatin modifications. In yeast, it was shown that Asf1 promotes histone H3 acetylation at lysine 56 by the histone acetyltransferase Rtt109 [57,86,87]. ...
Background
Fungal fruiting bodies are complex three-dimensional structures that are formed to protect and disperse the sexual spores. Their morphogenesis requires the concerted action of numerous genes; however, at the molecular level, the spatio-temporal sequence of events leading to the mature fruiting body is largely unknown. In previous studies, the transcription factor gene pro44 and the histone chaperone gene asf1 were shown to be essential for fruiting body formation in the ascomycete Sordaria macrospora. Both PRO44 and ASF1 are predicted to act on the regulation of gene expression in the nucleus, and mutants in both genes are blocked at the same stage of development. Thus, we hypothesized that PRO44 and ASF1 might be involved in similar aspects of transcriptional regulation. In this study, we characterized their roles in fruiting body development in more detail.
Results
The PRO44 protein forms homodimers, localizes to the nucleus, and is strongly expressed in the outer layers of the developing young fruiting body. Analysis of single and double mutants of asf1 and three other chromatin modifier genes, cac2, crc1, and rtt106, showed that only asf1 is essential for fruiting body formation whereas cac2 and rtt106 might have redundant functions in this process. RNA-seq analysis revealed distinct roles for asf1 and pro44 in sexual development, with asf1 acting as a suppressor of weakly expressed genes during morphogenesis. This is most likely not due to global mislocalization of nucleosomes as micrococcal nuclease-sequencing did not reveal differences in nucleosome spacing and positioning around transcriptional start sites between Δasf1 and the wild type. However, bisulfite sequencing revealed a decrease in DNA methylation in Δasf1, which might be a reason for the observed changes in gene expression. Transcriptome analysis of gene expression in young fruiting bodies showed that pro44 is required for correct expression of genes involved in extracellular metabolism. Deletion of the putative transcription factor gene asm2, which is downregulated in young fruiting bodies of Δpro44, results in defects during ascospore maturation.
Conclusions
In summary, the results indicate distinct roles for the transcription factor PRO44 and the histone chaperone ASF1 in the regulation of sexual development in fungi.
Electronic supplementary material
The online version of this article (10.1186/s12863-018-0702-z) contains supplementary material, which is available to authorized users.
... In addition to its role in nucleosome assembly, Asf1 also has a role in nucleosome disassembly and gene transcription. For instance, in budding yeast, Asf1 mediates nucleosome disassembly at promoter regions and is essential for transcriptional activation of yeast PHO5 and PHO8 genes (26)(27)(28). In Drosophila, Asf1 is recruited to specific target promoters, together with other histone modifiers, and regulates gene expression (29,30). ...
Significance
ES cells possess the unique capacity to self-renew as well as differentiate into specialized cell types. It is known that transcription factors and chromatin regulators regulate the cell-fate choices during differentiation. We report unexpectedly that Asf1a, a histone chaperone involved in nucleosome assembly, regulates mouse ES cell differentiation. Mechanistically, we show that Asf1a functions in nucleosome disassembly to resolve the bivalent chromatin domains at lineage-specific genes for gene activation during differentiation. These insights will likely be applicable for understanding human ES cell differentiation and regenerative medicine.
... Existing studies have elaborated that access to DNA in nucleosomes can be regulated via different mechanisms, e.g. chromatin remodelling (33)(34)(35), histone modifications (36,37), histone variants (5,6) and chaperones (38)(39)(40). These mechanisms differ in their ways and outcomes of triggering nucleosome destabilization, but ultimately, they all destabilize nucleosomes by altering DNA-histone interactions. ...
The first nucleosomes in the downstream of transcription starting sites are called +1 nucleosomes, which are expected to be readily unwrapped for DNA transcription. To investigate DNA accessibility in +1 nucleosomes, MNase-seq experiments were carried out with 20 reconstituted +1 nucleosomes of budding yeast. Although MNase has been known for its sequence preference in DNA digestions, we confirmed that this sequence preference is overwhelmed by DNA accessibility by identifying the sequence-driven and accessibility-driven cleavages. Specifically, we find that sequences favoured by MNase at the end regions such as TA dinucleotide are prohibited from cleavage at the internal sites in the early stage of digestion. Nevertheless, sequences less favoured by MNase at the end regions such as AA/TT dinucleotide are predominantly cleaved at the internal sites in the early stage of digestion. Since AA/TT is known as a rigid dinucleotide step resistant to DNA bending, these internal cleavages reflect the local site exposures induced by DNA mechanics. As the DNA entry site of +1 nucleosomes in yeast is found AA/TT-rich, this sequence element may play a role in gene activation by reducing DNA-histone affinities along the direction of DNA transcription.
... Appropriate acetylation of N-terminal tails of newly synthesized histones H3 and H4 is considered critical for histone nuclear import and nucleosome assembly [Burgess and Zhang, 2013;Fang et al., 2014]. Histone modifications affect chromatin assembly in various ways, including the regulation of histone folding and processing, histone nuclear import, and the interaction between histones and histone chaperones [Adkins et al., 2004;Groth et al., 2005;Zhang et al., 2012]. H4K5&K12Ac, a di-acetylation catalyzed by HAT1, is detected in newly synthesized histone H4 from yeast to human as an early modification occurring in H3-H4 [Ejlassi-Lassallette et al., 2011;Nagarajan et al., 2013]. ...
As the primary metabolite of alcohol and the most abundant carcinogen in tobacco smoke, acetaldehyde is linked to a number of human diseases associated with chronic alcohol consumption and smoking including cancers. In addition to direct DNA damage as a result of the formation of acetaldehyde‐DNA adducts, acetaldehyde may also indirectly impact proper genome function through the formation of protein adducts. Histone proteins are the major component of the chromatin. Post‐translational histone modifications (PTMs) are critically important for the maintenance of genetic and epigenetic stability. However, little is known about how acetaldehyde‐histone adducts affect histone modifications and chromatin structure. The results of protein carbonyl assays suggest that acetaldehyde forms adducts with histone proteins in human bronchial epithelial BEAS‐2B cells. The level of acetylation for N‐terminal tails of cytosolic histones H3 and H4, an important modification for histone nuclear import and chromatin assembly, is significantly downregulated following acetaldehyde exposure in BEAS‐2B cells, possibly due to the formation of histone adducts and/or the decrease in the expression of histone acetyltransferases. Notably, the level of nucleosomal histones in the chromatin fraction and at most of the genomic loci we tested are low in acetaldehyde‐treated cells as compared with the control cells, which is suggestive of inhibition of chromatin assembly. Moreover, acetaldehyde exposure perturbs chromatin structure as evidenced by the increase in general chromatin accessibility and the decrease in nucleosome occupancy at genomic loci following acetaldehyde treatment. Our results indicate that regulation of histone modifications and chromatin accessibility may play important roles in acetaldehyde‐induced pathogenesis. Environ. Mol. Mutagen., 2018. © 2018 Wiley Periodicals, Inc.
... Deletion of RTT106 or ASF1 alone also causes a growth phenotype when Cse4 is overexpressed. These three histone H3/H4 chaperones play roles in several chromatin dependent processes including promoter fidelity, heterochromatin silencing and histone gene transcription, and general H3 assembly and disassembly (2,(48)(49)(50)(51)(52)(53)(54)(55)(56). Disruption of any of these processes could negatively affect growth. ...
Correct localization of the centromeric histone variant CenH3/CENP-A/Cse4 is an important part of faithful chromosome segregation. Mislocalization of CenH3 could affect chromosome segregation, DNA replication and transcription. CENP-A is often overexpressed and mislocalized in cancer genomes, but the underlying mechanisms are not understood. One major regulator of Cse4 deposition is Psh1, an E3 ubiquitin ligase that controls levels of Cse4 to prevent deposition into non-centromeric regions. We present evidence that Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved histone H3/H4 chaperone with subunits shown previously to interact with CenH3 in flies and human cells, regulates Cse4 deposition in budding yeast. yCAF-1 interacts with Cse4 and can assemble Cse4 nucleosomes in vitro. Loss of yCAF-1 dramatically reduces the amount of Cse4 deposited into chromatin genome-wide when Cse4 is overexpressed. The incorporation of Cse4 genome-wide may have multifactorial effects on growth and gene expression. Loss of yCAF-1 can rescue growth defects and some changes in gene expression associated with Cse4 deposition that occur in the absence of Psh1-mediated proteolysis. Incorporation of Cse4 into promoter nucleosomes at transcriptionally active genes depends on yCAF-1. Overall our findings suggest CAF-1 can act as a CenH3 chaperone, regulating levels and incorporation of CenH3 in chromatin.
... Histone chaperones (e.g., Asf1p) are known to have both nucleosome assembly and disassembly activities [32][33][34][35] , and to promote adenosine triphosphate (ATP)-dependent remodeling in vitro and in vivo [36][37][38] . Since our results suggest that Dot1p is likely to be a histone chaperone that is involved in histone exchange and cryptic transcription, we hypothesized that Dot1p may stimulate ATP-dependent chromatin remodelers in vitro. ...
Dot1 (disruptor of telomeric silencing-1, DOT1L in humans) is the only known enzyme responsible for histone H3 lysine 79 methylation (H3K79me) and is evolutionarily conserved in most eukaryotes. Yeast Dot1p lacks a SET domain and does not methylate free histones and thus may have different actions with respect to other histone methyltransferases. Here we show that Dot1p displays histone chaperone activity and regulates nucleosome dynamics via histone exchange in yeast. We show that a methylation-independent function of Dot1p is required for the cryptic transcription within transcribed regions seen following disruption of the Set2-Rpd3S pathway. Dot1p can assemble core histones to nucleosomes and facilitate ATP-dependent chromatin-remodeling activity through its nucleosome-binding domain, in vitro. Global analysis indicates that Dot1p appears to be particularly important for histone exchange and chromatin accessibility on the transcribed regions of long-length genes. Our findings collectively suggest that Dot1p-mediated histone chaperone activity controls nucleosome dynamics in transcribed regions.
... However, we found that SPN1 interacts genetically with CAC1 (a CAF-1 subunit gene), ASF1 and RTT106 all of which are known to encode proteins that function in DNA repair and/or replication (50). While Asf1 and Rtt106 also function in transcription (63,64), CAF-1 does not, indicating the growth defects seen with spn1 141-305 and cac1Δ are most likely due to negative effects on DNA replication or repair. Finally, spn1 141-305 was essentially synthetically lethal when it was combined with deletion of HIR1 and HIR2, which encode subunits of the HIR complex (65,66). ...
The process of transcriptional elongation by RNA polymerase II (RNAPII) in a chromatin context involves a large number of crucial factors. Spn1 is a highly conserved protein encoded by an essential gene and is known to interact with RNAPII and the histone chaperone Spt6. Spn1 negatively regulates the ability of Spt6 to interact with nucleosomes, but the chromatin binding properties of Spn1 are largely unknown. Here, we demonstrate that full length Spn1 (amino acids 1-410) binds DNA, histones H3-H4, mononucleosomes and nucleosomal arrays, and has weak nucleosome assembly activity. The core domain of Spn1 (amino acids 141-305), which is necessary and sufficient in Saccharomyces cerevisiae for growth under ideal growth conditions, is unable to optimally interact with histones, nucleosomes and/or DNA and fails to assemble nucleosomes in vitro. Although competent for binding with Spt6 and RNAPII, the core domain derivative is not stably recruited to the CYC1 promoter, indicating chromatin interactions are an important aspect of normal Spn1 functions in vivo. Moreover, strong synthetic genetic interactions are observed with Spn1 mutants and deletions of histone chaperone genes. Taken together, these results indicate that Spn1 is a histone binding factor with histone chaperone functions.
... The in vivo nucleosome eviction activity of such chromatin remodelling complexes has been shown to require histone chaperones to tether the evicted histones [7]. One of the best characterised histone chaperones is Asf1, which coordinates nucleosome dynamics at gene promoters and open reading frames [33,34]. Indeed, Swi-Snf has been shown to cooperate with, or work in parallel to, Asf1 to displace nucleosomes at the HO promoter and PHO5/PHO8 promoters, respectively [35,36]. ...
Recently, we reported that a major function of histone acetylation at the yeast FLO1 gene was to regulate transcription elongation. Here, we discuss possible mechanisms by which histone acetylation might regulate RNA polymerase II processivity, and comment on the contribution to transcription of chromatin remodelling at gene coding regions and promoters.
... We find it striking, in this regard, that À1 FN promoters are characterized by an expansion of poly(dA:dT) elements and G/C rich motifs, rather than any clear increase in TF site density. This regulatory strategy contrasts with that observed at highly induced genes (Fig. 4), for example the well characterized PHO5 gene, where the active state is driven by nucleosome removal, which requires multiple binding sites for the Pho2 and Pho4 TFs, as well as the cooperation of the Asf1 histone chaperone [66,67]. ...
Improvements in deep sequencing, together with methods to rapidly deplete essential transcription factors (TFs) and chromatin remodelers, have recently led to a more detailed picture of promoter nucleosome architecture in yeast and its relationship to transcriptional regulation. These studies revealed that ?40% of all budding yeast protein-coding genes possess a unique promoter structure, where we propose that an unusually unstable nucleosome forms immediately upstream of the transcription start site (TSS). This "fragile" nucleosome (FN) promoter architecture relies on the combined action of the essential RSC (Remodels Structure of Chromatin) nucleosome remodeler and pioneer transcription factors (PTFs). FNs are associated with genes whose expression is high, coupled to cell growth, and characterized by low cell-to-cell variability (noise), suggesting that they may promote these features. Recent studies in metazoans suggest that the presence of dynamic nucleosomes upstream of the TSS at highly expressed genes may be conserved throughout evolution.
... The kinetics of histone turnover highlight enhancers and promoters as major sites of histone replacement, whereas gene bodies have slower rates of histone turn over (reviewed in REF. 2). Asf1 can aid histone eviction at promoters and in coding regions [183][184][185] . In moderately transcribed genes, a single H2A-H2B dimer may be displaced per nucleosome, whereas highly transcribed genes are characterized by more pronounced nucleo some disruption (reviewed in REF. 2). ...
The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly and genomic localization. In this way, the chaperoning of soluble histones is a key determinant of histone availability and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin.
... Pho4 activates PHO genes transcription (e.g. the phosphatase gene PHO5) in response to phosphate scarcity conditions (Oshima, 1997). Pho4 binding triggers alterations in the nucleosome positioning at the PHO5 promoter and the Asf1 histone chaperone removes histones from the promoter region, thereby opening the TATA box (Adkins et al., 2004;Svaren and Hörz, 1997). However, a repressing function of activator Pho4 could be also detected, since it binds to the promoter of SNZ1 (a gene expressed in the stationary phase) and inhibits its expression in the absence of Pho85 (Nishizawa et al., 2008). ...
... expression which means that Sro9 is associated with actively transcribed genes (Rother et al.,2010).Moreover, Sro9 shares with Slf1 approximately 29.8% similarity throughout their amino acid sequence(Wolfe and Shields, 1997), and both Sro9 and Slf1 proteins are RNA-binding proteins which belong to a highly conserved La motif-containing proteins family that are found in all eukaryotes(Yu et al., 1994;Yoo andWolin, 1994 andCedervall, 2002).Rtt109 is a specific histone acetyltransferas (HAT) that is expressed in S-phase, and has been found to acetylate H3-K56 and H3-K9(Fillingham et al., 2008). Acetylation of H3-K9 suggest that Rtt109 has an important role in transcription of S-phase specific genes and likely H3 genes, which has variants H3.1 and H3.3 that are different in their expression (Gunjan et al., 2005). The synthetic genetic interaction of the rtt109Δ was observed with deletions of the transcription-related genes (Fillingham et al., 2008). ...
STRUCTURE/FUNCTION ANALYSIS OF THE HIF1 HISTONE CHAPERONE IN SACCHAROMYCES CEREVISIAE
... There is ample evidence that the H3-H4 chaperone Asf1 is involved directly and/or indirectly in chromatin disassembly during transcription. Yeast Asf1 is found in transcribed regions with elongating RNA Pol II (Schwabish and Struhl 2006 ) and facilitates chromatin disassembly at inducible promoters during transcriptional activation (e.g., PHO5, PHO8, GAL1-10, and HO ) (Adkins et al. 2004 ;Korber et al. 2006 ;Schwabish and Struhl 2006 ;Adkins et al. 2007 ;Gkikopoulos et al. 2009 ;Takahata et al. 2009 ). Loss of Asf1 reduces nucleosome remodeling at the HO promoter (Gkikopoulos et al. 2009 ), in a specifi c region ), resulting in decreased cell cycle-dependent transcription of HO (Gkikopoulos et al. 2009 ). ...
As we learned in the previous chapter, the eukaryotic genome exists in our cells as the nucleoprotein complex chromatin. A human cell contains approximately 40 million nucleosomes, which are the fundamental repeating units of chromatin. As expected for a highly conserved structure, such as that of the nucleosome, its assembly is tightly orchestrated. In this chapter, we learn how histone chaperone proteins help establish the formation of the nucleosomal structure from the core histones and DNA. One profound consequence of chromatin formation is blocked access of the cellular machineries that require intimate access to DNA for DNA replication, DNA repair and transcription. We present the current state of knowledge regarding how chromatin is locally disassembled to allow genomic processes to occur, and how it is then rapidly reassembled back into chromatin. As a whole, chromatin assembly not only enables the genome to be packaged to fit into our cells but also enables the regulation of all genomic functions through dynamic chromatin disassembly and reassembly. © 2014 Springer Science+Business Media New York. All rights reserved.
... Chromatin is a key regulator of gene expression, influencing factor binding, transcription initiation, and elongation (Li et al. 2007). Nucleosomes can occlude transcription factor binding sites, and at some promoters nucleosomes must be disassembled by transcriptional coactivators prior to effective protein binding (Verdone et al. 2002;Adkins et al. 2004;Schwabish and Struhl 2007;Biddick et al. 2008;Takahata et al. 2009). At most promoters, however, transcription factor binding is much less complex due to natural depletion of nucleosomes from their binding sites. ...
Nucleosome-depleted regions (NDRs) are present immediately adjacent to the TSS in most eukaryotic promoters. Here we show that NDRs in the upstream promoter region can profoundly affect gene regulation. Chromatin at the yeast HO promoter is highly repressive and numerous coactivators are required for expression. We modified the HO promoter with segments from the well-studied CLN2 NDR, creating chimeric promoters differing in nucleosome occupancy but with binding sites for the same activator, SBF. Nucleosome depletion resulted in substantial increases in both factor binding and gene expression and allowed activation from a much longer distance, probably by allowing recruited coactivators to act further downstream. Nucleosome depletion also affected sequential activation of the HO promoter; HO activation typically requires the ordered recruitment of activators firstly to URS1, secondly to the left-half of URS2 (URS2-L), and finally to the right-half of URS2 (URS2-R), with each region representing distinct gates that must be unlocked in order to achieve activation. The absence of nucleosomes at URS2-L resulted in promoters no longer requiring both the URS1 and URS2-L gates, as either gate alone is now sufficient to promote binding of the SBF factor to URS2-R. Furthermore, nucleosome depletion at URS2 altered the timing of HO expression and bypassed the regulation that restricts expression to mother cells. Our results reveal insight into how nucleosomes can create a requirement for ordered recruitment of factors to facilitate complex transcriptional regulation.
... Disassembly and reassembly of histones is required for efficient repair of DNA damage [2,7]. Furthermore, transcriptional modulation is intimately linked to the histone density at the corresponding loci [6,[33][34][35]. Interestingly, several studies reported functions of chromatin-remodeling factors in the regulation of stressresponsive genes [33,[36][37][38]. ...
Human fungal pathogens like Candida albicans respond to host immune surveillance by rapidly adapting their transcriptional programs. Chromatin assembly factors are involved in the regulation of stress genes by modulating the histone density at these loci. Here, we report a novel role for the chromatin assembly-associated histone acetyltransferase complex NuB4 in regulating oxidative stress resistance, antifungal drug tolerance and virulence in C. albicans. Strikingly, depletion of the NuB4 catalytic subunit, the histone acetyltransferase Hat1, markedly increases resistance to oxidative stress and tolerance to azole antifungals. Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment. Furthermore, hat1Δ/Δ cells, despite showing growth defects in vitro, display reduced susceptibility to reactive oxygen-mediated killing by innate immune cells. Thus, clearance from infected mice is delayed although cells lacking Hat1 are severely compromised in killing the host. Interestingly, increased oxidative stress resistance and azole tolerance are phenocopied by the loss of histone chaperone complexes CAF-1 and HIR, respectively, suggesting a central role for NuB4 in the delivery of histones destined for chromatin assembly via distinct pathways. Remarkably, the oxidative stress phenotype of hat1Δ/Δ cells is a species-specific trait only found in C. albicans and members of the CTG clade. The reduced azole susceptibility appears to be conserved in a wider range of fungi. Thus, our work demonstrates how highly conserved chromatin assembly pathways can acquire new functions in pathogenic fungi during coevolution with the host.
... The eviction of old histones by ASF1 also facilitates transcription factor or RNA polymerase II entry to the transcription start site at various promoter regions [15]. In addition, ASF1 mediates chromatin disassembly on gene promoter regions during transcriptional activation and elongation in budding yeast [16]. It has been reported that ASF1 binds to histones not bound to DNA, indicating its role in chromatin higher-order organization [17][18][19]. ...
Chromatin is a highly organized and dynamic structure in eukaryotic cells. The change of chromatin structure is essential in many cellular processes, such as gene transcription, DNA damage repair and others. Anti-silencing function 1 (ASF1) is a histone chaperone that participates in chromatin higher-order organization and is required for appropriate chromatin assembly. In this study, we identified the E2 ubiquitin-conjugating enzyme RAD6 as an evolutionary conserved interacting protein of ASF1 in D. melanogaster and H. sapiens that promotes the turnover of ASF1A by cooperating with a well-known E3 ligase, MDM2, via ubiquitin-proteasome pathway in H. sapiens. Further functional analyses indicated that the interplay between RAD6 and ASF1A associates with tumorigenesis. Together, these data suggest that the RAD6-MDM2 ubiquitin ligase machinery is critical for the degradation of chromatin-related proteins.
... Upon gene activation, activators are recruited to the promoter. This binding can occur on DNA packaged with nucleosomes (Adkins et al. 2004), but it is further stimulated by the activity of chromatin-remodeling complexes (Utley et al. 1997). Upon activator binding to the promoter, coactivators, such as chromatin-remodeling complexes (e.g. ...
Die MYST-HAT Sas2 in Saccharomyces cerevisiae acetyliert Histon H4 an Lysin 16 (H4 K16Ac), was die Heterochromatinausbreitung an Telomeren begrenzt. Sas2 interagiert mit den Histonchaperonen Asf1 und CAF-1. Da CAF-1 während der DNA-Replikation in der S-Phase aktiv ist, ergab sich die Hypothese, dass Sas2-katalysiertes H4 K16Ac genomweit während der S-Phase ins Chromatin eingebaut wird. Durch Aktivierung von Sas2 stieg die Menge von H4 K16Ac in der S-Phase, aber nicht in der G1-Phase, in Abhängigkeit von Asf1 und CAF-1 an. Dieses H4 K16Ac wurde jedoch nicht ins Chromatin eingebaut. Dies deutete auf die Existenz eines H4 K16Ac-Pools hin. Sas2-katalysiertes H4 K16Ac hat auch eine genomweite Funktion, da das H4 K16Ac-Niveau an ORFs in sas2D Zellen vermindert ist. Eine Hypothese ist, dass H4 K16Ac euchromatische Gene vor SIR-vermittelter transkriptioneller Stilllegung schützt. Entsprechend ist das H4 K16Ac-Niveau an schwach transkribierten Genen hoch und an stark transkribierten Genen niedrig. Wir konnten zeigen, dass sich H4 K16Ac an GAL-Genen während deren Repression anreichert. Dieser H4 K16Ac-Einbau ist abhängig vom Histonchaperon Spt6. In spt6-1004 Zellen war das H4 K16Ac-Niveau an stark sowie an schwach transkribierten Genen höher als im Wildtyp, während die H4-Menge an diesen Genen reduziert war. Dies weist auf einen indirekten Effekt von Spt6 hin, indem es das H4 K16Ac-Niveau durch den Einbau von K16-unacetyliertem H4 während der Transkription reguliert. Die Abwesenheit anderer Histonchaperone (Asf1, CAF-1, HIR, Rtt106) hatte keinen Einfluss auf den repressionsgekoppelten H4 K16Ac-Einbau. Weiterhin konnten wir zeigen, dass H4 K16Ac nicht notwendig ist, um die Bindung des SIR-Komplexes an euchromatischen Genen zu verhindern, da das verminderte H4 K16Ac-Niveau an ORFs in sas2D Zellen nicht auf Deacetylierung durch Sir2 zurückzuführen war. Daher verhindert Sas2-katalysiertes H4 K16Ac die SIR-Bindung nur an subtelomerischen Loci, jedoch nicht in genomweitem Maßstab.
... Apart from their role in histone deposition, histone chaperones are emerging as a class of proteins involved in many aspects of chromatin dynamics (Loyola and Almouzni, 2004 (Gamble et al, 2005). The antisilencing function 1 protein Asf1 was required for activation of the PHO5 and PHO8 genes through chromatin disassembly (Adkins et al, 2004). It also synergizes with CAF-1 in histone deposition during replication (Tyler et al, 1999). ...
MacroH2A (mH2A) is an unusual histone variant which consists of a histone-like domain and a non-histone region (NHR). Immunofluorescence data suggested that macroH2A is accumulated at the inactive X chromosome. In this work we have used chromatin immunoprecipitation (ChIP) analysis, combined with human and mouse genome-wide array hybridization (ChIP on CHIP), to investigate the association of mH2A with the inactive X chromosome. The mH2A enrichment is moderate, suggesting a non-essential mH2A participation to the X inactivation
We describe a novel function of mH2A, namely its involvement in DNA repair. In vivo mH2A1 nucleosomes are found associated with PARP-1 and in vitro experiments demonstrate that the NHR domain of mH2A1 is essential for this interaction. The siRNA suppression of the expression of mH2A1 affects cell survival after oxidative DNA damage and inhibition of PARP-1 enzymatic activity abolishes this effect. The absence of mH2A1 results in overactivation of PARP-1 and compromises severely DNA repair after oxidative damage. Rescue experiments with silent resistant mutants of mH2A1 evidence that the NHR, but not the H2A-like domain of mH2A1, is required for the efficient repair of DNA. These data show that the involvement of mH2A1 in the repair of DNA is realized through a PARP-1 repair pathway.
... nucleosomes are subsequently reassembled following the passage of the transcriptional or replication complexes. 29,30 This dynamic of histone assembly and disassembly is reminiscent of the mercurial nature of H2B ubiquitylation. Indeed, H2Bub appears to contribute to nucleosome assembly during both transcription and DNA replication; in yeast, histone occupancy at early origins of replication under HU was reduced by elimination of H2Bub, and histone occupancy at the coding region of the GAL1 gene during transcription is reduced in double mutants of H2Bub and the histone chaperone Spt16. ...
Abstract The reversible ubiquitylation of histone H2B has long been known to regulate gene transcription, and is now understood to modulate DNA replication as well. In this review, we describe how recent, genome-wide analyses have demonstrated that this histone mark has further reaching effects on transcription and replication than once thought. We also consider the ongoing efforts to elucidate the molecular mechanisms by which H2B ubiquitylation affects processes on the DNA template, and outline the various hypothetical scenarios.
... One mechanism of regulating transcription factor binding is by controlling binding site accessibility through alterations of chromatin (1). At some promoters, nucleosomes can occlude transcription factor binding sites, and nucleosomes must be remodeled or evicted by transcriptional coactivators to permit protein binding (2)(3)(4)(5). Three transcriptional coactivators promote chromatin changes at many promoters in yeast to facilitate gene activation: SWI/SNF, SAGA, and Mediator. These three transcriptional coactivators are conserved from yeast to humans and are required for activation of many eukaryotic genes (6), including the yeast HO gene (7). ...
Promoters often contain multiple binding sites for a single factor. The yeast HO gene contains nine highly conserved binding sites for the SBF complex (Swi4-Swi6) in the 700 bp URS2 promoter region. Here we show that the distal and proximal SBF sites in URS2 function differently. ChIP experiments show SBF binds preferentially to the left side of URS2 (URS2-L), despite equivalent binding to the left-half and right-half SBF sites in vitro. SBF binding at URS2-L sites depends on prior chromatin remodeling events at the upstream URS1 region. These signals from URS1 influence chromatin changes at URS2, but only at sites within a defined distance. SBF bound at URS2-L, however, is unable to activate transcription, but instead facilitates SBF binding to sites in the right-half (URS2-R) which are required for transcriptional activation. Factor binding at HO therefore follows a temporal cascade with SBF bound at URS2-L serving to relay a signal from URS1 to the SBF sites in URS2-R that ultimately activate gene expression. Taken together, we describe a novel property of a transcription factor that can have two distinct roles in gene activation, depending on its location within a promoter.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which employs a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in Saccharomyces cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.
Multi-cellular organisms such as humans contain hundreds of cell types that share the same genetic information (DNA sequences), and yet have different cellular traits and functions. While how genetic information is passed through generations has been extensively characterized, it remains largely obscure how epigenetic information encoded by chromatin regulates the passage of certain traits, gene expression states and cell identity during mitotic cell divisions, and even through meiosis. In this review, we will summarize the recent advances on molecular mechanisms of epigenetic inheritance, discuss the potential impacts of epigenetic inheritance during normal development and in some disease conditions, and outline future research directions for this challenging, but exciting field.
As cells replicate their DNA, there is a need to synthesize new histones with which to wrap it. Newly synthesized H3 histones that are incorporated into the assembling chromatin behind the replication fork are acetylated at lysine 56. The acetylation is removed by two deacetylases, Hst3 and Hst4. This process is tightly regulated and any perturbation leads to genomic instability and replicative stress. We recently showed that Dun1, a kinase implicated mainly in the regulation of dNTPs, is vital in cells with hyper-acetylation, to counteract Rad53′s inhibition on late-firing origins of replication. Our work showed that ∆hst3 ∆hst4 cells depend on late origin firing for survival, and are unable to prevent Rad53′s inhibition when Dun1 is inactive. Thus, our work describes a role for Dun1 that is independent on its known function as a regulator of dNTP levels. Here we show that Mrc1 (Claspin in mammals), a protein that moves with the replicating fork and participates in both replication and checkpoint functions, plays also an essential role in the absence of H3K56Ac deacetylation. The sum of the results shown here and in our recent publication suggests that dormant origins are also utilized in these cells, making Mrc1, which regulates firing from these origins, also essential when histone H3 is hyper-acetylated. Thus, cells suffering from hyper-acetylation of H3K56 experience replication stress caused by a combination of prone-to-collapse forks and limited replication tracts. This combination makes both Dun1 and Mrc1, each acting on different targets, essential for viability.
Enzymatic activity of histones
Eukaryotic histones serve as structural elements to package DNA. However, they contain a copper-binding site for which the biological relevance is unknown. Copper homeostasis is critical for several fundamental eukaryotic processes, including mitochondrial respiration. Attar et al. hypothesized that histones may play a critical role in cellular copper utilization (see the Perspective by Rudolph and Luger). Using a multifaceted approach ranging from in vitro biochemistry to in vivo genetic and molecular analyses, they found that the histone H3-H4 tetramer is an oxidoreductase enzyme that catalyzes reduction of cupric ions, thereby providing biologically usable cuprous ions for various cellular processes. This work opens a new front for chromatin biology, with implications for eukaryotic evolution and human biology and disease.
Science , this issue p. 59 ; see also p. 33
Cell senescence is accompanied, and in part mediated, by changes in chromatin, including histone losses, but underlying mechanisms are not well understood. We reported previously that during yeast cell senescence driven by telomere shortening, the telomeric protein Rap1 plays a major role in reprogramming gene expression by relocalizing hundreds of new target genes (called NRTS, for new Rap1 targets at senescence) to the promoters. This leads to two types of histone loss: Rap1 lowers histone level globally by repressing histone gene expression, and it also causes local nucleosome displacement at the promoters of upregulated NRTS. Here, we present evidence of direct binding between Rap1 and histone H3/H4 heterotetramers, and map amino acids involved in the interaction within the Rap1 SANT domain to amino acids 392-394 (SHY). Introduction of a point mutation within the native RAP1 locus that converts these residues to alanines (RAP1SHY ), and thus disrupts Rap1-H3/H4 interaction, does not interfere with Rap1 relocalization to NRTS at senescence, but prevents full nucleosome displacement and gene upregulation, indicating direct Rap1-H3/H4 contacts are involved in nucleosome displacement. Consistent with this, the histone H3/H4 chaperone Asf1 is similarly unnecessary for Rap1 localization to NRTS but is required for full Rap1-mediated nucleosome displacement and gene activation. Remarkably, RAP1SHY does not affect the pace of senescence-related cell cycle arrest, indicating that some changes in gene expression at senescence are not coupled to this arrest.
Epigenetic modifications regulate plant genes to cope with a variety of environmental stresses. Chorispora bungeana is an alpine subnival plant with strong tolerance to multiple abiotic stresses, especially cold stress. In this study, we characterized the alcohol dehydrogenase 1 gene from Chorispora bungeana, CbADH1, that is up-regulated in cold conditions. Overexpression of CbADH1 in Arabidopsis thaliana improved cold tolerance, as indicated by a decreased lethal temperature (LT50). Chromatin immunoprecipitation assays showed that histone H3 is removed from the promoter region and the middle-coding region of the gene. H3K9 acetylation and H3K4 trimethylation increased throughout the gene and in the proximal promoter region, respectively. Moreover, increased Ser5P and Ser2P polymerase II accumulation further indicated changes in the transcription initiation and elongation of CbADH1 were due to the cold stress. Taken together, our results suggested that CbADH1 is highly expressed during cold stress, and is regulated by epigenetic modifications. This study expands our understanding of the regulation of gene expression by epigenetic modifications in response to environmental cues.
Anti-silencing function 1 (ASF1) is an evolutionarily conserved histone H3-H4 chaperone involved in nucleosome assembly/disassembly and histone modification. Two paralogous genes exist in the mouse genome: Asf1a and Asf1b. Asf1a is ubiquitously expressed and its loss causes embryonic lethality. Conversely, Asf1b expression is more restricted and it has been less studied. To determine the in vivo function of Asf1b, we generated an Asf1b-deficient mouse line (Asf1bGT(ROSAgeo)437) in which expression of the lacZ reporter gene is driven by the Asf1b promoter. Analysis of -galactosidase activity at early embryonic stages indicated a correlation between Asf1b expression and cell differentiation potential. In the gonads of both sexes, Asf1b expression was specifically detected in the germ cell lineage with a peak correlated with meiosis. The viability of Asf1b null mice suggests that Asf1b is dispensable for mouse development. However, these mice showed reduced reproductive capacity compared with wild type controls. We present evidence that the timing of meiotic entry and the subsequent gonad development are affected more severely in Asf1b null female than male mice. In females, in addition to subfertility related to altered gamete formation, variable defects compromising the development and/or survival of their offspring were also observed. Altogether, our data indicate the importance of Asf1b expression at the time of meiotic entry, suggesting that chromatin modifications may play a central role in this process.
Gene expression is the process whereby DNA sequence information is converted into a functional transmitter or player, namely, mRNA, and then a major functional player, namely, protein. Transcription is the first step in gene expression. Since the temporal and spatial regulation of gene expression define cellular identity, transcription is the most critical and fundamental step in the cellular functions of a gene. We have classified transcriptional regulation into three functional stages on the basis of the complexity of the DNA structures involved. The first level concerns the activation/inactivation of promoters on naked DNA, the second level entails activation/inactivation of nucleosomes, while the third level involves the activation/inactivation of chromosomal regions (Fig. 1). We denote the components that determine which genes are activated or repressed at each of these levels as gene selectors. We have categorized the gene selectors into three main groups, namely, DNA-binding proteins, histones (non-specific DNA-binding proteins), and histone-binding proteins. These three types of gene selectors work in cooperation to select the genes that are to be expressed (Fig. 2).
Long non-coding RNAs have in recent years emerged as regulatory molecules in their own right impacting transcriptional regulation at the level of chromatin. Long non-coding RNAs have also been implicated in regulation of embryogenesis and tumor initiation, progression and metastasis. Regulation of gene transcription in yeast underpins a diverse array of cellular processes including metabolic regulation, sporulation and growth responses to nutrient deprivation. For most of these cases the transcription factors that regulate these processes have served as paradigms for our understanding of gene regulation in yeast and mammalian cells. More recently, an additional layer of transcriptional control in yeast has been uncovered in the form of long non-coding RNAs which originate as anti-sense transcripts of known genes or as intergenic transcripts overlapping gene promoters. These long non-coding RNAs and their transcription through promoter regions exhibits complex effects that directly affect promoter conformation at the level of histone modifications and chromatin structure. In this review we summarize some of the best characterized examples of transcriptional control through long non-coding RNAs and suggest that studies in yeast will greatly inform our understanding of the mechanisms of action of long non-coding RNAs in human cells. © 2014, Croatian Society of Natural Sciences. All rights reserved.
The genetic information encoded in enormous length of DNA is packaged and compartmentalized into the nucleus of eukaryotes as chromatin. Chromatin consists of nucleosomes as the fundamental unit, where ~146 bp of DNA is wrapped around an octamer of histones in nearly two superhelical turns. Within the histone octamer, two copies of H2A-H2B and H3-H4 dimer pairs form the core histones, whereas, histone H1, also called as linker histone, locks the DNA at the either end of the nucleosome and, along with other architectural proteins, folds the chromatin into more condensed and yet poorly defined higher order structures (see Chap. 1). In almost all nuclear processes involving DNA as a substrate, such as transcription, replication, recombination, and repair, the packaging of the genome in chromatin presents inherent barriers that restrict the access of DNA to processing enzymes. Therefore, to access DNA within a chromatin context, the chromatin is reversibly and locally unfolded by counteracting these chromatin constraints during the nuclear process and refolded back after the process is completed. In this regard, the eukaryotic cell has developed two fundamental chromatin modification strategies that includes: (1) Covalent modification of histones catalyzed by histone-modifying enzyme complexes and (2) ATP-dependent perturbations of histone-DNA interactions catalyzed by the SWI/SNF family of ATP-dependent chromatin remodeling complexes. The covalent modification of histone residues that primarily occurs at the N-terminal region of histones can disrupt histone interaction with DNA or alternatively serve as the binding sites for chromatin-associated factors (Jenuwein and Allis 2001). However, the mechanism employed by ATP-dependent chromatin remodeling complexes uses the energy of ATP hydrolysis to alter the positions or composition of nucleosomes in chromatin (Eberharter and Becker 2004). Much of what we currently know about the biological roles of these two classes of chromatin-modifying factors has come from research on the transcriptional regulatory mechanisms that occur during gene activation, whereas studies from the past decade have also shown the link between chromatin modifications and other nuclear events such as DNA repair and replication. Both covalent modification of histones and ATP-dependent chromatin remodeling have been shown to maintain genome integrity and transmit the genetic and epigenetic information to the next generation. This chapter elaborates how the ATP-dependent chromatin remodeling complexes employ mechanisms that work in concert with the DNA repair and replication processes. © 2014 Springer Science+Business Media New York. All rights reserved.
Chaperones, nucleosome remodeling complexes and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these co-factors function ubiquitously, and the impact of nucleosome eviction on transcription genome-wide, are poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple co-factors to address these issues for ~200 genes belonging to the Gcn4 transcriptome, of which ~70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 co-chaperone Ydj1, and chromatin-associated factor Yta7 are required downstream of Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC and H2AZ are dispensable, for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double and triple mutants implicated Gcn5, Snf2 and Ydj1 in H3 eviction at most, but not all Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these 3 co-factors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in co-factor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes, but that other steps in gene activation are more rate-limiting for most other yeast genes.
Crystal structures of proteins and their complexes have become critical information for molecular-based life science. Biochemical and biological analysis based on tertiary structural information is a powerful tool to unveil complex molecular processes in the cell. Here, we present two examples of the structure-based life science study, structural biology studies of CagA, an effector protein from Helicobacter pylori, and histone chaperone CIA/ASF1, which is involved in transcription initiation.
That thesis is about the role of ASF1 in histone deposition during replication.
Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication- coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.
Nucleic Acids Research, 20, pp. 1031–1038 (1992)
The authors wish to acknowledge Professor Y.Oshima for having provided plasmids pALlOl and pAL144 containing the PH08 gene which were the source of all PH08 DNA fragments used. This study would not have been possible without his previous work on the PHO8 locus.
We have analyzed the chromatin structure of a phosphate-repressible acid phosphatase gene (PHO5) within yeast nuclei. Under derepressed conditions (low Pi media), the gene is much more sensitive to either DNAse I or micrococcal nuclease digestion than is the repressed gene. We have mapped DNase I hypersensitive sites unique to the active gene near the 5'-end of the acid phosphatase mRNA and within a region presumed to function in the regulation of the gene by Pi. Although the gene is packaged into regularly spaced nucleosomes, no detectable phase relationship exists between nucleosomes and DNA sequence under derepressed conditions, whereas in the repressed state the nucleosomes occur in one predominant phase. These results demonstrate reversible changes in the chromatin structure of a eukaryotic gene system that directly correlate with the functional state of the gene.
PHO4, a transcription factor required for induction of the PHO5 gene in response to phosphate starvation, is phosphorylated by the PHO80-PHO85 cyclin-CDK (cyclin-dependent kinase) complex
when yeast are grown in phosphate-rich medium. PHO4 was shown to be concentrated in the nucleus when yeast were starved for
phosphate and was predominantly cytoplasmic when yeast were grown in phosphate-rich medium. The sites of phosphorylation on
PHO4 were identified, and phosphorylation was shown to be required for full repression of PHO5 transcription when yeast were grown in high phosphate. Thus, phosphorylation of PHO4 by PHO80-PHO85 turns off PHO5 transcription by regulating the nuclear localization of PHO4.
Genetic analysis has implicated SPT6, an essential gene of Saccharomyces cerevisiae, in the control of chromatin structure. Mutations in SPT6 and particular mutations in histone genes are able to overcome transcriptional defects in strains lacking the Snf/Swi protein complex. Here it is shown that an spt6 mutation causes changes in chromatin structure in vivo. In addition, both in vivo and in vitro experiments provide evidence that Spt6p interacts directly with histones and primarily with histone H3. Consistent with these findings, Spt6p is capable of nucleosome assembly in vitro.
The X-ray crystal structure of the nucleosome core particle of chromatin shows in atomic detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it. Both histone/histone and histone/DNA interactions depend on the histone fold domains and additional, well ordered structure elements extending from this motif. Histone amino-terminal tails pass over and between the gyres of the DNA superhelix to contact neighbouring particles. The lack of uniformity between multiple histone/DNA-binding sites causes the DNA to deviate from ideal superhelix geometry.
Chromatin structure plays important roles in regulating many DNA-templated processes, such as transcription, replication, and recombination. Considerable progress has recently been made in the identification of large, multisubunit complexes dedicated to these nuclear processes, all of which occur on nucleosomal templates. Mapping specific genomic loci relative to the position of selectively modified or unique histone variants or nonhistone components provides valuable insights into how these proteins (and their modifications) function in their normal chromatin context. Here we describe a versatile and high-resolution method which involves two basic steps: (1) in vivo formaldehyde cross-linking of intact cells followed by (2) selective immunoprecipitation of protein-DNA complexes with specific antibodies. This method allows for detailed analyses of protein-DNA interactions in a native chromatin environment. Recently, this technique has been successfully employed to map the boundaries of specifically modified (e.g., acetylated) histones along target genes, to define the cell cycle-regulated assembly of origin-dependent replication and centromere-specific complexes with remarkable precision, and to map the in vivo position of reasonably rare transcription factors on cognate DNA sites. Thus, the basic chromatin immunoprecipitation technique is remarkably versatile and has now been used in a wide range of cell types, including budding yeast, fly, and human cells. As such, it seems likely that many more studies, centered around chromatin structure and protein-DNA interactions in its native setting, will benefit from this technique. In this article, a brief review of the history of this powerful approach and a discussion of the basic method are provided. Procedures for protein recovery as well as limitations and extensions of the method are also presented.
Yeast defective in the checkpoint kinase Rad53 fail to recover from transient DNA replication blocks and synthesize intact chromosomes. The effectors of Rad53 relevant to this recovery process are unknown. Here we report that overproduction of the chromatin assembly factor Asf1 can suppress the Ts phenotype of mrc1rad53 double mutants and the HU sensitivity of rad53 mutants. Eliminating silencing also suppresses this lethality, further implicating chromatin structure in checkpoint function. We find that Asf1 and Rad53 exist in a dynamic complex that dissociates in response to replication blocks and DNA damage. Thus, checkpoint pathways directly regulate chromatin assembly to promote survival in response to DNA damage and replication blocks.
The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To
gain a better understanding of the assembly of chromatin during DNA synthesis, we identified, cloned, and characterized the
180- and 105-kDa polypeptides of Drosophila chromatin assembly factor 1 (dCAF-1). The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled
chromatin assembly. Furthermore, we have established that the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated
form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant
dCAF-1 revealed an interaction between dCAF-1 and theDrosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1
is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, we
observed that dASF1 and dCAF-1 p105 colocalized in vivo inDrosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed
between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin.
The PHO5 gene promoter is an important model for the study of gene regulation in the context of chromatin. Upon PHO5 activation the chromatin structure is reconfigured, but the mechanism of this transition remains unclear. Using templates
reconstituted into chromatin with purified recombinant yeast core histones, we have investigated the mechanism of chromatin
structure reconfiguration on the PHO5 promoter, a prerequisite for transcriptional activation. Footprinting analyses show that intrinsic properties of the promoter
DNA are sufficient for translational nucleosome positioning, which approximates that seenin vivo. We have found that both Pho4p and Pho2p can bind their cognate sites on chromatin-assembled templates without the aid of
histone-modifying or nucleosome-remodeling factors. However, nucleosome remodeling by these transcriptional activators requires
an ATP-dependent activity in a yeast nuclear extract fraction. Finally, transcriptional activation on chromatin templates
requires acetyl-CoA in addition to these other activities and cofactors. The addition of acetyl-CoA results in significant
core histone acetylation. These findings indicate that transcriptional activation requires Pho4p, Pho2p, nucleosome remodeling,
and nucleosome acetylation. Furthermore, we find that DNA binding, nucleosome remodeling, and transcriptional activation are
separable steps, facilitating biochemical analysis of the PHO5regulatory mechanism.
De novo chromatin assembly into regularly spaced nucleosomal arrays is essential for eukaryotic genome maintenance and inheritance. The Anti-Silencing Function 1 protein (ASF1) has been shown to be a histone chaperone, participating in DNA-replication-coupled nucleosome assembly. We show that mutations in the Drosophila asf1 gene derepress silencing at heterochromatin and that the ASF1 protein has a cell cycle-specific nuclear and cytoplasmic localization. Furthermore, using both genetic and biochemical methods, we demonstrate that ASF1 interacts with the Brahma (SWI/SNF) chromatin-remodelling complex. These findings suggest that ASF1 plays a crucial role in both chromatin assembly and SWI/SNF-mediated chromatin remodelling.
Previous studies have suggested that transcription elongation results in changes in chromatin structure. Here we present studies of Saccharomyces cerevisiae Spt6, a conserved protein implicated in both transcription elongation and chromatin structure. Our results show that, surprisingly, an spt6 mutant permits aberrant transcription initiation from within coding regions. Furthermore, transcribed chromatin in the spt6 mutant is hypersensitive to micrococcal nuclease, and this hypersensitivity is suppressed by mutational inactivation of RNA polymerase II. These results suggest that Spt6 plays a critical role in maintaining normal chromatin structure during transcription elongation, thereby repressing transcription initiation from cryptic promoters. Other elongation and chromatin factors, including Spt16 and histone H3, appear to contribute to this control.
De novo chromatin assembly maintains histone density on the daughter strands in the wake of the replication fork. The heterotrimer chromatin assembly factor 1 (CAF-1) couples DNA replication to histone deposition in vitro, but is not essential for yeast cell proliferation. Depletion of CAF-1 in human cell lines demonstrated that CAF-1 was required for efficient progression through S-phase. Cells lacking CAF-1 accumulated in early and mid S-phase and replicated DNA slowly. The checkpoint kinase Chk1, but not Chk2, was phosphorylated in response to CAF-1 depletion, consistent with a DNA replication defect. CAF-1-depleted cell extracts completely lacked DNA replication-coupled chromatin assembly activity, suggesting that CAF-1 is required for efficient S-phase progression in human cells. These results indicate that, in contrast to yeast, human CAF-1 is necessary for coupling chromatin assembly with DNA replication.
Drosophila melanogaster is a proven model system for many aspects of human biology. Here we present a two-hybrid–based protein-interaction map of
the fly proteome. A total of 10,623 predicted transcripts were isolated and screened against standard and normalized complementary
DNA libraries to produce a draft map of 7048 proteins and 20,405 interactions. A computational method of rating two-hybrid
interaction confidence was developed to refine this draft map to a higher confidence map of 4679 proteins and 4780 interactions.
Statistical modeling of the network showed two levels of organization: a short-range organization, presumably corresponding
to multiprotein complexes, and a more global organization, presumably corresponding to intercomplex connections. The network
recapitulated known pathways, extended pathways, and uncovered previously unknown pathway components. This map serves as a
starting point for a systems biology modeling of multicellular organisms, including humans.
An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5⁺ or Escherichia coli kanr gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae. © 1998 John Wiley & Sons, Ltd.
PHO8 encodes an alkaline phosphatase in Saccharomyces cerevisiae whose transcription is regulated by the phosphate concentration in the medium. This occurs through the action of several
positive and negative regulatory proteins, also involved in the regulation of other members of the phosphatase gene family.
A central role is played by PH04, the gene encoding a DNA binding regulatory protein. Digestion experiments with DNasel, micrococcal
nuclease and 20 different restriction nucleases show that under conditions of PHO8 repression, there is a highly ordered chromatin
structure at the promoter consisting of three hypersensitive regions, approximately 820 to 690, 540 to 510, and 230 to 160
bp upstream of the initiation codon. These hypersensitive sites are surrounded by DNA organized in nucleosomes. Gel shift
analysis and in vitro footprlnting revealed the presence of two PHO4 binding sites at the PH08 promoter: a low affinity site at − 728 and a high
affinity site at −532. Each one is located within a hypersensitive site. Upon derepression of PHO8, the chromatin structure
changes significantly: The two upstream hypersensitive sites containing the PHO4 binding sites merge, resulting in a long
region of hypersensitivity. This transition is PHO4 dependent. However, not all of the promoter becomes nucleosome free. Instead,
as a novel feature, regions of intermediate accessibility are generated upstream and downstream of the third hypersensitive
site, the latter region encompassing the TATA-box. The available data fit best into a concept that these regions are organized
in unstable or partly unfolded nucleosomes.
Chromatin is a highly dynamic structure that plays an essential role in regulating all nuclear processes that utilize the DNA template including DNA repair, replication, transcription and recombination. Thus, the mechanisms by which chromatin structures are assembled and modified are questions of broad interest. This minireview will focus on two groups of proteins: (a) histone chaperones and (b) ATP-dependent chromatin remodeling machines, that co-operate to assemble DNA and histone proteins into chromatin. The current understanding of how histone chaperones and ATP-dependent remodeling machines coordinately assemble chromatin in vitro will be discussed, together with the growing body of genetic evidence that supports the role of histone chaperones in the cell.
An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5(+) or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae. (C) 1998 John Wiley & Sons, Ltd.
Recent work has shown that the yeast histone H4 N-terminus, while not essential for viability, is required for repression of the silent mating loci and activation of GAL1 and PHO5 promoters. Because histone H3 shares many structural features with histone H4 and is intimately associated with H4 in the assembled nucleosome, we asked whether H3 has similar functions. While the basic N-terminal domain of H3 is found to be non-essential (deletion of residues 4-40 of this 135 amino acid protein allows viability), its removal has only a minor effect on mating. Surprisingly, both deletions (of residues 4-15) and acetylation site substitutions (at residues 9, 14 and 18) within the N-terminus of H3 allow hyperactivation of the GAL1 promoter as well as a number of other GAL4-regulated genes including GAL2, GAL7 and GAL10. To a limited extent glucose repression is also alleviated by H3 N-terminal deletions. Expression of another inducible promoter, PHO5, is shown to be relatively unaffected. We conclude that the H3 and H4 N-termini have different functions in both the repression of the silent mating loci and in the regulation of GAL1.
The purification and characterization of a replication-dependent chromatin assembly factor (CAF-I) from the nuclei of human cells is described. CAF-I is a multisubunit protein that, when added to a crude cytosol replication extract, promotes chromatin assembly on replicating SV40 DNA. Chromatin assembly by CAF-I requires and is coupled with DNA replication. The minichromosomes assembled de novo by CAF-I consist of correctly spaced nucleosomes containing the four core histones H2A, H2B, H3, and H4, which are supplied in a soluble form by the cytosol replication extract. Thus, by several criteria, the CAF-I-dependent chromatin assembly reaction described herein reflects the process of chromatin formation during DNA replication in vivo.
The chromatin structure of two tandemly linked acid phosphatase genes (PHO5 and PHO3) from Saccharomyces cerevisiae was analyzed under conditions at which the strongly regulated PHO5 gene is repressed. Digestion experiments with DNase I, DNase II, micrococcal nuclease and restriction nucleases reveal the presence of five hypersensitive sites at the PHO5/PHO3 locus, two of them upstream of PHO5 at distances of 920 and 370 bp, one in between the two genes and two downstream of PHO3. Specifically positioned nucleosomes are located next to these hypersensitive sites as shown by indirect end-labeling experiments. The positions deduced from these experiments could be verified by monitoring the accessibility of various restriction sites to the respective nucleases. Sites within putative linker regions were about 50-60% susceptible, whereas sites located within nucleosome cores were resistant. Hybridizing micrococcal nuclease digests to a probe from in between the two upstream hypersensitive sites leads to an interruption of an otherwise regular nucleosomal DNA pattern. This shows directly that these hypersensitive sites represent gaps within ordered nucleosomal arrays.
We have previously constructed a yeast strain (UKY403) whose sole histone H4 gene is under control of the GAL1 promoter. This yeast arrests in G2 upon glucose treatment as a result of histone H4 depletion. The yeast PHO5 gene contains phase nucleosomes covering promoter (UAS) sequences in the PHO5 repressed state and it has been suggested that nucleosomes prevent the binding of positively acting factors to these UAS sequences. Using UKY403 we examined the length of polynucleosomes and nucleosome phasing in the PHO5 upstream region by the use of micrococcal nuclease and indirect end-labeling. It was found that glucose arrest led to a severe disruption in PHO5 chromatin structure and that most nucleosomes had their position altered or were lost from the PHO5 promoter region. Cell undergoing nucleosome depletion synthesized large quantities of accurate PHO5 transcripts even under repressive, high inorganic phosphate conditions. Histone H4 depletion did not appear to affect the repression or activation of another inducible yeast gene, CUP1. Arrest with landmarks in early G1 (in the cell division cycle mutant cdc28) or in various stages of G2 (in cdc15, cdc17 and cdc20) does not activate PHO5; nor does arrest due to chromosome topology changes (in top2 or the top1top2 topoisomerase mutants). cdc14, which has its arrest landmark at a similar point in the cell cycle as cdc15, does derepress PHO5. However, since it also leads to derepression of CUP1 it is probably functioning through an independent mechanism. Therefore, our data suggest that nucleosomes regulate PHO5 transcription.
The chromatin fine structure in the promoter region of PHO5, the structural gene for a strongly regulated acid phosphatase in yeast, was analyzed. An upstream activating sequence 367 bp away from the start of the coding sequence that is essential for gene induction was found to reside in the center of a hypersensitive region under conditions of PHO5 repression. Under these conditions three related elements at positions -469, -245 and -185 are contained within precisely positioned nucleosomes located on both sides of the hypersensitive region. Upon PHO5 induction the chromatin structure of the promoter undergoes a defined transition, in the course of which two nucleosomes upstream and two nucleosomes downstream of the hypersensitive site are selectively removed. In this way approximately 600 bp upstream of the PHO5 coding sequence become highly accessible and all four elements are free to interact with putative regulatory proteins. These findings suggest a mechanism by which the chromatin structure participates in the functioning of a regulated promoter.
Biochemical and x-ray diffraction results concerning the oligomeric structure of the histones are presented. The results show pairwise associations in solution, two types of histone forming a tetramer and two other types of histone forming a different oligomer. The same pairwise associations appear to occur in chromatin.
Activation of the Saccharomyces cerevisiae PHO5 gene by phosphate starvation is accompanied by the disappearance of two pairs of positioned nucleosomes that flank a short hypersensitive region in the promoter. The transcription factor Pho4 is the key regulator of this transition. By in vitro footprinting it was previously shown that there is a low affinity site (UASp1) which is contained in the short hypersensitive region in the inactive promoter, and a high affinity site (UASp2) which is located in the adjacent nucleosome. To investigate the interplay between nucleosomes and Pho4, we have performed in vivo footprinting experiments with dimethylsulfate. Pho4 was found to bind to both sites in the active promoter. In contrast, it binds to neither site in the repressed promoter. Lack of binding under repressing conditions is largely due to the low affinity of Pho4 for its binding sites under these conditions. Despite the increased affinity of Pho4 for its target sites under activating conditions, binding to UASp2 is prevented by the presence of the nucleosome and can only occur after prior disruption of this nucleosome in a process that requires UASp1. Protection of the PHO5 UASp2 by the nucleosome is not absolute, however, since overexpression of Pho4 can disrupt this nucleosome even when UASp1 is deleted. Also under these conditions, with only UASp2 present, all four nucleosomes at the PHO5 promoter are disrupted, whereas no chromatin change at all is observed when both UAS elements are destroyed.
The PHO5 promoter from Saccharomyces cerevisiae can exist in two chromatin configurations depending on its state of activity. In the repressed promoter a short hypersensitive site containing a binding site for the transcription factor PHO4 is flanked by specifically positioned nucleosomes. After induction two nucleosomes upstream and two downstream of the hypersensitive site are disrupted, and the entire promoter becomes accessible. We have investigated mechanisms responsible for setting up the structure of the repressed state and for the transition. Episomal centromeric plasmids bearing the PHO5 promoter show the same chromatin structure as the endogenous chromosomal copy arguing that the chromosomal context is not essential and that the nucleosomal organization is not set up from a distance. Deleting most of the hypersensitive region including the PHO4 binding site also leaves the positioning of the adjacent nucleosomes in the repressed promoter unchanged indicating that histone-DNA interactions play an important role in setting up nucleosome positions. However, when half of the DNA of a nucleosome is deleted a new nucleosome forms at the same location with respect to the neighboring nucleosome indicating that boundary effects also contribute to nucleosome positioning in the native promoter. Disruption of the nucleosomes under activating conditions is shown to require interaction of PHO4 with its binding site located within the hypersensitive region. This disruption takes place also in two independent constructs in which the TATA box had been deleted and as a result the gene was not transcribed. This result shows for the first time that the generation of active chromatin at a regulated promoter is not the result of gene expression but occurs prior to transcription.
Histone acetyltransferase (HAT) activity has been demonstrated for several transcriptional activators, formally connecting chromatin modification with gene regulation. However, no effect on chromatin has been demonstrated. We have investigated the role of the HAT Gcn5 at the nucleosomally regulated PHO5 promoter. Under conditions of constitutive submaximal activation (i.e., in the absence of the negative regulator Pho80), deletion of Gcn5 determines a novel randomized nucleosomal organization across the promoter and leads to a dramatic reduction in activity. Furthermore, mutation of amino acids critical for Gcn5 HAT activity is sufficient to generate this structure. This intermediate state in chromatin opening gives way to the fully open structure upon maximal induction (phosphate starvation), even in the absence of Gcn5. Thus, Gcn5 is shown to affect directly the remodeling of chromatin in vivo.
Chromatin assembly factor I (CAF-I) is a three-subunit histone-binding complex conserved from the yeast Saccharomyces cerevisiae to humans. Yeast cells lacking CAF-I (cacΔ mutants) have defects in heterochromatic gene silencing. In this study, we showed that deletion of HIRgenes, which regulate histone gene expression, synergistically reduced gene silencing at telomeres and at the HM loci incacΔ mutants, although hirΔ mutants had no silencing defects when CAF-I was intact. Therefore, Hir proteins are required for an alternative silencing
pathway that becomes important in the absence of CAF-I. Because Hir proteins regulate expression of histone genes, we tested
the effects of histone gene deletion and overexpression on telomeric silencing and found that alterations in histone H3 and
H4 levels or in core histone stoichiometry reduced silencing in cacΔ mutants but not in wild-type cells. We therefore propose that Hir proteins contribute to silencing indirectly via regulation
of histone synthesis. However, deletion of combinations of CAC and HIR genes also affected the growth rate and in some cases caused partial temperature sensitivity, suggesting that global aspects
of chromosome function may be affected by the loss of members of both gene families.
An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kan(r) gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae.
The study of chromatin, once thought to be a purely structural matrix serving to compact the DNA of the genome into the nucleus, is of increasing value for our understanding of how DNA functions in the cell. This article provides two basic procedures for the study of chromatin in vivo. The first is a DNase I-based method for the treatment of isolated nuclei to resolve the chromatin structure of a particular region; the second employs dimethyl sulfate footprinting of whole cells in vivo to determine the binding of factors to cis elements in the locus of interest. Specific examples illustrating the techniques described are given from our work on the regulation of the yeast PHO8 gene, but have also been successfully and reliably applied to the study of many other yeast loci. These procedures make it possible to correlate the binding of a transactivator with an altered or perturbed chromatin organization at a specific locus.
This chapter describes the mapping of chromatin structure in yeast and discusses three basic techniques that provide a high degree of reproducibility to determine the nucleosomal organization of yeast chromatin at various loci. The first employs DNase I with yeast nuclei to determine the presence of positioned nucleosomes or hypersensitive sites within a specific region of chromatin. A micrococcal nuclease-based method that assays for the presence or absence of a nucleosome on a particular stretch of DNA is discussed in the chapter. Although these techniques can provide important structural information, the level of hypersensitivity or the extent to which chromatin structure is perturbed is hard to assess by such methods. The chapter also discusses a complementary technique that employs restriction enzymes with yeast nuclei to provide quantitative accessibility data. Thus, the combination of these methods presents an effective diagnostic tool for the study of chromatin structure in yeast.
The evolutionarily conserved yeast checkpoint protein kinase Rad53 regulates cell cycle progression, transcription, and DNA repair in response to DNA damage. To uncover potential regulatory targets of Rad53, we identified proteins physically associated with it in vivo using protein affinity purification and tandem mass spectrometry. Here we report that Rad53 interacts in a dynamic functional manner with Asf1, a chromatin assembly factor recently shown to mediate deposition of acetylated histones H3 and H4 onto newly replicated DNA. Biochemical and molecular genetic studies suggest that Asf1 is an important target of the Rad53-dependent DNA damage response and that Rad53 may directly regulate chromatin assembly during DNA replication and repair.
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.
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.
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.
Histone acetyltransferases (HATs) such as Gcn5 play a role in transcriptional activation. However, the majority of constitutive genes show no requirement for GCN5, and even regulated genes, such as the yeast PHO5 gene, do not seem to be affected significantly by its absence under normal activation conditions. Here we show that even though the steady-state level of activated PHO5 transcription is not affected by deletion of GCN5, the rate of activation following phosphate starvation is significantly decreased. This delay in transcriptional activation is specifically due to slow chromatin remodeling of the PHO5 promoter, whereas the transmission of the phosphate starvation signal to the PHO5 promoter progresses at a normal rate. Chromatin remodeling is equally delayed in a galactose-inducible PHO5 promoter variant in which the Pho4 binding sites have been replaced by Gal4 binding sites. By contrast, activation of the GAL1 gene by galactose addition occurs with normal kinetics. Lack of the histone H4 N-termini leads to a similar delay in activation of the PHO5 promoter. These results indicate that one important contribution of HATs is to increase the rate of gene induction by accelerating chromatin remodeling, rather than to affect the final steady-state expression levels.
Chromatin assembly factor I (CAF-I) is a conserved histone H3/H4 deposition complex. Saccharomyces cerevisiae mutants lacking CAF-I subunit genes (CAC1 to CAC3) display reduced heterochromatic gene silencing. In a screen for silencing-impaired cac1 alleles, we isolated a mutation that reduced binding to the Cac3p subunit and another that impaired binding to the DNA replication
protein PCNA. Surprisingly, mutations in Cac1p that abolished PCNA binding resulted in very minor telomeric silencing defects
but caused silencing to be largely dependent on Hir proteins and Asf1p, which together comprise an alternative silencing pathway.
Consistent with these phenotypes, mutant CAF-I complexes defective for PCNA binding displayed reduced nucleosome assembly
activity in vitro but were stimulated by Asf1p-histone complexes. Furthermore, these mutant CAF-I complexes displayed a reduced
preference for depositing histones onto newly replicated DNA. We also observed a weak interaction between Asf1p and Cac2p
in vitro, and we hypothesize that this interaction underlies the functional synergy between these histone deposition proteins.
Chromatin is a highly dynamic structure that plays an essential role in regulating all nuclear processes that utilize the DNA template including DNA repair, replication, transcription and recombination. Thus, the mechanisms by which chromatin structures are assembled and modified are questions of broad interest. This minireview will focus on two groups of proteins: (a) histone chaperones and (b) ATP-dependent chromatin remodeling machines, that co-operate to assemble DNA and histone proteins into chromatin. The current understanding of how histone chaperones and ATP-dependent remodeling machines coordinately assemble chromatin in vitro will be discussed, together with the growing body of genetic evidence that supports the role of histone chaperones in the cell.
The mammalian HIRA gene encodes a histone-interacting protein whose homolog in Xenopus laevis is characterized here. In vitro, recombinant Xenopus HIRA bound purified core histones and promoted their deposition onto plasmid DNA. The Xenopus HIRA protein, tightly associated with nuclear structures in somatic cells, was found in a soluble maternal pool in early embryos. Xenopus egg extracts, known for their chromatin assembly efficiency, were specifically immunodepleted for HIRA. These depleted extracts were severely impaired in their ability to assemble nucleosomes on nonreplicated DNA, although nucleosome formation associated with DNA synthesis remained efficient. Furthermore, this defect was largely corrected by reintroduction of HIRA along with (H3-H4)(2) tetramers. We thus delineate a nucleosome assembly pathway that depends on HIRA.
Two very similar H3 histones-differing at only four amino acid positions-are produced in Drosophila cells. Here we describe a mechanism of chromatin regulation whereby the variant H3.3 is deposited at particular loci, including active rDNA arrays. While the major H3 is incorporated strictly during DNA replication, amino acid changes toward H3.3 allow replication-independent (RI) deposition. In contrast to replication-coupled (RC) deposition, RI deposition does not require the N-terminal tail. H3.3 is the exclusive substrate for RI deposition, and its counterpart is the only substrate retained in yeast. RI substitution of H3.3 provides a mechanism for the immediate activation of genes that are silenced by histone modification. Inheritance of newly deposited nucleosomes may then mark sites as active loci.
General transcription initiation factor IID (TFIID) plays a central and critical role in transcription initiation from both naked and chromatin templates. Although interaction between several DNA-binding proteins and TFIID were identified and well characterized, functional linkage between TFIID and chromatin factors has remained to be elucidated. Here we show the identification and characterization of human CIA/hASF1 (identified previously as a histone chaperone) as an interactor of two tandem bromodomain modules of human (h)TAF(II)250/CCG1, the largest subunit of TFIID. Although yeast (y)TAF(II)145, a homologue of hTAF(II)250/CCG1 in Saccharomyces cerevisiae, lacks bromodomains, glutathione S-transferase pull-down and immunoprecipitation assays revealed that Asf1p (antisilencing function 1), the counterpart of CIA in S. cerevisiae, interacts with Bdf1p (bromodomain factor 1), which is reported to serve as the missing bromodomain in yTAF(II)145. Furthermore, yeast strain lacking the BDF1 gene shows the Spt phenotype that is shown also by the ASF1 gene disruptant, and a double-knockout strain of both genes shows synthetic lethality, indicating that ASF1 genetically interacts with bromodomains associated with yTFIID. We also found that Asf1p coprecipitates with yTFIID subunits from yeast whole-cell extract, and overexpression of yTFIID subunits suppress the Spt phenotype caused by gene disruption of the ASF1. This study describes the functional linkage between TFIID and a histone chaperone.
The histone tails on the nucleosome surface are subject to enzyme-catalyzed modifications that may, singly or in combination, form a code specifying patterns of gene expression. Recent papers provide insights into how a combinatorial code might be set and read. They show how modification of one residue can influence that of another, even when they are located on different histones, and how modifications at specific genomic locations might be perpetuated on newly assembled chromatin.
Chromatin is a highly dynamic structure that plays a key role in the orchestration of gene expression patterns during cellular differentiation and development. The packaging of DNA into chromatin generates a barrier to the transcription machinery. The two main strategies by which cells alleviate chromatin-mediated repression are through the action of ATP-dependent chromatin remodeling complexes and enzymes that covalently modify the histones. Various signaling pathways impinge upon the targeting and activity of these enzymes, thereby controlling gene expression in response to physiological and developmental cues. Chromatin structure also underlies many so-called epigenetic phenomena, leading to the mitotically stable propagation of differential expression of genetic information. Here, we will focus on the role of SWI/SNF-related ATP-dependent chromatin remodeling complexes in developmental gene regulation. First, we compare different models for how remodelers can act in a gene-selective manner, and either cooperate or antagonize other chromatin-modulating systems in the cell. Next, we discuss their functioning during the control of developmental gene expression programs.
Approximately 800 transcripts in Saccharomyces cerevisiae are cell cycle regulated. The oscillation of ∼40% of these genes, including a prominent subclass involved in nutrient acquisition,
is not understood. To address this problem, we focus on the mitosis-specific activation of the phosphate-responsive promoter,
PHO5. We show that the unexpected mitotic induction of the PHO5 acid phosphatase in rich medium requires the transcriptional activators Pho4 and Pho2, the cyclin-dependent kinase inhibitor
Pho81, and the chromatin-associated enzymes Gcn5 and Snf2/Swi2. PHO5 mitotic activation is repressed by addition of orthophosphate, which significantly increases cellular polyphosphate. Polyphosphate
levels also fluctuate inversely with PHO5 mRNA during the cell cycle, further substantiating an antagonistic link between this phosphate polymer and PHO5 mitotic regulation. Moreover, deletion of PHM3, required for polyphosphate accumulation, leads to premature onset of PHO5 expression, as well as an increased rate, magnitude, and duration of PHO5 activation. Orthophosphate addition, however, represses mitotic PHO5 expression in a phm3Δ strain. Thus, polyphosphate per se is not necessary to repress PHO transcription but, when present, replenishes cellular phosphate during nutrient depletion. These results demonstrate a dynamic
mechanism of mitotic transcriptional regulation that operates mostly independently of factors that drive progression through
the cell cycle.
We have analyzed the histone modification status of the PHO5 promoter from yeast by the ChIP technology and have focused on changes occurring upon activation. Using various acetylation-specific antibodies, we found a dramatic loss of the acetylation signal upon induction of the promoter. This turned out to be due, however, to the progressive loss of histones altogether. The fully remodeled promoter appears to be devoid of histones as judged by ChIP analyses. Local histone hyperacetylation does indeed occur, however, prior to remodeling. This can explain the delay in chromatin remodeling in the absence of histone acetyltransferase activity of the SAGA complex that was previously documented for the PHO5 promoter. Our findings shed new light on the nucleosomal structure of fully remodeled chromatin. At the same time, they point out the need for novel controls when the ChIP technique is used to study histone modifications in the context of chromatin remodeling in vivo.
It has long been known that promoter DNA is converted to a nuclease-sensitive state upon transcriptional activation. Recent findings have raised the possibility that this conversion reflects only a partial unfolding or other perturbation of nucleosomal structure, rather than the loss of nucleosomes. We report topological, sedimentation, nuclease digestion, and ChIP analyses, which demonstrate the complete unfolding of nucleosomes at the transcriptionally active PHO5 promoter of the yeast Saccharomyces cerevisiae. Although nucleosome loss occurs at all promoter sites, it is not complete at any of them, suggesting the existence of an equilibrium between the removal of nucleosomes and their reformation.
Given the "beads-on-a-string" nature of chromatin, scientists have long pondered how the large RNA polymerase II complex accesses the DNA during transcription. In his Perspective, [Svejstrup][1] discusses new work from three groups ([ Belotserkovskaya et al .][2], [ Kaplan et al .][3], and [ Saunders et al .][4]) revealing that two elongation factors, Spt6 and FACT, are responsible for displacing histone proteins ahead of the polymerase and redepositing them on the DNA in its wake.
[1]: http://www.sciencemag.org/cgi/content/full/301/5636/1053
[2]: http://www.sciencemag.org/cgi/content/short/301/5636/1090
[3]: http://www.sciencemag.org/cgi/content/short/301/5636/1096
[4]: http://www.sciencemag.org/cgi/content/short/301/5636/1094
Rad53 and Mec1 are protein kinases required for DNA replication and recovery from DNA damage in Saccharomyces cerevisiae. Here, we show that rad53, but not mec1 mutants, are extremely sensitive to histone overexpression, as Rad53 is required for degradation of excess histones. Consequently, excess histones accumulate in rad53 mutants, resulting in slow growth, DNA damage sensitivity, and chromosome loss phenotypes that are significantly suppressed by a reduction in histone gene dosage. Rad53 monitors excess histones by associating with them in a dynamic complex that is modulated by its kinase activity. Our results argue that Rad53 contributes to genome stability independently of Mec1 by preventing the damaging effects of excess histones both during normal cell cycle progression and in response to DNA damage.
Regu-lation of PHO4 nuclear localization by CDK complex
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Transcription elon-in vivo to a critical target site in the PHO5 promoter. EMBO J. gation factors repress transcription initiation from cryptic sites
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