The histone chaperone Asf1 is dispensable for direct de novo histone deposition in <Emphasis Type="Italic">Xenopus</Emphasis> egg extracts

Histone chaperones that escort histones during their overall lifetime from synthesis to sites of usage can participate in various tasks. Their requirement culminates in the dynamic processes of nucleosome assembly and disassembly. In this context, it is important to define the exact role of the histone chaperone Asf1. In mammals, Asf1 interacts with two other chaperones, CAF-1 and HIRA, which are critical in DNA synthesis-coupled and synthesis-uncoupled nucleosome assembly pathways, respectively. A key issue is whether Asf1 is able or not to deposit histones onto DNA by itself in both pathways. Here, to delineate the precise role of Asf1 in chromatin assembly, we used Xenopus egg extracts as a powerful system to assay de novo chromatin assembly pathways in vitro. Following characterization of both Xenopus Asf1 and p60 (CAF-1), we used immunodepletion strategies targeting Asf1, HIRA, or CAF-1. Strikingly, the depletion of Asf1 led to the simultaneous depletion of HIRA and consequently impaired the DNA synthesis-independent nucleosome assembly pathway. The rescue of nucleosome assembly capacity in such extracts was effective when adding HIRA along with H3/H4 histones, yet addition of Asf1 along with H3/H4 histones did not work. Moreover, nucleosome assembly coupled to DNA repair was not affected in these Asf1/HIRA-depleted extracts, a pathway impaired by CAF-1 depletion. Thus, these data show that Asf1 is not directly involved in de novo histone deposition during DNA synthesis-independent and synthesis-dependent pathways in egg extracts. Based on our results, it becomes important to consider the implications for Asf1 function during early development in Xenopus.     

Identification of small molecules that inhibit the histone chaperone Asf1 and its chromatin function

The eukaryotic genome is packed into chromatin, which is important for the genomic integrity and gene regulation. Chromatin structures are maintained through assembly and disassembly of nucleosomes catalyzed by histone chaperones. Asf1 (anti-silencing function 1) is a highly conserved histone chaperone that mediates histone transfer on/off DNA and promotes histone H3 lysine 56 acetylation at globular core domain of histone H3. To elucidate the role of Asf1 in the modulation of chromatin structure, we screened and identified small molecules that inhibit Asf1 and H3K56 acetylation without affecting other histone modifications. These pyrimidine-2,4,6-trione derivative molecules inhibited the nucleosome assembly mediated by Asf1 in vitro, and reduced the H3K56 acetylation in HeLa cells. Furthermore, production of HSV viral particles was reduced by these compounds. As Asf1 is implicated in genome integrity, cell proliferation, and cancer, current Asf1 inhibitor molecules may offer an opportunity for the therapeutic development for treatment of diseases. [BMB Reports 2015; 48(12): 685-690]     

Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity

The process of coordinated DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3–H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three his-tone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3–H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3–H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.Key words: Gen5, Rtt109, chromatin, nucleosome assembly, genome integrity     

The activity of the histone chaperone yeast Asf1 in the assembly and disassembly of histone H3/H4-DNA complexes

The deposition of the histones H3/H4 onto DNA to give the tetrasome intermediate and the displacement of H3/H4 from DNA are thought to be the first and the last steps in nucleosome assembly and disassembly, respectively. Anti-silencing function 1 (Asf1) is a chaperone of the H3/H4 dimer that functions in both of these processes. However, little is known about the thermodynamics of chaperone–histone interactions or the direct role of Asf1 in the formation or disassembly of histone–DNA complexes. Here, we show that Saccharomyces cerevisiae Asf1 shields H3/H4 from unfavorable DNA interactions and aids the formation of favorable histone–DNA interactions through the formation of disomes. However, Asf1 was unable to disengage histones from DNA for tetrasomes formed with H3/H4 and strong nucleosome positioning DNA sequences or tetrasomes weakened by mutant (H3K56Q/H4) histones or non-positioning DNA sequences. Furthermore, Asf1 did not associate with preformed tetrasomes. These results are consistent with the measured affinity of Asf1 for H3/H4 dimers of 2.5 nM, which is weaker than the association of H3/H4 for DNA. These studies support a mechanism by which Asf1 aids H3/H4 deposition onto DNA but suggest that additional factors or post-translational modifications are required for Asf1 to remove H3/H4 from tetrasome intermediates in chromatin.     

Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity

The coordinated process of DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3-H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three histone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3-H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3-H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.     

Structure of the histone chaperone ASF1 bound to the histone H3 C-terminal helix and functional insights

Asf1 is a histone chaperone that favors histone H3/H4 assembly and disassembly. We solved the structure of the conserved domain of human ASF1A in complex with the C-terminal helix of histone H3 using nuclear magnetic resonance spectroscopy. This structure is fully compatible with an association of ASF1 with the heterodimeric form of histones H3/H4. In our model, ASF1 substitutes for the second H3/H4 heterodimer that is normally found in heterotetrameric H3/H4 complexes. This result constitutes an essential step in the fundamental understanding of the mechanisms of nucleosome assembly by histone chaperones. Point mutations that perturb the Asf1/histone interface were designed from the structure. The decreased binding affinity of the Asf1-H3/H4 complex correlates with decreased levels of H3-K56 acetylation and phenotypic defects in vivo.     

Codanin-1, mutated in the anaemic disease CDAI, regulates Asf1 function in S-phase histone supply

Efficient supply of new histones during DNA replication is critical to restore chromatin organization and maintain genome function. The histone chaperone anti-silencing function 1 (Asf1) serves a key function in providing H3.1-H4 to CAF-1 for replication-coupled nucleosome assembly. We identify Codanin-1 as a novel interaction partner of Asf1 regulating S-phase histone supply. Mutations in Codanin-1 can cause congenital dyserythropoietic anaemia type I (CDAI), characterized by chromatin abnormalities in bone marrow erythroblasts. Codanin-1 is part of a cytosolic Asf1-H3.1-H4-Importin-4 complex and binds directly to Asf1 via a conserved B-domain, implying a mutually exclusive interaction with the chaperones CAF-1 and HIRA. Codanin-1 depletion accelerates the rate of DNA replication and increases the level of chromatin-bound Asf1, suggesting that Codanin-1 guards a limiting step in chromatin replication. Consistently, ectopic Codanin-1 expression arrests S-phase progression by sequestering Asf1 in the cytoplasm, blocking histone delivery. We propose that Codanin-1 acts as a negative regulator of Asf1 function in chromatin assembly. This function is compromised by two CDAI mutations that impair complex formation with Asf1, providing insight into the molecular basis for CDAI disease.     

Asf1, a loveseat for a histone couple

In this issue of Cell, English et al. present the first crystal structure of a histone chaperone (Asf1) bound to histones (the H3/H4 heterodimer). The structure provides insights into how histone chaperones participate in nucleosome disassembly. It reveals that Asf1 physically blocks (H3/H4)(2) tetramer formation and that the C terminus of H4 undergoes a dramatic conformational change upon binding to Asf1.     

Histone chaperones, a supporting role in the limelight

In eukaryotic cells, highly basic histone proteins are associated with the DNA to form the nucleosome, the fundamental unit of chromatin. Histones are closely escorted by histone chaperones from their point of synthesis up to their delivery site. We will present an overview of the histone chaperones identified to date with their various roles, in an attempt to highlight their importance in cellular metabolism. Nucleoplasmin will illustrate a role in histone storage and Nap-1, a histone translocator. CAF-1 and Hira will provide examples of distinct histone deposition factors coupled to and uncoupled from DNA synthesis, respectively, while Asf1 could act as a histone donor. We then will illustrate with two examples how histone chaperones can be associated with chromatin remodeling activities. Finally, we will discuss how the RbAp46/48 proteins, as escort factors, are part of multiple complexes with various functions. Based on these examples, we will propose a scheme in which the diverse roles of histone chaperones are integrated within an assembly line for chromatin formation and regulation. Finally, we discuss how these chaperones may have more than a supporting role in a histone metabolic pathway.     

Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis

Deposition of the major histone H3 (H3.1) is coupled to DNA synthesis during DNA replication and possibly DNA repair, whereas histone variant H3.3 serves as the replacement variant for the DNA-synthesis-independent deposition pathway. To address how histones H3.1 and H3.3 are deposited into chromatin through distinct pathways, we have purified deposition machineries for these histones. The H3.1 and H3.3 complexes contain distinct histone chaperones, CAF-1 and HIRA, that we show are necessary to mediate DNA-synthesis-dependent and -independent nucleosome assembly, respectively. Notably, these complexes possess one molecule each of H3.1/H3.3 and H4, suggesting that histones H3 and H4 exist as dimeric units that are important intermediates in nucleosome formation. This finding provides new insights into possible mechanisms for maintenance of epigenetic information after chromatin duplication.     

Chromatin Assembly Factor I Mutants Defective for PCNA Binding Require Asf1/Hir Proteins for Silencing

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.     

综述: 组蛋白变体的染色质组装

真核细胞的染色质组装是组蛋白和DNA有序地形成核小体和染色质的过程．通过调节DNA的开放或折叠状态,染色质组装不但影响遗传信息的编码和存储,也决定了遗传信息的提取和解读．作为染色质组装的重要调控因子,组蛋白变体和组蛋白伴侣在与DNA相关的生命活动进程中发挥着至关重要的作用．本文综述了组蛋白变体H2A.Z以及CENP-A进行染色质组装的研究进展,并着重讨论了组蛋白变体和组蛋白伴侣在染色质组装中的重要作用．