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.     

The radioresistance kinase TLK1B protects the cells by promoting repair of double strand breaks

Background  

The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila or expression of a dominant negative mutant in mouse cells causes loss of nuclear divisions and missegregation of chromosomes probably, due to alterations in chromatin remodeling capacity. Overexpression of TLK1B, a spliced variant of the TLK1 mRNA, in a model mouse cell line increases it's resistance to ionizing radiation (IR) or the radiomimetic drug doxorubicin, also likely due to changes in chromatin remodeling. TLK1B is translationally regulated by the availability of the translation factor eIF4E, and its synthesis is activated by IR. The reason for this mechanism of regulation is likely to provide a rapid means of promoting repair of DSBs. TLK1B specifically phosphorylates histone H3 and Asf1, likely resulting in changes in chromatin structure, particularly at double strand breaks (DSB) sites.     

Utilizing Targeted Mass Spectrometry to Demonstrate Asf1-Dependent Increases in Residue Specificity for Rtt109-Vps75 Mediated Histone Acetylation

In Saccharomyces cerevisiae, Rtt109, a lysine acetyltransferase (KAT), associates with a histone chaperone, either Vps75 or Asf1. It has been proposed that these chaperones alter the selectivity of Rtt109 or which residues it preferentially acetylates. In the present study, we utilized a label-free quantitative mass spectrometry-based method to determine the steady-state kinetic parameters of acetylation catalyzed by Rtt109-Vps75 on H3 monomer, H3/H4 tetramer, and H3/H4-Asf1 complex. These results show that among these histone conformations, only H3K9 and H3K23 are significantly acetylated under steady-state conditions and that Asf1 promotes H3/H4 acetylation by Rtt109-Vps75. Asf1 equally increases the Rtt109-Vps75 specificity for both of these residues with a maximum stoichiometry of 1:1 (Asf1 to H3/H4), but does not alter the selectivity between these two residues. These data suggest that the H3/H4-Asf1 complex is a substrate for Rtt109-Vps75 without altering selectivity between residues. The deletion of either Rtt109 or Asf1 in vivo results in the same reduction of H3K9 acetylation, suggesting that Asf1 is required for efficient H3K9 acetylation both in vitro and in vivo. Furthermore, we found that the acetylation preference of Rtt109-Vps75 could be directed to H3K56 when those histones already possess modifications, such as those found on histones purified from chicken erythrocytes. Taken together, Vps75 and Asf1 both enhance Rtt109 acetylation for H3/H4, although via different mechanisms, but have little impact on the residue selectivity. Importantly, these results provide evidence that histone chaperones can work together via interactions with either the enzyme or the substrate to more efficiently acetylate histones.     

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]     

Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B)

The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.     

Histone acetyltransferase-1 regulates integrity of cytosolic histone H3-H4 containing complex

Amounts of soluble histones in cells are tightly regulated to ensure supplying them for the newly synthesized DNA and preventing the toxic effect of excess histones. Prior to incorporation into chromatin, newly synthesized histones H3 and H4 are highly acetylated in pre-deposition complex, wherein H4 is di-acetylated at Lys-5 and Lys-12 residues by histone acetyltransferase-1 (Hat1), but their role in histone metabolism is still unclear. Here, using chicken DT 40 cytosolic extracts, we found that histones H3/H4 and their chaperone Asf1, including RbAp48, a regulatory subunit of Hat1 enzyme, were associated with Hat1. Interestingly, in HAT1-deficient cells, cytosolic histones H3/H4 fractions on sucrose gradient centrifugation, having a sedimentation coefficient of 5–6S in DT40 cells, were shifted to lower molecular mass fractions, with Asf1. Further, sucrose gradient fractionation of semi-purified tagged Asf1-complexes showed the presence of Hat1, RbAp48 and histones H3/H4 at 5–6S fractions in the complexes. These findings suggest the possible involvement of Hat1 in regulating cytosolic H3/H4 pool mediated by Asf1-containing cytosolic H3/H4 pre-deposition complex.     

CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA

Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1–H3/H4 or H3/H4–DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)₂ tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.     

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.     

Crystal structures of fission yeast histone chaperone Asf1 complexed with the Hip1 B-domain or the Cac2 C terminus

The assembly of core histones onto eukaryotic DNA is modulated by several histone chaperone complexes, including Asf1, CAF-1, and HIRA. Asf1 is a unique histone chaperone that participates in both the replication-dependent and replication-independent pathways. Here we report the crystal structures of the apo-form of fission yeast Asf1/Cia1 (SpAsf1N; residues 1-161) as well as its complexes with the B-domain of the fission yeast HIRA orthologue Hip1 (Hip1B) and the C-terminal region of the Cac2 subunit of CAF-1 (Cac2C). The mode of the fission yeast Asf1N-Hip1B recognition is similar to that of the human Asf1-HIRA recognition, suggesting that Asf1N recognition of Hip1B/HIRA is conserved from yeast to mammals. Interestingly, Hip1B and Cac2C show remarkably similar interaction modes with Asf1. The binding between Asf1N and Hip1B was almost completely abolished by the D37A and L60A/V62A mutations in Asf1N, indicating the critical role of salt bridge and van der Waals contacts in the complex formation. Consistently, both of the aforementioned Asf1 mutations also drastically reduced the binding to Cac2C. These results provide a structural basis for a mutually exclusive Asf1-binding model of CAF-1 and HIRA/Hip1, in which Asf1 and CAF-1 assemble histones H3/H4 (H3.1/H4 in vertebrates) in a replication-dependent pathway, whereas Asf1 and HIRA/Hip1 assemble histones H3/H4 (H3.3/H4 in vertebrates) in a replication-independent pathway.     

Characterization of two different Asf1 histone chaperones with distinct cellular localizations and functions in Trypanosoma brucei

The anti-silencing function protein 1 (Asf1) is a chaperone that forms a complex with histones H3 and H4 facilitating dimer deposition and removal from chromatin. Most eukaryotes possess two different Asf1 chaperones but their specific functions are still unknown. Trypanosomes, a group of early-diverged eukaryotes, also have two, but more divergent Asf1 paralogs than Asf1 of higher eukaryotes. To unravel possible different functions, we characterized the two Asf1 proteins in Trypanosoma brucei. Asf1A is mainly localized in the cytosol but translocates to the nucleus in S phase. In contrast, Asf1B is predominantly localized in the nucleus, as described for other organisms. Cytosolic Asf1 knockdown results in accumulation of cells in early S phase of the cell cycle, whereas nuclear Asf1 knockdown arrests cells in S/G2 phase. Overexpression of cytosolic Asf1 increases the levels of histone H3 and H4 acetylation. In contrast to cytosolic Asf1, overexpression of nuclear Asf1 causes less pronounced growth defects in parasites exposed to genotoxic agents, prompting a function in chromatin remodeling in response to DNA damage. Only the cytosolic Asf1 interacts with recombinant H3/H4 dimers in vitro. These findings denote the early appearance in evolution of distinguishable functions for the two Asf1 chaperons in trypanosomes.     

Replication-independent histone deposition by the HIR complex and Asf1

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

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.     

Chaperone control of the activity and specificity of the histone H3 acetyltransferase Rtt109

Acetylation of Saccharomyces cerevisiae histone H3 on K56 by the histone acetyltransferase (HAT) Rtt109 is important for repairing replication-associated lesions. Rtt109 purifies from yeast in complex with the histone chaperone Vps75, which stabilizes the HAT in vivo. A whole-genome screen to identify genes whose deletions have synthetic genetic interactions with rtt109Delta suggests Rtt109 has functions in addition to DNA repair. We show that in addition to its known H3-K56 acetylation activity, Rtt109 is also an H3-K9 HAT, and we show that Rtt109 and Gcn5 are the only H3-K9 HATs in vivo. Rtt109's H3-K9 acetylation activity in vitro is enhanced strongly by Vps75. Another histone chaperone, Asf1, and Vps75 are both required for acetylation of lysine 9 on H3 (H3-K9ac) in vivo by Rtt109, whereas H3-K56ac in vivo requires only Asf1. Asf1 also physically interacts with the nuclear Hat1/Hat2/Hif1 complex that acetylates H4-K5 and H4-K12. We suggest Asf1 is capable of assembling into chromatin H3-H4 dimers diacetylated on both H4-K5/12 and H3-K9/56.     

The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours

Background  

Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9) and at lysine 20 of histone H4 (H4K20) governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability. Therefore, it is essential to analyze mechanisms responsible for high order chromatin packing and in particular the interplay between enzymes involved in histone modifications, such as histone methyltransferases and proteins that recognize these epigenetic marks.