InteractiveFly: GeneBrief

anti-silencing factor 1: Biological Overview | References

Gene name - anti-silencing factor 1

Synonyms -

Cytological map position - 76B9-76B9

Function - chromatin protein

Keywords - chromatin assembly, histone chaperone, Notch pathway, DNA replication

Symbol - asf1

FlyBase ID: FBgn0029094

Genetic map position - 3L: 19,611,992..19,613,501 [+]

Classification - Anti-silencing protein

Cellular location - nuclear

NCBI links: Precomputed BLAST | EntrezGene

The histone chaperone Asf1 assists in chromatin assembly and remodeling during replication, transcription activation, and gene silencing. However, it has been unclear to what extent Asf1 could be targeted to specific loci via interactions with sequence-specific DNA-binding proteins. This study shows that Asf1 contributes to the repression of Notch target genes, as depletion of Asf1 in cells by RNAi causes derepression of the E(spl) Notch-inducible genes. Conversely, overexpression of Asf1 in vivo results in decreased expression of target genes and produces phenotypes that are strongly modified (enhanced and suppressed) by mutations affecting the Notch pathway, but not by mutations in other signaling pathways. Asf1 can be coprecipitated with the DNA-binding protein Su(H) and the corepressor Hairless and interacts directly with two components of this complex, Hairless and SKIP. Thus, in addition to playing more general roles in chromatin dynamics, Asf1 is directed via interactions with sequence-specific complexes to mediate silencing of specific target genes (Goodfellow, 2007).

Modulation of the chromatin structure is a key feature in transcriptional regulation. Chromatin remodeling by ATP-dependent enzymes and posttranslational histone modifications are two important mechanisms that affect transcriptional activity, by influencing the accessibility of upstream regions and promoters. A third mechanism involves the breakdown and reassembly of nucleosomes on the DNA, a process that also allows for the incorporation of histone variants, such as H3.3. Histone chaperones, which bind to histone heterodimers, are required both for nucleosome assembly and for their disassembly. They include the H3/H4 chaperone Anti-silencing factor 1 (Asf1), which has roles in replication-dependent and replication-independent chromatin dynamics (Goodfellow, 2007).

In yeast, extensive Asf1-mediated exchange of histones that is independent of replication and of transcription has been detected at gene promoters and is likely to be highly significant in maintaining the balance between induction and silencing of genes (Schermer, 2005). Indeed, there are now several examples of yeast Asf1 contributing to chromatin disassembly at promoters to facilitate binding of the RNA-polymerase complex (Adkins, 2004; Adkins, 2007). Conversely, Asf1 also plays important roles in gene silencing (Sharp, 2001) when the reassembly of nucleosomes accompanies transcriptional repression. For example, in the absence of Asf1, there is a delay in promoter closure at the PHO5 gene (Schermer, 2005). However, it remains unclear whether Asf1-mediated nucleosome reassembly occurs via a targeted mechanism, involving sequence-specific DNA-binding proteins, or whether it occurs constitutively by default (Goodfellow, 2007).

A strong correlation between histone loss and gene activation has emerged from genome-wide studies in Drosophila, as it has in yeast, suggesting that transcription in higher eukaryotes is also likely to be regulated by histone loss and replacement at the promoter. However, thus far, the contribution of Asf1 to dynamic gene regulation during cell signaling in multicellular organisms has not been examined. One cell-signaling pathway with very direct effects on transcription is the highly conserved Notch pathway. Activation of the receptor results in the release of a nuclear-targeted intracellular fragment (Nicd), which binds directly to the CSL DNA-binding protein (Suppressor of Hairless, Su(H), in Drosophila) and recruits the coactivator Mastermind, resulting in the activation of target genes. CSL proteins also contribute to the silencing of target genes in the absence of Nicd, through adaptor-mediated recruitment of corepressors such as Groucho (Gro), CtBP, and SMRT. Previous analysis indicates that the activity of Notch target genes correlates with a reduction in histone H3 density (Krejci, 2007), suggesting that nucleosome disassembly and reassembly is likely to be involved in their regulation, and prompting an investigatation of whether Asf1 could play a role (Goodfellow, 2007).

This study shows that Asf1 contributes to the repression of Notch target genes, and that it is recruited to the DNA through interactions with the Su(H)/H complex. Thus, Asf1 is targeted to specific loci by binding to sequence-specific DNA-binding complexes, where it can promote gene silencing during development (Goodfellow, 2007).

To investigate whether Asf1 contributes to the regulation of inducible genes in Drosophila, RNA interference (RNAi) was used to deplete S2-N cells and the levels of transcription were analyzed from the 11 well-characterized Notch target genes clustered in the E(spl) complex. Conditions were established for activating Notch in these cells and it was shown (Krejci, 2007) that activation results in Su(H)-dependent stimulation of E(spl) gene transcription (Goodfellow, 2007).

Unlike knockdown of the other chromatin regulators tested, depletion of Asf1 led to a 4-fold increase in E(spl)m7 mRNA levels, but it had no effect on the housekeeping genes rp49 and EF2B. More extensive analysis revealed that mRNA levels for all E(spl) genes were increased after Asf1 depletion in the absence of Notch activation; some showed a greater than 10-fold change in expression, suggesting that these Notch targets are derepressed as they are when the corepressor Hairless is depleted. In contrast, there was little effect of Asf1 depletion on several other repressed genes, including a phagocytosis receptor gene, nimrod. In addition to the derepression observed in resting cells, Asf1 depletion also altered the responsiveness to Notch activation. Many more of the E(spl) genes were susceptible to Notch activation in Asf1-depleted cells; for example, 5 of the 11 genes were expressed at greater than 20-fold higher levels after Asf1 RNAi. There was comparatively little change at the genes, such as E(spl)m3, which normally has the most robust response to Notch and is depleted for histones (Krejci, 2007). Thus, it appears that Asf1 makes important contributions to the silencing of Notch target genes (Goodfellow, 2007).

Previous studies showed that overexpression of Asf1 in the Drosophila eye (ey::Gal4 UAS::asf1/+) causes a 'small-eye' phenotype in which the eye is reduced in size and ommatidia are disorganized (Moshkin, 2002). If these small-eye phenotypes are a consequence of Asf1 altering the transcription of Notch targets, they may be modified when combined with mutations in the Notch pathway. To investigate this possibility, flies overexpressing Asf1 were crossed to alleles affecting genes central to Notch or to other signaling pathways, and the eye size was analyzed in the heterozygous progeny (Goodfellow, 2007).

The first dramatic result was that the heterozygous combination of a Notch loss-of-function allele (N55e11) and Asf1 overexpression caused a severe reduction in the eye/head capsule ('pin-head') and resulted in lethality. Thus, the effects of Asf1 overexpression were strongly enhanced by a decrease in Notch function. Significant enhancement of the Asf1 phenotype also occurred with Delta loss-of-function alleles, but not with alleles affecting Hedgehog (smo), EGF-R (Egfr), or Wingless (arm, arrow) pathways or with alleles affecting the SET domain protein Trithorax-related (trr), the histone exchange factor Domino (dom), or the cell adhesion protein Pawn (pwn). Complementary results were obtained by using mutant alleles that increase Notch signaling: both a loss-of-function Hairless (H) allele and a gain-of-function Notch allele (NMcd1) suppressed the small-eye defect caused by Asf1 overexpression. These findings are fully consistent with the results of RNAi-mediated Asf1 depletion, and they suggest that Asf1 is involved in repression of Notch target genes. As asf1 mutant cells failed to proliferate, it was not possible to obtain clones of homozygous mutant cells to test the effects of eliminating Asf1 on Notch target genes in the eye (Goodfellow, 2007).

To investigate whether interactions between Notch and Asf1 occur in other tissues, it was asked whether Asf1 overexpression also perturbed Notch function in the Drosophila wing. Expression of Asf1 in the developing wing pouch (sd::Gal4/+; UAS::asf1/+) resulted in margin loss/wing nicks and mild vein thickening, characteristics of reduced Notch function (Notch/+ heterozygous flies have mild wing nicks due to reduced signaling at the dorsal/ventral (d/v) organizer of the wing). The Asf1 overexpression phenotypes were strongly enhanced when the levels of Notch were reduced; thus, wings had extensive scalloping/margin loss and more extensive vein thickening. Wing phenotypes, similar to the eye phenotypes, produced by Asf1 expression were thus enhanced by reduced Notch (Goodfellow, 2007).

To further assess whether Asf1 affects expression of target genes regulated by Notch (e.g., cut) or by other pathways (e.g., spalt), the effects of overexpressing Asf1 in wing discs was analyzed. In wild-type discs, Notch-dependent expression of Cut is detected in a stripe along the d/v boundary. This was interrupted and reduced in discs in which Asf1 was overexpressed. In contrast, there was no visible effect on Spalt under these conditions. Similar results were obtained when Asf1 was expressed in a more limited domain (by using ptc::Gal4), where a local loss of Cut, but not Spalt, expression was seen. Stronger expression of Asf1 resulted in more pronounced Notch-like phenotypes and loss of Cut expression, which could be rescued by a reduction in Hairless function. Under these conditions, where Asf1 was expressed more strongly, some more generalized effects of Asf1 were sometimes detected, compatible with its proposed role as a histone chaperone during replication. The replication defects became more severe at even higher levels of expression (29°C). Similarly, clones of cells mutant for asf1 failed to proliferate. Thus, as in yeast, Asf1 appears to have roles in replication-dependent as well as replication-independent chromatin dynamics in Drosophila. By moderating the levels of Asf1 expression, it was possible to uncouple these requirements, revealing a contribution to repression of Notch target genes (Goodfellow, 2007).

Complexes implicated in repression at Notch targets are formed by the CSL/Su(H) DNA-binding protein in conjunction with adaptor proteins, such as SKIP and Hairless, which recruit general corepressors, including SMTR or Gro and CtBP. On polytene chromosomes from Drosophila salivary glands, Asf1 is detected at most Su(H)-enriched sites, suggesting that these proteins are present at the same loci. Asf1 is also bound at many other loci, and it is strongly enriched at centromeres and telomeres, reflecting its multiple roles in chromatin dynamics (Goodfellow, 2007).

The colocalization of Su(H) and Asf1 on polytene chromosomes prompted a test of whether Su(H) and/or associated factors could copurify with Asf1 in immunoprecipitation (IP) experiments. For these experiments, extracts prepared from Drosophila embryos, and Su(H) or Asf1 was immunoprecipitated by using moderate salt conditions. Under these conditions, Asf1 was detected in Su(H) IP experiments, and, conversely, Su(H) was precipitated with Asf1, as was the corepressor Gro, but not CtBP. To exclude the possibility that the interaction between Asf1 and the Su(H) complex was mediated by the independent binding of both protein complexes to DNA, IP experiments were performed in the presence of ethidium bromide (EtBr), a DNA-intercalating drug that dissociates proteins from DNA. This treatment did not affect the interaction of Asf1 with Su(H). Thus, these data suggest that Asf1 is present in protein complexes containing the sequence-specific DNA-binding protein Su(H) and the Gro corepressor. A significant suppression of the Asf1-induced small-eye phenotype was observed in flies that were also heterozygous for a strong gro allele (groE48) and an enhancement was seen by Hairless proteins that retained a Gro-binding domain, agreeing with a model linking Gro to Asf1-mediated repression. Therefore whether any of the proteins in the Su(H) repression complex are able to bind to bacterially produced Asf1 (fused to glutathione S-transferase, GST), was examined. Of those tested, both Hairless and the adaptor protein SKIP were bound to GST-Asf1, but not to GST alone or to GST-CAF1p55 (a component of chromatin assembly factor 1). Neither Gro nor Su(H) itself showed direct interactions with Asf1 in this assay (Goodfellow, 2007).

Finally, to test whether Hairless contributes to the recruitment of Asf1 in vivo, chromatin immunoprecipitation (ChIP) was performed with anti-Asf1 antibodies in cells with and without RNAi-mediated depletion of Hairless and association with two E(spl) genes, m3 and m7, was assayed. The E(spl)m7 gene is silenced in the S2 cells and is strongly affected by Asf1 depletion, whereas E(spl)m3 is expressed in S2 cells, is highly induced by Notch activation, and is more mildly affected by Asf1 depletion. Of the two genes, the greatest effects were seen for E(spl)m7; binding of Asf1 to both enhancer and ORF fragments strongly decreased in ChIP after Hairless depletion. A decrease was also seen at the E(spl)m3 ORF region, but not at the E(spl)m3 enhancer. This enhancer is found to have very low histone coverage in these cells, and it was found that it shows only small Asf1 occupancy levels. The decrease in Asf1 from ORFs of both E(spl)m3 and E(spl)m7 after Hairless depletion may indicate that Asf1 spreads from the site of recruitment. Binding of Asf1 to E(spl)m7 and E(spl)m3 regions was confirmed by using affinity-purified anti-Asf1 antibodies raised in a different species. Loss of Hairless does not affect the binding of Asf1 to other loci that do not require Su(H)/H for their regulation, such as eiger or snRNP69D. Similarly, there was no change in the levels of Polycomb protein associated with bxd-PRE after Hairless knockdown. Together, these data support the model that recruitment of Asf1 to Notch targets requires Hairless (Goodfellow, 2007).

The density and precise positioning of nucleosomes are important factors in determining the transcriptional activity of a locus. It is now evident that most nonnucleosomal histones in cells are likely to be complexed with chaperones. It is therefore not surprising that the histone chaperone Asf1 is important for chromatin dynamics and has been shown to have multiple roles in transcription as well as in the disassembly and reassembly of chromatin during replication (Mousson, 2007). These include gene-specific roles in repression, activation, and transcription elongation (Sutton, 2001; Adkins, 2004; Zabaronick, 2005; Schwabish, 2006). For example, Asf1 is required for nucleosome disassembly and transcription activation at the yeast PHO5, PHO8, ADY2, and ADH2 promoters (Adkins, 2004; Adkins, 2007). However, the mechanisms responsible for targeting Asf1 to these loci remain unclear. This study has demonstrated that Asf1 can be specifically recruited to target loci by interactions with sequence-specific DNA-binding transcription factors. Asf1 is present in a complex with Su(H), the central DNA-binding protein in the Notch pathway, and that it interacts directly with two proteins found in CSL complexes, Hairless and SKIP. Importantly, it was found that Asf1 plays a significant role in the repression of Notch target genes. Thus, contrary to effects at many of the inducible loci examined in yeast, these data demonstrate a requirement for Asf1 in silencing rather than in activation of these inducible genes (Goodfellow, 2007).

As the global corepressor Gro is also coprecipitated with Asf1 and is implicated in Asf1-mediated repression through genetic interactions, Gro and Asf1 may cooperate in the repression of Notch target genes. Gro has been postulated to exert long-range repressive effects by nucleating a transcriptionally silent chromatin state, in a similar manner to its yeast relative Tup1. For example, at the STE6 locus, Tup1 recruitment results in increased nucleosomal density and local nucleosome positioning. The recruitment of the histone chaperone Asf1 with Gro to Su(H)/H DNA-binding complexes could facilitate a similar localized increase in histone deposition and participate in the spreading of repressed chromatin. Furthermore, since (H)/H complexes engage in comparatively low-stability interactions with target loci (Krejci, 2007), it is suggested that Asf1 could be critical for translating these transient interactions into stable silencing. However, thus far, the analysis has focused on relatively few targets and tissues; thus, it remains to be determined whether Asf1 is recruited to all targets regulated by Su(H)/H, or whether there are additional factors that influence its recruitment at specific loci. Similarly, it will be important to determine whether other sequence-specific complexes are able to bind directly to Asf1 (Goodfellow, 2007).

In conclusion, these results show that the histone H3/H4 chaperone Asf1 contributes to selective silencing of genes in Drosophila, through interactions with the Su(H)/H DNA-binding protein complexes. In this way, chaperones can act as gene-selective regulators that contribute to the control of gene expression by developmental signaling pathways (Goodfellow, 2007).

The Drosophila DAXX like protein (DLP) cooperates with ASF1 for H3.3 deposition and heterochromatin formation

Histone variants are non-allelic isoforms of canonical histones and they are deposited, in contrast to canonical histones, in a replication-independent (RI) manner. RI deposition of H3.3, a histone variant from the H3.3 family, is mediated in mammals by distinct pathways involving either the histone regulator A (HIRA) complex or the death-associated protein (DAXX)/alpha-thalassemia X-linked mental retardation protein (ATRX) complex. This study investigated the function of Drosophila DAXX Like Protein (DLP) by using both fly genetics approaches and protein biochemistry. DLP specifically interacts with H3.3 and shows a prominent localization on the base of the X chromosome, where it appears to act in concert with XNP the Drosophila homolog of ATRX, in heterochromatin assembly and maintenance. The functional association between DLP and XNP is further supported by a series of experiments, which illustrate genetic interactions and DLP-XNP-dependent localization of specific chromosomal proteins. In addition, DLP both participates in RI deposition of H3.3 and associates with the anti-silencing factor-1 (ASF1). It is suggested, in agreement with a recently proposed model, that DLP and ASF1 are part of a pre-deposition complex, which is recruited by XNP and is necessary to prevent DNA exposure in the nucleus (Fromental-Ramain, 2017).

This study has identified DLP as the Drosophila homolog of DAXX. DLP is involved, likely in concert with XNP/dATRX, in the formation of pericentric heterochomatin of the X chromosome. Moreover, DLP is implicated in RI deposition of the histone variant H3.3 and may constitute with ASF1 the central core of a pre-deposition complex, recruited to chromatin gaps by XNP. The existence of such complex was recently suggested (Fromental-Ramain, 2017 and references therein).

In spite of the fact that both proteins do not molecularly associate as their mammal homologs do, this study provides evidence that DLP and XNP functions are closely linked. DLP and XNP are located on the base of the X chromosome and analysis of animals simultaneously mutant for both dlp and xnp revealed that DLP and XNP likely act together during heterochromatin formation. In addition, both DLP and XNP are located next to distal heterochromatic marker HP1 on the X chromosome of larvae carrying the ln(1)wm4h rearrangement. Functional interactions between DLP and XNP 55 were also supported by the similar behavior of DLP and XNP in H3.3 deficient cells. In wild-type cells, in addition to the base of the X chromosome, expression of XNP is detected at many sites across the chromosome arms where DLP is not observed. In H3.3 knock-down-cells, DLP and XNP are present at many euchromatic sites of the chromosomes and are simultaneously associated with nucleolar chromatin of the rDNA. Finally, overexpressed DLP binds to many interbands on the polytene chromosomes, suggesting that DLP may also be involved in chromatin organization at euchromatic sites in addition to the pericentric heterochromatin. However, this latter observation should be viewed with caution since it cannot be ruled out that over-expressed DLP is not present in its usual complex and is consequently mis-targeted. Additional support for functional interactions between XNP and DLP is provided by genetic interactions between xnp and dlp. Indeed, loss of xnp function is characterized by reduced viability, which is further aggravated when dlp function is simultaneously reduced, strongly indicating that xnp and dlp may functionally cooperate during regulation of common targets. How XNP is recruited to nucleosome-depleted chromatin remains an important issue. XNP may be recruited by transcriptional machinery to active genes. Alternatively, XNP may bind structural motifs common to chromatin gaps, or may simply bind exposed DNA. The homologous ATRX contains a PHD domain that can bind DNA or histones tails. Recent work demonstrates that mammalian Hira may bind exposed DNA at chromatin gaps. Moreover, Hira and XNP bind active regions independently of one another. Hence, there may be multiple ways that RI assembly factors recognize exposed DNA (Fromental-Ramain, 2017).

In Drosophila, loss of H3.3 has a large impact on viability and fertility of both males and females. The Drosophila genome encompasses two single copy genes, H3.3A and H3.3B, which code for the same protein. H3.3 is highly expressed in mitotic, meiotic and post-meiotic male germ cells, probably reflecting high transcriptional activity. Interestingly, high dlp expression is observed in primary spermatocytes, in meiotic spermatocytes and also in the germinal vesicle, suggesting that it may have important functions during development of germ cells. In Drosophila testis, H3.3 disappears with the bulk of histones, prior to accumulation of protamine and other sperm-specific nuclear basic proteins, leading to sperm DNA compaction at late stages of spermiogenesis. At fertilization, assembly of nucleosomes on paternal DNA immediately follows the rapid loss of protamines from the decondensing male nucleus and is dependent on maternally provided factors like Hira and YEM. HIRA and YEM are crucial since male pronuclei fail to decondense at the pronuclear stage in eggs derived from female mutants for HIRA and YEM. Hence, function of HIRA/YEM at fertilization represents a unique example where deficient chromatin activity cannot be compensated by other redundant factors (Fromental-Ramain, 2017).

In contrast, many examples suggest that H3.3 chaperones/chromatin remodeling complexes may display functional redundancy as mutants in these factors have limited phenotypes. In this context, DLP may be viewed as a typical example. The null allele dlpG and the dlp45 allele encoding a truncated protein lacking the C-terminal DHR necessary for H3.3 binding are viable and fertile, indicating that DLP and other chromatin factors may share common functions. Alternatively, DLP may display accessory functions during development of germ cells. Characterization of the phenotypes of double-mutant animals during germ cell development would help to resolve this important issue (Fromental-Ramain, 2017).

H3.3 was initially seen as a characteristic of active genes with histone turnover occurring as a consequence of transcription. More recent studies revealed that H3.3 is widespread within the genome. In particular, H3.3 is deposited by ATRX/DAXX at telomeres and pericentric repeats. Interestingly, ATRX and DAXX are components of the same chromatin-remodeling complex and physically interact. Recently, Schneiderman (2012) proposed a model on how XNP, the Drosophila homolog of ATRX, and HIRA identify nucleosome-depleted DNA following gene activation, and promote nucleosome assembly through a three steps process. Initially, XNP and HIRA bind exposed DNA at chromatin gaps where nucleosomes have been displaced. Subsequently they co-operate to recruit a predeposition complex including ASF1 and histones. In the final step, XNP and HIRA assist the transfer of histones from delivery factors to DNA and are released when nucleosome assembly is complete. Even if XNP and DLP do not physically interact, this study provides several evidences suggesting that DLP could be a component of the predeposition complex recruited by XNP/HIRA (Fromental-Ramain, 2017).

Both HIRA and XNP have been implicated in RI nucleosome assembly, but mutants of these factors have only limited phenotypes, revealing that they have redundant functions. Thus, single mutants of either xnp or hira weakly affect H3.3 deposition, which is abolished in a double-mutant of xnp and hira. This observation highlights the need for two distinct pathways during RI nucleosome assembly, one mediated by HIRA/YEM and the other by XNP. This study assigns a role to DLP during RI H3.3 deposition since H3.3 incorporation is affected in animals lacking DLP. DLP is thought to co-operate with XNP and it was surprising to observe H3.3 deposition in animals lacking HIRA and DLP. Hence, it is speculated that the pathway mediated by XNP is always functional, although less efficient. It has been recently proposed that XNP recognizes exposed DNA when a nucleosome has been displaced, and serves as a binding platform for the recruitment of H3.3 predeposition complexes to chromatin gaps. Such complexes are believed to contain (H3.3-H4) heterodimers, ASF1 and additional factors. In line with this the data revealed physical interactions between ASF1 and DLP in protein extracts made from baculovirus-infected Sf9 cells co-expressing DLP and ASF1, suggesting that DLP may be one of these additional factors (Fromental-Ramain, 2017).

These data provides additional links between HIRA, DLP, ASF1 and XNP during H3.3 incorporation but how they functionally interact during development remains an open question. Identification of their genomic targets and characterization of their activities during H3.3 deposition would obviously help to resolve this important issue (Fromental-Ramain, 2017).

Histone chaperones ASF1 and NAP1 differentially modulate removal of active histone marks by LID-RPD3 complexes during NOTCH silencing

Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, the interaction networks of histone chaperones ASF1 (Anti-silencing factor 1), CAF1, HIRA, and NAP1 were identified. This study analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) Notch-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. It is concluded that histone chaperones ASF1 and NAP1 differentially modulate local chromatin structure during gene-selective silencing (Moshkin, 2009).

Regulated modulation of the chromatin structure is essential for the transmission, maintenance, and expression of the eukaryotic genome. The combined actions of ATP-dependent chromatin-remodeling factors (remodelers), histone chaperones, and histone-modifying enzymes drive chromatin dynamics. Histones are subjected to a wide range of reversible posttranslational modifications, including acetylation, phosphorylation, methylation, and ubiquitylation. Histone modifications, in turn, can promote the recruitment of selective regulatory factors and modulate chromatin accessibility. Chromatin remodelers control DNA accessibility by mediating nucleosome mobilization either through sliding or by nucleosome (dis)assembly (Moshkin, 2009).

Whereas originally considered mainly as mere chaperones, it has become clear that histone chaperones play diverse roles during chromatin transactions. Histone chaperones bind selective histones and include the highly conserved H3/H4 chaperones ASF1, CAF1, HIRA, and Spt6 and the H2A/H2B chaperones NAP1, Nucleoplasmin, and FACT. Although their biochemical activity, binding and release of histones, appears rather mundane, in conjunction with other factors, histone chaperones participate in a variety of chromatin transactions and other cellular tasks. For example, yeast NAP1 participates in an extensive interaction network including a diverse set of transcription initiation/elongation factors, chromatin remodelers, RNA-processing factors, cell-cycle regulators, and other proteins (Moshkin, 2009).

ASF1 is one of the major H3/H4 chaperones, and through association with other proteins, it contributes to diverse chromatin transactions. (1) In conjunction with CAF1 and the MCM2-7 DNA helicase, ASF1 participates in replication-coupled chromatin assembly. (2) When associated with HIRA, ASF1 participates in replication-independent chromatin assembly and histone replacement. (3) DNA-repair-associated chromatin assembly requires the cooperation between ASF1 and the H3K56 acetyltransferase Rtt109. (4) ASF1 functionally cooperates with the Drosophila BRM chromatin remodeler, and (5) interaction of ASF1 with transcription activators stimulates histone eviction from promoter areas and facilitates recruitment of chromatin-specific coactivator complexes. (6) ASF1 itself is one of the targets of Tousled-like kinase (TLK), which controls cell-cycle progression and chromatin dynamics. (7) Finally, ASF1 is involved in developmental gene expression control by mediating transcriptional repression of Notch target genes. ASF1 is recruited to E(spl) genes by the sequence-specific DNA-binding protein Su(H) and its associated corepressor complex, harboring Hairless (H) and SKIP (Moshkin, 2009).

Notch is the central component of a highly conserved developmental signaling pathway that is present in all metazoans. Notch is a single-pass transmembrane protein that is activated through ligand binding, resulting in the release of the Notch intracellular domain (Nicd), which is targeted to the nucleus to activate gene expression. The CSL (CBF1, Su(H), and Lag1) family of sequence-specific DNA-binding proteins is the key targeting factor of Nicd and coactivators and, in the absence of Nicd, corepressors. The repression of Notch target genes involves multiple chromatin-modifying activities including histone deacetylases, H3K9 methyltransferases, CtBP, NcoR/SMRT, and Goucho (GRO). In the absence of the Nicd, loss of ASF1 leads to derepression of the E(spl) genes, revealing its essential role in silencing (Moshkin, 2009).

The molecular mechanism by which ASF1 achieves gene-specific transcription repression and the potential roles of other histone chaperones in developmental gene regulation remains largely unknown. To address these issues, a proteomics survey was performed of the protein interaction networks of ASF1, CAF1, HIRA, and NAP1 in Drosophila embryos. This analysis revealed that ASF1 and NAP1 interact with two related but distinct corepressor complexes: LAF and RLAF. LAF, comprising LID/KDM5 SIN3A, PF1, EMSY, and MRG15, associates with ASF1 (forming LAF-A). RLAF, comprising LAF plus RPD3, interacts with NAP1 (forming RLAF-N). Through a combination of biochemistry and developmental genetics, it was established that LAF-A and RLAF-N are tethered to Notch target genes by the Su(H)/H complex and mediate gene-selective silencing. Both ASF1 and NAP1 are required for the targeted removal of the positive H3K4me3 mark by facilitating LID/KDM5 recruitment to chromatin. Furthermore, NAP1 mediates nucleosome assembly at regulatory elements of Notch target genes and histone deacetylation by RLAF. These results uncover extensive crosstalk between distinct histone chaperones and histone-modifying enzymes in developmental gene regulation (Moshkin, 2009).

These results emphasize that, rather than generic, redundant factors, histone chaperones play highly specialized roles in gene-specific regulation. This study has dissected the molecular mechanism underpinning coordinate silencing of Notch target genes by the histone H3/H4 chaperone ASF1 and the H2A/H2B chaperone NAP1. ASF1 interacts with LAF, comprising SIN3A, PF1, EMSY, MRG15, and the histone H3K4me2/3 demethylase LID/KDM5, forming LAF-A. A closely related complex, RLAF that includes the deacetylase RPD3, does not bind ASF1. Instead, RLAF associates with NAP1, forming RLAF-N. The chaperones ASF1 and NAP1 link, respectively, LAF and RLAF to the Su(H)/H DNA-binding complex, tethering them to the E(spl) genes. Both ASF1 and NAP1 bind the SKIP subunit of the Su(H)/H complex (Goodfellow, 2007). Thus, at least in part, ASF1 and NAP1 facilitate H3K4me3 demethylation activity at the E(spl) genes through LID recruitment. Other LAFs might provide additional links to the Su(H)/H complex by contacting GRO and CtBP, which themselves associate with the Su(H)/H complex. For example, mammalian PF1, MRG15, and SIN3A have been reported to bind GRO. This study identified CtBP in LID, PF1, and NAP1 immunopurifications, providing an additional contact between the Su(H)/H complex and (R)LAF (Moshkin, 2009).

ASF1 does not bind RLAF and has no effect on histone H3 deacetylation by RPD3. In contrast, NAP1 does associate with RLAF and stimulates both H3K4 demethylation by LID and H3 deacetylation by RPD3. SIN3A had a mild effect, but the other LAF subunits played no apparent role in deacetylation. Finally, NAP1 depletion caused a dramatic loss of histones at the E(spl) regulatory elements, whereas ASF1 depletion had no effect on local histone density (Moshkin, 2009).

ASF1 has been proposed to function in chromatin assembly by acting as a donor that hands off the H3/H4 tetramer to either CAF1 or HIRA (De Koning, 2007). Because LAF-A does not associate with either CAF1 or HIRA, this might explain that ASF1 does not modulate nucleosome density at the E(spl) genes. In conclusion, the H3/H4 chaperone ASF1 mediates silencing of Notch target genes by (1) providing a connection between LAF and the Su(H)/H tether and (2) facilitating H3K4 demethylation by LID. The H2A/H2B chaperone NAP1 participates in E(spl) silencing by (1) linking RLAF to Su(H)/H, (2) facilitating H3K4 demethylation by LID, (3) facilitating H3 deacetylation by RPD3, and (4) directing high nucleosome density at repressed loci. The functioning of the H2A/H2B chaperone NAP1 in demethylation and deacetylation of histone H3 provides an example of trans-histone regulation (Moshkin, 2009).

LID and its interacting factors appear to work in a context-dependent manner. For example, LID facilitates activation of dMYC target genes in a manner independent of its demethylase activity. Suggestively, this study observed a genetic interaction between ASF1 and dMYC, indicating a potential role for LAF-A. Recently, it has been suggested that selective RLAF subunits could interact with a homolog of GATA zinc-finger domain-containing protein 1 to facilitate expression of targets by inhibition of RPD3 activity. In mammalian cells, LID homolog RBP2 and MRG15 have been implicated in transcription elongation by restricting H3K4me3 levels within transcribed regions. Identification of SIN3A as a LAF and RLAF subunit provides a molecular explanation for the recent observation that SIN3A is involved in genome-wide removal of both H3K4 methyl and acetyl marks. Collectively, these findings suggest that LID and RPD3 enzymatic activities can be modulated through association with specific partners. The proteomics analysis of the LID, PF1, and EMSY interaction networks further emphasizes the diverse involvement of LAFs in regulation of chromatin dynamics (Moshkin, 2009).

In conclusion, these results emphasize the close interconnectivity between distinct chromatin transactions and reveal cooperation between histone chaperones and targeted histone modifications during developmental gene control. The proteomic survey of ASF1, CAF1, HIRA, and NAP1 provides a starting point for the functional analysis of the regulatory networks in which these chaperones participate. As illustrated by the analysis of LAF-A and RLAF-N, specific protein-protein associations and gene targeting provide an intricate network of combinatorial gene expression control (Moshkin, 2009).

The assembly of chromatin during DNA synthesis

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, the Caf1-180 and Caf1-105 subunits of Drosophila chromatin assembly factor 1 (dCAF-1: see Drosophila Chromatin assembly factor 1 subunit) have been identified, cloned, and characterized. The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled chromatin assembly. Furthermore, 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 the Drosophila 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, dASF1 and dCAF-1 p105 colocalize in vivo in Drosophila 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 (Tyler, 2001).

The analysis of factors that are required in addition to CAF-1 for DNA replication-coupled chromatin assembly led to the identification of RCAF. RCAF comprises the Drosophila homolog of the yeast anti-silencing function 1 protein (dASF1) and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones that are assembled onto newly replicated DNA. RCAF functions synergistically with CAF-1 in the assembly of chromatin in DNA replication-chromatin assembly reactions. The study of yeast strains that are lacking CAF-1 and/or RCAF further suggested that CAF-1 and RCAF have both common and unique functions in the cell. RCAF-mediated chromatin assembly appears to be essential for normal progression through the cell cycle, gene expression, DNA replication, and DNA repair. Furthermore, it appears that the checkpoint kinase Rad53 may regulate the chromatin assembly function of ASF1 during DNA replication and repair (Tyler, 2001 and references therein).

To analyze the biochemical properties of dCAF-1, the p180, p105, and p55 proteins were synthesized in Sf9 cells by using baculovirus expression vectors. The p180 subunit contained a C-terminal FLAG epitope tag and was thus designated as p180-FLAG. The p105 subunit contained a C-terminal His6 tag and was therefore termed p105-His6. Different combinations of dCAF-1 subunits were synthesized and purified by either anti-FLAG or Ni(II) affinity chromatography. When p180-FLAG, p105-His6, and p55 were cosynthesized and subjected to anti-FLAG immunoaffinity chromatography, the purified p180+p105+p55 dCAF-1 complex was obtained. Similarly, cosynthesis of p180-FLAG with either p105-His6 or p55 yielded p180+p105 and p180+p55 subcomplexes. Although the three-subunit p180+p105+p55 complex can be purified by Ni(II) affinity chromatography via p105-His6, cosynthesis of p105-His6 and p55 and subsequent Ni(II) affinity chromatography yielded only p105. Hence, these findings indicate that dCAF-1 p180 interacts with both p105 and p55, but that p105 and p55 do not interact with one another (Tyler, 2001).

To test whether the p180, p105, and p55 subunits are required for chromatin assembly, DNA replication-chromatin assembly reactions were performed with partial and complete (i.e., p180+p105+p55) dCAF-1 complexes. These experiments revealed that the purified recombinant p180+p105+p55 dCAF-1 complex possesses a specific activity for DNA replication-coupled chromatin assembly that is comparable to that of native dCAF-1, as demonstrated by plasmid supercoiling analysis. It was further confirmed that dCAF-1-mediated plasmid supercoiling is a consequence of chromatin assembly by using micrococcal nuclease digestion analysis. In addition, the two-subunit p180+p105 subcomplex is fully active for chromatin assembly. In contrast, neither the p180 subunit alone nor the p105 subunit alone is sufficient for chromatin assembly. These results thus indicate that the p180 and p105 subunits are each essential for DNA replication-coupled chromatin assembly by dCAF-1 (Tyler, 2001).

It is relevant that the DNA replication extract used in these experiments contains significant amounts of hCAF-1 p60 and hCAF-1 p48 (also known as RbAp48), which are homologous to dCAF-1 p105 and dCAF-1 p55, respectively. Based on the requirement of dCAF-1 p105 for chromatin assembly, it appears that the hCAF-1 p60 subunit cannot function with the Drosophila CAF-1 polypeptides. However, the lack of a requirement for dCAF-1 p55 may be due to the ability of the hCAF-1 p48 subunit, which is about 87% identical to dCAF-1 p55, to function with the dCAF-1 p180 and p105 subunits in lieu of dCAF-1 p55. It is also possible, however, that the dCAF-1 p180+p105 subcomplex has the intrinsic ability to mediate chromatin assembly. It has not been possible to immunodeplete the hCAF-1 p48 protein from the DNA replication extract to differentiate between these possibilities. It is noteworthy, however, that the Arabidopsis equivalent of dCAF-1 p55 is required for DNA replication-coupled chromatin assembly with the same assay (Tyler, 2001).

The assembly of newly replicated DNA into chromatin requires both dCAF-1 and the RCAF chromatin assembly factor, which comprises Drosophila ASF1 (dASF1) and specifically acetylates histones H3 and H4. To investigate this effect further, coimmunoprecipitation analyses was performed with a crude Drosophila embryo extract. In these experiments, it was observed that immunoprecipitation with anti-dASF1 results in the coimmunoprecipitation of dCAF1 p180, p105, and p55, but not dCAF-1 p75. Conversely, immunoprecipitation with anti-p105 or with anti-p55 results in the coimmunoprecipitation of dASF1. Thus, these findings indicate that native dASF1 interacts with the native p180+p105+p55 form of dCAF-1 but not with the p75-containing form of dCAF-1. Immunoprecipitation of dCAF-1 with anti-p105+p75 does not result in the coimmunoprecipitation of dASF1, which suggests that the anti-p105+p75 antibodies destabilize the interaction between dASF1 and the p180+p105+p55 form of dCAF-1 (Tyler, 2001).

To summarize, the p75 subunit of dCAF-1 appears to be a C-terminally truncated form of p105 and there are distinct forms of dCAF-1 that contain either the p105 subunit or the p75 subunit. The p105-containing form of dCAF-1 comprises the p180, p105, and p55 proteins. The purified recombinant p180+p105+p55 dCAF-1 complex is as active for DNA replication-coupled chromatin assembly as native dCAF-1. Both the p180 and p105 subunits are essential for chromatin assembly. A preexisting interaction between dCAF-1 and the dASF1 chromatin assembly factor has been discovered in crude extracts. This dCAF-1-ASF1 interaction occurs via the dCAF-1 p105 subunit, and this interaction appears to be direct. dASF1 and dCAF-1 p105 colocalize in vivo in Drosophila polytene chromosomes. These results suggest that there is physical cooperation between dCAF-1 and dASF1 during chromatin assembly. The p105 and p75 subunits of dCAF-1 are closely related, and dCAF-1 is not a single four-subunit complex but rather a three-subunit p180+p105+p55 complex and a presumed p180+p75+p55 complex. Thus, the basic three-subunit structure of CAF-1 is conserved among yeast, Drosophila, and humans. The presence of multiple forms of dCAF-1 is of particular interest. Because dCAF-1 was isolated from whole embryos instead of a specific cell line, there is potential for considerable diversity in the range of functions that may be performed by the different forms of dCAF-1. It is possible, for instance, that the p105-containing form of dCAF-1 functions in ASF1-dependent processes, whereas the p75-containing form of dCAF-1 may function in ASF1-independent processes. Alternatively, the activity of dCAF-1 may be regulated during embryogenesis by processing the p105 polypeptide into p75 (Tyler, 2001).

This physical 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 coupling of DNA synthesis and chromatin assembly appears to require a specific interaction between CAF-1 and PCNA. The results presented in this work further extend this model to include the binding of ASF1 to CAF-1. It is possible, for instance, that a complex of RCAF and CAF-1 is recruited to sites of DNA synthesis via the interaction of CAF-1 with PCNA. In the future, it will be interesting to study how RCAF and CAF-1 mediate the formation of nucleosomes in conjunction with the other components of the chromatin assembly machinery (Tyler, 2001).

Histone chaperone ASF1 cooperates with the Brahma chromatin-remodeling machinery

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. Mutations in the Drosophila asf1 gene derepress silencing at heterochromatin and the ASF1 protein has a cell cycle-specific nuclear and cytoplasmic localization. Using both genetic and biochemical methods, it has been demonstrated 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 (Moshkin, 2002).

Assembly of newly synthesized DNA into chromatin requires both nucleosome assembly activities and ATP-dependent chromatin-remodelling. Nucleosome assembly is the process by which newly synthesized histones are loaded onto naked DNA. This function is performed primarily by histone chaperones like Chromatin Assembly Factor-1 (CAF-1) and Nucleosome Assembly Protein-1 (NAP-1). However, nucleosome assembly factors alone are unable to efficiently produce long and regularly spaced nucleosomal arrays. To perform this function properly requires the recruitment of ATP-dependent chromatin-remodelling factors (Moshkin, 2002).

The asf1 gene was originally identified in yeast by its ability, when overexpressed, to repress silencing at the HMR and HML mating-type loci and at telomeres. Interestingly, it has also been shown that loss-of-function mutations in the yeast asf1 gene derepress transcription from silenced loci, when combined with mutations in the largest subunit of the yeast CAF-1 complex. Because of this, the role of ASF1 in silencing is thought to be in the assembly of silenced chromatin (Moshkin, 2002).

Recently, ASF1 has been shown to participate in the process of nucleosome assembly during DNA replication. Both biochemical and genetic studies have shown that ASF1 acts as a histone chaperone (Tyler, 1999, 2001; Munakata, 2000), which in concert with another histone chaperone, CAF-1, is thought to deposit histones H3 and H4 tetramers onto naked DNA. The assembly of nucleosome particles is completed by the addition of two dimers of histones H2A and H2B, probably by the histone chaperone, NAP-1 (Moshkin, 2002).

Although most studies on ASF1 have focused on its role in nucleosome assembly, recent data have shown that the yeast ASF1 is required for the proper transcriptional repression and activation of the histone genes (Sutton, 2001). This role in transcription raises the possibility that ASF1 may play a role in chromatin remodelling, as well as nucleosome assembly. This study explores the function of ASF1 in chromatin dynamics; ASF1 is shown to directly associated with the Brahma chromatin-remodelling machinery in flies (Moshkin, 2002).

During an EMS saturation screen over the deficiency Df(3L)kto2, which removes the 76BD region of the third chromosome, two mutations were identified in Drosophila asf1 gene (asf11 and asf12). The asf11 mutation deletes two nucleotides in the open reading frame (ORF) at base pair 380 relative to the 'start' codon, creating a premature 'stop' codon and resulting in the truncation of approximately half of the ASF1 protein. The protein synthesized from asf11 mutant allele seems to be unstable. Although this protein still contains major epitopes recognized by polyclonal anti-ASF1 antibodies, it cannot be detected in crude protein extracts from heterozygous asf11 embryos. Hemizygous asf11 mutants are embryonic or larval lethal; loss of maternal ASF1 function completely blocks oogenesis as revealed by asf11 germ-line clones (Moshkin, 2002).

The asf12 removes 24 nucleotides from the ORF of asf1 at base pair 54 after the 'start' codon, resulting in an 8-amino-acid deletion in the protein. Because of the slight size difference between the mutant and wild-type proteins, it was not possible to determine whether the ASF12 protein is present in heterozygous embryos. Histone-binding experiments, however, indicate that the mutated ASF1 protein produced by asf12 allele shows markedly reduced binding to Drosophila histones H3 and H4 (Moshkin, 2002).

Because ASF1 is involved in the assembly of silenced chromatin in yeast (Tyler, 1999; Sharp, 2001), tests were performed to see whether ASF1 is able to affect the silenced chromatin state at pericentric heterochromatin. The In(1)wm4h and In(1)wm4 mutant lines, which carry an inversion on the X chromosome juxtaposing the white gene to centromeric heterochromatin, were used. This inversion leads to a classic position effect variegation (PEV) phenotype. The cell-autonomous inactivation of the white gene is thought to occur via the occasional spreading of the heterochromatic compaction of the DNA into the white gene. In flies heterozygous for the asf11 or asf12 mutations, it was observed that the white gene expression is strongly derepressed in comparison to flies carrying two wild-type asf1 alleles. The dominant suppression of PEV caused by mutations in the asf1 gene strongly suggests a function for ASF1 in the formation of silenced chromatin in Drosophila (Moshkin, 2002).

To gain more insight into ASF1 cellular function an antibody directed against the full-length ASF1 protein was raised and affinity purified. This antibody recognizes a single band of 26 kD in embryonic nuclear and crude extracts, which coincides with the predicted size of ASF1 and the size of bacterially expressed ASF1 protein (Moshkin, 2002).

ASF1 localization on polytene chromosomes was examined. ASF1 is strongly associated with multiple sites along the polytene chromosomes. Among them are many decondensed and transcriptionally active regions such as interbands and developmental puffs. Besides this, there is distinct staining of the chromocenter and the partially heterochromatic fourth chromosome, supporting the role of ASF1 in heterochromatin-mediated gene silencing. A particularly strong signal was observed at the 39DE region. The 39DE region is the location of the histone gene cluster. Interestingly, ASF1 is known to be involved in the control of the histone genes expression in yeast, and the staining of the 39DE region may point to a similar role in flies (Moshkin, 2002).

The intracellular localization of ASF1 protein was examined in the early Drosophila embryo. During the first hours of development, embryos undergo 13 cycles of nearly synchronous accelerated mitotic nuclear divisions, in which the G1 and G2 phases of the cell cycle are eliminated and cells only go through the S and M phases. Immunostaining with the anti-ASF1 antibody of these early embryos reveals that during S phase, ASF1 protein is primarily concentrated in the nucleus with only diffuse cytoplasmic staining. Because staining of the interphase cells of the salivary gland shows that nuclear ASF1 is associated with the chromosomes, it is likely that the early S phase embryonic staining is also chromosomal. Upon the commencement of mitosis, however, ASF1 nuclear staining fades and is not detected on the condensed chromatin (Moshkin, 2002).

To further explore ASF1 function in the regulation of chromatin dynamics and to identify potential interacting partners, the eyeless-GAL4, UAS-Asf1 strain was created, which over-expresses asf1 cDNA in the eye. This strain has a rough-eye phenotype, which allows an assay of genetic interactions between asf1 and genes known to be involved in the regulation of chromatin structure such as the Polycomb Group (PcG) and the Trithorax Group (TrxG) genes. Among the tested mutations [brm1, brm2, mor1, osa2, Df(3R)red-P6, kto1, taraL4, AsxXf23, ph410, Pc3, PclD5, Psc1, E(z)Su301], it was found that only mutations in the brahma (brm), moira (mor), and osa (osa) genes suppress the ASF1-mediated rough-eye phenotype. Interestingly, the proteins encoded by these genes are parts of the Brahma chromatin-remodelling complex (Moshkin, 2002).

To confirm the genetic interaction between ASF1 and the Brahma complex, a reciprocal analysis was performed. Transgenic flies overexpressing a dominant-negative form of brm (brmK804R) in the eye were used; this results in a rough-eye phenotype, similar to asf1 overexpression. In this assay, brm and mor mutations aggravate the effect of brmK804R over-expression, substantiating the dominant-negative nature of the brmK804R allele. Similarly, the asf11 mutation significantly enhances the rough-eye phenotype caused by overexpression of the dominant-negative brmK804R allele. These two complementary genetic assays strongly suggest that ASF1 functions in vivo in the Brahma chromatin-remodelling pathway (Moshkin, 2002).

Because the genetic data show that ASF1 acts in the Brahma chromatin-remodelling pathway, whether ASF1 directly interacts with the Brahma complex was tested. Although the ASF1 protein is not found tightly associated with a highly purified Brahma complex, the BRM and its associated MOR proteins are coimmunoprecipitated with anti-ASF1 antibodies from embryonic nuclear extracts suggesting that ASF1 does physically interact with the Brahma chromatin-remodelling complex. To test whether ASF1 can bind directly to the Brahma complex, GST pull-down experiments were performed using a bacterially expressed and purified ASF1-GST fusion protein and purified Brahma complex. Western blot analysis of pulled down material reveals that BRM, the ATPase subunit of the Brahma complex, is among the ASF1-interacting molecules, suggesting that ASF1 binds directly to the Brahma complex (Moshkin, 2002).

Therefore, Drosophila ASF1 plays a role in the formation of silenced chromatin similar to its yeast counterpart (Tyler, 1999). Although it is not yet clear how this is accomplished, the data re-emphasize the importance of chromatin assembly factors in the formation of silenced chromatin. Because regularly spaced nucleosomal arrays are a landmark of silenced heterochromatin, it is believed that ASF1 contributes to silencing through its nucleosome assembly activity (Tyler, 1999). Therefore, the reduction of silencing in asf11 mutants may result from the disruption of the nucleosome array at heterochromatin. This interpretation is supported by the chromocentric localization of the ASF1 protein on polytene chromosomes (Moshkin, 2002).

ASF1 protein has a cell cycle-specific chromosomal and cytoplasmic localization reminiscent of another histone chaperone protein, NAP-1. It has been speculated that the NAP-1 localization pattern could reflect a role for NAP-1 in binding newly synthesized histones in the cytoplasm and delivering them to the sites of chromatin assembly and/or remodelling. It is believed that ASF1 may play a similar role in histone shuttling to sites of chromatin assembly (Moshkin, 2002).

Furthermore, the data suggest a dualistic function for the histone chaperone ASF1 in both histone deposition during chromatin assembly and histone displacement during chromatin-remodelling. ASF1 interacts genetically and biochemically with the Brahma chromatin-remodelling complex. The Drosophila Brahma complex is a member of the SWI/SNF ATP-utilizing chromatin-remodelling factors conserved in yeast, flies, and mammals. Since the Brahma complex participates in both the initiation and the repression of transcription, it is believed that ASF1 may also function in transcriptional control. Although a direct role for ASF1 in transcription has not been firmly established, recent evidence supports this hypothesis: (1) mutation of the yeast asf1 gene results in the suppression of S-phase-specific histone genes activation (Sutton, 2001); (2) it was shown that ASF1 interacts with bromodomain-containing subunits of TFIID (Moshkin, 2002 and references therein).

The association of ASF1 with the chromatin-remodelling machinery raises several intriguing possibilities for ASF1 function in chromatin-remodelling. As a histone chaperone, ASF1 could facilitate chromatin-remodelling by attenuating the strong electrostatic histone-DNA contacts, in effect, lubricating the chromatin for remodelling factors. Recently, it has been shown that the disruption of a single histone-DNA contact by a mutation in the SIN domain of histone H4 results in an increased rate of remodelling by the yeast SWI/SNF complex. In a similar fashion, ASF1 may weaken the contacts of histones H3 and H4 with DNA, creating an altered nucleosome structure favorable for translocation by remodelling factors (Moshkin, 2002 and references therein).

However, ASF1 could function in targeting chromatin-remodelling factors to the sites of newly assembled chromatin. Since assembly of long and regularly spaced nucleosome arrays cannot be achieved by histone chaperones alone and some chromatin assembly complexes contain ATP-dependent nucleosome spacing activity, an interaction between ASF1 and chromatin-remodelling factors could indicate a mechanism by which functional chromatin is assembled after DNA replication (Moshkin, 2002).

Tousled-like kinase functions with the chromatin assembly pathway regulating nuclear divisions

Tousled-like kinases (TLKs) constitute a family of serine/threonine kinases conserved in plants and animals that act in a cell cycle-dependent manner. In mammals, their activity peaks during S phase, when they phosphorylate the antisilencing function protein 1 (ASF1), a histone chaperone involved in replication-dependent chromatin assembly. This study shows that Drosophila ASF1 is also a phosphorylation target of TLK, and that the two components cooperate to control chromatin replication in vivo. By altering TLK activity through loss-of-function mutations, it was shown that nuclear divisions are arrested at interphase, followed by apoptosis. Overexpression of TLK alters the chromatin structure, suggesting that TLK mediates the activity of chromatin proteins. These results suggest that TLK coordinates cell cycle progression through the regulation of chromatin dynamics (Carrera, 2003; full text of article).

Structural basis for the histone chaperone activity of Asf1

Anti-silencing function 1 is a highly conserved chaperone of histones H3/H4 that assembles or disassembles chromatin during transcription, replication, and repair. The structure of the globular domain of Asf1 bound to H3/H4 determined by X-ray crystallography to a resolution of 1.7 Å shows how Asf1 binds the H3/H4 heterodimer, enveloping the C terminus of histone H3 and physically blocking formation of the H3/H4 heterotetramer. Unexpectedly, the C terminus of histone H4 that forms a mini-β sheet with histone H2A in the nucleosome undergoes a major conformational change upon binding to Asf1 and adds a β strand to the Asf1 β sheet sandwich. Interactions with both H3 and H4 are required for Asf1 histone chaperone function in vivo and in vitro. The Asf1-H3/H4 structure suggests a 'strand-capture' mechanism whereby the H4 tail acts as a lever to facilitate chromatin disassembly/assembly that may be used ubiquitously by histone chaperones (English, 2006).

The ubiquitous function of Asf1 in eukaryotes is highlighted by the sequence conservation of the residues involved in the interactions between Asf1 and histones H3/H4. Budding yeast Asf1 is 56% identical to Xenopus Asf1 in the conserved core, and the Xenopus histones are 88% and 92% identical to H3 and H4 from yeast, respectively. Only the following three residues of Xenopus H3 that contact Asf1 differ in other species: C110 (Ala in yH3), Q125 (Lys in yH3), and I130 (Leu in yH3); these substitutions would appear to cause only minor and possibly compensated differences in interprotein packing. Furthermore, none of these interspecies differences occur in residues of H4 that contact Asf1. Therefore, the interactions observed in this structure will likely be applicable to Asf1-histone H3/H4 complexes from different species (English, 2006).

The Asf1 histone chaperone forms extensive contacts with both histones H3 and H4. The Asf1-H3/H4 structure reveals the details of the interface between Asf1 and a3 of H3 and has identified a new interaction between Asf1 and a2 of H3. The implications of the mutagenesis study, with regard to Asf1 and H3, are that disruption of this intricate interface has severe consequences in the context of the cellular activity. For example, mutations in the regions of Asf1 that bind to only H3 (R145E/S48R, Y112A/R145E, V94R, and S48R) or the region of H3 that binds to Asf1 (K115 and K122) weakened the interaction between Asf1 and H3 and disrupted Asf1 function in vivo and in vitro. As such, the interaction between histone H3 and Asf1 is clearly critical for its cellular functions (English, 2006).

The Asf1-H3/H4 structure shows extensive contacts between Asf1 and histone H4. This interface has two parts: (1) the globular core of Asf1 interacts with the C-terminal tail of H4 to form a strand-swapped dimer and (2) the C-terminal tail of Asf1 binds to the histone-fold region (a3) of histone H4. These interactions are also important because mutations in residues of Asf1 that contact H4 (T147, L6, V109, and V146) weaken histone binding and alter the functions of Asf1 in yeast. Similarly, mutation of histone H4 residues R92, H75, Y72, Y88, and F100 that contact Asf1 in the Asf1-H3/H4 structure reduces the chromatin assembly and/or disassembly functions of Asf1 in vivo. Clearly, interactions of Asf1 with both histones H3 and H4 are required for Asf1 function, and neither interaction is sufficient (English, 2006).

The mutations that affect the interaction between Asf1 and H3/H4 fall into two distinct functional classes; (1) those that reduce the function of Asf1 and (2) those that cause a gain-of-function phenotype. The former was expected, but the latter uncovered specific mutations that overcome the requirement for CAF-1 in transcriptional silencing. These include Asf1 S48R, V109M, Y112E, and V146L that weaken the interaction with histones H3/H4 in vivo and in vitro. Interestingly, the histone H4 H75Y mutation that had reduced Asf1-mediated chromatin-disassembly activity and Zeocin sensitivity has also been shown to bypass the requirement for CAF-1 in silencing. The same ability to bypass the requirement for CAF-1 in silencing has been demonstrated by truncations or insertion mutations in the C terminus of Asf1. Specifically, inactivation of CAF-1 leads to reduced histone deposition onto DNA, while additional mutations in the C terminus of Asf1 restores the histone deposition onto DNA. Although the C terminus of Asf1 is not present in the determined structure, it may extend toward histone H4 from its current location in the structure and may contribute further to histone binding affinity. It is possible that the Asf1 L6M, S48R, V109M, Y112E, V146L, and T147E mutations enhance transcriptional silencing by the same mechanism as the C-terminal mutations in Asf1 (English, 2006).

The histone chaperone ASF1 localizes to active DNA replication forks to mediate efficient DNA replication

The packaging of the eukaryotic genome into chromatin is likely to regulate all processes that occur on the DNA template. The assembly and disassembly of chromatin structures from histone proteins and DNA are mediated by histone chaperones, including the histone H3/H4 chaperone anti-silencing function 1 (ASF1). To address the function of ASF1 in metazoan cells, RNA interference-mediated knockdown of Drosophila melanogaster ASF1 (dASF1) was carried out. Cells lacking dASF1 accumulate in S phase of the cell cycle as determined by flow cytometry analysis of DNA content and quantitation of the proportion of cells with replication foci. In agreement, bromodeoxyuridine (BrdU) pulse-chase analysis demonstrates that the absence of ASF1 leads to delayed progression through S-phase. Furthermore, the absence of ASF1 leads to a reduced ability to incorporate the nucleoside analog BrdU, indicating that ASF1 is required for efficient DNA replication. dASF1 was found to colocalize with DNA replication foci throughout S phase by immunofluorescence analysis, and these dASF1 foci are disrupted upon inhibition of DNA replication by treatment of cells with hydroxyurea. As such, these results demonstrate that dASF1 is present at active, but not stalled, replication forks. It is proposed that dASF1 has a direct role in modifying chromatin structure during DNA replication and that this function of dASF1 is important for the processivity of the replication machinery (Schulz, 2006).

The replication defect found apon dASF1 depletion is most consistent with a problem with replication elongation, since PCNA is known to be present during replication elongation and cells lacking dASF1 accumulate with PCNA foci. Furthermore, twice as many cells were observed with replication foci when dASF1 was absent as compared with the control cells, yet only slightly more cells lacking dASF1 were BrdU positive as compared with control cells. These data indicate that even though more cells have the replication machinery loaded onto the DNA when dASF1 is absent, there are not more cells undergoing active replication. Finally, the anomalous BrdU arc in cells lacking dASF1 is consistent with a defect in replication elongation, not initiation. Consistent with a role for dASF1 during replication elongation is the finding that yeast with ASF1 deleted has genomic instability during S phase, which may be a consequence of the stalling of replication forks resulting in DNA double-strand breaks and genomic instability (Schulz, 2006).

Why would the absence of dASF1 lead to defects in replication elongation? Having shown that dASF1 localizes to replication foci, one possibility is that dASF1 assembles the newly-replicated DNA into chromatin in vivo as it does in vitro (Tyler, 1999). There is precedent for chromatin assembly factors influencing DNA replication, since RNAi-mediated knockdown of CAF-1 results in S phase arrest and defects in BrdU incorporation. Alternatively, ASF1 has recently been shown to be a global chromatin disassembly factor in yeast (Adkins, 2004), such that dASF1 may disassemble chromatin before DNA replication (Schulz, 2006).

Importantly, this study found that dASF1 colocalizes with active replication forks throughout S phase. This suggests that ASF1 has a general function during DNA replication that is not specific, for example, to replicating heterochromatin late in S phase. Furthermore, this result suggests that at least some of the functions of ASF1 are coupled to ongoing DNA replication, as would be expected for a protein involved in chromatin assembly and/or disassembly. It will be important to determine the replication fork components that mediate the localization of ASF1 to the replication foci. One obvious candidate is CAF-1, which localizes to replication foci via its interaction with PCNA, and is known to directly bind to ASF1. Another possibility is that RFC may recruit Asf1 to replication forks in vivo; it has been recently shown that RFC binds to ASF1 in vitro and can recruit Asf1 to DNA in vitro (Franco, 2005). It is also possible that Asf1 simply localizes to regions of active chromatin assembly in a nonspecific fashion (Schulz, 2006).

Treatment of cells with hydroxyurea disrupts dASF1 foci, suggesting that dASF1 is lost from stalled replication forks. How the loss of chromatin-bound dASF1 from stalled replication forks is regulated remains to be determined. It is possible that ASF1 localization is regulated by the S phase checkpoint, as human and Drosophila ASF1 are phosphorylated by tousled-like kinases (tlks) during S phase of the cell cycle (Carrera, 2003; Sillje, 2001), and this phosphorylation is inhibited by activation of the S phase checkpoint (Groth, 2003). However, both phosphorylated and nonphosphorylated ASF1 functionally interact with CAF-1 (Mello, 2002), suggesting that checkpoint activation would not cause ASF1 to be lost from replication forks if its localization was via its interaction with CAF- 1/PCNA. Recent data show that during treatment with hydroxyurea human ASF1 joins a multichaperone complex in the cytosol and that this process is not checkpoint mediated (Groth, 2005). It is possible that the same unknown regulatory mechanism that governs the localization of ASF1 to the cytosol during replication stress also controls localization of ASF1 to the replication fork (Schulz, 2006).

In summary, this study found that Drosophila ASF1 localizes to active replication forks and that the loss of dASF1 results in defective replication and the accumulation of cells in S phase of the cell cycle. These results suggest that dASF1 may have a direct and important role in the modulation of chromatin structure during DNA replication in vivo (Schulz, 2006).

Human Asf1 regulates replication fork progression

DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. This study characterized a complex in which the human histone chaperone Asf1 and MCM2-7, the putative replicative helicase, are connected through a histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and similar defects arise from overproduction of new histone H3-H4 that compromises Asf1 function. These data link Asf1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. It is proposed that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork progression and histone supply and demand (Groth, 2007).

When one parental nucleosome is disrupted ahead of the moving replication fork, two new nucleosomes, using new and recycled histones, must assemble on the daughter strands to reproduce nucleosomal density. The regulatory link between histone biosynthesis and DNA replication ensures the supply of new histones at the global level. However, an additional layer of regulation must be at play locally at individual replication forks to ensure balanced deposition of new and parental histones on the daughter strands. This may involve histone chaperones, such as Asf1 (antisilencing function 1), that can participate in both nucleosome assembly and disassembly. Human Asf1a and Asf1b exist in two pools, a highly mobile (cytosolic) pool that buffers excess soluble histones during replication stress and a salt-extractable pool in nuclear extracts. How the latter relates to other chromatin proteins and contributes to nuclear function remains open (Groth, 2007).

In vivo complexes containing Asf1a or Asf1b were isolated and characterized using stable HeLa S3 cell lines expressing tagged Asf1 (e-Asf1). Mass spectrometry and Western blotting revealed the presence of Mcm2, 4, 6, and 7 in the nuclear e-Asf1 (a and b) complexes, together with histone H3 and H4. By comparison, only Mcm2 was associated with cytosolic e-Asf1 (a and b) complexes. Antibodies against Mcm6 coimmunoprecipitated Asf1 and histone H3-H4 from nuclear extracts, whereas Mcm2, 4, and 7 were retrieved from both cytosolic and nuclear fractions. Given that this set of MCM proteins copurifies on histone H3-H4 columns, tests were performed to see whether Asf1 associates with Mcm2, 4, 6, and 7 through histone H3-H4 by isolating complexes containing e-Asf1a mutated in the histone-binding domain, by replacement of valine at codon 94 with arginine (V94R). e-Asf1a V94R did not bind histones H3-H4, as expected, and concomitantly MCMs were lost from the complex, which implicated histone H3-H4 in bridging the interaction between Asf1 and MCMs. To further confirm the chromatin link and to avoid the use of salt-extraction, which disrupts MCM2-7 hexamers into subcomplexes, deoxyribonuclease (DNase I)-solubilized chromatin was used. Again, Mcm2, 4, 6, and 7 coimmunoprecipitated with Asf1, and Mcm6 antibodies retrieved Asf1. Under these conditions, which preserve the hexameric MCM2-7 complex, Mcm3 and Mcm5 coimmunoprecipitated with Asf1, which was also confirmed by epitope tag purification of e-Asf1 complexes from chromatin. This interaction on chromatin occurred in S phase, which suggested a role in DNA replication (Groth, 2007).

S-phase defects have been reported in various systems upon interference with Asf1 function. Human cells depleted of Asf1 (a and b) accumulate in S phase with reduced 5-bromo-2'-deoxyuridine (BrdU) incorporation. However, the appearance and distribution of replication factories marked by proliferating cell nuclear antigen (PCNA) and the pattern of chromatin-bound Mcm2 were unchanged, which was consistent with findings in Drosophila (Schulz, 2006). Given the link with MCM2-7, considered to be the replicative helicase, it was wondered whether inefficient replication could reflect problems of unwinding DNA in the context of chromatin. If so, the level of single-stranded DNA (ssDNA) at replication sites might be reduced. To monitor ssDNA at replication sites, two markers, replication protein A (RPA), which binds ssDNA, and PCNA, a polymerase accessory factor, were used. In control cells, both RPA and PCNA showed characteristic replication patterns. Although PCNA patterns were unchanged in Asf1-depleted cells, RPA replication patterns were barely detectable. Some nonextractable RPA localized to bright nuclear foci, which were identified as promyelocytic leukemia (PML) nuclear bodies, clearly distinct from PCNA replication foci. It was verified that Asf1 depletion did not affect RPA expression or its ability to bind ssDNA. Thus, absence of RPA replication profiles is consistent with the hypothesis of a helicase defect (Groth, 2007).

To examine helicase function, DNA unwinding in the absence of polymerase progression was analyzed by treating cells with hydroxyurea (HU) to deplete the nucleotide pool, which inhibits the DNA polymerase and leads to formation of ssDNA. In Xenopus, this response is dependent on MCM2-7 function. Formation of ssDNA was measured ahead of the polymerase by detection of BrdU-substituted DNA and acute accumulation of RPA at replication sites. In control cells treated with HU, 75% of cells in S phase showed formation of ssDNA and recruitment of RPA to these ssDNA patches at replication sites. This response was dramatically reduced when Asf1 (a and b) (Asf1 knockdown) was depleted, indicating that impaired replication reflects a DNA unwinding defect and implying that DNA in chromatin cannot be properly unwound by the replicative helicase. This could reflect a direct effect of Asf1 on DNA unwinding and fork progression or indirect effects, involving DNA damage at the replication fork, replisome collapse, and/or checkpoint signaling. However, no evidence was found of DNA damage or checkpoint activation upon Asf1 knockdown, and consistently, checkpoint abrogation by caffeine did not rescue the unwinding defect. Instead, induction of gamma-H2AX (phosphorylation of a histone 2A variant) in response to HU treatment was impaired in Asf1-depleted cells, which was consistent with a role of ssDNA in checkpoint signaling. Furthermore, expression and chromatin association of several key replication factors remained unchanged upon Asf1 knockdown (Groth, 2007).

To explore whether a direct role of Asf1 in facilitating DNA unwinding could involve interaction with histones and MCM2-7, the Asf1-(H3-H4)-MCM complex was followed when helicase progression was uncoupled from the polymerase. Nuclear Asf1 bound significantly more Mcm2, 4, 6, and 7 and histone H3-H4 in HU-treated cells, and within this complex, phosphorylated forms of Mcm2 were prominent (Ser108 and Ser139, putative targets of ATR and Cdc7-Dbf4), which underlined a connection to replication control. During HU treatment, continued unwinding of nucleosomal DNA ahead of the fork without DNA synthesis creates a situation where displaced parental histones cannot immediately be recycled. The accumulation of Asf1-(H3-H4)-MCM complexes under such conditions suggests that this complex could be an intermediate in parental histone transfer. Within these complexes, histone modifications could be detected, H4 with acetylated lysine 16 (H4K16Ac) and H3 with trimethylated lysine 9 (H3K9me3). This further substantiates the hypothesis, since these chromatin marks are poorly represented on newly synthesized histones (Groth, 2007).

These results suggest that Asf1 coordinates histone supply (parental and new) with replication fork progression. To manipulate new histone supply, a conditional cell line was generated for coexpression of tagged histone H3.1 and H4. About 50% of the cells expressed H3.1-H4 when tetracycline was removed, and Asf1 bound the exogenous histones. After induction, the nonnucleosomal histone pool increased two- to threefold, a range that is comparable to histone overload during a replication block. Increasing new histone supply interfered with DNA replication and caused acute accumulation of H3.1-H4 overexpressing cells in S phase (tracked by the green fluorescent protein tag on H4). At later time points, the majority of GFP-positive cells arrested in late S/G2. Focus was placed on the S-phase defect, to address whether H3-H4 excess mimicked Asf1 depletion. The moderate increase in H3-H4 expression did not cause DNA damage monitored by gamma-H2AX. Thus analyzed RPA and PCNA profiles were analyzed using GFP-negative cells (no H3.1-H4 induction) as an internal control for proper localization. Again, as in Asf1-depleted cells, RPA replication patterns in histone-overexpressing cells were barely visible, with some RPA localized to bright nuclear foci mainly corresponding to PML bodies. Furthermore, as for Asf1 knockdown, an excess of new H3-H4 histones impaired ssDNA formation and RPA accumulation at replication sites, as well as checkpoint activation in response to HU. Together, these data indicate that overproduction of histone H3-H4 impairs replication by impeding DNA unwinding. Consistent with the possibility that this results from interference with Asf1 function, it was found that ectopic expression of Asf1a partially alleviated the inhibitory effect of histone excess on S-phase progression. Moreover, Asf1 depletion aggravated the S-phase defect resulting from histone H3-H4 excess, in that progression into G2 was delayed even further (Groth, 2007).

Together, these results show that replication fork progression is dependent on the histone H3-H4 chaperone, Asf1, and on an equilibrium between histone supply and demand. This dependency could ensure that replication only proceeds when nucleosomes are being formed behind the fork with a proper balance between new and parental histones H3-H4. In the most parsimonious view, a model is proposed in which Asf1 uses its properties as a histone acceptor and donor to facilitate unwinding of the parental chromatin template in coordination with nucleosome assembly on daughter strands. Nucleosome disruption during replication fork passage would involve the histone-binding capacity of the MCM2-7 complex and transfer of parental histones to Asf1 through the Asf1-(H3-H4)-MCM intermediate, followed by their deposition onto daughter strands. In parallel, Asf1 would provide the additional complement of histones through its established role as a new histone donor. Asf1 knockdown will impair histone transfer and disruption of parental nucleosomes that thus present an impediment to unwinding and replication fork progression. Similarly, because of the dual function of Asf1, an excess of new histones will not leave Asf1 available for parental transfer, which impairs unwinding. On the basis of structural data, this model implies that parental histones (H3-H4)2, like new histones, go through a transient dimeric state during transfer. Furthermore, the MCM-(H3-H4)-Asf1 connection opens new angles to understand MCM2-7 function in chromatin. In conclusion, having Asf1 deal with both new and parental histones could provide an ideal means to fine-tune de novo deposition and recycling with replication fork progression. By offering a mechanism to coordinate new and parental histones during replication, this model should pave the way to addressing key questions regarding chromatin-based inheritance, including transmission of histone modifications (Groth, 2007).

Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork

MCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nucleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. This study solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 Å resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure, but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 heteromeric complex. Thermodynamic analysis of the quaternary complex together with structural modeling support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway (Richet, 2015).

UNC-85, a C. elegans homolog of the histone chaperone Asf1, functions in post-embryonic neuroblast replication

Normal animal development requires accurate cell divisions, not only in the early stages of rapid embryonic cleavages, but also in later developmental stages. The C. elegans unc-85 gene is implicated in cell divisions that occur only post-embryonically, primarily in terminal neuronal lineages. Variable post-embryonic cell division failures in ventral cord motoneuron precursors result in uncoordinated locomotion of unc-85 mutant larvae by the second larval stage. These neuroblast cell division failures often result in unequally sized daughter nuclei, and sometimes in nuclear fusions. Using a combination of conventional mapping techniques and microarray analysis, the unc-85 gene was cloned; it encodes one of two C. elegans homologs of the yeast Anti-silencing function 1 (Asf1) histone chaperone. The unc-85 gene is expressed in replicating cells throughout development, and the protein is localized in nuclei. Examination of null mutants confirms that embryonic neuroblast cell divisions occur normally, but post-embryonic neuroblast cell divisions fail. Analysis of the DNA content of the mutant neurons indicates that defective replication in post-embryonic neuroblasts gives rise to ventral cord neurons with an average DNA content of approximately 2.5 n. It is concluded that UNC-85 functions in post-embryonic DNA replication in ventral cord motor neuron precursors (Grigsby, 2008).

Acetylated lysine 56 on Histone H3 drives chromatin assembly after repair and signals for the completion of repair

DNA damage causes checkpoint activation leading to cell cycle arrest and repair, during which the chromatin structure is disrupted. The mechanisms whereby chromatin structure and cell cycle progression are restored after DNA repair are largely unknown. Chromatin reassembly following double-strand break (DSB) repair requires the histone chaperone Asf1 and that absence of Asf1 causes cell death, as cells are unable to recover from the DNA damage checkpoint. Asf1 contributes toward chromatin assembly after DSB repair by promoting acetylation of free histone H3 on lysine 56 (K56) via the histone acetyl transferase Rtt109. Mimicking acetylation of K56 bypasses the requirement for Asf1 for chromatin reassembly and checkpoint recovery, whereas mutations that prevent K56 acetylation block chromatin reassembly after repair. These results indicate that restoration of the chromatin following DSB repair is driven by acetylated H3 K56 and that this is a signal for the completion of repair (Chen, 2008).

Chromatin is taken apart and reassembled during DNA replication and transcription by chromatin assembly factors, including histone chaperones, and this is also likely to the be the case during double-strand DNA repair. The histone chaperone Anti-silencing Function 1 (Asf1) was identified biochemically by its ability to deposit histones H3 and H4 onto newly replicated DNA in vitro. Yeast deleted for ASF1 are highly sensitive to DNA damaging agents, which is likely to reflect a direct role for Asf1 in modulating chromatin structure during repair. Indeed, human Asf1 is required for the assembly of nucleosomes following nucleotide excision repair in vitro. Furthermore, yeast asf1 mutants have elevated rates of genomic instability. Furthermore, there exists a dynamic interaction between Asf1 and the Rad53 DNA damage checkpoint kinase, which suggests that activation of Asf1 may be an important cellular response to DNA damage. In addition to its role in chromatin assembly and disassembly, Asf1 is also essential for stimulating the acetylation of free histone H3 on lysine 56 (K56) by the histone acetyl transferase (HAT) Rtt109 (Recht, 2006; Tsubota, 2007). Despite its occurrence in eukaryotes from yeast to humans, the molecular function of acetylation of H3 K56 remains unknown (Chen, 2008).

Although chromatin disassembly has been previously documented at a site of double-strand DNA damage, chromatin reassembly following double-strand DNA repair has not been reported. This work set out to discover why the Asf1 histone chaperone is required for rapid growth after DSB repair. In addition to finding a role for Asf1 in chromatin reassembly following DSB repair, a role was discovered for Asf1 in recovery and adaptation to the DNA damage checkpoint following repair, explaining why asf1 mutant yeast die after DNA repair. These roles for Asf1 can be bypassed by a mimic of permanent acetylation of histone H3 on lysine 56, whereas deletion of the gene encoding the K56 histone acetyl transferase, RTT109, also leads to persistent DNA damage checkpoint activation following DNA repair. As such, acetylated K56 on H3 is required to reinstate the chromatin structure over the repaired DNA, which, in turn, is a critical signal for turning off the DNA damage checkpoint, allowing cell cycle re-entry following DNA repair (Chen, 2008).

Acetylation of Histone H3 lysine 56 regulates replication-coupled nucleosome assembly

Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. This study shows that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106 (Li, 2008).

In the yeast S. cerevisiae, three histone chaperones, CAF-1, Asf1, and Rtt106, have been implicated in the assembly of H3-H4 into nucleosomes. However, how the roles of these histone chaperones are coordinated to promote nucleosome assembly is largely unknown. The results suggest that these histone chaperones function in a hierarchical manner to promote nucleosome assembly. First, Asf1 binds to newly synthesized H3-H4 dimers and presents those dimers for acetylation of H3K56 by the Rtt109-Vps75 complex. H3K56Ac-H4 complexes are then transferred to Rtt106 and CAF-1 for deposition onto DNA and subsequent nucleosome formation (Li, 2008).

While the mechanism of parental histones segregation is still debated and it is not known whether this process requires CAF-1, it is clear that deposition of newly synthesized histones requires CAF-1 and Asf1 in yeast cells. In the crystal structures of Asf1-H3-H4 complexes, Asf1 binds to a surface of H3 that is critical for formation of (H3-H4)2 tetramers. In vitro, Asf1 disrupts (H3-H4)2 tetramers and forms Asf1-H3-H4 heterotrimeric complexes. Thus, it may not be energetically and/or kinetically possible for Asf1 alone to deposit histones onto replicating DNA for formation of (H3-H4)2 tetramers, the first building blocks needed for rapid de novo nucleosome assembly during S phase of the cell cycle. H3 lysine 56 is far away from the surface involved in the formation of (H3-H4)2 tetramers. Thus, the transfer of H3K56Ac-H4 dimers from Asf1 to CAF-1 and Rtt106, which bind preferentially to H3K56Ac, may ensure rapid formation of (H3-H4)2 tetramers and subsequent formation of nucleosomes (Li, 2008).

Binding of the histone chaperone ASF1 to the CBP bromodomain promotes histone acetylation

The multifunctional Creb-binding protein (CBP) protein plays a pivotal role in many critical cellular processes. This study demonstrate that the bromodomain of CBP binds to histone H3 acetylated on lysine 56 (K56Ac) with higher affinity than to its other monoacetylated binding partners. Autoacetylation of CBP is critical for the bromodomain-H3 K56Ac interaction, and it is proposed that this interaction occurs via autoacetylation-induced conformation changes in CBP. Unexpectedly, the bromodomain promotes acetylation of H3 K56 on free histones. The CBP bromodomain also interacts with the histone chaperone anti-silencing function 1 (ASF1) via a nearby but distinct interface. This interaction is necessary for ASF1 to promote acetylation of H3 K56 by CBP, indicating that the ASF1-bromodomain interaction physically delivers the histones to the histone acetyl transferase domain of CBP. A CBP bromodomain mutation manifested in Rubinstein-Taybi syndrome has compromised binding to both H3 K56Ac and ASF1, suggesting that these interactions are important for the normal function of CBP (Das, 2014).


Search PubMed for articles about Drosophila Asf1

Adkins, M. W., Howar, S. R. and Tyler, J. K. (2004). Chromatin disassembly mediated by the histone chaperone Asf1 is essential for transcriptional activation of the yeast PHO5 and PHO8 genes. Mol. Cell 14: 657-666. PubMed citation: 15175160

Adkins, M. W., et al. (2007). Chromatin disassembly from the PHO5 promoter is essential for the recruitment of the general transcription machinery and coactivators. Mol. Cell. Biol. 27: 6372-6382. PubMed citation: 17620413

Carrera, P., Moshkin, Y. M., Gronke, S., Sillje, H. H., Nigg, E. A., Jackle, H., and Karch, F. (2003). Tousled-like kinase functions with the chromatin assembly pathway regulating nuclear divisions. Genes Dev. 17: 2578-2590. PubMed citation: 14561777

Chen, C.-C., et al. (2008). Acetylated lysine 56 on Histone H3 drives chromatin assembly after repair and signals for the completion of repair. Cell 134: 231-243. PubMed ID: 18662539

Das, C., Roy, S., Namjoshi, S., Malarkey, C. S., Jones, D. N., Kutateladze, T. G., Churchill, M. E. and Tyler, J. K. (2014). Binding of the histone chaperone ASF1 to the CBP bromodomain promotes histone acetylation. Proc Natl Acad Sci U S A 111: E1072-1081. PubMed ID: 24616510

De Koning, L., Corpet, A. Haber, J. E. and Almouzni, G. (2007). Histone chaperones: an escort network regulating histone traffic. Nat. Struct. Mol. Biol. 14: 997-1007. PubMed ID: 17984962

English, C. M., Adkins, M. W., Carson, J. J., Churchill, M. E. and Tyler, J. K. (2006). Structural basis for the histone chaperone activity of Asf1. Cell 127(3): 495-508. PubMed citation: 17081973

Franco, A. A., Lam, W. M., Burgers, P. M. and Kaufman, P. D. (2005). Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C. Genes Dev. 19: 1365-1375. PubMed citation: 15901673

Fromental-Ramain, C., Ramain, P. and Hamiche, A. (2017). The Drosophila DAXX like protein (DLP) cooperates with ASF1 for H3.3 deposition and heterochromatin formation. Mol Cell Biol [Epub ahead of print]. PubMed ID: 28320872

Goodfellow, H., et al. (2007). Gene-specific targeting of the histone chaperone Asf1 to mediate silencing. Dev. Cell 13: 593-600. PubMed citation: 17925233

Groth, A., Lukas, J., Nigg, E. A., Sillje, H. H., Wernstedt, C., Bartek, J., and Hansen, K. (2003). Human Tousled like kinases are targeted by an ATM- and Chk1-dependent DNA damage checkpoint. EMBO J. 22: 1676-1687. PubMed citation: 12660173

Groth, A., Ray-Gallet, D., Quivy, J. P., Lukas, J., Bartek, J., and Almouzni, G. (2005). Human Asf1 Regulates the Flow of S Phase Histones during Replicational Stress. Mol. Cell 17: 301-311. PubMed citation: 15664198

Groth, A., et al. (2007). Regulation of replication fork progression through histone supply and demand. Science 318: 1928-1931. PubMed citation: 18096807

Goodfellow, H., Krejcí, A., Moshkin, Y., Verrijzer, C. P., Karch, F. and Bray, S. J. (2007). Gene-specific targeting of the histone chaperone asf1 to mediate silencing. Dev. Cell 13(4): 593-600. PubMed citation: 17925233

Grigsby, I. F. and Finger, F. P. (2008). UNC-85, a C. elegans homolog of the histone chaperone Asf1, functions in post-embryonic neuroblast replication. Dev. Biol. 319(1): 100-9. PubMed ID: 18490010

Krejci, A. and Bray, S. J. (2007). Notch activation stimulates transient and selective binding of Su(H)/CSL to target enhancers. Genes Dev. 21: 1322-1327. PubMed citation: 17545467

Li, Z., et al. (2008). Acetylation of Histone H3 lysine 56 regulates replication-coupled nucleosome assembly. Cell 134: 244-255. PubMed ID: 18662540

Mello, J. A., Sillje, H. H., Roche, D. M., Kirschner, D. B., Nigg, E. A. and Almouzni, G. (2002). Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway. EMBO Rep. 3: 329-334. PubMed citation: 11897662

Moshkin, Y. M., et al. (2002). Histone chaperone ASF1 cooperates with the Brahma chromatin-remodelling machinery. Genes Dev. 16: 2621-2626. 12381660

Moshkin, Y, M., et al. (2009). Histone chaperones ASF1 and NAP1 differentially modulate removal of active histone marks by LID-RPD3 complexes during NOTCH silencing. Mol. Cell 35(6): 782-93. PubMed ID: 19782028

Mousson, F., Ochsenbein, F. and Mann, C. (2007). The histone chaperone Asf1 at the crossroads of chromatin and DNA checkpoint pathways. Chromosoma 116: 79-93. PubMed citation: 17180700

Munakata, T., Adachi, N., Yokoyama, N., Kuzuhara, T. and Horikoshi, M. (2000). A human homologue of yeast anti-silencing factor has histone chaperone activity. Genes Cells 5(3): 221-33. PubMed ID: 10759893

Recht, J., et al. (2006). Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis. Proc. Natl. Acad. Sci. 103: 6988-6993. PubMed ID: 16627621

Richet, N., Liu, D., Legrand, P., Velours, C., Corpet, A., Gaubert, A., Bakail, M., Moal-Raisin, G., Guerois, R., Compper, C., Besle, A., Guichard, B., Almouzni, G. and Ochsenbein, F. (2015). Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork. Nucleic Acids Res 43: 1905-1917. PubMed ID: 25618846

Schermer, U. J., Korber, P. and Horz, W. (2005). Histones are incorporated in trans during reassembly of the yeast PHO5 promoter. Mol. Cell 19: 279-285. PubMed citation: 16039596

Schneiderman, J. I., Orsi, G. A., Hughes, K. T., Loppin, B. and Ahmad, K. (2012). Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant. Proc Natl Acad Sci U S A 109(48): 19721-19726. PubMed ID: 23150573

Schulz, L. L. and Tyler, J. K. (2006). The histone chaperone ASF1 localizes to active DNA replication forks to mediate efficient DNA replication. FASEB J. 20(3): 488-90. PubMed citation: 16396992

Schwabish, M. A. and Struhl, K. (2006). Asf1 mediates histone eviction and deposition during elongation by RNA polymerase II. Mol. Cell 22: 415-422. PubMed citation: 16678113

Sharp, J. A., et al. (2001). Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing, Curr. Biol. 11: 463-473. PubMed citation: 11412995

Sillje, H. H., and Nigg, E. A. (2001). Identification of human Asf1 chromatin assembly factors as substrates of Tousled-like kinases. Curr. Biol. 11: 1068-1073. PubMed citation: 11470414

Sutton, A., Bucaria, J., Osley, M. A., and Sternglanz, R. (2001). Yeast ASF1 protein is required for cell cycle regulation of histone gene transcription. Genetics 158: 587-596. 11404324

Tsubota, T., et al. (2007). Histone H3-K56 acetylation is catalyzed by histone chaperone-dependent complexes. Mol. Cell 25: 703-712. PubMed ID: 17320445

Tyler, J. K., Adams, C. R., Chen, S. R., Kobayashi, R., Kamakaka, R. T., and Kadonaga, J. T. (1999). The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402: 555-560. PubMed citation: 10591219

Tyler, J. K., et al. (2001). Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors. Mol. Cell. Bio. 21: 6574-6584. 11533245

Zabaronick, S. R. and Tyler, J. K. (2005). The histone chaperone anti-silencing function 1 is a global regulator of transcription independent of passage through S phase. Mol. Cell. Biol. 25: 652-660. PubMed citation: 15632066

Biological Overview

date revised: 10 August 2017

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