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Potential role of the histone chaperone, CAF-1, in transcription

The eukaryotic genome forms a chromatin structure that contains repeating nucleosome structures. Nucleosome packaging is regulated by chromatin remodeling factors such as histone chaperones. The Saccharomyces cerevisiae H3/H4 histone chaperones, CAF-1 and Asf1, regulate DNA replication and chromatin assembly. CAF-1 function is largely restricted to non-transcriptional processes in heterochromatin, whereas Asf1 regulates transcription together with another H3/H4 chaperone, HIR. This study examined the role of the yeast H3/H4 histone chaperones, Asf1, HIR, and CAF-1 in chromatin dynamics during transcription. Unexpectedly, CAF-1 was recruited to the actively transcribed region in a similar way to HIR and Asf1. In addition, the three histone chaperones genetically interacted with Set2-dependent H3 K36 methylation. Similar to histone chaperones, Set2 was required for tolerance to excess histone H3 but not to excess H2A, suggesting that CAF-1, Asf1, HIR, and Set2 function in a related pathway and target chromatin during transcription. [BMB reports 2009; 42(4): 227-231]


INTRODUCTION
The eukaryotic genome is packed into chromatin by formation of a nucleosome, a repeating unit containing two copies each of histones H2A, H2B, H3, and H4 (1). Hence, functions such as transcription, replication, repair, and recombination are strongly influenced by the packaging state of DNA into chromatin.
The key players in these processes are the histone chaperones that mediate histone assembly and disassembly (2)(3)(4).
The HIR genes, HIR1, HIR2, HIR3, and HPC2, genetically interact with the transcription-coupled H2A/H2B chaperone, FACT (a complex of Spt16, Spt6, and Pob3) (10). ASF1 has a similar genetic interaction profile with the HIR genes and is directly involved in PHO gene activation (11,12). ASF1 is also important for repression of internal aberrant transcription and for the release and deposition of histones during transcriptional elongation (13)(14)(15), suggesting that HIR and Asf1 are important chromatin regulators during transcription. In contrast, the role of CAF-1 during transcription is unclear, as no CAF-1 subunits have significant genetic interactions with these elongation factors (10). Although Asf1 and CAF-1 work together in heterochromatin function, their role in euchromatin has not been elucidated. This study examined the role of the yeast H3/H4 histone chaperones, Asf1, HIR, and CAF-1 in chromatin dynamics during transcription. Unexpectedly, CAF-1 was recruited to the actively transcribed region, as was HIR and Asf1. In addition, three histone chaperones genetically interacted with Set2-dependent H3 K36 methylation, implying that Set2, Asf1, HIR, and CAF-1 function in a related pathway. Similar to histone chaperones, Set2 was required for tolerance to excess histone H3 but not to excess H2A, suggesting that they all play a role in transcriptional regulation via chromatin.

RESULTS AND DISCUSSION
The histone chaperone, CAF-1, is recruited to the transcriptional region H3/H4 chaperones, Asf1, and the HIR complex are recruited to the gene during transcription. Here, we analyzed the replication-dependent H3/H4 chaperone, CAF-1, as well as Asf1 and HIR. To monitor protein occupancy, we performed a chromatin immunoprecipitation (ChIP) assay with S. cerevisiae expressing each subunit of histone chaperone complex, Cac1-Flag, Asf1-TAP, Hir1-Flag and Hir2-Flag, from their genomic loci. We examined the PMA1 gene, which is constitutively expressed and widely used for the detection of transcription factors (Fig. 1A). Interestingly, Cac1 as well as Asf1, Hir1, and Hir2, cross-linked at high levels to the promoter, coding, and 3'UTR of PMA1 (Fig. 1B, upper panel), indicating that CAF-1, Asf1, and HIR were associated with the transcribed region. HIR is a complex consisting of Hir1, Hir2, Hir3, and Hpc2, while CAF-1 is composed of Cac1, Cac2, and Cac3. The cross-linking of other subunits of each complex was also examined to confirm transcription-dependent recruitment. http://bmbreports.org  They all similarly cross-linked to PMA1 ( Supplementary Fig. 1). Their recruitment profile was further analyzed by normalization with pol II (Fig. 1B, bottom panel), and resembled the association pattern of the elongation factors, Spt2, Spt4, Spt5, Spt6, Spt16, and Elf1, all involved in chromatin dynamics, whose association persists over the 3'UTR (14,(16)(17)(18).
The HIR complex associates with a discrete region of the histone genes during the cell cycle, and CAF-1 is involved in replication-coupled chromatin assembly Asf1 is physically and functionally linked to both activities, but recruitment of the CAF-1 complex to the transcribed gene is unexpected, despite a similar role for CAF-1 and Asf1 in protein-protein interactions in heterochromatin. CAF-1, Asf1, and HIR all cross-linked specifically to the entire region of the actively transcribed gene, indicating that they function as general transcription elongation factors as well as transcriptional corepressor (HIR/Asf1) and replication-coupled chromatin assembly factor (CAF-1/Asf1). In fact, none of them are essential and are largely redundant in yeast chromatin function.

Recruitment of histone chaperones does not depend on Rtf1, Ctk1, or Set2
Histone chaperones must be recruited to the chromatin upon transcription. Asf1 recruitment was unaffected by hir1Δ or cac1Δ, indicating that protein-protein interactions between Asf1 and HIR or CAF-1 are not needed for Asf1 recruitment (Fig. 2), despite their known interactions (9,19). Similarly, the recruitment of Hir2 and Cac1 was unaffected by asf1Δ, which suggests that their recruitment is not dependent on each other (data not shown). To determine whether elongation factors target histone chaperones to the chromatin, we constructed a set of mutants lacking rtf1Δ (a component of the PAF1 elongation complex), ctk1Δ (a kinase of the CTD of elongating polymerase), or set2Δ (a CTD-associated histone H3 methyltransferase). The mutants, rtf1Δ, ctk1Δ, or set2Δ, did not change Hir1, Hir2, http://bmbreports.org BMB reports or Cac1 recruitment ( Supplementary Fig. 2). These data suggest that the transcription elongation factors, Rtf1, Ctk1, and Set2, are not required for HIR and CAF-1 recruitment to PMA1.
In yeast, the Swi/Snf complex alters nucleosome positioning at transcription sites (20). It travels with elongating Pol II and mediates activator-dependent removal of H3 (21 and references therein). In the absence of Snf2, H3 removal is delayed and Hir2 occupancy is decreased, indicating that ATP-dependent chromatin remodeling factors such as the Swi/Snf complex can initiate chromatin change during transcription. As such, the histone chaperones may be recruited to the transcribed genes by recognizing the initial changes in chromatin structure introduced by other chromatin remodeling factors, such as Swi/Snf or polymerase II itself, and then facilitate the process of chromatin change.

The function of histone chaperones is related to SET2-dependent H3 K36 methylation
CAF-1 does not show any significant genetic interactions with elongation factors, unlike Asf1/HIR (10). However, the localization of CAF-1 suggests that it might be related to transcription.
Set2 is a histone H3 methyltransferase and contributes to transcriptional elongation through H3 K36 methylation. The phenotype of set2Δ can be reproduced by the mutation of lysine 36 to alanine. To examine the role of CAF-1 in transcription, we constructed yeast strains with each histone chaperone subunit deleted in the histone shuffle background, and then transformed them to introduce the H3 wild type or K36A allele on the TRP plasmid. Each transformant was grown in SC-TRP medium and spotted serially on a YPD plate with or without 5-fluoro-orotic acid (FOA). FOA counter-selects for yeast that have lost the original source of wild type H3 carried on the URA plasmid.
Interestingly, deletion of CAF-1 subunits, HIR subunits, or ASF1 slowed growth on FOA medium when combined with H3 K36A (Fig. 3). This growth phenotype was specific to K36A because there was no growth defect with K4A or K79A except asf1Δ, which was sensitive to K4A in addition to K36A (Supplementary Fig. 3). Thus, yeast with the histone chaperone mutation prefers to retain wild type H3 rather than K36A. In contrast, a combination of set2Δ and H3 K36A did not develop further growth defects, indicating that K36A is not intrinsically defective in growth on FOA (Fig. 3). In addition, SPT10, a histone acetyltransferase that regulates the expression of histone genes and affects global transcription (22,23), did not slow growth on FOA with K36A, indicating that not all histone-related factors have a growth phenotype in combination with K36A. These results suggest that CAF-1, Asf1, HIR, and Set2 might function in a similar pathway in transcriptional elongation but do not overlap completely.

Set2 is required for tolerance to excess histones
Transcription by pol II on the chromatin template is accompanied by dynamic changes in chromatin structure. Therefore, this process can be affected by excess free histones, because the chromatin histones and free histones compete for chaperone binding. To understand the basis of the genetic interaction between H3 K36A and H3/H4 chaperones, we tested whether Set2 is implicated in histone chaperone function by measuring the growth of yeast over-expressing H3 or H2A under the GAL1 promoter. H3 or H2A overexpression on galactose medium did not affect the growth of the wild type (Fig. 4). However, as a control, asf1Δ was sensitive to excess H3 but not H2A (Fig. 4A and B). Interestingly, SET2 deletion also rendered the cells sensitive to excess H3, but not H2A, which was not the case for the other H3 methyltransferase mutants, set1Δ or dot1Δ (Fig. 4A). This indicates that all histone methyltransferases are not sensitive to excess H3, even with similar H3 expression levels (Fig. 4B). Set2 was not needed for the recruitment of Hir1 or Asf1 (Supplementary Fig. 2 and data not shown), but was required for tolerating excess histones, specifically H3. Our data indicate that Set2 or K36 methylation and http://bmbreports.org H3/H4 histone chaperones play a common role in the pathway affected by excess H3.
Asf1 and HIR play a role in transcription, but CAF-1 does not. Mutations in HIR and ASF1 subunits produce synthetic growth defects with components of the FACT or PAF1 complex, and hir1Δ is lethal when combined with some SPT4, SPT5, and SPT6 alleles (10,24). Asf1 is a central factor in the activation of PHO genes. However, the role of CAF-1 in transcription is indirect, such that it contributes to gene silencing by maintaining heterochromatin. Although CAF-1 has contact with Asf1, it seems to be dispensable for Asf1-dependent transcriptional activation in PHO genes. However, CAF-1 is recruited to a transcriptionally active region and functionally required when H3 K36 is mutated, suggesting that CAF-1 might be also involved in transcription-coupled chromatin dynamics. H3 K36 is selectively methylated in actively transcribed regions and is required to keep transcribed chromatin in a repressed state to inhibit internal cryptic transcription (25,26). The ability of Set2 to repress aberrant chromatin opening may relate to its ability to cope with excess H3, as does CAF-1. However, more work will be needed to determine the precise role of CAF-1 and Set2 in chromatin dynamics and transcription.

Growth conditions and analysis
Yeasts were grown at 30 o C in either YPD media or in synthetic minimal media lacking the nutritional supplements required for plasmid maintenance. For histone expression, yeasts were transformed with the plasmids HTH-HHT2/pYES2, pGALHA-H2A, or the empty vector (pYES2). Yeasts were grown overnight in SC-URA containing 2% raffinose, serially diluted 10fold, and spotted on SC-Uracil with either glucose or galactose as the carbon source. The plates were incubated for 3-4 days at 30 o C before analysis. For the genetic interaction assay between histone chaperones and H3 alleles, each histone shuffle strain carrying the histone chaperone mutation was transformed with the plasmids individually and then tested for the ability to grow on YPD with 5-fluoroorotic acid (FOA).

Immunoblotting
To analyze the overexpression of histones, yeast cultures were harvested and boiled in a cracking buffer (8 M urea, 40 mM Tris-HCl, pH 8.0, 5% SDS, 0.1 M EDTA, 0.01% β-mercaptoethanol, 0.001% bromophenol blue supplemented with complete protease inhibitors) for 10 min after 1 min of vortexing. The resulting lysate was clarified by centrifugation and separated on a 15% denaturing polyacrylamide gel and analyzed by immunoblotting with HA-antibody (Roche).

Chromatin Immunoprecipitation (ChIP)
The ChIPs were performed essentially as described (13). The PCR signals were quantified using a Phosphoimager (Fujix BAS 2040) and normalized to the input DNA and the intergenic control. The PCR primers were described previously (28). http://bmbreports.org