Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability

The replisome functions in a dynamic environment that is at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the action of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (Hat1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12 that accompanies replication-coupled chromatin assembly. Analysis of the role of Hat1 in replication-coupled chromatin assembly demonstrates that Hat1 also physically associates with chromatin near sites of DNA replication. The association of Hat1 with newly replicated DNA is transient but can be stabilized by replication fork stalling. The association of Hat1 with nascent chromatin may be functionally relevant as loss of Hat1 results in a decrease in replication fork progression and an increase in replication fork stalling. In addition, in the absence of Hat1, stalled replication forks are unstable and newly synthesized DNA becomes susceptible to Mre11-dependent degradation. These results suggest that Hat1 links replication fork function to the proper processing and assembly of newly synthesized histones.


INTRODUCTION
The central event in the division of a cell is the duplication of its chromosomes.
Chromosome duplication requires the proper functioning of two interconnected processes. The first is the replication of the genomic DNA. The second is the duplication of the chromatin structure that governs the correct packaging and architecture of the chromosomes in the nucleus. The successful coordination and completion of these processes is essential to ensure genome stability, maintain correct patterns of gene expression and properly regulate cell proliferation.
DNA replication occurs in a unique and highly dynamic chromatin environment. The replication fork must navigate through the chromatin structure in front of the fork by disrupting nucleosomes in its path. In the wake of the replication fork, the nascent daughter duplexes must be rapidly assembled into nucleosomes. Nascent chromatin on the daughter duplexes is assembled from two distinct pools of histones; parental and newly synthesized.
The parental histones are derived from the nucleosomes disrupted during the passage of the replication fork. These nucleosomes dissociate into stable H3/H4 tetramers and H2A/H2B dimers. Regulation of parental histone recycling, mediated by the histone chaperone Asf1, is critical for proper replication fork function and stability (1). Asf1 functions in conjunction with the MCM2-7 replicative helicase and RPA to remove parental histones H3 and H4 from in front of the replication fork and transfer them to the newly replicated DNA near their original genomic location (2)(3)(4)(5)(6). Loss of Asf1 or disruption of Asf1 activity through histone over-expression impedes DNA unwinding and replication fork progression (7)(8)(9). Other factors, such as FACT and the POLE3-POLE4 complex are also involved in processing parental histones at the replication fork and may be involved in the association of H2A/H2B dimers with the H3/H4 tetramers (10)(11)(12).
Several recent studies have shown that replication-coupled chromatin assembly is required for replication fork function. In these studies, the supply of histones to the replication fork was blocked, either by preventing new histone protein synthesis or disrupting histone deposition by depleting CAF-1 or Asf1 (39,(48)(49)(50)(51). There is also evidence that the post-translational modifications on newly synthesized histones can influence replication fork function. Histone H3 lysine 56 acetylation has been shown to positively regulate binding of histones to CAF-1 (38). Consistent with this, loss of H3 lysine 56 acetylation and CAF-1 have similar effects on DNA replication in S. cerevisiae (49). Histone deacetylases, HDAC1 and HDAC2, which have been proposed to deacetylate newly synthesized histones following their assembly into chromatin, have been shown to be important for replication fork function and for the stabilization of stalled replication forks in conjunction with the WRN helicase (52)(53)(54)(55)(56).
Recent evidence suggests that Hat1 also has the potential to influence replication fork function. Studies in a wide range of eukaryotes show that loss of Hat1 sensitizes cells to DNA double strand breaks and causes HU sensitivity and genome instability in mammalian cells (19,57,58). In addition, it was recently reported that Hat1 is transiently recruited to chromatin during replication-coupled chromatin assembly and affects the protein composition of nascent chromatin (59). Therefore, we investigated whether Hat1 provides a link between the processing and assembly of newly synthesized histones and replication fork function. We confirm that Hat1 transiently associates with newly replicated DNA. We show that loss of Hat1 induces a dramatic reduction in replication fork progression and increases replication fork stalling. We also demonstrate that stalling of replication forks stabilizes the association of Hat1 with newly replicated DNA and that loss of Hat1 leads to destabilization of stalled forks and MRE11-dependent degradation of newly synthesized DNA.

MATERIALS AND METHODS
Cell culture conditions. Mouse embryonic fibroblasts were prepared as previously described (19). Cells were grown in DMEM (Sigma) supplemented with 10%FBS (Sigma) and Penicillin/Streptomycin (Gibco). Images were acquired using MetaMorph version 7.8.10 and quantification was completed using ImageJ version 1.52t according to a previously described protocol (63).
DNA fiber assay. DNA was labeled with 50µM and 250µM for 20 minutes each. HU (Sigma) was used at 4mM for 5 hours; Mirin (Sigma) was used at 100µM for 5 hours.
After labeling and treatment, cells were collected by trypsinization and resuspended in PBS. 2µL of the cells suspension were spotted on a glass slide and lysed with lysis buffer (0.5% SDS, 200 mM Tris-HCl, pH 7.4, 50 mM EDTA) for 10min, slides were then tilted to 15° to stretch the DNA fibers and fixed with Methanol/Acetic Acid (3:1) overnight at 4 degrees. Next day DNA was denatured with 2.5N HCl for 30min and wash several times with PBS before blocking with 1%BSA/PBS for 30min. Rat anti-BrdU (1:50, AbD Serotec) was used to detect CldU, and mouse anti-BrdU (1:20, Becton Dickinson) to detect IdU. Antibodies were diluted in blocking buffer and incubated for 1 hour at room temperature. AlexaFluor 594-conjugated anti-rat (1:250, Molecular Probes) and AlexaFluor 488-conjugated anti-mouse (1:250, Molecular Probes) were used as secondary antibodies and incubated for 1 hour at room temperature. Slides were mounted with Vectashield with DAPI.
Immunofluorescence. Cells were seeded on coverslips and allowed to attach for 24 hours. Next day the cells were fixed with 4% PFA at room temperature for 10 minutes, washed several times with PBS and permeabilized with 0.5% Triton X-100/PBS for 15 minutes at room temperature, after several PBS washes cells were blocked with 5% BSA in PBS for 30 minutes at room temperature. Anti-phosphorylated ATR (Ser 428) (Cell Signaling #2853 1/100) was incubated overnight at 4 degrees. Next day after several washes, secondary AlexaFluor 594-conjugated anti-rabbit was diluted 1/250 and incubated 1 hour at room temperature. Antibody excess was extensively washed and slides were mounted with Vectashield with DAPI.
Comet Assay. The Comet Assay kit (Trevigen, Gaitherburg,MD) was used according to the manufacture instructions. Briefly, MEFs were resuspended in ice cold PBS (Ca2+ and Mg2+ free) to a concentration of 1 × 10 5 cells/ml. 5 µl cells were mixed with 50 µl of warm low melting Agarose and 50 µl were evenly spread onto the special comet slides.
Slides were stored at 4 °C in the dark and transferred to pre-chilled lysis solution for 60 minutes at 4 °C. Next, slides were transferred to alkali unwinding solution at room temperature for 60 minutes. Slides were transferred to electrophoresis tank which contained pre-chilled Alkaline electrophoresis solution and run at 1 Volt/cm, 300 mA for 45 minutes at 4 degrees. The slides were immersed twice in deionized water for 5 minutes intervals and washed in 70% ethanol for 5 minutes. Then cells were stained with 100 µl of SYBR Green I for 5 minutes in the dark and slides were analyzed under Zeiss Axiophot fluorescence microscope. Images were taken using Metavue software version 6.3r2 software and comet tails were analyzed using opencomet by Imagej.

RESULTS
Hat1 transiently localizes to newly replicated DNA. Current models of replicationcoupled chromatin assembly predict that Hat1 associates with, and modifies, newly synthesized histone H4 in the cytoplasm before transferring the modified histones to Asf1 for subsequent nuclear import and deposition. However, recent results using iPOND (isolation of proteins on nascent DNA) suggested that Hat1 becomes transiently associated with newly replicated DNA (59). As this has the potential to significantly expand the role of Hat1 in genome duplication, we sought to confirm this observation.
Proximity ligation assay-based chromatin assembly assays (CAAs) have recently been developed and serve as a powerful method for analyzing protein dynamics on newly replicated DNA (60)(61)(62)(63). The proximity ligation technique determines whether two molecules reside close to each other in the cell by employing two species-specific secondary antibodies that are fused to oligonucleotides. If the secondary antibodies recognize primary antibodies that are in close proximity, the oligonucleotides can both bind to a nicked circular DNA, creating a template for rolling circle replication. This amplifies sequences that can be bound by a fluorescent probe and visualized. To adapt this for use as a chromatin assembly assay, newly replicated DNA is labeled by the incorporation of the thymidine analog IdU. The proximity of proteins to newly replicated DNA is detected using antibodies against the protein of interest and antibodies recognizing IdU. To validate the CAA, we monitored the localization of PCNA, H4 lysine 5 acetylation and H4 lysine 12 acetylation to newly replicated DNA in Hat1 +/+ and Hat1 -/-MEFs (mouse embryonic fibroblasts). As seen in Figure 1A, quantitation of the CAA precisely mirrored the results previously obtained with iPOND. The localization of PCNA to newly replicated DNA was Hat1-independent and the acetylation of H4 lysines 5 and 12 required Hat1 (19,59).
Using α-Hat1 antibodies, we tested whether Hat1 is in proximity to newly replicated DNA. There is abundant CAA signal in Hat1 +/+ cells and only background in the Hat1 -/cells ( Figure 1B). We next asked whether Hat1 is transiently associated with newly synthesized DNA or whether it is stably bound to chromatin. We performed CAA assays immediately following a pulse of IdU and after 15, 30 and 60 minutes of a thymidine chase. As seen in Figure 1C, the level of Hat1 on newly replicated DNA is significantly reduced after a 15 minute chase and is completely lost after 30 minutes.
Intriguingly, if replication forks are stalled by the addition of HU, Hat1 association with newly replicated DNA is stabilized for extended periods of time (at least 5 hours). These data verify that Hat1 is transiently associated with nascent chromatin near sites of DNA replication and becomes stably associated when replication forks stall.

Hat1 is required for normal replication fork progression. The physical association
of Hat1 with the highly dynamic chromatin at sites of DNA replication greatly expands the spectrum of potential functions for this enzyme in genome duplication. In particular, this raises the possibility that Hat1 plays a direct role in replication fork function or stability. To test this, we used DNA fiber analysis in Hat1 +/+ and Hat1 -/-MEFs ( Fig. 2A).
Hat1 +/+ and Hat1 -/cells were incubated with CldU, followed by IdU incubation for equal times and replication fork progression was measured by DNA fiber analysis in which antibodies targeting the CldU (red) and IdU (green) are used to label the newly replicated DNA with different colors. The relative rates of replication fork progression were determined by measuring the lengths of the IdU tracts that are located at junctions with CldU labeled DNA, as this ensures that the replication fork was functional at the beginning of the IdU incubation. We observed a significant decrease in the length of labeled DNA fibers in the Hat1 -/cells, indicating that DNA replication progressed more slowly in the absence of Hat1. Consistent with an effect of Hat1 loss on replication fork function, analysis of PCNA dynamics at the replication fork by CAA showed that PCNA dissociation is significantly delayed in the absence of Hat1 ( Figure 2B).

Loss of Hat1 increases replication fork stalling. A decreased rate of DNA replication
can be due to decreases in the velocity of the replication fork or increases in the frequency of replication fork stalling. To test the latter possibility, we stained Hat1 +/+ and Hat1 -/cells with antibodies against phosphorylated ATR (Ser428). ATR is recruited to single stranded DNA at sites of replication fork stalling where it is activated by phosphorylation. Loss of Hat1 resulted in an increased number of cells positive for phospho-ATR foci (Fig. 3A).
To confirm the increase in replication fork stalling, we used the CAA to measure the association of Rad51 with the single strand DNA that is created at stalled replication forks (64). As seen in Figure 2B, there was a significant increase in the association of Together, these data indicate that Hat1 is necessary for proper replication fork function and the prevention of replication stress.
Hat1 is critical for the stability of stalled replication forks. As seen in Figure 1C, Hat1 is stably associated with stalled replication forks. To determine whether Hat1 is involved in maintaining the stability of stalled replication forks, we analyzed the stability of newly replicated DNA at stalled replication forks using the DNA fiber assay. Hat1 +/+ and Hat1 -/cells were treated with CldU and IdU sequentially for equal lengths of time.
HU was then added to induce replication fork stalling. After 5 hours, the lengths of the IdU and CldU tracts were measured. If the stalled replication forks remain stable, the ratio of IdU tract length to CldU tract length will be 1. If the newly replicated DNA (represented by the IdU labeled DNA) at the stalled forks is unstable, the ratio of IdU tract length to CldU tract length will be less than 1. As seen in Figure 4A, there was a significant decrease in the IdU tract length in the absence of Hat1. We conclude that Hat1 is required for the protection of newly replicated DNA at stalled replication forks.
The degradation of newly replicated DNA at stalled replication forks is the result of Mre11 nuclease activity (66)((67). To determine whether the instability of nascent DNA in the absence of Hat1 is also Mre11-dependent, Hat1 +/+ and Hat1 -/cells were sequentially treated with CldU and IdU for equal lengths of time. The cells were then treated with HU in the presence of Mirin, a specific inhibitor of Mre11 activity. As seen in Figure 4B, newly replicated DNA is equally stable in Hat1 +/+ and Hat1 -/cells when Mre11 activity is inhibited. These data indicate that Hat1 functions to protect newly replicated DNA from Mre11-mediated degradation.
We used a comet assay to determine whether Hat1-dependent replication fork instability led to a decrease in the ability of cells to recover from replication stress. Hat1 +/+ and Hat1 -/cells were treated with HU for 3 hours and then allowed to recover for 12 hours in the absence of HU. As seen in Figure 4C, Hat1 +/+ cells were better able to recover from prolonged replication stress than the knock out cells, consistent with a loss of replication fork integrity in the absence of Hat1.

DISCUSSION
Contrary to the predictions of current models of replication-coupled chromatin assembly, our results demonstrate that Hat1 localizes to chromatin at sites of DNA replication.
There are several models to explain the localization of Hat1 to nascent chromatin. First, Hat1 may not transfer H3/H4 dimers to Asf1. Rather, Hat1 may remain associated with the H3/H4 dimers throughout the entire replication-coupled chromatin assembly process and load onto newly replicated DNA through CAF-1-mediated deposition of H3/H4/Hat1 complexes. This model is consistent with numerous proteomic studies that have identified the Hat1 complex as major components of soluble H3 and H4 complexes (22,26,(68)(69)(70)(71)(72). Alternatively, following transfer of H3/H4 dimers to Asf1, Hat1 may enter the nucleus independently and bind to nascent chromatin after histone deposition. Finally, Hat1 may participate in an additional chromatin assembly pathway distinct from the Asf1/CAF-1 pathway. One potential pathway may utilize the histone chaperone NASP. A distinct nuclear yeast Hat1 complex contains histones H3 and H4 and a histone chaperone, Hif1, which is the yeast homolog of NASP (15,21,68).
Subsequent experiments have shown that NASP also interacts with the Hat1 complex in mammalian cells (22). NASP is important for buffering the pools of soluble H3/H4, particularly under conditions of replication stress, and can form a multi-chaperone complex with Asf1. Several studies have shown that NASP can function as a nucleosome assembly factor in vitro (73)(74)(75). Hence, this model predicts that Hat1 localizes to newly replicated DNA in conjunction with NASP-mediated deposition of H3/H4. It is clear that DNA replication is coupled to newly synthesized histone deposition. This link was originally suggested by studies demonstrating that DNA replication required active protein synthesis(76-78). More recently, histone supply was more directly linked to replication fork function by experiments that specifically limited histone production (48,50). The assembly of newly synthesized histones into chromatin was directly implicated in replication fork function through the identification of DNA replication defects in cells lacking components of the replication-coupled chromatin assembly pathway, such as Asf1 and CAF-1 (39,49,51).  (59). Decreased levels of these proteins in the proximity of replication forks may create an altered chromatin structure that negatively affects replication fork function or they may be directly involved in replisome function. Indeed, it was recently shown that Brd2, Brd3 and Brd4 function at the replication fork to antagonize the ATAD5-mediated unloading of PCNA, which is consistent with our observation that PCNA unloading is slowed in Hat1 -/cells (79). Finally, the presence of Hat1 on nascent chromatin near replication forks suggests that Hat1 may directly modify and regulate components of the replisome.
The association of Hat1 with nascent chromatin is transient but becomes stable if replication forks stall. The physical association of Hat1 with stalled forks is likely to be functionally relevant as Hat1 is required for the stability of stalled replication forks. An attractive mechanism for the role of Hat1 in replication fork stabilization involves the recruitment of Rad51 to stalled forks. Rad51 binds to single strand DNA at stalled replication forks and plays a central role in maintaining replication fork stability. Hat1 forms an S-phase-specific complex with Rad51 and is involved in the recruitment of Rad51 to DNA double strand breaks (80). However, we do not detect any decrease in Rad51 localization to stalled replication forks in Hat1 -/cells, suggesting that the mechanisms for Rad51 recruitment to DNA double strand breaks and stalled replication forks are distinct.
It has also been suggested that Hat1 is involved in the initiation of DNA replication.
Studies in yeast showed that Hat1 physically interacts with the origin recognition complex (ORC). In addition, combining mutations in Hat1 with temperature sensitive alleles of ORC components or CDC45 resulted in synthetic growth defects. Hat1 was also recruited to origins of replication at the time of origin activation. Despite these connections, there were no defects in replication origin firing in Hat1 mutants in yeast(81).
Our results suggest an update to current models of replication-coupled chromatin assembly to incorporate the localization of Hat1 to nascent chromatin at sites of DNA replication. In addition, our results indicate that Hat1 lays a direct and integral role in both genome and epigenome duplication.

Acknowledgements
This work was support by a grant form the National Institutes of Health (R01 GM062970 to M.R.P.)). Microscopy was supported by a grant from the NIH/NINDS (P30 NS104177).