Transcription is regulated both by the recruitment of RNAP II to promoters and by the recruitment of factors that regulate transcription elongation (45
). Many of these factors alter chromatin structure, allowing RNAP II to move through the chromatin barrier; others recruit proteins necessary to complete mRNP biogenesis (27
). Here, we show that Nap1 associates with chromatin and is necessary for normal transcription of an activated gene. We propose that Nap1 plays an important role as a histone chaperone during transcription elongation, and our evidence suggests that Nap1 functions in chromatin reassembly after the passage of the polymerase. Nap1 has genetic and physical interactions with components of the RNA TREX complex as well as with the export factor Mex67. Our results suggest a model whereby the TREX complex helps target Nap1 to elongating genes, where it acts as a chromatin assembly factor. Nap1 therefore represents a new connection between the chromatin remodeling and mRNP biogenesis machineries (Fig. ).
FIG. 7. Model of Nap1 function during transcription. The TREX complex component Yra1 directly recruits Nap1 to a site of active transcription. (A) Nap1 facilitates the reincorporation of H2A/H2B dimers and octamer reassembly on the open template following RNAP (more ...)
We showed that combined loss of NAP1
and deficiencies in TREX complex components, or MEX67
, led to exacerbated growth and bud morphology defects. The genetic interaction with TREX complex components and Mex67 suggested that Nap1 may share an important role in transcription or mRNP maturation. Nap1 interacts directly with Yra1, but so far we have no evidence that there is a direct physical interaction with the other components. There is no evidence that Nap1 is directly contributing to mRNA export, as deletion or overexpression of NAP1
did not result in changes in global mRNA export. While Nap1 is a nuclear shuttling protein, it is predominantly in the cytoplasm at steady state (36
). A proportion of Nap1 is found at the incipient bud site and bud neck, and loss of Nap1 leads to a long bud phenotype in a proportion of cells, suggesting a delay in the activation of the mitotic cyclin Clb2. We considered the possibilities as to why loss of Nap1 and different TREX complex components would lead to a dramatic exacerbation of the elongated bud phenotype, as well as growth defects. We cannot exclude the possibility that in the Δnap1
strain, specific loss of Nap1 from the bud neck is responsible for the bud morphology defect, and this phenotype is exacerbated in the double mutants by the reduction of transcripts that are also important for G2
/M progression. However, we favor the model in which Nap1 nuclear functions also regulate bud morphology. For example, Yra1 and Mex67 are physically associated with transcripts involved in cell wall development and other metabolic processes, and the concomitant absence of Nap1 and these proteins may affect the transcription of important G2
/M regulators (16
). It has also been shown that Rad53, Tel1, and Mec1, components of the DNA damage repair pathway, are requisites for proper bud formation (10
). Interestingly, deletion of these components results in hyperelongated buds that resemble those of the yra1
strain, giving further evidence that these buds are the product of defects in chromatin-templated processes.
A hallmark of genes involved in transcription elongation is the sensitivity of corresponding mutants to 6-AU and DNA damage (12
). Consequently, we show that not only are Δnap1
cells are sensitive to 6-AU but also this effect is compounded for both yra1
cells. This indicates that the Nap1 histone chaperone activity and TREX complex/Mex67 mRNP functions are linked, providing further evidence that intersecting nuclear functions are responsible for the phenotypes observed. Additionally, it has been shown that yra1
cells are more susceptible to genomic instability (19
). The observation that yra1
cells are more sensitive to DNA damage by 4-NQO than either single mutant also supports our hypothesis. The double mutants likely have defects in nucleosome positioning, histone mobilization, and transcription elongation. The net effect of the defects likely results in abrogated DNA damage repair and genomic instability.
Many nuclear functions have been proposed for Nap1, and different chromatin assembly and disassembly activities have been observed in vitro (40
). Remarkably, there has been no evidence in S. cerevisiae
of Nap1 association with chromatin in vivo. Here, we demonstrate that S. cerevisiae
Nap1 is associated with chromatin. Importantly, we determined that Nap1 was associated with some of the same ORFs as Yra1, in addition to associating with nontranscribed regions and promoters. Furthermore, using a yra1
mutant strain grown at the restrictive temperature, we showed that Nap1 recruitment to these regions appeared to be dependent on Yra1. Previous studies have indicated that the yra1-1 mutant has five amino acid substitutions, with F223S being critical for the temperature-sensitive phenotype. This mutant is slightly overexpressed at 23°C, indicative of an inability to autoregulate expression (41
). Additionally, this mutation lies within a region important for binding Mex67 and the TREX complex component Sub2 (49
). We showed that at the permissive temperature, this mutant was still able to interact with Nap1. At 30°C, yra1
mutant cells are also more prone to transcription-associated recombination defects (19
). The precise mechanism whereby this mutation leads to lethality at 37°C is not known. It is possible that the protein is less stable, or that the protein no longer interacts with chromatin, or that the protein is unable to interact with a subset of binding partners, particularly Sub2 and Mex67. The concomitant loss of Nap1 from the same regions of chromatin at 37°C is likely due to the loss of Yra1 function via one of the above mechanisms. We determined that that RNAP II association with the ORF also decreased in a Yra1-dependent fashion. This is not surprising, as cells lacking the TREX complex component Hpr1 demonstrate a loss of association of RNAP II with the 3′ end of ORFs, and it was proposed that the absence of Hpr1 impairs RNAP II processivity (32
). We cannot rule out the possibility that RNAP II also coordinates Nap1 recruitment to ORFs. In this model, Nap1 could be recruited to both promoters and ORFs by the RNAP II-dependent disruption of chromatin, where it would be involved in replacing nucleosomes displaced by transcription. However, in this model, the observed role of Yra1 in Nap1 recruitment is unclear, unless it is an indirect effect of transcription not proceeding into the downstream regions of the gene. However, because of the direct physical interaction reported here, as well as the genetic interactions with YRA1
and other TREX complex components, we favor the model in which Yra1 is recruiting Nap1 to specific ORFs.
Nap1 has diverse functions in the cell; however, its precise role in nucleosome assembly in vivo is largely uncharacterized. One function that may be dependent on Nap1-mediated nucleosome rearrangement is chromatin remodeling at an activated promoter. Our data demonstrate that PHO5
are examples of genes where Nap1 is recruited to the promoter regions upon transcription activation. Moreover, Nap1 recruitment to the promoter is occurring independently of Yra1. This suggests that in vivo, Nap1 also has chromatin functions that are distinct from Yra1 and are regulated by other mechanisms. The function of Nap1 at the promoter is likely also tied to nucleosome mobilization. The eviction and incorporation of histones at the PHO5
promoter occurs in trans
and can be mediated by the histone chaperones Asf1, Hir1, and Spt6 as well as by the SWI/SNF chromatin remodeling complex (2
). Our data show that Nap1 is required for normal transcription of PHO5
, although measurements of mRNA levels, phosphatase activity assays, and H2B ChIP suggested that Nap1 is not a requisite for PHO5
transcription initiation. However, recruitment of Nap1 to promoter regions suggests that Nap1 is involved in chromatin remodeling of the promoter. Interestingly the S. pombe
homologs of Nap1 and the ATP-dependent remodeling factor Chd1 have also recently been shown to be associated with both coding and nontranscribed regions. They are believed to cooperate to displace histones from promoters in vivo (53
). One role for S. cerevisiae
Nap1 at the PHO5
promoter could be in the mobilization of the H2A variant H2A.Z. This histone is poised at the repressed PHO5
promoter and then removed during activation (44
). Nap1 and Chz1 are redundant histone chaperones for H2A.Z; therefore, the loss of NAP1
alone may not alter PHO5
transcription initiation (31
). The identification of Nap1 at chromatin regions independently of Yra1 raises the question of how Nap1 is targeted to the correct chromatin domain. We speculate that this may involve the SWR1 or other protein complexes, or possibly Nap1 itself, via recognition of different modifications within the histone N-terminal tails.
The occupancy with Yra1 at the PHO5
ORF, the modest changes in PHO5
transcript level and phosphatase activity observed for Δnap1
cells, and the delay in H2B redeposition during transcription repression suggest that Nap1 may also be important for chromatin remodeling during transcription elongation. Our data are consistent with the model whereby the net function of Nap1 is to facilitate nucleosome reassembly. This function is in contrast to the role of Nap1 with Chd1 in S. pombe
, where it was shown to function in chromatin disassembly, suggesting that at specific chromatin domains, Nap1 may act both in assembly and disassembly (53
). Asf1p has been shown to mediate both the eviction and the deposition of H3 during elongation, and both Spt6 and the FACT complex have chromatin reassembly activity (45
). We propose that Nap1 serves that same purpose for H2A/H2B dimers and that chromatin reassembly is important during elongation to prevent transcription initiation within the coding region. Nap1 interacts with histones directly but must be targeted to the correct chromatin domains. We propose a model whereby during elongation, Yra1 recruits Nap1 to the actively transcribed region where Nap1-mediated H2A/H2B mobilization occurs (Fig. ). Nap1 may be either a histone acceptor and donor or an active participant in the disassembly and reconstitution phases during RNAP II progression. Given the redundancy of histone chaperones, Nap1 may facilitate disassembly reactions during elongation in concert with other factors. A likely mechanism for Nap1 histone chaperone activity during elongation would therefore also involve interactions with other factors, like Chd1, the RSC complex, or the FACT complex.
In summary, we present evidence that suggests that Nap1 functions in transcription elongation and that recruitment of Nap1 to some chromatin domains is dependent on the TREX complex. Furthermore, at an activated gene, Nap1 appears to be important for chromatin reassembly. Generally, factors involved in pre-mRNA processing are recruited by the RNAP II C-terminal domain, and the Yra1-Nap1 interaction would demonstrate a unique mechanism of recruitment of a histone chaperone to an actively transcribed gene (4
). Thus, Nap1 represents a new connection between chromatin assembly and mRNP biogenesis.