Histone H2A.Z is strongly implicated in the activation of transcription initiation (19
). The results of the experiments reported here argue that H2A.Z also influences one or more steps in transcription elongation. First, htz1
Δ produces strong synthetic growth defects in combination with mutations in genes for the transcription elongation factors Spt5 and Spt16. Second, htz1
Δ single mutants are hypersensitive to elongation inhibitors, and our genetic results suggest that the mechanism of this sensitivity is at the level of transcription elongation. The mutations in SPT5
that suppress 6-AU sensitivity create dominant alleles, consistent with active suppression of an htz1
Δ-dependent elongation defect. Third, direct biochemical measurements at GAL10p-VPS13
show that htz1
Δ reduces the rate of RNA Pol II elongation runoff by approximately 24% compared to that for the wild-type HTZ1
strain. Fourth, transcription-dependent nucleosome remodeling over the GAL10p-VPS13
ORF requires Htz1. Together, these results indicate that budding yeast cells experience actual defects in Pol II transcription elongation in the absence of histone H2A.Z nucleosomes.
The specific mutations in SPT5
reported here fit well with their known functions and domains. The recessive synthetic lethal allele, spt5
, expresses a truncation of Spt5 that is missing the C-terminal repeat (CTR) domain. In human cells this region is phosphorylated by P-TEFb and is critical for the elongation activation role of DSIF (106
). In yeast, the BUR kinase is important for the phosphorylation of Spt5 in vivo
and can also phosphorylate the Spt5 CTR in vitro
). It has been suggested that the CTR may serve as a platform to recruit other elongation factors such as the PAF complex (106
). The dominant suppressor SPT5
encodes a sequence with two mutations immediately upstream of the CTR, one of which introduces an acidic charge that theoretically could serve as a phosphomimetic. The second dominant suppressor, SPT5
, also encodes a sequence with two amino acid substitutions. One of these, Q342R, falls in the NGN domain, which serves as the binding interface between Spt5 and Spt4 (31
). We suggest that the Q342R substitution might further stabilize the NGN domain and activate the positive elongation function of the Spt4-Spt5 complex.
allele is particularly intriguing. Aside from encoding two conservative amino acid substitutions, its main feature is that it encodes a truncation that deletes 357 residues from the C-terminal end of the protein. The truncation in Spt16-18 deletes a conserved acidic C-terminal domain that is shared with histone chaperones such as Rtt106. Deletion of this domain from Spt16 destroys the ability of reconstituted FACT to bind mononucleosomes and stimulate Pol II transcription on chromatin templates in vitro
While the phenotypic properties of SPT5
suggest functional interactions with htz1Δ
during Pol II elongation, it is important to note that SPT
genes have been linked to transcription initiation and that, in particular, FACT has been shown to have roles in establishing transcription initiation complexes as well as modulating promoter accessibility (11
There are at least two models for how Htz1 might positively regulate Pol II transcription elongation. First, we suggest that Htz1 may facilitate Pol II elongation by helping to establish the complete assembly, or correct modification state, of the elongation complex. Acting within promoter-proximal chromatin, the positional cues established by Htz1-containing nucleosomes (2
) or direct factor recruitment (1
) could provide space, time, or cofactors needed to assemble a fully functional elongation complex. In support of this model we have detected physical alterations in the elongation complexes formed in htz1
Δ mutants, including increased association of Spt5 and decreases in both the phosphorylation of CTD Ser2 and the loading of Elongator. This model also fits well with the preferential occupancy of Htz1 within promoter chromatin.
A second model for the function of Htz1 in elongation is that it serves to modulate the properties of the nucleosomes encountered by Pol II as it traverses the open reading frame. Although Htz1 is generally concentrated at promoters genome wide, it is not entirely absent from ORFs and can actually be enriched within the ORFs of individual genes (2
). Acetylated Htz1-K14ac has also been observed over the ORF and positively correlates with the level of transcription (66
). The altered stability of H2A.Z nucleosomes could then facilitate Pol II elongation just as it is thought to help remodel promoter chromatin for initiation (39
). Consistent with this model, we found that the transcription-dependent decrease in nucleosome occupancy is strongly attenuated in htz1
Δ cells. These results are in agreement with genome-wide studies which found that the global turnover of histone H3 is decreased in htz1
Δ cells, especially in regions with the highest turnover (18
). This dynamic cycling of nucleosomes could be driven by the exchange of Htz1 into canonical nucleosomes through the action of the SWR1 complex (60
), and this activity provides an attractive explanation for the partial role for SWR1
in specific elongation phenotypes of htz1
Δ that we observed.
These models are not mutually exclusive and could be interrelated. For example, it is formally possible that the increased nucleosome density in htz1Δ cells causes the change in the composition of the Pol II elongation complex. Alternatively, the defective elongation complex could be less efficient in remodeling nucleosomes for eviction, resulting in a higher nucleosome occupancy in ChIP assays. Further studies focused on the physical and temporal relationships between Htz1 and elongation cofactors and modifications will help to discriminate among alternate models. Nevertheless, the results of the genetic and molecular experiments reported here support the hypothesis that Htz1-containing chromatin has an important direct mechanistic role in facilitating Pol II transcription elongation.