Depletion of FACT affects transcription of genes differentially.
Pol II cannot carry out productive transcription of DNA templates organized in chromatin, both at initiation and at elongation phases, in the absence of additional factors. In vitro transcription experiments have demonstrated the ability of FACT to stimulate transcription elongation of chromatin templates by Pol II (42
) in cooperation with H2B monoubiquitination (45
). In those in vitro experiments, the activity of FACT as a chromatin-dependent elongation factor was demonstrated by comparing its effects on naked DNA and chromatin. The same kind of experiment is not possible in vivo since cell DNA is always organized in chromatin. However, the features of chromatin organization vary throughout the genome. In animal cells, some genomic regions show a higher tendency to establish translationally positioned nucleosomes than others (66
). In Saccharomyces cerevisiae
, 65% of the genome shows translationally positioned nucleosomes; promoter-proximal sequences within coding regions show a higher tendency toward nucleosome positioning, but coding regions almost entirely covered by nonpositioned nucleosomes are also found (69
). Following an in vivo depletion strategy, we have tested the consequences of FACT scarcity on the expression of several genes. In this kind of in vivo experiment, it is formally impossible to exclude the involvement of additional elements. However, the simultaneity between the decrease of Spt16 below the wild-type level and the documented effects on mRNA levels, transcription rates, and Pol II occupancies indicates that our results describe direct effects of FACT depletion on gene transcription.
We have shown here that FACT is required for the transcription of three coding regions driven by the GAL1 promoter (GAL1, PHO5, and LAC4), but it is significantly less necessary for the transcription of two others also driven by the GAL1 promoter (YAT1 and lacZ). We first showed that mRNA levels of these five genes were differentially affected by Spt16 depletion. We then showed by ChIP that increased amounts of Pol II were associated to the 5′ end of the transcribed regions of GAL1, GAL1pr::PHO5, and GAL1pr::LAC4 in response to Spt16 depletion, whereas no significant change in Pol II distribution was found in GAL1pr::lacZ or GAL1pr::YAT1. Furthermore, the results of run-on experiments also reflected lower densities of elongating Pol II after Spt16 depletion at GAL1 and GAL1pr::PHO5 than at GAL1pr::YAT1. The phenomenon described in this article is not restricted to genes driven by the GAL1 promoter. We have shown that the expression of the native SR09 gene is negatively affected by Spt16 depletion. In contrast, the expression levels of CIT2, CUP1, and YAT1, driven by their own promoters, were not. Altogether, our results indicate that the effects of FACT depletion on transcription are gene specific.
We have found a good correlation in the set of analyzed genes between the sensitivity to Spt16 depletion and the translational positioning of nucleosomes at the 5′ end of the coding regions. A relationship between translational positioning and nucleosome stability, shown in vitro by challenging reconstituted nucleosomes with high salt concentrations or temperature, has been observed elsewhere (16
). At least in some cases, the specific interactions between the DNA sequence and the histone octamer determine both positioning and stability (62
). This provides the simplest explanation for the connection between nucleosome positioning and Spt16-dependent transcription. We find it reasonable that the translationally positioned nucleosomes that we have detected at the coding regions of the studied genes, or at least a subset of them, are more reluctant to slide or to be transferred than those nucleosomes occupying nonpositioned genes. These stable nucleosomes would require the octamer disassembly-reassembly activity of FACT.
The connection between positioning and nucleosome stability is also supported by the phenotype of some histone mutants which show defects in nucleosome positioning in vivo (65
) and an increased nucleosome mobility in vitro (15
), due to alterations of the histone-DNA interactions on the surface of the nucleosome (40
). We have used one of these histone mutants (hhf2-13
) to test our hypothesis, and we found that the impairment of GAL1
transcription after Spt16 depletion was clearly suppressed by hhf2-13
, making GAL1
expression insensitive to Spt16. It is the central DNA wrap of the nucleosome which is affected by hhf2-13
). The same region of nucleosomal DNA is also perturbed by FACT action, according to the in vitro studies of FACT-nucleosome interaction (48
). We find it full of sense, therefore, that hhf2-13
suppresses the absence of FACT at a gene displaying positioned nucleosomes. It is theoretically possible that this suppression is not caused by the histone mutation itself but by the increase in histone dosage produced by the introduction of extra copies of the H3 and H4 coding genes (12
). We do not favor this interpretation, since it has been shown that histone imbalance does not affect GAL1
chromatin organization and does not derepress GAL1
). But even if this were true, the results of this experiment would support that the gene-specific effect of FACT depletion is mediated by chromatin.
The analysis of the nucleosomal organization of GAL1
in glucose and in galactose indicates a deep reorganization of the 5′ end of the coding region after activation, in agreement with the inverse correlation between histone-DNA interactions (measured by ChIP) and transcriptional activity reported for GAL
). It is worth mentioning that the transcription-dependent changes in chromatin structure that we have detected at the GAL1
coding region were not observed in a previous analysis of GAL1
). The main difference between the two analyses was the genetic background. In the previous study, we used W303-derived strains, whereas in the present study, all strains were isogenic to BY4741. Further studies would be needed to clarify this striking difference. In any case, in both BY4741 and W303 cells grown in galactose, the 5′ end of the GAL1
coding region is not nucleosome free. At least one positioned nucleosome is present during transcription in that region, suggesting that the reported decrease in histone occupancy of GAL
genes during transcription (54
) does not involve a random nucleosomal distribution.
Finally, an important piece of evidence connecting FACT and positioned nucleosomes at the 5′ end of the coding region comes from the insertion of two positioned nucleosomes between the promoter and the transcribed region of the Spt16-independent GAL1pr::YAT1 transcription unit. The resulting GAL1pr::GAL1(5′)-YAT1 became sensitive to Spt16 depletion. Altogether, our results suggest that FACT is required for the transcription of those genes whose transcribed region is organized into positioned nucleosomes at the 5′ end.
It has been shown that FACT plays a role in preventing the activation of cryptic initiation sites by contributing to the proper reposition of nucleosomes after the passage of elongating Pol II (26
). We have indeed shown here the slight activation of a cryptic initiation site present in FLO8
12 h after adding doxycycline. However, 8 or 10 h after doxycycline was added, times chosen for the functional analyses of this study, the cryptic initiation site present in FLO8
was not active yet, and the nucleosomal organization of GAL1
was similar to that of the wild type. We have shown that the negative effect of Spt16 shortage on the accumulation of GAL1
, and LAC4
mRNAs was not due to the activation of cryptic initiation sites within their coding regions. Moreover, the results of the run-on experiments show a lower density of elongating Pol II at PHO5
and do not support secondary transcripts emerging in these genes. Mason and Struhl (36
) suggested that the overall negative effect of Spt16 depletion on transcription might be due to a competition between normal promoters and cryptic initiation sites for the transcriptional machinery. According to this hypothesis, the higher number of initiation sites originated in the cell by the depletion of Spt16 might affect the GAL1
promoter due to a subsequent scarcity of general transcription factors. Since the five genes driven by the GAL1
promoter do not behave the same, we can also exclude this explanation for the phenomenon described here, unless the sequences located downstream differentially affect the activity of the promoter (see below).
FACT has also been involved in PIC assembly by facilitating TATA-binding protein binding to the TATA box in the context of a nucleosome. According to this, FACT depletion might also affect transcription initiation in a promoter-specific manner. However, it is difficult to explain all of the results shown in this work in terms of transcription initiation. We have shown here that several transcription units driven by the same promoter (GAL1pr) exhibit different degrees of sensitivity to Spt16 depletion. The diverse nucleosomal distributions at the coding regions might differentially affect the chromatin organization of the GAL1 promoter. However, we did not find such differences at the nucleosomal mapping that we have carried out, although subtle differences cannot be completely ruled out. We would then expect a decrease in Pol II recruitment. In contrast, we have found accumulation of Pol II at the 5′ end of the Spt16-dependent genes. We cannot exclude that a part of this accumulation corresponds to initiating Pol II due to the inherent inaccuracy of the ChIP technique, but in that case, the results would be compatible with a role of FACT in the transition from initiation to elongation and not in PIC assembly.
Although we cannot completely rule out an initiation component, the simplest interpretation of our results suggests that an involvement of FACT in transcription elongation may be immediately after initiation has occurred. According to this perspective, FACT would be required for facilitating transcription through those nucleosomes less prompt to slide or to be transferred. In the absence of FACT, Pol II would pause in front of such nucleosomes and would eventually become arrested. The comparison of the patterns of Pol II distribution after Spt16 depletion, obtained by ChIP and by run-on, detects a difference: in GAL1
, the amounts of immunoprecipitated Pol II located at 5′ are higher than in the rest of the gene; in contrast, the densities of active Pol II are roughly similar at the 5′ and 3′ ends of both genes. The simplest explanation for this phenomenon would be that the excess of Pol II present at 5′ became arrested after suffering backtracking and therefore was undetectable by a run-on assay. This hypothesis is also in agreement with published results showing how nucleosomes induce Pol II arrest in vitro by stabilizing its backtracked conformation (27
polytene chromosomes, Saunders et al. (51
) have shown that FACT is not recruited to RNA polymerase III-dependent genes, which are known to undergo nucleosome transfer rather than disassembly during in vitro transcription elongation. It may be possible that FACT-dependent nucleosome disassembly/reassembly would be required only by Pol II to transcribe positioned nucleosomes, whereas those nucleosomes not exhibiting a fixed translational positioning might be more likely to transfer or slide during transcription elongation. We do not have data to distinguish which of the two proposed functions of FACT, disassembly or reassembly, is critical for transcription of GAL1
and the other Spt16-dependent genes studied here. However, if we consider positioning as an indication of nucleosome stability, as discussed before, it seems more likely that transcription elongation of highly organized chromatin requires the nucleosome disassembly activity of FACT. If this interpretation is true, an explanation must be provided for the predominant requirement for FACT at the 5′ end of the coding regions. Either nucleosomes positioned at these regions are particularly stable, or the capability of Pol II machinery to interact with nucleosomes changes along the transcribed region by including perhaps other histone chaperones such as Asf1, also acting during transcription elongation (53
). Alternatively, the accumulation of positive DNA supercoiling ahead of Pol II might facilitate nucleosome reorganization once genes have been transcribed to some extent, as it has been shown elsewhere (31
), reducing the FACT requirement at these regions.
Does every gene require its own menu of factors after transcription initiation?
Biochemical and genetic analyses during the last 15 years have described a numerous set of factors playing auxiliary roles in Pol II-dependent mRNA biogenesis after transcription initiation, favoring mainly processivity (35
). However, little is known about the relative importance of each of these factors in terms of the number of genes that requires their function. Since they measure the combination of initiation, elongation, and mRNA stability, global transcriptome analyses have not been very useful in this respect. In this work, by comparing five genes under the control of the same promoter, we have shown that FACT is not equally required for all genes during transcription. It was recently reported that, although recruited to the transcribed region of the human p21 gene in a carboxyl-terminal domain (CTD) kinase-dependent manner when it becomes activated by p53, FACT is dispensable for p21 expression (21
). In fact, p21 transcription does not require CTD phosphorylation at Ser2, indicating that the requirement of P-TEFb for transcription elongation is also gene specific (21
). It is worth mentioning that CUP1
, one of the genes whose expression is not affected by Spt16 depletion, can be transcribed by a mutant version of Pol II that lacks the CTD (37
). Altogether, these elements suggest a relationship between the requirements for CTD phosphorylation and FACT. In this respect, it would be interesting to analyze the nucleosomal organization of p21 and other possible P-TEFb- and FACT-independent mammalian genes.
By using the same five transcription units driven by the GAL1
promoter that have been analyzed in this work, we have shown elsewhere that the THO complex, involved in the connection between transcription elongation and mRNA transport, is also not uniformly needed for all of them (11
). It is interesting that those transcription units whose elongation is highly dependent on FACT are not strongly affected by tho
mutations; this is the case for GAL1
, and GAL1
. However, GAL1
, dramatically affected by tho
mutants, are only mildly affected by Spt16 depletion. According to the chromatin analysis presented here, the THO complex seems to be specially needed at genes with random chromatin organization. Since THO plays a role in preventing the formation of R loops by nascent mRNA (23
), a contribution of positioned nucleosomes in preventing R loops can be suggested.
Another gene-specific factor involved in postinitiation events is TFIIS, an elongation factor dispensable for the expression of most genes, which plays a capital role in transcriptional activation of Drosophila hsp70
. It does so by releasing promoter-proximal paused Pol II from arrest (1
). Pol II pausing in hsp70
at the transcription elongation step seems to be influenced by the nucleosomal organization of the promoter-proximal region (8
). Considering FACT, P-TEFb, THO, and TFIIS, the emerging picture is that the intrinsic properties of the transcribed region of a given gene determine the set of factors required for its proper mRNA biogenesis.