Transcription Elongation Factors Repress Transcription Initiation from Cryptic Sites

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Science  22 Aug 2003:
Vol. 301, Issue 5636, pp. 1096-1099
DOI: 10.1126/science.1087374


Previous studies have suggested that transcription elongation results in changes in chromatin structure. Here we present studies of Saccharomyces cerevisiae Spt6, a conserved protein implicated in both transcription elongation and chromatin structure. Our results show that, surprisingly, an spt6 mutant permits aberrant transcription initiation from within coding regions. Furthermore, transcribed chromatin in the spt6 mutant is hypersensitive to micrococcal nuclease, and this hypersensitivity is suppressed by mutational inactivation of RNA polymerase II. These results suggest that Spt6 plays a critical role in maintaining normal chromatin structure during transcription elongation, thereby repressing transcription initiation from cryptic promoters. Other elongation and chromatin factors, including Spt16 and histone H3, appear to contribute to this control.

Although the molecular function of Spt6 in vivo is not understood, evidence suggests that it is involved in transcription elongation, mRNA processing, and interaction with nucleosomes [reviewed in (1)]. The function of Spt6 seems to be closely related to that of Spt4 and Spt5 (26), and to Spt16, Spt10, Bur1, Bur2, and core histones (1, 714).

To characterize the requirement for Spt6 in RNA polymerase (Pol) II transcription in S. cerevisiae, we performed microarray analysis of a temperature-sensitive spt6 mutant, spt6-1004, and an isogenic wild-type strain [Materials and Methods, Supporting Online Material]. These experiments revealed a large number of mRNAs whose levels were altered in the spt6-1004 mutant (15). For many of the genes whose expression appeared to be increased by the spt6-1004 mutation, Northern analysis detected new transcripts of smaller size (Fig. 1, A and B). Using a combination of 5′ and 3′ specific probes, we determined that the shorter transcripts observed for at least two of these genes, RAD18 and FLO8, correspond to the 3′ portions of the genes (Fig. 1C). Single-stranded probes showed that these shorter transcripts are on the same strand as the full-length transcripts (15).

Fig. 1.

Production of aberrant transcripts in spt6-1004 at 39°C. (A) Total RNA from wild-type or spt6-1004 strains, grown at 30°C or after an 80-min shift to 39°C, was analyzed by Northern blot for FLO8 and RAD18 RNAs. Ribosomal RNA (rRNA) is shown as a control for the amount of RNA in each lane. ACT1 mRNA levels are reproducibly decreased in spt6-1004, but ACT1 does not exhibit shorter transcripts (15); therefore, ACT1 serves as an internal control for an spt6-1004 mutant phenotype. (B) Northern analysis for SPB4, STE11, and VPS72 in strains grown at the nonpermissive temperature (39°C). (C) Northern analysis of FLO8 and RAD18 with probes specific for the 5′ and 3′ coding regions of each gene.

To examine this effect in greater detail, we focused on the short FLO8 transcript. To map the 5′ end of the short FLO8 transcript, we used primer extension and RACE (rapid amplification of cDNA ends) analysis [Fig. 2A and (15)]. By primer-extension analysis, we detected a cluster of 5′ ends for FLO8 at +1679, +1684, and +1685 with respect to the FLO8 ATG (Fig. 2A). These 5′ ends were the only ones observed by primer extension between positions +1500 and +1820. RACE analysis also identified 5′ ends in this region (15).

Fig. 2.

Characterization of the FLO8 short transcript. (A) Primer extension of the FLO8 short transcript was performed to detect FLO8 transcripts with 5′ ends in the coding region. The level of ADH1 mRNA was measured as a control for the amounts of RNA used for the SPT6 and spt6-1004 strains. The FLO8 coding sequence that contains the internal initiation site is shown below the gels. The 5' ends found by primer-extension analysis are indicated by the arrows. The consensus TATA box is underlined. (B) ChIP analysis of the FLO8 internal promoter. ChIP analysis was performed for TBP and RNA Pol II. A schematic of FLO8 is shown, with the black bars representing the regions amplified by polymerase chain reaction (PCR). The control region is on chromosome V, in a region lacking open reading frames (32). The graphs represent the ratio [(ChIP region n/ChIP control) ±SD of three independent experiments (30°C) or six independent experiments (39°C)]. Student's t test (two-tailed, equal variance) P values for spt6-1004 ChIPs for TBP and Rpb3 at 39°C, relative to ChIP values in SPT6 at 30°C or 39°C or spt6-1004 at 30°C, are all <0.006. (C) Northern analysis of a flo8 internal TATA mutation. The flo8-100 mutation changes the internal FLO8 TATA sequence at position +1626 from TATAAA to CCTAGG.

Several lines of evidence suggest that the FLO8 short transcript arises from initiation by RNA Pol II rather than by processing of the wild-type FLO8 mRNA. First, examination of the FLO8 DNA sequence identified a consensus TATA sequence starting 54 base pairs 5′ of the +1679 site (Fig. 2A). In addition, our microarray analysis was performed with poly(A)+ selected mRNA, and the RACE protocol relies on the presence of a 5′ methylated guanosine (5′-meG-cap) and 3′ poly(A) tail. Finally, we performed chromatin immunoprecipitation (ChIP) to examine the association of RNA Pol II and the TATA box–binding protein (TBP) near the FLO8 putative internal initiation sites. The results showed a modest, but statistically significant, increase in the levels of both RNA Pol II and TBP association with the internal FLO8 initiation region specifically in the spt6-1004 mutant after incubation at nonpermissive temperature (Fig. 2B). In addition, we tested whether the production of the short FLO8 transcript requires the internal TATAAA sequence at position +1626. Mutation of this TATAAA to CCTAGG abolishes production of the short transcript in an spt6-1004 mutant (Fig. 2C). These data suggest that in spt6-1004, TBP binding to the TATAAA at +1626 allows aberrant transcription initiation within FLO8.

To determine whether these aberrant transcripts were specific to spt6 mutants, we tested other mutants for production of the short FLO8 transcript. We examined several mutants that have been shown to allow activation of the S. cerevisiae SUC2 promoter lacking its upstream activation sequence (UAS) region (SUC2ΔUAS) (11), as both production of the FLO8 short transcript and expression of SUC2ΔUAS appear to be cases of expression from minimal promoters. Northern analysis shows that mutations in SPT10, SPT21, SPT16, BUR1, BUR2, and the histone H3-encoding gene HHT1 cause production of the short FLO8 transcript (Fig. 3). In contrast, mutations in SPT4, SPT5, MOT1, and BUR6 do not [Fig. 3 and (15)].

Fig. 3.

Northern analysis of FLO8 transcripts in mutant strains related to spt6. The top panel shows RNA prepared from strains incubated at 39°C for 80 min after growth at 30°C and the bottom panel is RNA from strains grown at 30°C. For the spt6-1004 and spt16-197 temperature-sensitive mutants, the level of the short FLO8 transcript is greater at 39°C.

Because Spt6 has been shown to interact with histones (16, 17) and spt6 mutations have been shown to affect chromatin structure (16), we examined whether spt6-1004 causes an alteration in chromatin structure at the nonpermissive temperature, coincident with cryptic promoter usage. To do this, we assayed chromatin from an spt6-1004 strain for sensitivity to micrococcal nuclease (MNase) when incubated at nonpermissive temperature. The results show that spt6-1004 chromatin exhibits an increased sensitivity to MNase compared with SPT6 chromatin (Fig. 4A). This increased MNase sensitivity is similar to that observed in previous studies when histones were depleted (1820).

Fig. 4.

MNase and ChIP analysis. Cells were grown at 30°C, shifted to 39°C, and samples were prepared as described in Materials and Methods and table S1. The undigested DNA is present owing to unspheroplasted cells that are inaccessible to MNase. (A) MNase analysis of total chromatin from SPT6, rpb1-1, and spt6-1004 strains. Inverse images of agarose gels stained for DNA with ethidium bromide are shown. (B) MNase analysis of rpb1-1, spt6-1004, and rpb1-1 spt6-1004 strains. The left panel was probed with FLO8, the middle panel was probed with GAL1, and the right panel is total chromatin, stained for DNA with ethidium bromide. Gels were loaded to obtain equal levels of MNase-accessible chromatin. (C) ChIP analysis of histone H4 levels over FLO8 and GAL1. The values shown are calculated from three independent experiments as described in the legend for Fig. 2B. M, molecular mass markers.

Spt6 has been shown to be physically associated with transcribed regions (35, 13, 15). Furthermore, it has been proposed that Spt6 facilitates RNA Pol II passage through nucleosomes or restores normal chromatin structure in the wake of RNA Pol II transcription (2). Therefore, we tested whether transcription is required to cause the increased MNase sensitivity observed in spt6-1004 mutants. First, we examined the MNase sensitivity of FLO8 and an untranscribed gene, GAL1. Second, we constructed double mutants containing spt6-1004 and rpb1-1, a temperature-sensitive mutation in the gene encoding the largest subunit of RNA Pol II. After a shift to the nonpermissive temperature, the rpb1-1 mutation causes a rapid shutoff of transcription (21). Our results (Fig. 4B) show that the spt6-1004–dependent MNase hypersensitivity is severe over FLO8, but it does not significantly occur over the repressed GAL1 gene. Furthermore, the hypersensitivity is suppressed by the rpb1-1 mutation, evident both in total chromatin (Fig. 4B, right panel) and at FLO8 (Fig. 4B, left panel). The rpb1-1 mutation alone causes no detectable effect on MNase sensitivity (Fig. 4A). In contrast, inhibition of translation by cycloheximide has no effect on MNase sensitivity in an spt6-1004 background (Materials and Methods; fig. S1). These results are consistent with the hypothesis that the increased MNase sensitivity observed in the spt6-1004 mutant is dependent on RNA Pol II transcription.

The MNase hypersensitivity in an spt6-1004 mutant could be caused by either loss of nucleosomes or an altered nucleosome structure. To distinguish between these possibilities, we measured the level of histone H4 at FLO8 by ChIP in wild-type and spt6-1004 strains. Our results demonstrate that the level of H4 is significantly reduced over FLO8 specifically in the spt6-1004 mutant (Fig. 4C, compare lanes 3 and 4 to 7 and 8). Furthermore, this loss does not occur at GAL1, a sequence that is not transcribed.

Our results show that the elongation factor Spt6 is required to repress transcription initiation from a cryptic promoter within FLO8 and that such repression likely occurs at cryptic promoters throughout the genome. These findings support a model in which Spt6 promotes the restoration of normal chromatin structure in the wake of RNA Pol II elongation (2, 22). By this model, in an spt6 mutant, RNA Pol II elongation causes a global disruption of chromatin structure, creating a permissive chromatin environment for transcription initiation from cryptic sites that fortuitously contain a TATA element and proximal initiation site. Other investigators have also proposed that the passage of RNA Pol II alters chromatin structure [reviewed in (1, 2326)]. Our model is also analogous to that in studies demonstrating that intergenic transcription in the human β-globin locus remodels chromatin to allow gene expression (27).

Our model for transcription from cryptic initiation sites can be applied to previously observed phenotypes for spt6 mutants. First, the initiation from cryptic promoters within coding regions is similar to the previous demonstration that mutations in SPT6 and several other genes allow transcription initiation from a yeast promoter lacking its UAS (6, 11, 28, 29). Our model may also explain the isolation of mutations that impair transcription elongation factors, including Spt6, as suppressors of a transcription initiation defect of his4-912δ, an insertion of the long terminal repeat of a Ty retrotransposon in the HIS4 promoter (30, 31). Similar to transcription initiation from within coding regions, suppression of his4-912δ may also be a case of promoter activation by transcription elongation–mediated changes that occur in spt6 mutants.

Our results indicate that chromatin-mediated repression of promoter usage operates on a scale larger than previously thought and that RNA Pol II and transcription elongation factors play important roles in determining chromatin structure within transcribed regions. By maintaining a specific chromatin structure over transcribed regions, RNA Pol II, Spt6, and other factors prevent improper initiation that would be harmful to normal gene expression. Although our study was performed exclusively in S. cerevisiae, prevention of such aberrant transcription is likely to be at least as important in larger eukaryotes with more complex genomes.

Supporting Online Material

Materials and Methods

Fig. S1


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