The nucleosome DNA entry-exit site is important for transcription termination in Saccharomyces cerevisiae

Compared to other stages in the RNA polymerase II transcription cycle, the role of chromatin in transcription termination is poorly understood. Through a genetic screen, we identified histone mutant strains that exhibit transcriptional readthrough of terminators in vivo. Amino acid subtitutions map to the nucleosome DNA entry-exit site. On a genome-wide scale, the strongest H3 mutants revealed increased sense-strand transcription upstream and downstream of Pol II transcribed genes, increased antisense transcription overlapping gene bodies, and reduced nucleosome occupancy particularly at the 3’ ends of genes. Replacement of the native sequence downstream of a gene with a sequence that increases nucleosome occupancy in vivo reduced readthrough transcription and suppressed the effect of a DNA entry-exit site substitution. Our results suggest that nucleosomes can facilitate termination by serving as a barrier to RNA polymerase II progression and highlight the importance of the DNA entry-exit site in maintaining the integrity of the transcriptome.


Introduction
altering specific H2A residues near the DNA entry-exit site on SNR gene transcription by northern blot analysis ( Figure 1F). We observed varying degrees of terminator readthrough in 105 these H2A mutant strains but consistently saw the strongest defects in the H2A H113A, H2A 106 L117A and H2A S121A mutants. The amino acids altered in these H2A mutants map near the 107 positions of the amino acids in H3 that, when mutant, cause strong readthrough phenotypes 108 ( Figure 1D-E).

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To identify the source of the intergenic nascent transcript density in the H3 T45A and H3 R52A 176 mutants, we mapped the spike-in normalized, stranded 4tU-seq reads in the wild-type and 177 mutant strains to protein-coding genes sorted by their relative orientation ( Figure 4A). The data 178 support the conclusion that disruption of the DNA entry-exit site leads to elevated transcription

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In addition to its roles in suppressing sense cryptic initiation, H3 K36me 3 is also required for 218 repressing transcription of a class of antisense transcripts, termed the Set2-repressed antisense 219 transcripts (SRATs) (48). We, therefore, analyzed our RNA-seq data for the H3 T45A and R52A 220 mutants for changes in SRAT expression ( Figure 5B). Both DNA entry-exit site mutants showed 221 upregulation of SRAT expression by greater than four-fold relative to a wild-type control strain

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We noticed that the severity of the termination defects of the H3 T45A and H3 R52A mutants 227 did not correlate with the strength of their H3 K36me 3 defects ( Figures 1D and 5A) Figure 5D). We confirmed that the inviability was not due to lack of expression of the

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In this context, cells retained the ability to express the double mutant H3 protein ( Figure 5E).

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These results argue that the phenotypes of the H3 R52A mutant are, at least in part, 244 independent of its roles in H3 K36me 3 .

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Structural studies implicate histone residues at the DNA entry-exit site in physically interacting 247 with Rtt109, the histone acetyltransferase that catalyzes the modification of H3 at lysine 56 (H3 248 K56ac) (58). This mark on newly synthesized H3 is coupled to histone deposition after DNA 249 repair and replication, replication-independent nucleosome assembly during transcription 250 elongation, and regulation of promoter accessibility during transcription initiation (2). To 251 determine whether residues at the DNA entry-exit site required for transcription termination are 252 also required for H3 K56ac, we assessed the mutant strains for levels of this modification by     Informed by our MNase-seq data, we hypothesized that nucleosome occupancy at termination 323 sites is important for proper termination by Pol II. To directly test this, we integrated a 133 bp 324 "superbinder" DNA sequence, which has high affinity for histones (68), in place of the natural 325 DNA sequence downstream of the SNR48 termination site, as determined by our de novo 326 transcriptome assembly data ( Figure 7A). To monitor nucleosome occupancy at this location, 327 we performed H2A ChIP coupled to qPCR using primers conserved in strains containing and 328 lacking the superbinder sequence ( Figure 7B). These data show that the superbinder sequence 329 increases nucleosome occupancy approximately 3-fold in wild-type cells and 2.5-fold in H3 at the natural sequence between the H3 R52A mutant and the wild-type strain ( Figure 7A

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Using an unbiased genetic screen of a comprehensive histone mutant library (46) and a well-      (29), we note that these mutants differ significantly in the global levels of this mark and 400 that H3 K36A was not identified as a strong candidate in our termination screen. These 401 observations suggest that the DNA entry-exit site contributes to transcription fidelity through a 402 mechanism distinct from and in addition to promoting H3 K36 methylation. In support of this, we 403 found that the H3 R52A substitution is synthetically lethal with deletion of SET2 or the H3 K36A 404 substitution, indicating that these H3 residues function, at least in part, through separate 405 pathways.

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Several residues at the DNA entry-exit site are post-translationally modified. Acetylation of H3 408 K56 is involved in nucleosome assembly following DNA replication, repair, and transcription (2).

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Although structural evidence shows that the acetyltransferase for H3 K56, Rtt109, binds to the 410 DNA entry-exit site near residues identified here (58), our mutants do not display global 411 reductions in H3 K56ac, and the H3 K56A mutant was not identified as termination-defective in 412 our screen. Among the residues identified in our screen, H3 K42 can be methylated in S.

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cerevisiae and replacement of this residue with alanine has been reported to cause a genic transcripts in the H3 T45A and H3 R52A mutants, and likely the H3 K42A strain (60)                            while the long exposure (right) reveals the read-through transcripts (blue arrow). As in Figure   6D, suppression of transcription read-through by the SB sequence is observed.