Comparison of nucleosome occupancy around TSSs between S. pombe and S. cerevisiae revealed both similarities and differences [8]. While in S. cerevisiae
nucleosomal arrays are present both up- and downstream of the TSS in TSS-aligned composite nucleosome occupancy plots, a nucleosomal array solely downstream of the TSS is present in S. pombe. When plotting nucleosome occupancy for a large number of genes aligned at TSSs, upstream nucleosomal arrays are only visible when they are in register with the alignment point, i.e. the TSS. This means that even if upstream nucleosomal arrays are not present in composite nucleosome occupancy plots, they can still be present at individual loci, but just not in register with the TSS. Thus, from S. pombe composite nucleosome occupancy plots one cannot judge if upstream nucleosomal arrays are really not present at individual loci or if they are just not in register with the TSS.
Detailed transcriptome mapping of S. cerevisiae wildtype and exosome mutant (rrp6Δ) strains uncovered that transcription is quite pervasive in the S. cerevisiae
genome [217]. For example, 36% of all annotated elements were found to start within a shared 5’ NDR. Therefore, we wondered if appearance of upstream nucleosomal arrays in S. cerevisiae can be explained by upstream transcription. Thus, we took the annotation of transcriptional elements either initiating at the same NDR as downstream elements initiate (i.e. bidirectional/divergent genes) or ceasing at the same NDR as downstream elements initiate (tandem genes) of Xu et al. [217] and plotted in vivo (Fig. 7A) and in vitro reconstituted (Fig. 7B) S. cerevisiae nucleosome occupancy data [2] separately for genes of these two categories and for genes which do not fall in any of these two categories. Genes with bidirectional and tandem partners showed indeed very clear nucleosomal arrays, while upstream nucleosomal arrays of genes without such a partner were rather faint.
We next asked if the absence of upstream nucleosomal arrays in S. pombe can be explained accordingly. For this analysis we employed a list of TSSs annotated for open reading frames (ORFs) in wildtype [8], a list of TSSs annotated for non-coding transcripts in wildtype [218] and a list of cryptic unstable transcripts (CUTs) in a rrp6Δ mutant for a large part of chromosome II [219]. Rrp6 is a ribonuclease subunit of the exosome [220]. The exosome is responsible for RNA degradation connected to all kinds of cellular processes like RNA maturation, RNA quality control or degradation of unstable RNAs. Destruction of functional exosomes by depletion of one of its subunits allows mapping of transcripts, which are usually rapidly degraded by the exosome, so called CUTs. We first computationally defined transcripts either initiating within a common 500 bp window (potential bidirectional/divergent genes) or initiating and ceasing within a common 500 bp window (potential tandem genes). Subsequently, for the coding and non-coding genes (not for CUTs as TSSs were not
Results
assigned), we used our nucleosome occupancy data to check by eye if those transcripts share a NDR, and thus are really bidirectional or tandem genes. From this analysis we obtained a list of 564 bidirectional genes (both bidirectional partners counted separately) or 282 bidirectional promoters (shared promoters counted) and 386 tandem genes. Furthermore, for 128 ORFs, CUTs lying within a 500 bp window from the ORF’s TSS could be mapped. Thus, only about 15% of annotated elements seem to share a 5’ NDR in S. pombe.
However, for the analysis a list of CUTs annotated for only a large part of chromosome II and not the whole genome was used. Furthermore, also the list of non-coding transcripts is very likely not complete. To make reliable statements on the pervasiveness of transcription in the S. pombe genome, more complete transcriptome data are needed.
As with the S. cerevisiae data, we plotted nucleosome occupancy separately for bidirectional elements, tandem elements and remaining elements (Fig. 7C). For nucleosome occupancy data generated by MNase-chip upstream nucleosomal arrays of genes with bidirectional or tandem partners were only slightly pronounced compared to genes without such partners. We wondered if TSS-TSS distances for bidirectional promoters and TTS-TSS distances for tandem genes are too heterogeneous in S. pombe, and thus generating nucleosomal arrays of different registers, which would cancel each other. Therefore, we binned bidirectional or tandem transcripts according to their TSS-TSS or TTS-TSS distances, respectively, and plotted nucleosome occupancy for genes in the respective bins. Indeed, such binning uncovered upstream nucleosomal arrays for both, bidirectional and tandem genes (Figure 7E, F). As the employed Affymetrix tiling arrays have only a resolution of 20 bp and as we could detect faint upstream nucleosomal arrays in composite plots of all genes aligned at TSSs when using nucleosome occupancy maps generated by Illumina sequencing, we plotted our high-resolution nucleosome occupancy data for the three transcript classes as well (Fig. 7D). And in fact, upstream nucleosomal arrays were more pronounced for elements with bidirectional partners, however not so much for elements with tandem partners.
Another reason for the worse correlation of two TSSs in bidirectional NDRs and TTS and TSS in tandem NDRs observed in S. pombe in comparison to S. cerevisiae, could also be ascribed to improper TSSs annotations of S. pombe transcriptional elements.
Figure 7 Upstream nucleosomal arrays can be mainly ascribed to upstream transcriptional elements sharing the 5’ NDR of the downstream transcriptional element. (A,B) Nucleosome occupancy profiles divided into 2535 bidirectional genes, 551 tandem genes and 4160 genes which are neither bidirectional nor tandem for (A) S. cerevisiae wt in vivo and for (B) S. cerevisiae in vitro chromatin reconstituted with cell extract and ATP [2]. (C,D) Nucleosome occupancy profiles divided into 274 bidirectional promoters, 276 tandem genes and 3465 genes which are neither for (C) S. pombe wt (Hu303, 5 replicates) mapped by MNase-chip and (D) S. pombe wt (Hu303) mapped by MNase-ChIP- seq. (E,F) Nucleosome occupancy profiles for (E) 39 bidirectional genes with TSS-TSS distance of 1-100 bp and (F) 81 tandem genes with TTS-TSS distance of 101-200 bp for S. pombe wt (Hu303, 5 replicates) mapped by MNase-chip. Nucleosome occupancy plots for genes in remaining bins are not shown.