A group of interesting papers popped up on ScienceExpress this past week. These papers (by Core et al., Seila et al., He et al., and Preker et al.) all describe characterizations of unusual patterns of transcription in human cells. The bottom line (well, one bottom line – there are lots of interesting data in these studies, and the nuances may take readers in slightly different directions) is that, for numerous promoters, transcription extends in both directions, not just in the one direction that is usually associated with productive (= leading to synthesis of a processed and translated mRNA) transcription. Moreover, this bidirectional transcription is quite distinct from that associated productive transcription, in that it yields short and relatively unstable RNAs. More elaboration follows below the fold. As always, enjoy.
These four reports describe different approaches towards detailed analyses of transcription. Core et al. purified RNA being synthesized by transcriptionally-engaged RNA polymerases by incorporating a nucleotide analog (5-bromouridine) that could be purified using antibodies specific for the analog; this is an adaptation of the commonly-used nuclear run-on method. The purified RNA was then worked up for sequencing using Solexa technology, and the results “placed” on the human genome. Seila et al. came across this phenomenon in the course of deep-sequencing of small RNAs in human cells; in this case, RNAs were size-selected and adapted (by various and sundry steps of ligation, cDNA synthesis, and amplification) for Solexa sequencing. Because they were interested in microRNAs, they initially focused on molecules about 21 nts in length; however, they later found that the subjects of interest were more disperse in length than the typical microRNA (and were synthesized independent of Dicer, thus indicating that they were not canonical small RNAs). Preker et al. compared the expression of genes present on the so-called ENCODE tiling array in cells containing and lacking a core subunit of the so-called exosome (the complex of 3′->5′ exonucleases that helps to break down RNAs in the cell). Their goal was to get a better idea of the scope of transcription that gives rise to unstable RNAs (analogous to the Cryptic Unstable Transcripts found by Wyers et al. in yeast; see this essay for more on such studies). Finally, He et al. sequenced bisulfite-treated RNA so as to easily identify the strand to which the RNA correspond. The idea is that bisulfite treatment converts C’s to U’s; after cDNA synthesis and sequencing of treated RNAs (again by Solexa sequencing), the sequences will only match one of the two strands of the corresponding gene, and only after the gene has been “modified” by the computer.
The theme that runs through these four papers is fascinating, as it suggests that transcription may proceed bidirectionally at most promoters, even those that are not parts of bidirectional transcription units (pairs of adjacent genes that are transcribed into translated RNAs in opposite directions). The unusual transcription products these groups find are short, but are not canonical small RNAs. Moreover, based on the similarity with CUTs, it would seem as if these small RNAs themselves are not “functional” in the sense usually associated with mRNAs. Rather, this phenomenon seems to hint at hitherto overlooked aspects of promoter and chromatin structure and function. Along these lines, the summary paragraphs of the four papers are interesting to ponder and compare.
As stated by Core et al.:
“We envision several possible functions for divergent transcription. First, the act of transcription itself could be crucial for granting access of transcription factors to control elements that reside upstream of core promoters, possibly by creating a barrier that prevents nucleosomes from obstructing transcription factor binding sites (20, 21). Second, as proposed by Seila et al. (14), negative supercoiling produced in the wake of transcribing polymerases could facilitate initiation in these regions. Third, these short nascent RNAs could themselves be functional–through either Argonaute -dependent (22) or –independent (23) pathways. Upcoming challenges will be to decipher whether the widespread transcriptional activity that lies upstream but divergent from the direction of coding genes positively or negatively regulates transcription output, and how promoter or unknown DNA elements are designed to distinguish between productive elongation in one direction versus the other.”
Seila et al. say:
“RNAPII initiation complex polarity at promoters is thought to be established by TFIID/TBP complex binding together with TFIIB (14). RNAPII/TFIIF binding and DNA unwinding by the TFIIH helicase then gives rise to the open preinitiation complex (9). The prevalence of divergently oriented RNAPII at most promoters suggests a more complex situation. We hypothesize that transcription factors first nucleate a sense oriented preinitiation complex at the TSS. Transcription by this complex generates at least two signals that could subsequently promote upstream antisense paused polymerase. First, the RNAPII carboxy-terminal domain and other initiation complex components can activate transcription when tethered to DNA, suggesting that the sense complex may promote antisense preinitiation complex formation in the upstream region (15). Second, as RNAPII elongates the sense transcript, negative supercoiling of the DNA will occur upstream, perhaps promoting the antisense initiation process (16). This divergent transcription could structure chromatin and nascent RNA at the TSS for subsequent regulation. “
He et al. say:
“Our results raise many questions about the genesis and metabolism of antisense transcripts. It has been hypothesized that antisense transcripts are widely and promiscuously expressed, perhaps because of weak promoters distributed throughout the genome [reviewed in (25, 26)]. Our data argue against this hypothesis in human cells: Promiscuous expression would lead to a uniform distribution of antisense tags across the genome, whereas the observed distribution was nonrandom, localized to genes and within particular regions of genes, much like sense transcripts (Fig. 1 and figs. S2 and S8). This distribution is consistent with a model wherein many antisense transcripts initiate and terminate near the terminators and promoters, respectively, of the sense transcripts. Some of the apparent antisense transcripts from a gene on the plus-strand could actually be sense transcripts originating from unterminated transcription of a downstream gene on the minus-strand (or vice versa). However, this idea is not generally supported by the poor correlation between antisense tag density within a gene and the density of sense tags from the closest downstream gene (fig. S13). One explanation for the higher density of antisense tags in transcribed regions is that transcription of the sense transcripts from correct initiation sites would reduce nucleosome density throughout the entire transcribed region, thereby increasing DNA accessibility and hence the likelihood of nonspecific transcription (26). This is unlikely, given that genes with high sense tag densities did not generally have high antisense densities. There is substantial evidence that sense transcripts can be negatively regulated by antisense transcripts (3–7). Such regulation can occur either by transcriptional interference or through posttranscriptional mechanisms involving splicing or RNA-induced silencing complexes (RISC). Our data support the possibility that antisense-mediated regulation affects a large number of genes. “
Finally, Peker et al. note:
“PROMPTs may arise wherever open chromatin presents itself, possibly as the byproduct of an as yet unexplored aspect of the mechanism of gene transcription. Evolution, being an opportunistic force, may then have co-opted at least some of these PROMPTs as part of regulatory mechanisms (fig. S11). One such molecular system could involve the control of CpG (de)methylation, an as of now poorly understood process (13). An alternative, but not mutually exclusive, possibility is that PROMPT transcription may have a more general function by providing reservoirs of RNAPII molecules, which can facilitate rapid activation of the downstream gene and/or by serving to alter chromatin structure. Clearly, the generality of the PROMPT phenomenon hints at a more complex regulatory chromatin structure around the TSS than was previously anticipated. “
(PROMPTs = Promoter Upstream Transcripts)
I’ll update this essay with citations and links when the papers hit the newstand.
The papers are out, in the Dec. 19 isue of Science. The links above are still valid.