We have previously shown that tiRNAs isolated from THP-1 cells (a human monocytic leukemia cell line) are systematically enriched at white blood cell CTCF binding sites [9
]. To examine if this relationship is preserved across cell and tissue types, and multiple species, we interrogated small RNA enrichments at CTCF binding sites in MCF-7 breast cancer cells and mouse embryonic stem cells (mESCs) (for a full list of data sources please see (Additional file 1
, Table S1).
Consistent with prior work we found that tiRNAs derived from both MCF-7 and mESCs are enriched approximately sixfold at CTCF binding sites that sit outside TSSs or other annotated genomic features (see Methods and Figure ), and show the characteristic 18 nucleotide tiRNA peak (Figure ). When CTCF binding sites were further refined to include only sites coincident with RNA polymerase II binding (CTCF-RNAPII sites), tiRNA enrichments increased considerably, to approximately 45-fold. Indeed, more than 50% and 20% of MCF-7 and mESC CTCF-RNAPII sites intersect with tiRNAs, respectively (Tables and ). This relationship appears to bridge the reports indicating that tiRNA biogenesis is a direct result of RNAPII backtracking and nascent transcript cleavage [4
], and recent studies showing that CTCF is directly involved in RNAPII function. Indeed, it has now become clear that (i) a subpopulation of CTCF directly interacts with the large subunit of RNAPII through it's phosphorylated C-terminal tail [21
], (ii) that in some cases a single CTCF site is both necessary and sufficient to drive RNAPII transcription in the absence of canonical promoters by recruitment of RNAPII [21
], and (iii) that CTCF specificity for, and regulation of, transcriptionally competent complexes also extends to RNA polymerase I [18
Figure 1 Enrichment of transcription initiation (ti)RNAs at CCCTC-binding factor-RNA polymerase II (CTCF-RNAPII) sites in human MCF-7 and mouse embryonic stem (mES) cells. (a) Enrichment of tiRNAs at CTCF sites in MCF-7 and mES cells compared to 1,000 simulated (more ...)
MCF-7 CCCTC-binding factor (CTCF), RNA polymerase II (RNAPII) and CTCF-RNAPII site intersections with small RNAs (sRNAs)
Mouse embryonic stem (mES) CCCTC-binding factor (CTCF), RNA polymerase II (RNAPII) and CTCF-RNAPII site intersections with small RNAs (sRNAs)
To examine if the association between tiRNAs, CTCF and RNAPII extends beyond MCF-7 and mESCs we identified CTCF sites conserved across an additional eight human cell lines (GM12878, HepG2, HMEC, HSMM, HUVEC, K562, NHEK, and NHLF cells) [23
] and RNAPII sites conserved across three (K562, GM12878, and HUVEC), and intersected them with nuclear and cytoplasmic small RNAs (sRNAs) from THP-1 and 5-8f cells (a nasopharyngeal carcinoma cell line [25
]) and MCF-7 total sRNAs. Despite the fact that these datasets are derived from disparate origins, nuclear sRNAs from THP-1 and 5-8f are 33-fold and 16-fold enriched, respectively, and total sRNAs from MCF cells are 31-fold enriched at conserved CTCF-RNAPII sites (Additional file 2
, Figure S1a). Additionally, like the MCF-7 and mESC datasets discussed above, the small RNAs that overlap CTCF-RNAPII sites are dominantly 18 nucleotides, indicating they are tiRNAs (Additional file 2
, Figure S1b-d). Overall, greater than 10% of the conserved CTCF sites, and 60% of conserved CTCF-RNAPII sites, overlap with sequences that generate tiRNAs (Additional file 3
, Table S2). To further ensure that the tiRNA enrichment at CTCF-RNAPII sites was robust we parsed the conserved human CTCF sites into two groups by origin, 'cancer' and 'normal', and removed all CTCF sites that overlapped with TSSs, repeat masker annotations and small RNAs. Using the most robust RNAPII datasets in each group (MCF-7 and HUVEC for 'cancer' and 'normal', respectively), we found that this dramatically reduced set still shows robust enrichment for tiRNAs at CTCF-RNAPII sites (Additional file 4
, Figure S2).
To experimentally interrogate the tiRNA-CTCF-RNAPII relationship we queried for sites in clinically relevant genes and identified a CTCF-RNAPII site with tiRNAs in the first intron of p21
, which is conserved across both multiple human cell types (Figure ) and mammalian species (Additional file 5
, Figure S3). CDKN1A/p21 is a significant tumor suppressor that acts at the G1 checkpoint to inhibit cell cycle progression [26
], and its downregulation (but not mutation) is a common feature of many cancers [30
]. In addition to p21
mRNA, the p21
locus encodes a number of other transcripts, including alternative p21 transcripts (p21alt
) that originate from a unique promoter located approximately 2 kb upstream of the canonical p21
transcription start site and include the majority of the p21
coding regions in their final spliced products [35
], and a long non-coding antisense RNA (bx332409
) that regulates local epigenetic states [36
] (Figure ).
Figure 2 Schematic depicting the p21 (cyclin-dependent kinase inhibitor 1A gene, also known as CDKN1A) locus. The blue lines, boxes and arrows at the top of the image show the p21 and antisense transcripts. Small black boxes connected by black lines depict the (more ...)
locus encodes two tiRNA clusters, one at the TSS (tss-tiRNAs) and the other at the CTCF-RNAPII site (CTCF proximal (cp)-tiRNAs) that are antisense to one another. The tss-tiRNAs are sense to the gene (as observed generally), while the cp-tiRNAs are antisense. Both overlap distinct peaks of RNAPII binding, suggesting that their biogenesis is tied to RNAPII molecules heading in opposite directions, possibly linked to nucleosome position [4
], and this reinforces our previous finding that tiRNAs are found at sites of active RNAPII transcription initiation outside of canonical transcription start sites (Figure ).
To investigate the function of p21 tiRNAs, we utilized short antisense 'sponge' RNAs [37
] that were designed to bind and inhibit tss-tiRNAs and cp-tiRNAs (Figure ). MCF-7 cells transfected with the cp-tiRNA sponge demonstrated a significant increase of p21
mRNA and p21alt
expression, as measured by quantitative PCR (qPCR) (Figure ). In contrast, the tss-tiRNA sponge did not exhibit a detectable effect on p21
expression (Figure ), and thus cp-tiRNAs became the focus of the remainder of this study.
Figure 3 The effects of transcription initiation (ti)RNA sponges and mimics on p21 (cyclin-dependent kinase inhibitor 1A gene, also known as CDKN1A) expression. (a) Transfection by the CCCTC-binding factor (CTCF) proximal (cp)-tiRNA sponge resulted in an increase (more ...)
As reverse transcription in the qPCR samples was not specifically primed (Figure ), these transcripts might represent sense and/or antisense transcripts associated with these regions [36
] or any of the plethora of splice variants. To determine the extent of the effect that the cp-tiRNA sponge has on relative sense and antisense p21
transcript levels, strand-specific reverse transcription PCR (RT-PCR) was performed. Upon treatment with the cp-tiRNA sponge, p21
mRNA, sense p21alt
, and antisense p21alt
transcript levels increased, whereas transcripts antisense to p21
mRNA were unaffected (Figure ). These data indicate that CTCF-proximal tiRNAs may be involved in the negative regulation of p21.
We next performed the reciprocal experiment testing the effect that overexpression of CTCF-proximal tiRNA mimics has on p21 expression. Consistent with our speculation that tiRNAs are connected to transcriptional regulation, we found that overexpression of a set of four cp-tiRNA mimics resulted in a marked reduction of the p21
mRNA (Figure ). To confirm that the effect of the cp-tiRNA sponges and mimics was not restricted to MCF-7 cells we repeated these experiments in THP-1 cells and found that the principal results were recapitatulated (Additional file 6
, Figure S4), indicating that cp-tiRNAs have a regulatory effect on p21
transcription in multiple human cell systems.
To further investigate the effects of cp-tiRNA sponge and mimics on p21 transcription, elongating forms of RNAPII were assessed by chromatin immunoprecipitation-PCR (ChIP-PCR). The only signal increase appeared in regions overlapping p21alt, although that increase was modest (Figure ), suggesting that cp-tiRNAs do not function by affecting local RNAPII densities, but rather by directly or indirectly modulating local chromatin architecture.
Figure 4 RNA polymerase II (RNAPII) response to the p21 (cyclin-dependent kinase inhibitor 1A gene, also known as CDKN1A) CCCTC-binding factor (CTCF) proximal (cp)-transcription initiation (ti)RNA sponge. Active RNAPII enrichment sites in the p21 (CDKN1A) locus (more ...)
To explore this possibility we examined the effects of cp-tiRNAs sponge and mimic constructs on CTCF localization, and on epigenetic marks at various locations within the p21
locus by ChIP. The density of the silent state chromatin mark, H3K27me3, did not change upon introduction of cp-tiRNA sponge or mimic constructs at their perfectly complementary target sites (that is, at sites of tiRNA biogenesis; Figure ), as would be expected if the cp-tiRNA mimic or sponges were themselves altering local chromatin status, as has been observed previously with small non-coding RNAs associated with transcriptional gene silencing [38
]. However, H3K27me3 levels upstream of the p21alt
transcription start site were decreased upon cp-tiRNA sponge treatment (Figure ). Given that the distance between the site of tiRNA biogenesis and the p21alt
promoter is greater than 6 kilobases, we speculated that these effects are facilitated by tiRNA-mediated regulation of other epigenetic regulators capable of acting at long distances.
Figure 5 CCCTC-binding factor (CTCF) binding and H3K27 trimethylation (H3K27me3) at p21 (cyclin-dependent kinase inhibitor 1A gene, also known as CDKN1A) in response to the CTCF proximal (cp)-transcription initiation (ti)RNA sponge and mimics. (a) Transfection (more ...)
Consistent with this, treatment with the cp-tiRNA sponge resulted in a significant increase in CTCF binding (Figure ), and overexpression of the cp-tiRNA mimics exhibited a significant decrease of CTCF binding (Figure ). This indicates that the effect of cp-tiRNAs on p21 transcription is directly related to its ability to modulate CTCF binding, which may be involved in three-dimensional (re)ordering of the p21 locus. Indeed, western blot analysis showed that p21 protein levels were increased in samples treated with the cp-tiRNA sponge, and decreased in samples treated with the cp-tiRNA mimic constructs (Figure ). Taken together, these data suggest that one function of p21 cp-tiRNAs may be to inhibit CTCF binding to the p21 gene, possibly as a means to repress transcription and downstream translation.
To test whether cp-tiRNAs can modulate CTCF binding at other loci we generated sponges for cp-tiRNAs derived from an intergenic region downstream of the C2orf42
chromosome 2 open reading frame 42), and an intergenic site upstream of StAR-related lipid transfer domain containing 13 (STARD13
) (Additional file 7
, Figure S5). To ensure that selection bias did not affect our study, these sites were chosen at random from approximately 900 sites with strong CTCF binding and tiRNA conservation across cell lines (see Methods). Examination of the C2orf42
site revealed no significant effect of tiRNA sponges (Additional file 8
, Figure S6). However, we observed that STARD13
cp-tiRNA sponges resulted in a reduction in STARD13
mRNA expression, in spite of the fact that CTCF binding was largely unaffected (Figure ). This cp-tiRNA-mediated sponge effect is contrary to that observed for p21, which strongly increased p21 expression. To further investigate this we examined local nucleosome density at both loci and found that the p21
cp-tiRNA sponges induced increased nucleosomal localization, while the STARD13
cp-tiRNA induced a decrease in nucleosomal localization (Figure ). This is consistent with our hypothesis that cp-tiRNAs mimics and sponges facilitate condition dependent small-scale rearrangements to nucleosome order, and that this in turn leads to large-scale chromatin reorganization orchestrated by CTCF or other DNA binding and chromatin modifying complexes. Indeed, recent work has shown that an array of up to 20 well positioned nucleosomes enriched for the transcription initiation mark H3K4me3 flank CTCF sites, a phenomenon previously only observed downstream of TSSs [39
]. This finding not only potentially explains why tiRNAs are frequently found at CTCF sites, but also suggests that the contradictory p21
tiRNA sponge effects may result from changes to the local density of chromatin activating marks (Figure ).
Figure 6 The effect of a CCCTC-binding factor (CTCF) proximal (cp)-transcription initiation (ti)RNA sponge on StAR-related lipid transfer domain containing 13 (STARD13). (a) The effect of the STARD13 sponge on CTCF at the CTCF binding site. (b) The effects the (more ...)
Figure 7 A schematic representation of transcription initiation (ti)RNA sponge effects on the p21 (cyclin-dependent kinase inhibitor 1A gene, also known as CDKN1A) and StAR-related lipid transfer domain containing 13 (STARD13) loci. (a) The p21 CCCTC-binding factor (more ...)
The mechanism by which tiRNAs inhibit CTCF localization is unclear, although there are several obvious possibilities: (i) cp-tiRNAs spanning the CTCF binding site may coat local chromatin by binding nascent transcripts [36
] or chromatin associated RNAs [40
], which could sterically hinder CTCF from accessing its binding site; (ii) cp-tiRNAs may directly interact with CTCF and inhibit CTCF binding, although attempts to immunoprecipitate CTCF with biotin-linked cp-tiRNAs were unsuccessful (data not shown); (iii) cp-tiRNAs may bind to regulatory elements including cis-acting ncRNAs (for example, bx332409
) or polycomb group components and direct their action to specific sites; or (iv) cp-tiRNAs may serve as sequence-specific markers for chromatin modification complexes.