Our datasets and analyses address the scope and regulation of human RNA Polymerase III transcriptomes, providing multiple insights. First, we observe the close proximity of Pol III genes to Pol II genes genome-wide. This result is in keeping with previous work at the Pol III-transcribed U6
gene (a Type 3 gene), which has proximal Pol II that assists in Pol III expression34
. Second, we show genome-wide a highly significant overlap of Pol III with active chromatin. Here, previous work at U6
supports the notion that nearby chromatin remodeling promotes U6
, and our work extends this concept genome-wide to all Type 2 and 3 genes, and also to many active histone modifications and composition. However, most occupied Pol III genes reside at regions outside of annotated Pol II genes—yet those regions still bear high levels of H3K4me3 and Pol II protein, properties typical of promoters. Interestingly, these unannotated regions also have properties of enhancers27
, as they contain H3K4me1, H3K27ac, and overlap with enhancer-binding proteins and DNase I hypersensitive sites. In addition, we find few, if any, transcripts adjacent to most of these unannotated Pol II peaks, and they generally lack a long downstream open reading frame. Thus, it is not entirely clear whether one should consider these regions new unannotated promoters, or instead a sub-class of enhancers that contain Pol II and H3K4me3, resembling Pol II ‘poised’ promoters. Here, we speculate that the promoter-like chromatin formed nearby either ‘poised’ or active Pol II might be sufficient for enabling Pol III occupancy. In addition, it will be of interest to determine whether these unannotated promoters/enhancers produce a functional transcript in particular cell types, or whether they are typical enhancers, activating another Pol II gene in the larger region. Regardless, the overlap of Pol III-occupied tDNAs with active chromatin is striking. Notably, Pol III occupancy scaled with active chromatin marks and proximal Pol II, but not with RNA transcript levels for the Pol II gene, emphasizing the connection of Pol III occupancy with active chromatin. Finally, DNA hypomethylation may also contribute to the active chromatin state, as occupied Pol III genes correlated with high CpG content regions (which are typically unmethylated) and as STAT1 consensus sites are strongly correlated with DNA hypomethylation36
Occupied Pol III genes often reside 300–900 bp upstream of the Pol II TSS—close enough to overlap with promoter proximal chromatin, but generally not within the promoter proximal region where the Pol II basal machinery assembles. Furthermore, tRNAs residing in Pol II promoters are typically transcribed away from the Pol II gene (divergent orientation), with a significant bias (p-value 0.006). We suggest that these properties allow the Pol III gene to benefit from promoter chromatin dynamics while avoiding interference with the transcription of the Pol II gene itself. We note that previous Pol III transcriptomes in S. cerevisiae10,37,38
showed that virtually all predicted Pol III genes (including tDNAs) were occupied by Pol III, arguing against appreciable Pol III regulation by chromatin or position relative to Pol II genes. Moreover, S. cerevisiae
lacks key modifications of vertebrate heterochromatin (H3K9me3, H3K27me3, DNA methylation), suggesting that Pol III in human cells may encounter chromatin obstacles not present in lower yeasts.
Our work also reveals moderate variation in Pol III occupancy among cell types, and cell-type ‘specific’ occupancy of a small number of loci. Here, we note that specificity is defined using a stringent criterion, but very low occupancy of these ‘specific’ loci may exist in other cell types. A key issue is the basis for cell-type variation and specificity. One possibility is that as each cell type varies its repertoire of Pol II gene transcription and active enhancers, the Pol III genes overlapping that permissive chromatin gain access to their machinery. By this model, Pol III relies on both general Pol II transcription factors and cell-type specific factors to create open/active chromatin. Although active chromatin may gate Pol III access, it does not constitute all of Pol III regulation—the activity of occupied Pol III genes is likely still regulated by other factors such as the general Pol III repressor Maf139
. Furthermore, we emphasize that these models are based on extensive sets of strong correlations, but genetic experiments are required to determine their dependency relationships.
Interestingly, tDNAs are thought to have expanded in the genome via retrotransposition40
, and retrotransposons often insert in regions of open chromatin. One interpretation of our data is that the juxtaposition of active Pol III genes (~300) with active Pol II chromatin is largely a consequence of this initial accessibility during transposition, which would imply that these regions were accessible in the germline at some point during evolution. However, there are an additional ~1396 tRNA-derived elements in the genome, and these are generally not occupied by Pol III machinery (~0.1% occupied). Furthermore, as a class these elements are not coincident with active chromatin (data not shown), raising the possibility that transposition may have occurred in the germline into inactive chromatin, with inactive chromatin preventing their subsequent expression and contribution to fitness, allowing sequence drift. Alternatively, the transposition may have occurred into active chromatin, but these regions were later converted into heterochromatin, with similar consequences. Regardless, we observe active chromatin coincident with active Pol III genes, and not with tRNA-derived elements.
Our work also reveals many new Pol III-occupied loci in multiple cell types, which also require functional work in vivo
. For the three new loci clearly enriched with Pol III machinery (Supplementary Fig. 5
), we propose new names with a ‘P3’ (Pol III) designation: the MIR
in the POLR3E
; the chr8 locus conserved in primates, CPP3
; LINE L1M5
. Furthermore, we show the transcription of a miRNA, clarifying and extending earlier work30,32
. Here, it will be of interest to determine the Pol III transcriptomes of pluripotent cell lines and/or early embryos to determine if additional noncoding RNAs are produced by Pol III.