Pol II and its associated epigenetic marks are present at Pol III-transcribed noncoding RNA genes

Abstract

Epigenetic control is an important aspect of gene regulation. Despite detailed understanding of protein-coding gene expression, the transcription of noncoding RNA genes by RNA polymerase III (Pol III) is less well characterized. Here we profile the epigenetic features of Pol III target genes throughout the human genome. This reveals that the chromatin landscape of Pol III-transcribed genes resembles that of Pol II templates in many ways, although there are also clear differences. Our analysis also uncovered an entirely unexpected phenomenon: namely, that Pol II is present at the majority of genomic loci that are bound by Pol III.

Figure 1

Chromatin environment of pol III genes. (a,b) Expressed and silent copies of tRNA—Leu-TAA, (c) U6 RNA, (d) 7SK RNA, (e) HVG1 RNA, (f) RNase MRP RNA. The positions of the non-coding RNA genes are indicated at the bottom of each panel. Bars show number of ChIP-Seq tags in 200bp windows and RNA-Seq tags in 20bp windows.

Figure 2

Pol III target genes are associated with both similar and distinct chromatin features to pol II target genes. Average ChIP-Seq read density (reads per 100 bp) profiles at tRNA (left) and pol II (right) genes are plotted for (a) H3K4me3, (b) H3K9ac, (c) H2A.Z, (d) H3K27me3, (e)H3K79me2, (f) H3K36me3. Genes were imperfectly classified as expressed or silent based on presence of pol III for tRNA genes or pol II for pol II genes. Area surrounding TSS (TES for H3K36me3)- transcription start (end) site is shown. Profiles for other modifications are shown in Supplementary Fig. 1. (g) Histone H3 ChIP-Seq read densities surrounding the TSSs of expressed (red) and silent (blue) tRNA genes.

Figure 3

Tissue specific expression of tRNA genes. (a) Pie chart shows number of tRNAs expressed in HeLa cells, CD4⁺ T cells, both or neither. (b) Chromatin environment of tRNA—Glu-TTC in CD4⁺ T and HeLa cells.

Figure 4

Novel pol III binding sites. (a,b,c) Novel pol III sites were found in the vicinity of known pol II promoters (a,b) as well as in gene free areas. Presence of total RNA-Seq signals suggests that the binding of polymerase at these loci is productive. Absence of corresponding polyA RNA-seq signal in b and c suggests that these loci are transcribed by pol III rather than pol II. (d) TFIIIB and TFIIIC can be found at the majority of the novel pol III loci.

Figure 5

Pol II is present at pol III-transcribed genes. (a) Various pol II isoforms are present at tRNASer-TGA in human CD4⁺ Tcells, (b) pol II is present at two tRNAs in Drosophila S2 cells. (c) Recruitment of pol II and its factors to various Pol III-transcribed RNA genes. (d) ChIP-Seq tag density profiles of pol II and III in the vicinity of silent and expressed tRNA genes.

Figure 6

Levels of 5S rRNA and pre-tRNAs are decreased by 4 hour treatment with alpha-amanitin oleate. qPCR results show that levels of both pol II (ACTB, GAPDH and IRF2) and some pol III genes (5S rRNA and pre-tRNAs) are decreased as a result of short term treatment with pol II inhibitor alpha-amanitin oleate. Mean +/−SD are shown, n=3.