Gene duplication and neofunctionalization: POLR3G and POLR3GL

Abstract

RNA polymerase III (Pol III) occurs in two versions, one containing the POLR3G subunit and the other the closely related POLR3GL subunit. It is not clear whether these two Pol III forms have the same function, in particular whether they recognize the same target genes. We show that the POLR3G and POLR3GL genes arose from a DNA-based gene duplication, probably in a common ancestor of vertebrates. POLR3G- as well as POLR3GL-containing Pol III are present in cultured cell lines and in normal mouse liver, although the relative amounts of the two forms vary, with the POLR3G-containing Pol III relatively more abundant in dividing cells. Genome-wide chromatin immunoprecipitations followed by high-throughput sequencing (ChIP-seq) reveal that both forms of Pol III occupy the same target genes, in very constant proportions within one cell line, suggesting that the two forms of Pol III have a similar function with regard to specificity for target genes. In contrast, the POLR3G promoter--not the POLR3GL promoter--binds the transcription factor MYC, as do all other promoters of genes encoding Pol III subunits. Thus, the POLR3G/POLR3GL duplication did not lead to neo-functionalization of the gene product (at least with regard to target gene specificity) but rather to neo-functionalization of the transcription units, which acquired different mechanisms of regulation, thus likely affording greater regulation potential to the cell.

Figure 1.

Evolution of the POLR3G and POLR3GL genes. (A) The genomic organization of the POLR3G (top line) and POLR3GL (bottom line) genes is shown with coding parts of exons as thick boxes, noncoding parts of exons as thinner boxes, and introns as lines with the arrowheads indicating the sense of transcription. The corresponding POLR3G and POLR3GL protein sections are schematized in the middle of the panel. (B) Alignment of POLR3G and POLR3GL protein sequences showing the borders (arrowheads) of corresponding exons. (C) Number of POLR3G and BRF homologs in different species. Species were classified according to species phylogeny. The numbers of detected POLR3G and BRF-related genes are indicated on the right.

Figure 2.

POLR3G and POLR3GL occupy largely the same loci in human IMR90 cells. (A) POLR3G and POLR3GL occupy all three types of Pol III promoters. UCSC Browser view of type 1 (RN5S), type 2 (TRNA), and type 3 (RNU6ATAC) Pol III genes showing occupancy by BDP1, POLR3D, POLR3G, and POLR3GL, as well as the input. The x-axis shows the genomic location; the y-axis shows sequence tag accumulation. The scales on the y-axes are similar for all factors. (B) Spearman's rank correlation of the POLR3G versus POLR3GL scores. (c) x-axis, POLR3G scores; y-axis, POLR3GL scores; in blue the x = y line; in red, the regression line. (b) Correlation coefficient. (a) Distribution histogram representing, for each POLR3G score interval of 0.2 (see x-axis scale at the bottom of c), the number of genes in that interval (y-axis at the right of the panel: The numbers in green correspond to the lowest, middle, and highest number of genes). (d) As in a, but for each POLR3GL score interval of 0.2. (C) MvA plot with the score means [(POLR3GL score + POLR3G score)/2] on the x-axis and the score difference (POLR3GL score − POLR3G score) on the y-axis. All scores are in log₂ (see Supplemental Table S2).

Figure 3.

Pol III–occupied loci in mouse liver and Hepa 1-6 cells. (A) Spearman's rank correlation of scores obtained by Canella et al. (2012) and in this work. The loci considered include all tRNAs and SINEs. (c) x-axis, POLR3D scores in this work; y-axis, POLR3D scores by Canella et al. (2012); in blue, the x = y line; in red, the regression line. (b) Correlation coefficient. (a) Distribution histogram representing, for each POLR3D 2013 score interval of 1 (see x-axis at the bottom of c), the number of genes in that interval (y-axis at the right of the panel: The numbers in green correspond to the lowest, middle, and highest number of genes). (d) As in a but for each POLR3D 2012 score interval of 0.5. (B) List of additional, Pol III–occupied loci identified in this work compared with that of Canella et al. (2012). (C) Box plots showing scores in replicate 1 (Rep1) and replicate 2 (Rep2) samples from liver or Hepa 1-6 cells, as indicated on the x-axis. The y-axis shows scores in log₂. Genes with scores below the cutoff (see Methods) are represented by gray dots. The median is indicated by the black horizontal bar, the mean of genes above the cutoff by the red dot, and the mean of genes below the cutoff by the black dot. The genes shown on the various panels correspond to the lists on the various pages of Supplemental Table S3. (D) MvA plot illustrating differential Pol III occupation in liver versus Hepa 1-6 cells. The x-axis shows score means [(POLR3D mean score in liver + POLR3D mean score in Hepa 1-6 cells)/2], and the y-axis score differences (POLR3D mean score in Hepa 1-6 cells − POLR3D mean score in liver). All scores are in log₂ (see Supplemental Tables S3, S4).

Figure 4.

POLR3G and POLR3GL occupy largely the same loci. (A) Box plots showing scores in replicate 1 (Rep1) and replicate 2 (Rep2) samples from liver or Hepa 1-6 cells, as indicated on the x-axis. The y-axis shows scores in log₂. Genes with scores below the cutoff (see Methods) are represented by gray dots. The median is indicated by the black horizontal bar, the mean of genes above the cutoff by the red dot, and the mean of genes below the cutoff by the black dot. The four box plots on the left are reproduced from Figure 3C, upper left panel. (B) Spearman's rank correlation of POLR3D and POLR3G (d), POLR3D and POLR3GL (g), or POLR3G and POLR3GL (h) scores in liver cells: In blue, the x = y line; in red, the regression line. (b,c,f) The correlation coefficients corresponding to panels d, g, and h, respectively. (a) Distribution histogram representing, for each POLR3D score interval of 0.5 (see x-axis at the bottom of g), the number of genes in that interval (y-axis at the right of the panel: The numbers in green correspond to the lowest, middle, and highest number of genes). (e) As in a but for each POLR3G score interval of 0.5 (see x-axis at the bottom of h). (i) As in a but for each POLR3GL score interval of 075. (C) As in B but in Hepa 1-6 cells. (D) Box plots showing POLR3G-POLR3GL score differences (in log₂) in replicate 1 (Rep1) and replicate 2 (Rep2) samples from liver or Hepa 1-6 cells, as indicated on the x-axis. The y-axis shows score differences (in log₂). Genes with scores below the cutoff (see Methods) are represented by gray dots. The median is indicated by the black horizontal bar, the mean of genes above the cutoff by the red dot, and the mean of genes below the cutoff by the black dot.

Figure 5.

MYC binds to POLR3G but not to POLR3GL TSS. (A) Schematic of the POLR3G and POLR3GL genomic regions. (B) UCSC Browser views showing MYC, MAX, and Pol II (antibody directed against the N-terminus of POLR2A, Santa Cruz sc-899) tag accumulation, as indicated on the right, on the POLR3G and POLR3GL promoter regions, in P493-6 cells at time 0, 1 h, and 24 h after induction of MYC, as indicated on the left. The scales on the y-axes were adjusted to the maximum height of the peaks in each track, which is indicated on the right of each track, just above the track identity. (Based on the data of Lin et al. [2012]). (C) As in B but in U87 and MM.1S cells. (D) As in A but in SCLC H128_1 and SCLC H2171 cells.

Figure 6.

The POLR3G gene duplication leads to neo-functionalization of promoter sequences rather than gene product. (A) The POLR3G and POLR3GL promoters are differentially regulated, probably at least in part through exclusive binding of MYC to the POLR3G TSS. (B) Model of POLR3G and POLR3GL regulation. The model assumes a constant cellular level of POLR3GL and a variable level of POLR3G that allows adaptation of total Pol III levels to cell growth and proliferation conditions. If the cell loses POLR3G, the constant expression level of POLR3GL allows survival. If the cell loses POLR3GL, it can encounter conditions when POLR3G expression levels are too low for survival.