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. 2013 Jan 17;152(1-2):144-56.
doi: 10.1016/j.cell.2012.12.015. Epub 2012 Dec 27.

The RNA Pol II elongation factor Ell3 marks enhancers in ES cells and primes future gene activation

Chengqi Lin  1 , Alexander S Garruss, Zhuojuan Luo, Fengli Guo, Ali Shilatifard
Affiliations

The RNA Pol II elongation factor Ell3 marks enhancers in ES cells and primes future gene activation

Chengqi Lin et al. Cell. .

Abstract

Enhancers play a central role in precisely regulating the expression of developmentally regulated genes. However, the machineries required for enhancer-promoter communication have remained largely unknown. We have found that Ell3, a member of the Ell (eleven-nineteen lysine-rich leukemia gene) family of RNA Pol II elongation factors, occupies enhancers in embryonic stem cells. Ell3's association with enhancers is required for setting up proper Pol II occupancy at the promoter-proximal regions of developmentally regulated genes and for the recruitment of the super elongation complex (SEC) to these loci following differentiation signals. Furthermore, Ell3 binding to inactive or poised enhancers is essential for stem cell specification. We have also detected the presence of Pol II and Ell3 in germ cell nuclei. These findings raise the possibility that transcription factors could prime gene expression by marking enhancers in ES cells or even as early as in the germ cell state.

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Figures

Figure 1
Figure 1. Ell3 occupies enhancer regions in mouse embryonic stem cells
(A) Pie chart showing that the percentages of the Ell3 peaks that are overlap with a Transcription Start Site (TSS), within a gene, and upstream or downstream of the nearest gene. (B) Upstream and downstream peaks were further categorized by their distance from the TSS. (C) Ell3 co-localizes with p300 at enhancer regions. Genome browser track examples for the occupancy profiles: for Ell3; transcription factor, Oct4; histone modifications, H3K4me1, H3K27ac, H3K4me3, and H3K27me3; and transcriptional coactivator, p300 (Creyghton et al., 2010; Marson et al., 2008; Mikkelsen et al., 2007). (D) Binding profiles for Ell3, p300, H3K4me1, and H3K4me3 are shown for regions 50 kb upstream and downstream of all 5,253 high-confidence Ell3 peaks. Color indicates enrichment at FDR < 0.05. The majority of the Ell3-occupied regions are also enriched for the enhancer signature of p300 with H3K4me1, but not the H3K4me3 (Creyghton et al., 2010). (E–F) Profiles of p300, H3K4me1, and H3K4me3 centered on Ell3 peaks, show 5 kb around the Ell3 peak summit. Majority of Ell3 peaks (4,806) are found upstream or downstream of a TSS, and these are co-enriched for p300 and H3K4me1. In contrast, only 447 Ell3 peaks are found at a TSS that is enriched for p300 and H3K4me3. (G) Functional annotation of Ell3-bound non-TSS peaks, as reported by GREAT (McLean et al., 2010), indicates enrichment for developmental processes. The logarithmic x-axis values correspond to binomial FDR corrected –log10 q-values. See also Figure S1.
Figure 2
Figure 2. Ell3 binding to enhancers is required for the expression of a subset of neighboring genes
(A) Cluster diagram of the 3,272 nearest genes to high-confidence Ell3 peaks. ChIP-seq enrichment profiles of Ell3-associated genes for the factor or modification indicated was K-means (K=3) classified into three groups, A (Active), B (Basal), and C (Constrained), which are mainly distinguished by the profiles of H3K36me3 and H3K27me3 in ES cells (Marson et al., 2008; Mikkelsen et al., 2007). (B) Gene expression (RPKM) analyses of the Ell3 nearest Group A–C genes. Only genes with statistically sufficient coverage by RNA-seq are included in this plot (see Methods). (C–E) Genome browser track examples of Groups A–C genes. Ell3 co-localizes with p300 at enhancer regions. RNA-seq analysis shows reduced expression of the Group C gene, St3gal1 upon Ell3 knockdown. (F–H) Gene expression MA plots show the differential expression of Groups A–C genes in Ell3-depleted ES cells vs. control cells. Significantly changed genes as reported by Cufflinks are shown in color. Dotted lines indicate log2 fold changes of −0.5 and 0.5. See also Figure S2 and Table S1.
Figure 3
Figure 3. Ell3 regulates Pol II occupancy at promoter-proximal regions of neighboring genes
(A–C) Genome browser profiles of Pol II occupancy in control and Ell3-depleted cells. Pol II levels are reduced at the Rere and St3gal1 genes, but not significantly altered on the Nanog gene. (D–G) Average Pol II occupancy plots for the top 1,000 highly expressed genes and Ell3 nearest genes from the Figure 2A group analysis. Rank normalized average Pol II levels within 5 kb of the TSS are shown in control (black line) and Ell3 knockdown (red line) ES cells. Pol II is reduced at the TSS region of Ell3-associated genes, with strong effects on Group C genes. See also Figure S3.
Figure 4
Figure 4. Ell3-dependent enhancer-promoter communication requires the cohesin complex
(A) Genome browser profiles for Ell3, p300, cohesin (Nipbl, Smc1(a), and Smc3), Mediator components (Med1 and Med12), and Ctcf (Kagey et al., 2010). Ell3 is found to colocalize with cohesin at sites that are enriched for Mediator and have low Ctcf occupancy (blue boxes). Ell3 is not enriched at cohesin sites that have high Ctcf and low Mediator occupancy (green box). (B) Knockdown of cohesin components Smc1a or Smc3 does not affect cellular Pol II levels. The unphosphorylated (8wg16 antibody), Ser5 phosphorylated (H14), and Ser2 phosphorylated (H5) forms of RNA Pol II levels remain unchanged upon the knockdown of the cohesin components. Triangles indicate increasing amounts of cell lysates. Tubulin serves as a loading control. (C) Knockdown of the cohesin components reduces the promoter-proximal Pol II occupancy at many Ell3-responsive genes. Histone H1 (Histh1d) and alpha globin (Hba2) serve as highly expressed and non-expressed control genes. (D) Knockdown of Smc3 reduces Ell3 occupancy at the enhancer regions of Ell3-responsive genes. Ell3-bound putative enhancer regions were chosen based on the co-occupancy with p300, Cohesin, and H3K4me1. The Hba2 gene serves as a non-transcribed control gene. The error bars stand for the standard deviation of three independent measurements. See also Figure S4.
Figure 5
Figure 5. Ell3 binding at enhancers is required for future gene activation by SEC
(A–D) qRT-PCR analyses of the activation time-course of four bivalently marked genes in the control and Ell3 knockdown EBs. Control and Ell3 knockdown ES cells were induced to form EB for 0 (EB0), 3 (EB3), 5 (EB5), and 10 (EB10) days, as indicated. The expression levels were normalized to Actb. (E) Schematic model for Ell3 pre-binding at enhancers primes future gene activation by SEC. (F) Ell2 is recruited to the promoters of Ell3-regulated genes in 5-day EBs, as shown by ChIP. The Hba gene serves as a non-transcribed control gene. (G) Ell2 is required for the activation of many of the genes regulated by Ell3 in EBs. The control and Ell2 knockdown ES cells were induced to form EBs in the petri dishes for 5 days before the qRT-PCR analyses. The expression levels were normalized to Actb. (H) Ell3 binding to enhancers is required for the activation of Hox genes by retinoic acid (RA). Control and Ell3 knockdown ES cells were untreated (Control) or treated with RA for 24 hours (RA24) before harvesting for the qRT-PCR analysis. (I) Ell3 is required for the recruitment of SEC (Aff4) to the Hoxa1 gene after RA treatment. ChIP signal is normalized to the non-transcribed Hba2 gene. The error bar stands for the standard deviation of three independent measurements.
Figure 6
Figure 6. Ell3 is essential for stem cell specification
(A–C) qRT-PCR analyses of the activation kinetics of lineage-specific genes in control and Ell3-knockdown EBs. Control and Ell3-knockdown ES cells were induced to form EB for the indicated time points. Expression levels were normalized to Actb. The error bar stands for the standard deviation of three independent measurements. (D) Ell3 is essential for the proper neural differentiation of mouse ES cells. The 5-day differentiated EBs from control and Ell3-knockdown ES cells were further differentiated into neural cells by exposure to retinoic acid for 14 days. Neural-differentiation competence was visualized by immunostaining for the neuronal marker class III, β-tubulin (β-tub III, green), and the DNA marker, DAPI (blue). See also Figure S5 and Table S2.
Figure 7
Figure 7. Ell3 and Pol II are found in germ cell nuclei
(A) Immunofluorescence staining of Ell3 in mouse sperm. Mouse sperm were fixed and stained with antibodies raised against the C-terminus of mouse Ell3, and were counterstained with DAPI. (B) Immunogold labeling of Ell3 and Pol II in mouse sperm. Mouse sperm were fixed, cryo-sectioned, and double stained with Ell3 N-terminal and Pol II antibodies. Both Ell3 (red arrow, 6 nm gold particles) and Pol II (blue arrow, 12 nm gold particles) localize to the nucleus of the sperm. Co-localization of Ell3 and Pol II was largely not observed compared to the co-localization of the N- and C-terminally raised Ell3 antibodies, which are frequently found within 5–10 nm of each other (See also Figure S6). (C–E) A model for the enhancer-associated Ell3 in coordinated transcriptional induction by SEC. H3K4me1, p300, Mediator, and cohesin can be found with Ell3 at inactive, poised, and active enhancers. At inactive/dormant enhancers (Group D), Ell3 is prebound with Mediator and cohesin, but Pol II is not found at the promoter. In the poised state (Group B and C), a subset of developmental regulators is in a constrained state of expression, with both H3K4me3 and H3K27me3 at the promoter. Pol II's presence at these promoters depends on the interactions between cohesin, Mediator, and Ell3. Bottom panel, upon receiving the proper activating signals, SEC is recruited and stabilized at the promoter region through interaction with Mediator and Ell3 (Group A). SEC phosphorylates RNA Pol II CTD, Spt5, and Nelf, thus resulting in the release of Pol II and gene activation.
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