Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

TRIM28 regulates RNA polymerase II promoter-proximal pausing and pause release

Nature Structural & Molecular Biology volume 21, pages 876–883 (2014)Cite this article

Subjects

Abstract

Promoter-proximal pausing of RNA polymerase II (Pol II) is a major checkpoint in transcription. An unbiased search for new human proteins that could regulate paused Pol II at the HSPA1B gene identified TRIM28. In vitro analyses indicated HSF1-dependent attenuation of Pol II pausing upon TRIM28 depletion, whereas in vivo data revealed de novo expression of HSPA1B and other known genes regulated by paused Pol II upon TRIM28 knockdown. These results were supported by genome-wide ChIP-sequencing analyses of Pol II occupancy that revealed a global role for TRIM28 in regulating Pol II pausing and pause release. Furthermore, in vivo and in vitro mechanistic studies suggest that transcription-coupled phosphorylation regulates Pol II pause release by TRIM28. Collectively, our findings identify TRIM28 as a new factor that modulates Pol II pausing and transcriptional elongation at a large number of mammalian genes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Identification of TRIM28 bound at the HSPA1B promoter-proximal site and in vitro visualization of promoter-proximal pausing in the native human HSPA1B gene.
Figure 2: TRIM28 regulates Pol II promoter-proximal pausing upon HSPA1B activation.
Figure 3: TRIM28 KD increases the expression of paused genes in vivo.
Figure 4: TRIM28 regulates Pol II pause release.
Figure 5: Release of TRIM28-mediated transcriptional repression involves TRIM28 phosphorylation at S824.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Rahl, P.B. et al. c-Myc regulates transcriptional pause release. Cell 141, 432–445 (2010).

    Article  CAS  Google Scholar 

  2. Rasmussen, E.B. & Lis, J.T. In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes. Proc. Natl. Acad. Sci. USA 90, 7923–7927 (1993).

    Article  CAS  Google Scholar 

  3. Nechaev, S. et al. Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science 327, 335–338 (2010).

    Article  CAS  Google Scholar 

  4. Muse, G.W. et al. RNA polymerase is poised for activation across the genome. Nat. Genet. 39, 1507–1511 (2007).

    Article  CAS  Google Scholar 

  5. Core, L.J., Waterfall, J.J. & Lis, J.T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).

    Article  CAS  Google Scholar 

  6. Zeitlinger, J. et al. Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo. Genes Dev. 21, 385–390 (2007).

    Article  CAS  Google Scholar 

  7. Seila, A.C. et al. Divergent transcription from active promoters. Science 322, 1849–1851 (2008).

    Article  CAS  Google Scholar 

  8. Adelman, K. & Lis, J.T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat. Rev. Genet. 13, 720–731 (2012).

    Article  CAS  Google Scholar 

  9. Gaertner, B. et al. Poised RNA polymerase II changes over developmental time and prepares genes for future expression. Cell Reports 2, 1670–1683 (2012).

    Article  CAS  Google Scholar 

  10. Wu, C.H. et al. NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila. Genes Dev. 17, 1402–1414 (2003).

    Article  CAS  Google Scholar 

  11. Peterlin, B.M. & Price, D.H. Controlling the elongation phase of transcription with P-TEFb. Mol. Cell 23, 297–305 (2006).

    Article  CAS  Google Scholar 

  12. Gilchrist, D.A. et al. NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly. Genes Dev. 22, 1921–1933 (2008).

    Article  CAS  Google Scholar 

  13. Rabindran, S.K., Giorgi, G., Clos, J. & Wu, C. Molecular cloning and expression of a human heat shock factor, HSF1. Proc. Natl. Acad. Sci. USA 88, 6906–6910 (1991).

    Article  CAS  Google Scholar 

  14. Sun, J. & Li, R. Human negative elongation factor activates transcription and regulates alternative transcription initiation. J. Biol. Chem. 285, 6443–6452 (2010).

    Article  CAS  Google Scholar 

  15. Cheng, B. et al. Functional association of Gdown1 with RNA polymerase II poised on human genes. Mol. Cell 45, 38–50 (2012).

    Article  CAS  Google Scholar 

  16. Byun, J.S. et al. ELL facilitates RNA polymerase II pause site entry and release. Nat. Commun. 3, 633 (2012).

    Article  Google Scholar 

  17. Ulrich, H. DNA and RNA aptamers as modulators of protein function. Med. Chem. 1, 199–208 (2005).

    Article  CAS  Google Scholar 

  18. Hendrix, D.A., Hong, J.W., Zeitlinger, J., Rokhsar, D.S. & Levine, M.S. Promoter elements associated with RNA Pol II stalling in the Drosophila embryo. Proc. Natl. Acad. Sci. USA 105, 7762–7767 (2008).

    Article  CAS  Google Scholar 

  19. Lee, C. et al. NELF and GAGA factor are linked to promoter-proximal pausing at many genes in Drosophila. Mol. Cell. Biol. 28, 3290–3300 (2008).

    Article  CAS  Google Scholar 

  20. Iyengar, S., Ivanov, A.V., Jin, V.X., Rauscher, F.J. III & Farnham, P.J. Functional analysis of KAP1 genomic recruitment. Mol. Cell. Biol. 31, 1833–1847 (2011).

    Article  CAS  Google Scholar 

  21. Chen, L. et al. Tripartite motif containing 28 (Trim28) can regulate cell proliferation by bridging HDAC1/E2F interactions. J. Biol. Chem. 287, 40106–40118 (2012).

    Article  CAS  Google Scholar 

  22. Messerschmidt, D.M. et al. Trim28 is required for epigenetic stability during mouse oocyte to embryo transition. Science 335, 1499–1502 (2012).

    Article  CAS  Google Scholar 

  23. Hu, G. et al. A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal. Genes Dev. 23, 837–848 (2009).

    Article  CAS  Google Scholar 

  24. Chikuma, S., Suita, N., Okazaki, I.M., Shibayama, S. & Honjo, T. TRIM28 prevents autoinflammatory T cell development in vivo. Nat. Immunol. 13, 596–603 (2012).

    Article  CAS  Google Scholar 

  25. Vassylyev, D.G. et al. Structural basis for substrate loading in bacterial RNA polymerase. Nature 448, 163–168 (2007).

    Article  CAS  Google Scholar 

  26. Kininis, M., Isaacs, G.D., Core, L.J., Hah, N. & Kraus, W.L. Postrecruitment regulation of RNA polymerase II directs rapid signaling responses at the promoters of estrogen target genes. Mol. Cell. Biol. 29, 1123–1133 (2009).

    Article  CAS  Google Scholar 

  27. Zobeck, K.L., Buckley, M.S., Zipfel, W.R. & Lis, J.T. Recruitment timing and dynamics of transcription factors at the Hsp70 loci in living cells. Mol. Cell 40, 965–975 (2010).

    Article  CAS  Google Scholar 

  28. Gilchrist, D.A. et al. Regulating the regulators: the pervasive effects of Pol II pausing on stimulus-responsive gene networks. Genes Dev. 26, 933–944 (2012).

    Article  CAS  Google Scholar 

  29. Makalowski, W. & Boguski, M.S. Evolutionary parameters of the transcribed mammalian genome: an analysis of 2,820 orthologous rodent and human sequences. Proc. Natl. Acad. Sci. USA 95, 9407–9412 (1998).

    Article  CAS  Google Scholar 

  30. Ziv, Y. et al. Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway. Nat. Cell Biol. 8, 870–876 (2006).

    Article  CAS  Google Scholar 

  31. White, D. et al. The ATM substrate KAP1 controls DNA repair in heterochromatin: regulation by HP1 proteins and serine 473/824 phosphorylation. Mol. Cancer Res. 10, 401–414 (2012).

    Article  CAS  Google Scholar 

  32. Lee, D.H. et al. Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response. EMBO J. 31, 2403–2415 (2012).

    Article  CAS  Google Scholar 

  33. Li, X. et al. Role for KAP1 serine 824 phosphorylation and sumoylation/desumoylation switch in regulating KAP1-mediated transcriptional repression. J. Biol. Chem. 282, 36177–36189 (2007).

    Article  CAS  Google Scholar 

  34. Chen, B.P. et al. Ataxia telangiectasia mutated (ATM) is essential for DNA-PKcs phosphorylations at the Thr-2609 cluster upon DNA double strand break. J. Biol. Chem. 282, 6582–6587 (2007).

    Article  CAS  Google Scholar 

  35. Yoon, Y.J. et al. KRIBB11 inhibits HSP70 synthesis through inhibition of heat shock factor 1 function by impairing the recruitment of positive transcription elongation factor b to the hsp70 promoter. J. Biol. Chem. 286, 1737–1747 (2011).

    Article  CAS  Google Scholar 

  36. Wolf, D. & Goff, S.P. TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells. Cell 131, 46–57 (2007).

    Article  CAS  Google Scholar 

  37. Ivanov, A.V. et al. PHD domain-mediated E3 ligase activity directs intramolecular sumoylation of an adjacent bromodomain required for gene silencing. Mol. Cell 28, 823–837 (2007).

    Article  CAS  Google Scholar 

  38. Lee, W. et al. A high-resolution atlas of nucleosome occupancy in yeast. Nat. Genet. 39, 1235–1244 (2007).

    Article  CAS  Google Scholar 

  39. Herquel, B. et al. Transcription cofactors TRIM24, TRIM28, and TRIM33 associate to form regulatory complexes that suppress murine hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 108, 8212–8217 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We appreciate S. Elledge at the Harvard Medical School for mediating collaborations and R. Young and D. Orlando at Massachusetts Institute of Technology (MIT) for providing with helpful comments and perspectives for the manuscript. We thank A. York, A. Schubert, M. Knuesel, T. Westerling and J. Stinchfield for technical support and thank C. Wu (US National Cancer Institute (NCI)) for the HSF1 vector, R. Kelm Jr. (University of Vermont) for the PURb antibody and F. Zhu (Florida State University) for the GST-TRIM28 vectors. We thank the laboratories of S. Alper, M. Pollak and M. Brown at Beth Israel Deaconess Medical Center (BIDMC) for technical assistance, equipment and discussions. H.B. thanks M. Cross and M.A. Stevenson at BIDMC for administrative support and J. Park and D. Bunch for loving encouragement throughout the work. This study was supported by grants from the US National Institutes of Health (NIH) (RO-1CA047407) and the Harvard Joint Center for Radiation Therapy to S.K.C. and H.B., from the Koch Institute for Integrative Cancer Research at MIT (P30-CA14051) and the MIT Center for Environmental Health Sciences (P30-ES002109) to S.M. and S.L. and also from the NCI (R01CA127364) and the American Cancer Society (RSG 0927401DMC) to D.J.T. This research was supported in part by the Intramural Research Program of the NIH NIEHS (1ZIAES102745-02) to G.H. and X.Z. and to A.B. and D.F.

Author information

Author notes

  1. Xiaofeng Zheng and Adam Burkholder: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

    Heeyoun Bunch, Simon T Dillon, Gabriel Birrane & Stuart K Calderwood

  2. Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

    Xiaofeng Zheng & Guang Hu

  3. Integrative Bioinformatics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

    Adam Burkholder & David Fargo

  4. Genomics and Proteomics Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

    Simon T Dillon

  5. BioMicro Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Shmulik Motola & Stuart Levine

  6. Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

    Gabriel Birrane

  7. Department of Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, Colorado, USA

    Christopher C Ebmeier & Dylan J Taatjes

Authors
  1. Heeyoun Bunch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaofeng Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Adam Burkholder
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Simon T Dillon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shmulik Motola
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gabriel Birrane
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Christopher C Ebmeier
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stuart Levine
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David Fargo
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Guang Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Dylan J Taatjes
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Stuart K Calderwood
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.Z. and G.H. generated WT and TRIM28 KD mES-cell extracts for ChIP-seq and ChIP-qPCR. S.M. and S.L. processed ChIP-seq. A.B., G.H. and D.F. performed bioinformatics. S.T.D. and C.C.E. carried out MS. G.B. constructed TRIM28 plasmids. H.B., D.J.T. and S.K.C. designed the experiments and wrote the manuscript. D.J.T. and G.H. contributed equally to the analyses as jointly supervising authors.

Corresponding author

Correspondence to Stuart K Calderwood.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Oligonucleotide pulldown assay.

(a) Predicted secondary structures of HSPA1B and GREB1 non-template DNA oligos used in the pull-down assay. G-C richness of HSPA1B transcriptional start site results in unique hairpin structures from different sizes of the non-template DNA. On the other hand, GREB1, a non-pausing control gene, lacks the hairpin structure. N.T stands for the Non-Template DNA. These structures were obtained using oligo structure prediction software from Integrated DNA technologies. (b) Oligo pull-down assays. Immobilized non-template ssDNA segments of HSPA1B +1 to +80 and GREB1 +1 to +50 were incubated with HeLa nuclear extracts and subjected to different wash conditions, 0.1, 0.3, 0.5, and 1 M HEMG, in order to optimize the sample preparation. We chose the wash condition with 0.5 M HEMG for mass spectrometry and immunoblotting to identify proteins bound near the pausing site of HSPA1B. SM, Size Marker; c, GREB1 as a non-pausing control (Kininis, M. et al. Mol. Cell. Biol. 29, 1123-1133); h80, HSPA1B +1 to +80.

Supplementary Figure 2 Proteins, DNA and TRIM28-depleted NE prepared for this study.

(a) DNA template and purified proteins used in this study. a. TATA box binding mutant of HSPA1B (-467 and +216), gel purified. (b) 5′ end biotinylated WT HSPA1B (-467 to +216), gel-purified. (c) Silver stained gel of purified GST-tagged full-length (left) and truncated (right) TRIM28. d. Left, his6-tagged HSF1 stained with Coomassie Blue reagent (Biorad). Right, untagged HSF1, silver-stained and confirmed with western blot. e. TRIM28 immunodepletion from HeLa nuclear extracts. Bound proteins were eluted and separated on the SDS-PAGE gel, followed by silver-staining. SM, Size Marker; Lane 1, Proteins immunoprecipitated from the first round of Immunoprecipitation (3 hours); Lane 2, Proteins immunoprecipitated from the second round of Immunoprecipitation (16 hours); Lane 3, Proteins immunoprecipitated from the third round of Immunoprecipitation (2.5 hours). TRIM28 is effectively depleted as shown with an arrow just above 100 KDa of SM.

Supplementary Figure 3 Box plots and chromosome viewers comparing WT and TRIM28-KD mESCs.

(a) and (b) Change in Gene Body Pol II of TRIM28-bound and –unbound genes. Change in normalized, input-adjusted total PolII ChIP-seq gene body reads per kilobase on TRIM28 KD as a function of TRIM28 binding. Values are plotted for genes intersecting (a) and not intersecting CpG islands (b) (p-values derived from Wilcoxon Rank Sum test). (c) UCSC Browser views of paused genes in replicates. Chromosome views of EGR1 (c), JUN (d), and ERK1 (e) with normalized and input-adjusted Total PolII ChIP-seq read counts in WT and TRIM28 KD mES cells.

Supplementary Figure 4 Pausing-index changes in replicates.

(a) Scatter plots depict per-replicate TRIMKD vs. WT pausing index fold change on log2 scale, as a function of WT pausing index (N= 8,652 (Rep #1), 8655 (Rep#2)). Plots do not include genes with undefined pausing index resulting from zero promoter or gene body read density in either condition. (b) Zoom-In views of plots shown in a, between 0 and 300 in pausing index in WT. (c) Scatter plots of raw pausing indices of replicate 1 versus replicate 2 for WT and TRIM28 KD. Plots were generated combining the genes with RPKM greater than 1, for all samples in both the promoter and gene body window (N=601). (d) Summary of paused genes regulated by TRIM28. Paused genes are defined using the following criteria: gene body and promoter windows >=50% mappable and p-value <= 0.001 (poisson probability of observing X or greater promoter density given expected value equal to gene body density). Paused genes have pausing index equal or greater than 5.

Supplementary Figure 5 Individual heat maps with raw Pol II and S2 Pol II ChIP-seq signals and inputs in replicates.

(a) Replicate 1. (b) Replicate 2.

Supplementary Figure 6 UCSC Browser views of Ier5 and the DQ072391 locus, and UCSC browser views of normalized read coverage.

(a) input for IER5 (Immediate Early Response 5). (b) input at DQ072391 (c) total Pol II at DQ072391, illustrating facilitated Pol II entry downstream of the promoter-proximal pausing site in TRIM28 KD. TSS and orientation of IER5 and DQ072391 are marked with arrowheads and the regions with noticeable increase in Pol II occupancy in KD are embraced in a red box.

Supplementary Figure 7 Purified TRIM28 WT and S824 mutants and MS analysis to identify TRIM28-interacting kinases.

(a) Dot blots showing WT and TRIM28 KD mES cells in replicates and purified WT and S824 mutant TRIM28 proteins. WT, control mES cells; KD, TRIM28 KD mES cells. (b) TRIM28 interacting proteins with mild affinity. Proteins that interact with TRIM28 were immunoprecipitated (IP) and bound proteins were eluted and visualized on a 10% acrylamide gel stained with silver. Lane 1, Size marker; lane 2, 3 hours of IP; lane 3, 2 hours of IP; lane 4, overnight IP (lane 2-4, 8% of elution was loaded on the gel); lane 5, 3 hours of IP; lane 6, 2 hours of IP; lane 7, overnight IP (lane 5-7, 34% of elution was loaded on the gel); lane 8, beads after 3 hours IP and elution; lane 9, beads after 2 hours IP and elution; lane 10, beads after overnight IP and elution. Through this experiment, the sample shown in lane 5 was subjected to MS after TCA precipitation and in-gel digestion as described in methods. (c) Untagged TRIM28 WT, S824A, and S824D proteins purified and used for transcription assays. Purified proteins were run on a gel and stained with Coomassie Blue reagent (Biorad). SM, Size marker (from the top: 250, 150, 100, 75, 50, 37, 25, and 20 KDa). Trim28 is marked by asterisk.

Supplementary Figure 8 In vitro transcription assay to characterize WT and S824-mutant TRIM28 proteins.

(a) TRIM28 WT, S824A, and S824D proteins were added with WT or TRIM28ΔNE in the transcription assay. Untagged WT, S824A, and S824D TRIM28 were supplied to WT or TRIM28ΔNE (0, 100 ng, and 600 ng). HSF1 was introduced after PIC assembly, immediately before adding NTPs. Transcription was allowed to proceed for 30 mins. The paused transcripts were visualized on a 12% sequencing gel. (b) The function and efficiency of TRIM28 WT, S824A, and S824D proteins to stabilize Pol II pausing in vitro. Approximately 100 ng of WT, S824A, and S824D were supplied to WT or TRIM28ΔNE during the PIC assembly on the HSPA1B template. After PIC was formed, loosely bound proteins were washed off (see Methods). HSF1 was included immediately before NTP addition. Transcription reaction was allowed for 45 minutes before visualizing nascent RNA transcripts on a 12% sequencing gel.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Note (PDF 3004 kb)

Supplementary Data Set 1

TRIM28-interacting proteins 1 (MS) (XLS 82 kb)

Supplementary Data Set 2

TRIM28-interacting proteins 2 (MS) (XLS 107 kb)

Supplementary Data Set 3

TRIM28-interacting proteins with mild affinity (MS) (XLS 380 kb)

Supplementary Data Set 4

Uncropped gels and images to supplement main figures (PDF 15188 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bunch, H., Zheng, X., Burkholder, A. et al. TRIM28 regulates RNA polymerase II promoter-proximal pausing and pause release. Nat Struct Mol Biol 21, 876–883 (2014). https://doi.org/10.1038/nsmb.2878

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2878

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing