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R_loops_presentation_v4.0
- 1. R-loop: a double-edged sword? Inga Songailiene 2016-06-03 Watermark Watermark Watermark Watermark
- 2. R-loop R-loop Size range from 8 bp to 2 kbp RNA Pol II DNA/RNA hybrid 8bp 2 Watermark Watermark Watermark Watermark
- 3. Outline 1. Short history 2. R-loop formation mechanisms 3. Functions of R-loops in: • Replication • Recombination • Transcription • Epigenetics 3 Watermark Watermark Watermark Watermark
- 4. History of R-loop research RNA can hybridize to dsDNA by displacing the identical DNA strand in the presence of 70% formamide. More stable than dsDNA. 4 Groh, M. & Gromak, N. PLoS Genet. 10, e1004630 (2014). Watermark Watermark Watermark Watermark
- 5. History of R-loop research R-loop is a key intermediate of E.coli plasmid replication 5 Groh, M. & Gromak, N. PLoS Genet. 10, e1004630 (2014). Watermark Watermark Watermark Watermark
- 6. History of R-loop research 6 Groh, M. & Gromak, N. PLoS Genet. 10, e1004630 (2014). Watermark Watermark Watermark Watermark
- 7. History of R-loop research Microarray studies In vivo In vitro DRIP-sequencing 7 Watermark Watermark Watermark Watermark
- 8. History of R-loop research R-loops detected in E.coli plasmid transcription in vivo. TopA mutants contained hypernegatively supercoilded plasmids that formed R-loops. 8Groh, M. & Gromak, N. PLoS Genet. 10, e1004630 (2014). Watermark Watermark Watermark Watermark
- 9. Today Natural R-loops • Replication • Recombination • Transcription • Epigenetics Hazardous R-loops • Mutations • Genome instability 9 Watermark Watermark Watermark Watermark
- 10. R-loop formation mechanisms Transcription associated Independent from transcription 10Costantino, L. & Koshland, D. Curr. Opin. Cell Biol. 34, 39–45 (2015). Watermark Watermark Watermark Watermark
- 11. MechanismI. R-loop formation (-) supercoiling (+) supercoiling “Thread-back model” In cis and in trans 11 Costantino, L. & Koshland, D. Curr. Opin. Cell Biol. 34, 39–45 (2015). Watermark Watermark Watermark Watermark
- 12. I. R-loop formation Transcription associated 1. Negative supercoiling 12 Forth, S., Sheinin, M. Y., Inman, J. & Wang, M. D. Annu. Rev. Biophys. 42, 583–604 (2013). Watermark Watermark Watermark Watermark
- 13. I. R-loop formation Transcription associated 2. G-rich non-template strand 13 Skourti-stathaki, K. & Proudfoot, N. J. 1384–1396 (2014). doi:10.1101/gad.242990.114.Freely Watermark Watermark Watermark Watermark
- 14. II. R-loop formation Independent from transcription In trans RAD52 14 Szczelkun, M. D. et al. 6–11 (2014). doi:10.1073/pnas.1402597111 Costantino, L. & Koshland, D. Curr. Opin. Cell Biol. 34, 39–45 (2015). Watermark Watermark Watermark Watermark
- 15. Functions Natural R-loops • Replication • Recombination • Transcription • Epigenetics Hazardous R-loops • Genome instability • Mutations 15 Watermark Watermark Watermark Watermark
- 16. Functions Natural R-loops • Replication • Recombination • Transcription • Epigenetics Hazardous R-loops • Genome instability • Mutations 16 Watermark Watermark Watermark Watermark
- 17. R-loops Initiation of replication E.coli ColE1 plasmid replication • Senstive to rifampicin, RNAP inhibitor • RNAP-driven RNAII sequence forms R-loop on the leading DNA strand • RNAII is processed by RNase H to generate 3’ for DNA Pol Mitochondrial DNA replication • Reminiscent of ColE1 plasmid replication 17 Aguilera, A. & García-Muse, T. Mol. Cell 46, 115–124 (2012). Watermark Watermark Watermark Watermark
- 18. Functions Natural R-loops • Replication • Recombination • Transcription • Epigenetics Hazardous R-loops • Genome instability • Mutations 18 Watermark Watermark Watermark Watermark
- 19. R-loops Transcription associated recombination Transcription dependent DNA lesions cause the blockage of the replicative DNA polymerase. Template switching, DSB repair or fork reversal would be required to complete replication. The DNA lesions may lead to HR or NHEJ. -> Genome instability 19 Aguilera, A. & Gómez-González, B. Nat. Rev. Genet. 9, 204–217 (2008). HR or NHEJ Watermark Watermark Watermark Watermark
- 20. R-loops Transcription associated recombination AID – activation-induced cytidine deaminase 20 Aguilera, A. & García-Muse, T. Mol. Cell 46, 115–124 (2012). Watermark Watermark Watermark Watermark
- 21. R-loops Transcription associated recombination Ig class-switch recombination Somatic hypermutation Tumorigenesis 21 Aguilera, A. & García-Muse, T. Mol. Cell 46, 115–124 (2012). Watermark Watermark Watermark Watermark
- 22. Natural R-loops AID induced Ig class-switch recombination B-cells Variable region – antigen specificity VD(J)R recombination Constant region – different antibody functional properties Ig class-switching 22Murphy K., Immunology, 8th ed., 2012 Watermark Watermark Watermark Watermark
- 23. Natural R-loops AID induced Ig class-switch recombination R-loops are long >1kb and very stable 23 Santos-pereira, J. M. & Aguilera. Nat. Publ. Gr. 16, 583–597 (2015). Watermark Watermark Watermark Watermark
- 24. R-loops AID and Burkitt lymphoma Recognized as fastest growing tumor 24 WatermarkWatermark
- 25. R-loops AID and Burkitt lymphoma The translocation brings the strong IgH enhancers (yellow box) close to c-MYC, leading to its overexpression in Burkitt’s lymphoma. 25Groh, M. & Gromak, N. PLoS Genet. 10, e1004630 (2014). Watermark Watermark Watermark Watermark
- 26. Functions Natural R-loops • Replication • Recombination • Transcription • Epigenetics Hazardous R-loops • Genome instability • Mutations 26 Watermark Watermark Watermark Watermark
- 27. R-loops Transcriptional regulation How do flowers know it is summer? 27
- 28. R-loops Transcriptional regulation How do flowers know it is summer? R-loops 28
- 29. R-loops Transcriptional regulation Flowering locus C Transcription factor Arabidopsis thaliana 29 Ietswaart, R., Wu, Z. & Dean, C. Trends Genet. 28, 445–453 (2012). Watermark Watermark Watermark Watermark
- 30. R-loops Transcriptional regulation Flowering locus C repressor COOLAIR transcription is regulated by R-loop Transcription activation at the FLC locus Sun Q at al, 2013 30 Santos-pereira, J. M. & Aguilera. Nat. Publ. Gr. 16, 583–597 (2015). Watermark Watermark Watermark Watermark
- 31. R-loops Transcriptional regulation Flowering locus C repressor Transcription activation at the FLC locus Arabidopsis thaliana pCOOLAIR:Luc ΔNDX 31 Sun, Q. et al. Science. 340, 619–621 (2013). Transcription activation at the FLC locus Watermark Watermark Watermark Watermark
- 32. R-loops R-loops at COOLAIR TSS ΔNDX 32 Sun, Q. et al. Science. 340, 619–621 (2013). Watermark Watermark Watermark Watermark
- 33. R-loops Transcriptional regulation Flowering locus C repressor COOLAIR transcription is regulated by R-loop Transcription activation at the FLC locus 33 Arabidopsis thaliana Sun, Q. et al. Science. 340, 619–621 (2013). Transcription activation at the FLC locus Watermark Watermark Watermark Watermark
- 34. Functions Natural R-loops • Replication • Recombination • Transcription • Epigenetics Hazardous R-loops • Genome instability • Mutations 34 Watermark Watermark Watermark Watermark
- 35. R-loops Epigenetics Gene boundaries? R-loops are enriched at both gene ends (DRIP-Seq). In human coding genes, R-loops form over unmethylated CpG island promoters and G-rich terminator regions. Transcription termination siteTranscription start site 35 Skourti-stathaki, K. & Proudfoot, N. J. 1384–1396 (2014). doi:10.1101/gad.242990.114.Freely Watermark Watermark Watermark Watermark
- 36. R-loops Epigenetics – TSS CpG islands function as promoters for ~60% of human genes. Majority of promoter CpG islands are unmethylated in normal tissues. The presence of RNA Pol II and active transcription is associated with unmethylated state. Transcription termination siteTranscription start site 36 Watermark Watermark Watermark Watermark Watermark
- 37. R-loops Epigenetics – TSS Positive GC skew 37Ginno, P. A. et al., Mol. Cell 45, 814–825 (2012). Watermark Watermark Watermark Watermark Watermark
- 38. GC-skew and R-loops 38Ginno, P. A. et al., Mol. Cell 45, 814–825 (2012). Watermark Watermark Watermark Watermark
- 39. Positive GC-skew and R-loops Footprinting of R-loops at TSS TSS with a strong positive GC-skew are a feature of highly expressed genes that are unmethylated. 39 Ginno, P. A. et al., Mol. Cell 45, 814–825 (2012). Watermark Watermark Watermark Watermark
- 40. R-loops Proposed model R-loops could lead to activation of genes by protecting DNA from methylation or recruiting the protective histone 3 Lys4 trimethylation (H3K4me3) methyltransferases. Activation of transcription Epigenetics – TSS 40 Ginno, P. A. et al., Mol. Cell 45, 814–825 (2012). Watermark Watermark Watermark Watermark
- 41. R-loops Gene boundaries? R-loops are enriched at both gene ends (DRIP-Seq). In human coding genes, R-loops form over unmethylated CpG island promoters and G-rich terminator regions. Transcription termination siteTranscription start site Epigenetics – TTS 41 Skourti-stathaki, K. & Proudfoot, N. J. 1384–1396 (2014). doi:10.1101/gad.242990.114.Freely Watermark Watermark Watermark Watermark
- 42. R-loops Epigenetics – TTS Downstream terminator regions and R-loops. G-rich sequences downstream from the poly(A) signal are relatively common in mammalian genes. Hypothesis R-loops formed over these G-rich regions facilitate Pol II pausing downstream from poly(A) signal prior to transcription termination. 42Ginno, P. A. et al.,. Genome Res. 23 VN - r, 1590–1600 (2013). Watermark Watermark Watermark Watermark
- 43. R-loops Stopping Pol II is not an easy task! Once in a processive elongation mode, Pol II elongates at 4.3 kb/min (70 bp/sec). Epigenetics – TTS Transcription termination siteTranscription start site 43 Skourti-stathaki, K. & Proudfoot, N. J. 1384–1396 (2014). doi:10.1101/gad.242990.114.Freely Watermark Watermark Watermark Watermark
- 44. R-loops Transcription termination R-loops lead to recruitment of histone methyltransferases and formation of heterochromatin which is proposed to pause RNA Pol II. Also SETX DNA/RNA nuclease helicase resolves R-loops and promotes transcription termination. H3K9me2 Transcriptional termination Epigenetics – TTS 44Sollier, J. & Cimprich, K. Trends Cell Biol. 25, 514–522 (2015). Watermark Watermark Watermark Watermark
- 45. Review R-loops play role in… 45
- 46. Review Initiation of replication ColE1 plasmid, mitochondrial DNA replication R-loops play role in… 46 Watermark Watermark Watermark Watermark
- 47. Review Transcription associated recombination Genome instability R-loops play role in… 47 Watermark Watermark Watermark Watermark
- 48. Review Ig class switching in B-cells R-loops play role in… 48 Watermark Watermark Watermark Watermark
- 49. Review Transcriptional regulation Activation or silencing R-loops play role in… Flowering locus C repressor 49 Watermark Watermark Watermark Watermark
- 50. Review Epigenetics In promoter and terminator regions R-loops play role in… Activation of transcription (not always) Transcription termination H3K9me2 50 Watermark Watermark Watermark Watermark
- 51. Thank you 51
- 52. References 1. El Hage, A., Webb, S., Kerr, A. & Tollervey, D. Genome-wide distribution of RNA-DNA hybrids identifies RNase H targets in tRNA genes, retrotransposons and mitochondria. PLoS Genet. 10, e1004716 (2014). 2. Costantino, L. & Koshland, D. The Yin and Yang of R-loop biology. Curr. Opin. Cell Biol. 34, 39–45 (2015). 3. Forth, S., Sheinin, M. Y., Inman, J. & Wang, M. D. Torque measurement at the single-molecule level. Annu. Rev. Biophys. 42, 583–604 (2013). 4. Skourti-stathaki, K. & Proudfoot, N. J. A double-edged sword : R loops as threats to genome integrity and powerful regulators of gene expression. 1384–1396 (2014). doi:10.1101/gad.242990.114.Freely 5. Szczelkun, M. D., Tikhomirova, M. S., Sinkunas, T., Gasiunas, G. & Karvelis, T. Direct observation of R-loop formation by single RNA- guided Cas9 and Cascade effector complexes. 6–11 (2014). doi:10.1073/pnas.1402597111 6. Aguilera, A. & García-Muse, T. R Loops: From Transcription Byproducts to Threats to Genome Stability. Mol. Cell 46, 115–124 (2012). 7. Aguilera, A. & Gómez-González, B. Genome instability: a mechanistic view of its causes and consequences. Nat. Rev. Genet. 9, 204– 217 (2008). 8. Santos-pereira, J. M. & Aguilera, R loops : new modulators of genome dynamics and function. Nat. Publ. Gr. 16, 583–597 (2015). 9. Groh, M. & Gromak, N. Out of balance: R-loops in human disease. PLoS Genet. 10, e1004630 (2014). 10. Ietswaart, R., Wu, Z. & Dean, C. Flowering time control: Another window to the connection between antisense RNA and chromatin. Trends Genet. 28, 445–453 (2012). 11. Sun, Q., Csorba, T., Skourti-Stathaki, K., Proudfoot, N. J. & Dean, C. R-loop stabilization represses antisense transcription at the Arabidopsis FLC locus. Science (80-. ). 340, 619–621 (2013). 12. Ginno, P. A., Lott, P. L., Christensen, H. C., Korf, I. & Chedin, F. R-Loop Formation Is a Distinctive Characteristic of Unmethylated Human CpG Island Promoters. Mol. Cell 45, 814–825 (2012). 13. Ginno, P. A., Lim, Y. W., Lott, P. L., Korf, I. & Chédin, F. GC skew at the 5’ and 3' ends of human genes links R-loop formation to epigenetic regulation and transcription termination. TL - 23. Genome Res. 23 VN - r, 1590–1600 (2013). 14. Sollier, J. & Cimprich, K. a. Breaking bad: R-loops and genome integrity. Trends Cell Biol. 25, 514–522 (2015). 52