1. Recent discoveries in molecular biologyInstructor: Henry Levin henry_levin@nih.gov Websitehttps://science.nichd.nih.gov/confluence/display/biochem539/Home

2. Class Schedule Exam Exam

3. Career path

4. The function and impact of transposable elements

5. any genomic sequence that A transposable element is: possesses an intrinsic ability to mobilize. TE Target

6. What are some ways transposable elements can impact the host?

7. Transposable elements can disrupt coding sequences Huang, 2012, Annual review of genetics.

8. Transposable elements can reduce protein activity or mRNA splicing

9. Integration of Gret1 reduces expression of anthocyanin producing genes in grapes Vvmby1A Gret1

10. Transposon insertion can change patterns of tissue specific expression HLH transcription factor B-Bolivia causes expression of the b1 transcription factor to change from plant to seed.

11. Transposon insertion can change patterns of tissue specific expression Ruby (Myb transcription factor) Insertion of Rider causes cold dependent expression of Ruby in fruit of blood oranges.

12. Transposons can alter gene function in three key ways Disrupt coding sequence. Change transcription level or mRNA stability. Change tissue specificity of gene expression.

13. DNA “cut and paste” transposons rely on a single enzyme Autonomous examples Tn7 in E. coli P in drosophila Ac in maize Sleeping beauty in salmon Non-autonomous examples Ds in maize

14. Long terminal repeat retrotransposons have an RNA intermediate Autonomous examples Ty1 in S. cerevisiae Gypsy in drosophila IAP in mouse Non-autonomous examples ETn in mouse PbTRIM in Harvester Ant

15. Non-Long terminal repeat retrotransposons have an RNA intermediate and rely on target primed reverse transcription Autonomous examples L1 in human I factor in drosophila R1 and R2 in insects Non-autonomous examples Alu in human SVA in human

16. Review of the three types of transposons

17. The evolution of transposons DNA transposons RT IN RT Retro- transposons RT IN RT IN RT IN

18. Transposable elements have been extremely successful throughout evolution 50% 30% 45% 10% 80% 12% 0.3% Huang et al, 2012 Ann. Rev. Genet.

19. A burst in LTR-retrotransposons Maize diverged from sorghum about 16 million years ago. During this time the maize genome increased by three fold. This is entirely due to increased numbers of retrotransposons.

20. How are new transposons discovered? Analyze spontaneous changes in phenotypes for their genetic origin. This is how transposons were discovered by Barbara McClintock. Compare multiple genomes for polymorphic insertions or deletions. Analyze genome sequences for repeats. Search genomes for sequences similar to known transposons

21. Before the genome of S. pombe was sequenced we used DNA blots to look for repeat elements Tf1 probe Tf2 probe

22. Tf1 has sequence similar to other transposon proteins LTR LTR Gag PR RT IN

23. How would you test Tf1 for transposition activity? neo Tf1 Tf1 plasmid URA3 Tf1 neo Kick out plasmid With 5-FOA Tf1 neo Tf1 plasmid URA3 chromosome Induce expression of Tf1-neo Select for integration with G418 PR fs WT IN fs

24. LTR retrotransposons and retroviruses use a tRNA to prime reverse transcription Tf1 had no homology to any tRNA.What entity could prime Tf1 reverse transcription?

25. The priming of Tf1 reverse transcription . . . . . . . . . . . 3’ 5’ WT IN- 7th base 5' end 7th base PBS 7th base double mutant 5th base 5' end 5th base PBS 5th base double mutant

26. Assays with cloned copies of transposable elements • Can measure transposition activity. • Can reveal mechanistic details. • Can generate strains with new copies of the element.

27. How would you identify active L1 elements given that: • There are 880,000 copies of L1 in the human genome. • Approximately 99.7% are 5’ truncated. • Of the few full-length L1s, most had obvious mutations.

28. L1 insertions were found to cause several cases of diseases

29. Method for studying L1 transposition in human cells

30. The human genome sequence yielded only 90 L1s with intact ORFs, most of the activity comes from 6 L1s

31. L1 activity in human cells • There are about 880,000 copies of L1 in human cells. • 99.7% have deletions in their 5’ sequence. • Over 80% of the L1 activity is derived from 6 copies of L1.

32. Why have transposons been so successful throughout evolution? • Transposons are nothing more than molecular parasites that can efficiently propagate without destroying the host. Vs. • Barbara McClintock thought transposons are maintained because they are beneficial and can reorganize the host genome to increase survival.

33. How would you determine whether transposons are parasites that don’t benefit the cell or whether they provide important contributions to the cell? • Look for examples where transposons clearly provide an essential function.

34. Retrotransposons HeT-A and TART of Drosophila play an essential role in protecting the ends of chromosomes HeT-A and TART substitute for telomerase by inserting into chromosome ends But, this is the only known case of a transposon with an essential function.

35. Evidence that transposons are parasites includes the defense mechanisms that cells use to degrade transposon mRNA

36. Another reason transposons are thought to be parasites is because most insertions disrupt genes or are neutral. • In humans, there are at least 65 documented cases of diseases resulting from de novo TE insertions. • In a limited study of 42 de novo insertions of L1 in human cells 43% occurred in transposon repeats and 50% occurred in introns. The impact of these on expression was not determined. • More can be learned about the impact of integration from the study of model organisms.

37. Target site preferences of the LTR-retrotransposons in S. cerevisiae an S. pombe

38. Deep sequencing of DNA now determines sequence of 250 million DNAs per lane

39. Large numbers of insertions were generated neo Tf1 Tf1 plasmid URA3 Tf1 neo Kick out plasmid With 5-FOA Tf1 neo Tf1 plasmid URA3 chromosome Induce expression of Tf1-neo Select for integration with G418 PR fs WT IN fs

40. Mse I Spe I Mse I Mse I Mse I Mse I Mse I Mse I Mse I pombe genome Tf1 insertion pombe genome High throughput sequencing of insertion sites Cut with Mse I 5’ Tf1 3’ Add adapters NH2 5’ Tf1 NH2 5’ PCR amplify and gel purify Send sample to 454 Life Sciences with $$$ Get 500,000 high quality sequence reads

41. 454 sequencing of four independent experiments revealed 73,125 insertions Guo and Levin, 2010, Genome Research

42. 21,848 inserts clustered in promoter sequences upstream of ORFs Number of insertions 3.5% ORF Nucleotide

43. Deep sequencing of integration sites: • Positioned 78,000 independent insertions. • The insertions sites matched the positions from the target plasmid assays. • A reproducible measure of the integration in each of the 5,000 promoters of S. pombe. • Tf1 integration is high in approximately 1,000 promoters. • Stress response genes are favored targets. • These data suggested Tf1 integration could have a significant impact on the fitness of cells subjected to stress. Guo and Levin, 2010, Genome Research

44. Tf1 insertions increased expression of adjacent genes Gang et al, 2013, NAR.

45. Tf1 increased expression of adjacent genes by providing enhancer activity

46. The expression of Tf1 itself is induced by stress

47. The impact of Tf1 integration Tf1 integrates into the promoters of stress response genes. Tf1 does not reduce the expression of adjacent genes. Tf1 can increase the expression of adjacent genes. Tf1 transcription is increased by heat shock and oxidative stress.

48. Break

49. The impact of Tf1 integration Tf1 integrates into the promoters of stress response genes. Tf1 does not reduce the expression of adjacent genes. Tf1 can increase the expression of adjacent genes. Tf1 transcription is increased by heat shock and oxidative stress.

50. How can we test in a systematic way whether Tf1 integration benefits cells exposed to stress? Analyze cultures of cells with insertions to determine whether cells with specific insertions grow faster than others.