1. Andrea Hand BIOL 303

2. Large Intergenic Non-Coding RNAs Intergenic Non-Coding: transcribed from a region of DNA between two genes that are used to make proteins but itself does not code for a protein linc RNA

3. Determine the function of lincRNA in mouse embryonic stem cells. Objective:

4. The intergenic regions they come from are enhancer regions for the genes that surround them, and are byproducts only. They act in cis to activate transcription (for the genes right around them) They act in trans to repress transcription (in genes not localized around where they were transcribed) Theories:

5. They are pluripotent- c an change into multiple types of cells, specifically… Ectoderm- external layer of cells that become skin Neuroectoderm- type of ectoderm that becomes the nervous system Endoderm- forms digestive, endocrine, auditory, and respiratory systems Mesoderm- forms muscles, including cardiac muscles Trophectoderm- differentiates into placenta What makes Embryonic Stem (ES) Cells So Special?

6. They self-renew in culture. The methods through which they signal, transcribe, and regulate chromatin to control pluripotency is well known. What makes ES cells Appropriate?

7. They used short hairpin RNA (shRNA) targeting lincRNA sections of DNA that were snuck in by a lentivirus to knockdown effectiveness of lincRNA (if of course they have an effect). They put one shRNA into one set of cells. Methods

8. When a section of DNA that has the reversely transcribed shRNA in it is transcribed, the RNA from the shRNA section will bend and bond to itself as shRNA contains 2 sections of RNA that are can bond to itself and so forms a hairpin shape. The shRNA give the lincRNA a different shape and thus makes them unsuitable for its previous function. How shRNA work in Knockdowns

9. They made 5 shRNA for each of the 226 lincRNA that have been identified in ES cells. They found out which of the 5 shRNA was most effective and specific for knocking down the lincRNA through multiple trials and used the best in the final experiment. Second most effective gave similar results. The shRNA suppressed 147 different lincRNA by ~75% from their amounts in uninfected ES cells. Methods Continued

10. They targeted shRNA on sections of DNA that code for proteins that regulate pluripotency, knowing that this should affect pluripotency. They targeted ~50 proteins and 40 of the proteins were affected by the shRNA. Positive Control

11. They made 27 shRNA that would not be able to be spliced into any known section of DNA and spliced them into lentiviruses that infected ES cells. Knowing the shRNA should not affect the DNA and therefore not affect pluripotency. Negative Control

12. They disregarded any effects that the negative control had on the ES cells in their assessment of how lincRNA knockdown affected the ES cells. This should have prevented their being a reaction attributed to lincRNA knockdown that was really due to lentivirus infection. To determine how the knockdown of lincRNA affected ES cells…

13. In 93% of the cultures with changed lincRNA there was a significant change in gene expression with around 175 protein- coding transcripts being affected on average for each lincRNA knockdown. This was very similar to the positive control which had results that 95% of the cultures with disabled proteins relevant to pluripotency affected on average 207 genes each. Results Compared to byproduct theory

14. Although a few lincRNA only repress genes- and therefore in this experiment they were overexpressed, the knockdown of lincRNA activated and repressed genes relatively evenly. Therefore, lincRNA are not just byproducts but are functional parts of ES cells. Results vs. Byproduct Continued

15. While a few lincRNAs do act in cis, the knockdown of lincRNA only affected neighbouring genes for 2 lincRNA. Only 13 affected genes closer than 10 genes from themselves. The genes the lincRNA affected were similar distances from their locus as the distance at which proteins affect gene expression. Results in Relation to Cis versus Trans Theories

16. These results suggest the locations of the genes that lincRNAs affect is just as random as any other proteins or RNA. Therefore while some lincRNA affect close by gene expression levels, they mostly affect genes in Trans. Results Compared to Cis vs. Trans

17. They next tried to find a correlation between the affects of the lack of lincRNA and the regulation of the Embryonic Stem Cell state. This regulation encompasses maintaining pluripotency and repressing differentiation. Beyond the Theories

18. To further test lincRNA affects, they infected cells with a mutation for a reporter gene (indicates if a gene is being used) from the Nanog promoter with shRNA targeted at lincRNA. Nanog is a key transcription factor that marks and is required for a cell to be pluripotent. LincRNA affects on Maintenance of Pluripotent State

19. Excluded results when shRNA infection caused a reduction in cell numbers. 26 lincRNA knockdowns had similar affects on Nanog levels as the knockdown of genes that code for protein regulators of pluripotency- for example Oct4 and Nanog itself. This affect means lincRNA contribute to the maintenance of the pluripotent state. Results with Nanog Reporter

20. They knocked down lincRNAs in wild-type ES cells then measured multiple pluripotency markers after 8 days. Those markers were Oct4, Sox2, Nanog, Klf4, and Zfp42. For all 26 lincRNA there was a significant reduction in multiple markers, >90% significantly decreased Oct4 and Nanog levels. Using an alternative hairpin shRNA for 15 lincRNA there were similar results in the reduction of Oct4. Just to make Sure!

21. “>90% of lincRNA knockdowns affecting Nanog reporter levels led to loss of ES cell morphology” (Page 2). In other words, 90% of the 26 lincRNA knockdowns led to markers of the differentiation from embryonic stem cell form to a different one. This means that lincRNA do in fact contribute in a significant way to the maintenance of the pluripotent state in ES cells. The Conclusion for Nanog Exp.

22. They compared the final state of cells after knockdowns to what lincRNA were repressed with Nanog repression and Oct4 repression as controls. The control proteins differentiated to the expected state and so lincRNA knockdown affects were accepted as results of their absence not other affects of infection. Do they Repress Differentiation?

23. They discovered that specific lincRNA do suppress specific differentiated state traits, and so upon their knockdown the cells showed early signs of differentiation, but did not actually cause differentiation. So in the absence of lincRNA we see markers we use to determine what a cell is differentiating to. Repressing Continued

25. They Experimented and determined that 75% of the lincRNA promoters- were bound by at least one of 9 transcription factors that were already associated with pluripotency. Most commonly they were bound by 3 of the pluripotency factors. When those pluripotency transcription factors were knocked down, 50% of those lincRNA affected by these factors exhibited expression changes. How do they fit into our knowledge?

26. Specifically, do lincRNA associate with polycomb repressive complexes physically? These are proteins that reshape chromatin and are significant to ES cell regulation. ~10% of the lincRNA in the ES cells associated with the 2 polycomb components Suz12 and Ezh2. It was also determined that ~30% of the lincRNA interact with one or more of the twelve chromatin complexes they tested. Do they affect Chromatin Proteins?

27. lincRNA are not just byproducts They affect genes far away from their transcription location They help to maintain the pluripotent state The repress differentiation pathways Pluripotency associated transcription factors aid in the transcription of lincRNA lincRNA interact directly with chromatin proteins Conclusion:

28. Works Cited Guttman, Mitchell, Julie Donaghey, Bryce W. Carey, Manuel Garber, Jennifer K. Grenier, Glen Munson, Geneva Young, Anne B. Lucas, Robert Ach, Laurakay Bruhn, Xiaoping Yang, Ido Amit, Alexander Meissner, Aviv Regev, John L. Rinn, David E. Root, and Eric S. Lander. "LinRNAs Act in the Circuitry Controlling Pluripotency and Differentiation." Nature 000.00 (2011). Nature.com. Nature Publishing Group, 28 Aug. 2011. Web. 28 Aug. 2011. "Stem-loop." Wikipedia, the Free Encyclopedia. Web. 29 Aug. 2011.. Work Cited