BIOL 7.342 Reading the Blueprint of Life: Transcription,

1. FALL 2006 - ADVANCED UNDERGRADUATE SEMINAR BIOL 7.342 Reading the Blueprint of Life: Transcription, Stem Cells and Differentiation Fall 2006. Thursdays, 3 pm – 5 pm. Room 68-151 Instructors: Matthew Guenther (guenther@wi.mit.edu, Young Lab) Roshan Kumar (roshan@wi.mit.edu, Young Lab) Organizer: Bob Horvitz http://web.mit.edu/biology/www/undergrad/adv-ugsem.html The Players: Cells, chromatin and transcriptional regulators Course Description Stem cells have the unique ability to give rise to all human tissues and hold great potential for tissue regeneration and treating human disease. Realizing this potential will require an understanding of the fundamental mechanisms that allow stem cells to generate descendants that have a variety of fates and that lock in the specialized states and distinctive RNA and protein expression patterns of differentiated cells. Transcriptional regulation is believed to account for a large part of the specialized gene expression programs of cells. In this course, we will address how transcriptional regulators both prohibit and drive differentiation during the course of development. How does a stem cell know when to remain a stem cell and when to become a specific cell type? Are there global differences in the way the genome is read in multipotent and terminally differentiated cells? We will explore how stem cell pluripotency is preserved, how master regulators of cell-fate decisions execute developmental programs, and how chromatin regulators control undifferentiated versus differentiated states. Additionally, we will discuss how aberrant regulation of transcriptional regulators produces disorders such as developmental defects and cancer. The Tools: Genome-wide discovery by expression analysis, location analysis and more!

2. Outline Introductions and class structure Introduction to subject matter: Regulation of transcription Stem cells Finding and reading scientific literature

3. Background Name: Major: Year: How did you become interested in biology in general or the course matter specifically: Do you have research experience? If so, where? Do you have experience reading primary literature? E-mail:

4. Class Structure General: Critical reading of original scientific literature (2 per class) Attendance and participation in every class Each student should be prepared to explain any figure Assignments: Email 2 question/comments about the upcoming papers to Roshan and Matt each week (1 per paper by 1 PM) Write a 2 page research proposal 15 min oral presentation of proposal Office hours: On demand, contact Roshan and Matt

5. Class Website: http://openwetware.org/wiki/7.342

6. Outline Introductions and class structure Introduction to subject matter: Regulation of transcription Stem cells Finding and reading scientific literature

7. 1 Genome 1 Cell >200 Cell Types

8. Cell Type Specificity Cell uniqueness is determined by which portions of the genome are expressed Transcription mRNA Protein expression Unique function of cell

9. Central Questions How does a stem cell remain a stem cell (not want to differentiate) Once a stem cell differentiates to something else, why does it stay that way? Are there fundamental differences in the way the genome is read in stem cells versus differentiated cells? We will explore how regulation of transcription answers these questions

10. Cell Type Specificity DNA packaging (chromatin) DNA sequence Transcription Factors

11. Higher Order Chromatin start stop DNA 11 nm

12. Chromatin Structure Chromatin Structure 1974 - Chromatin subunit model Kornberg Marks et al., Nat Rev Cancer 1:194 (2001) 1977,1997 - Nucleosome crystal structure Richmond, Luger, Klug, Finch Luger et al., Nature 389:251 (1997)

13. Histone Tail Modifications

14. Transcription Factors

15. HMT HDAC HAT Pol II Putting DNA + chromatin + transcription factors together acetylation H3-K4 methylation

16. ORF SWI/SNF SAGA Nua4 ORF GTFs Pol II Mediator Isw1a ORF Set1 PAF1 ORF Me Set2 Isw1b ORF Me Transcription of Yeast Protein-Coding Genes Initiation Elongation Termination

17. Outline Introductions and class structure Introduction to subject matter: Regulation of transcription Stem cells Finding and reading scientific literature

18. Culturing Embryonic Stem Cells trophectoderm Inner cell mass blastocyst

19. Culturing Stem Cells: Pluripotency Oct4 Sox2 Nanog

20. Culturing Stem Cells: Pluripotency

21. Outline Introductions and class structure Introduction to subject matter: Regulation of transcription Stem cells Finding and reading scientific literature

22. Finding a Journal Article http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed

25. Reading the Paper Abstract Introduction Results Discussion Concluding remarks

26. Reading the Paper Be an active reader: write in margins, underline important phrases or statements, ask why a certain experiment was performed Ask yourself: What question(s) did the authors set out to answer? Ask yourself: Did the authors answer that question effectively? Were the results to be expected? Boring Were the results unexpected? Exciting What experiments would you have done differently? What experiments are missing? What experiments would you do next?

27. Structure of chromatin: “Beads-on-a-string”

28. Nuclease digestion of chromatin

29. Weintraub & Groudine Experiment

30. Harold Weintraub 1945–1995 “It is hard to complete a picture of Hal without describing his appearance: always sneakers, never a tie; no matter what the occasion, better dressed for basketball than the opera.” - Marc Kirschner • Structure of chromatin • Antisense regulation of gene expression • Discovery of MyoD

31. FRAP: Fluorescence Recovery After Photobleaching

32. FRAP: Fluorescence Recovery After Photobleaching