1. Biol/Chem 473 Schulze lecture 10: Stem cells and chromatin

3. Reproductive and therapeutic cloning begin the same way

4. A decade of reproductive cloning 1996 1998 2000 2001 2003 RETRACTED 2005 2005 2004

6. can’t culture can culture Many (all?) fates possible Limited set of fates possible

8. Two important histone methyltransferases Enhancer of Zeste (PcG) & trithorax (trxG) E(z) and orthologs recognize and methylate H3K27 (forms part of repressor complex PRC2 that maintains repression of homeotic and other genes in development) Trithorax and orthologs recognize and methylate H3K4 (forms part of an activating complex that maintains activation of homeotic and other genes in development)

10. HCNEs cluster near developmentally important genes in vertebrates Woolfe et al. (2005) PLOS Biology 3(1): 0116

11. A bivalent chromatin structure marks key developmental genes in embryonic stem cells Bernstein, B.E. Angelina Jolie, Brad Pitt, Jennifer Aniston et al. (2006) Cell 125: 315-326

15. Bivalent domains: repressive AND activating histone modifications H3K27me H3K4me Fig 1A A higher than expected incidence of bivalents occur in HCNE’s

18. Fig 2 Most bivalents in ES cells are either H3K27Me OR H3K4Me in differentiated cells

22. QPCR, like regular PCR, occurs in stages PCR just getting started: amount of product not proportional to amount of starting material (can’t measure it anyway) Linear phase: amount of product is proportional to amount of starting material End of reaction and PCR components depleted: amount of product not proportional to amount of starting material Initial phase Plateau phase Logarithmic phase

23. Real time measurement during log phase of PCR correlates with starting concentration http://www.dorak.info/genetics/realtime.html

24. Fig 3 H3K27Me only H3K4Me only Bivalent Differentiated cells

25. Bivalents keep genes silent, but “poised” for later expression H3K27Me is epistatic to H3K4Me Fig 4

26. Poised state to resolved state: differentiation in cell culture All genes associated with bivalent domains Fig 5 Bivalent marks resolve into monovalent K4Me or K27Me, depending on the transcriptional state after differentiation Reverse transcriptase PCR – ie., starting template is mRNA population (not the same as QPCR)