EPIGENETIKA MB150P85

1. Přírodovědecká fakulta UK EPIGENETIKA MB150P85 Petr Svoboda mail: svobodap@img.cas.cz tel: 241063147

2. A few comments on the course: - the goal of this course is to present you the latest original information on epigenetics, to give you some idea on how is such information obtained and to make you a better scientist. - this course is designed for advanced students, particularly for those who consider career in science. The course is modeled after advanced Msc./PhD. courses at the University of Pennsylvania and is likely very different from anything you have experienced at the university, so far. - this course requires active participation and is quite is demanding. You will have to read the original literature, make a few homeworks, take an exam and write a scientific text. You earn your credits. - the course is taught in English now. Mp3 records in Czech from 2007 Spring semester are available per request. - there are no stupid question. It is stupid not to ask. Exception: don’t ask if this or that is going to be included in the exam because the answer is always YES. - take the course as a challenge. Don’t take it if you don’t like to be challenged.

3. Resources Suggested reading: Allis et al., Epigenetics Alberts et al. Molecular Biology of the Cell Passarge E., Color Atlas of Genetics Tost, Epigenetics Original articles a reviews will be provided during the course (as .pdf )

4. Resources Suggested reading for minimalists:

5. NCBI - literature (Pubmed, OMIM), sequences (Genbank) a BLAST BioGPS – atlas of gene expression Ensembl - anotated sequences, data mining BCM Search Launcher – sequence analysis (old and decaying) Google and Wikipedia work very well too

6. Additional information Office hours: • no specific time (unless you insist), you can come anytime • to make sure I’ll have time, please, write me or call me in advance Course requirements and the final exam • “take-home” two week exam • the code of academic integrity will be strictly enforced

7. EPIGENETIKA B150P85 • Introduction • overview of the course, basic concepts of epigenetic marks, diversity of epigenetic mechanisms and effects • Histones I • - concept of chromatin structure. Heterochromatin and euchromatin. Core histones, linker histones, replacement histones, protamines. Methods for studying chromatin. • Histones II • - histone modifications, polycomb proteins, acetylation, fosforylation and histone methylations, effects on gene expression. • DNA methylation I • - molecular basis of DNA methylation. CpG and non-CpG methylation. Adenosin methylation. Metods for studying DNA methylation. Bisulfite sequencing. • DNA methylation II • - effects of DNA methylation on gene expression, Methyl-binding proteins and mechanisms of inhibition of gene expression, distribution of DNA methylation within genes and mammalian genomes. Lecture 1 24.2. 2011 Lecture 2 10.3. 2011

8. RNA silencing I – molecular machines for RNA silencing • “historical” introduction into RNA silencing. Post-transcriptional effects. Roles and effects of dsRNA. Proteins and complexes in RNA silencing. • RNA silencing II - RNAi technology • - experimental and therapeutic use. Design of RNAi experiments • RNA silencing III – roles of RNA silencing pathways • miRNA pathway, chromatin connection. • Imprinting • - concept of imprinting, mammalian imprinting. Molecular mechanisms of imprinting. Role of imprinting, Battle of the sexes. • X-inactivation • - principles and different strategies for dosage compensation. Control of X-inactivation in mammals. • Epigenetic reprogramming in mammalian life-cycle • integration of epigenetic modification in the mammalian life cycle. Reprogramming of gene expression during development, artificial reprogramming – the traditional view. • Chromatin in transcribed regions - journal club • Integrated view of epigenetic regulation of gene expression • establishment of pluripotency in ES cells and embryos • Course overview, feedback session Lecture 3 24.3. 2011 Lecture 4 7.4. 2011 Lecture 5 21.4. 2011 Lecture 6 5.5. 2011

9. EPIGENETICS Epigeneticsdeals withheritable information which is not encoded in the DNA sequence • Such information can be encoded in: • structure and chromatin modifications • DNA modifications • RNA molecules

10. HISTONES I(CHROMATIN STRUCTURE AND FUNCTION)

11. 42= 16 43= 64 44= 256 45= 1024 46= 4096 47= 16384 48= 65356 Regulation of complex genomes is a problem E. coli TF binding site length? Core promoter length? Homo sapiens chromatin represents a structural solution for maintaining and accessing complex genomes

12. Higher order structure of genetic information in Eucaryots

13. HETEROCHROMATIN vs. EUCHROMATIN Dyes, like carminic acetic acid or orceine can be used to stain certain domains of a chromosome. The resulting pattern is characteristic for the respective chromosome of a species. During interphase, the chromosomal structure is usually resolved. The intensity of the nuclear staining becomes feebler and less uniform than that of the chromosomes. The stainable substance has been called chromatin by E. HEITZ (formerly at the Botanical Institute of the University of Hamburg, 1927, 1929). He distinguished between heterochromatin and euchromatin. Heterochromatin are all the intensely stained domains, euchromatin the diffuse ones. Heterochromatin is usually spread over the whole nucleus and has a granular appearance. It is known today that the heterochromatic domains are those where the DNA is tightly packed (strongly condensed) which is the reason for their more intense staining. The euchromatic domains are less tightly packed. http://www.biologie.uni-hamburg.de/b-online/e11/11c.htm#05

14. CHROMOSOME BANDING TECHNIQUES Prior to 1960, when Moorehead and Nowell described the use of Giemsa in their chromosome preparations, conventional cytologic stains such as acetoorcein, acetocarmine, gentian violet, hematoxylin, Leishman's, Wright's, and Feulgen stains were used to stain chromosomes. The Romanovsky dyes (which include Giemsa, Leishman's, and Wright's stain) are now recommended for conventional staining, because the slides can be easily destained and banded by most banding procedures. Orcein-stained chromosomes cannot be destained and banded; therefore, orcein is generally not used in routine chromosome staining. Giemsa stain is now the most popular stain for chromosome analysis (Gustashaw, 1991). Banding protocols http://homepage.mac.com/wildlifeweb/cyto/text/Banding.html

15. http://www-biology.ucsd.edu/classes/bimm110.SP06/lectures_WEB/L08.05_Cytogenetics.htmhttp://www-biology.ucsd.edu/classes/bimm110.SP06/lectures_WEB/L08.05_Cytogenetics.htm

16. http://www-biology.ucsd.edu/classes/bimm110.SP06/lectures_WEB/L08.05_Cytogenetics.htmhttp://www-biology.ucsd.edu/classes/bimm110.SP06/lectures_WEB/L08.05_Cytogenetics.htm metaphase and prometaphase G-banded human chromosome 1 and the standard nomenclature for labeling the bands;short arm: p (petite); long arm: q;1 - 4 regions for each arm labeled from centromere towards telomereeach region has several bands, again numbered away from the centromere

17. http://fig.cox.miami.edu/~cmallery/150/proceuc/chromosome.jpghttp://fig.cox.miami.edu/~cmallery/150/proceuc/chromosome.jpg

18. Evolution of chromatin structure models Molecular Biology of the Cell 1994 Molecular Biology of the Cell 2002 Molecular Biology of the Cell 2007

19. Things to remember … Nucleosome H2A, H2B, H3, H4 – core histones H1 – linker histone http://en.wikipedia.org/wiki/File:Nucleosome.JPG

20. Things to remember … Closed Open http://sgi.bls.umkc.edu/waterborg/chromat/chroma09.html

21. Things to remember … apparent global chromatin patterns are underlied by repetitive sequences Martens 2005

22. NUCLEOSOME AND CORE HISTONES H2A, H2B, H3, H4 – core histones H1 – linker histone

23. Replication-dependent core histones - localized in large clusters (common chromatin domains? RNA processing?) - the major human cluster - 6p21(mouse chr. 13) - smaller clusters on 1p21 (mouse chr. 3) and 1q42 (mouse chr. 11) - the major cluster tends to colocalize with Cajal bodies (functional link isn‘t well understood) histone type cluster gene nomenclature family member HIST1H2AG older nomenclature and synonyms can be clarified at the GNF Symatlas and NCBI webpages Marzluff 2002

24. Expression of core histones cell-cycle dependent http://www.unc.edu/depts/marzluff/research.html specific 3‘ end processing CPSF-73 http://www.reactome.org/cgi-bin/eventbrowser?DB=gk_current&ID=77588&

25. Mammalian core histone variants H2A.X - estimated to make 10% of nuclear H2A in mammals - rapidly phosphorylated in a response to DNA damage CENP-A (variant of histone 3, Cid in Drosophila) - found at centromeric regions macroH2A - enriched on the inactive X chromosome H2A.Z - possibly involved in initial steps of gene activation in euchromatin H3.3 - deposited within chromatin independent on DNA replication - enriched at sites of transcription - accumulates in non-cycling cells H3.1 - synthesized and deposited during S-phase H2A.Bbd - excluded from the inactive X chromosome - H2A.Bbd histone octamer organizes only approximately 130 bp of DNA

26. Protamines - non-histone replacement in sperms Braun 2001

27. Methods to study chromatin – Immunofluorescence I • - Small resolution on mammalian chromosomes • useful of analysis of large domains (centomeres, rDNA arrays …) and global protein distribution • IF and FISH combination - colokalization B C A 349 350 HA 349 CENP-A UBF UBF 349 Merge Merge Merge HEK293

28. Methodsto study chromatin – Immunofluorescence I - Higher resolution in polytene chromosomes in Drosophila Polytene chromosomes (blue) stained for Hairy (green) and Groucho (red) binding

29. Methods to study chromatin – Chromatin IP • good resolution (typically 0.5 - 1.0 kb) • useful for analysisof individual genes, promoters • genome-scale analysis nowadays possible • relatively expensive tips and tricks • Detection: • qPCR • promoter/tiling microarray = ChIP-Chip • deep sequencing = ChIP-Seq

30. Methods to study chromatin – Chromatin IP human rDNA repeat A 10kb 5kb 15kb 20kb 0kb 25kb 30kb 35kb 40kb 43kb 5´ETS 18S 5.8S 28S 1kb 3kb 6kb 13kb 20kb 29kb 38kb 42kb B 14 12 349 10 unspecific antibody 8 % of input 6 4 2 0 GAPDH 1kb 3kb 6kb 13kb 20kb 29kb 38kb 42kb

31. HISTONES II(MODIFICATIONS AND THEIR INTERPRETATION)

32. http://biology.plosjournals.org/perlserv?request=get-document&doi=10.1371/journal.pbio.0020136http://biology.plosjournals.org/perlserv?request=get-document&doi=10.1371/journal.pbio.0020136

33. http://sgi.bls.umkc.edu/waterborg/chromat/chroma09.html

34. Proc Natl Acad Sci U S A. 1964 May; 51(5): 786–794.

36. http://biology.plosjournals.org/perlserv?request=get-document&doi=10.1371/journal.pbio.0020136http://biology.plosjournals.org/perlserv?request=get-document&doi=10.1371/journal.pbio.0020136

38. Kuo 1998 Histone acetylation - deacetylation

39. Kuo 1998 Histone acetylation - deacetylation

40. Kuo 1998 Histone acetylation - deacetylation Histone acetylation Deposition-related (B HATs) Transcription-related (A HATs)

41. Annemieke 2003 Histone deacetylases Trichostatin A is an inhibitor of histone deacetylases. + Sir2 family of deacetylases - target nonhistone proteins

42. http://www.imt.uni-marburg.de/bauer/research.html Histone methylation - lysine residues SET domain HMTs Bannister 2002

43. specific methylation level specific residue specific lysine residue specific effect specific sequence specific complex specific HMT specific protein specific structure modification specific effect specific locus must be maintained! (memory)

44. Histone methylation - lysine residues Bannister 2002

45. histone code concept

46. Shi 2007 Histone methylation is reversible! JmjC domain (JumanjiC)

47. Histone methylation - arginine residues http://www.imt.uni-marburg.de/bauer/research.html

48. Abcam = common source of Abs and information http://www.abcam.com/

49. Bannister 2005

50. Shi 2007 mono di tri and back