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EPIGENETIKA MB150P85

Přírodovědecká fakulta UK. EPIGENETIKA MB150P85. Petr Svoboda. mail : svoboda1@natur.cuni.cz tel: 24106 3147. Několik poznámek ke struktuře kursu:. - kurs je určen pro pokročilé studenty, zejména pro ty, kteří se chtějí věnovat profesionálnímu výzkumu.

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EPIGENETIKA MB150P85

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  1. Přírodovědecká fakulta UK EPIGENETIKA MB150P85 Petr Svoboda mail: svoboda1@natur.cuni.cz tel: 241063147

  2. Několik poznámek ke struktuře kursu: - kurs je určen pro pokročilé studenty, zejména pro ty, kteří se chtějí věnovat profesionálnímu výzkumu - důraz bude kladen na prezentaci původních výsledků, syntézu a aplikaci znalostí - kurs bude vyžadovat aktivní účast i během semestru - přednáška bude v češtině, ale většina materiálů bude v angličtině. - neexistují hloupé otázky. Je hloupé se nezeptat. Výjimka: Neptejte se, zda něco bude u zkoušky. Odpověď na takovou otázku je vždy ANO.

  3. Organizační záležitosti: Doporučená literatura: Alberts et al. Molecular Biology of the Cell Passarge E., Color Atlas of Genetics + Originální články a reviews, které budou poskytnuty během kursu (ve formátu .pdf – nutno mít počítač s nainstalovaným Adobe Acrobat Viewer) Li E. Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet. 2002 Sep;3(9):662-73. Konzultace: • není pevně daný termín (pokud na něm někdo nebude trvat) • není nutno se ohlásit předem • ohlášení předem zaručuje, že budu mít čas Formát zkoušky • bude upřesněn

  4. NCBI - literatura (Pubmed, OMIM), sekvence (Genbank) a BLAST GNF Symatlas - exprese genů v tkáních Ensembl - anotované sekvence genomů, vyhledávání BCM Search Launcher - analýza sekvencí

  5. DALŠÍ WEBOVÉ STRÁNKY Abcam http://www.abcam.com/index.html?pageconfig=resource&rid=10583&pid=5 The Epigenome Network of Excellence (NoE) http://www.epigenome-noe.net/ A Microscopists' View of Chromosome Organization http://cellbio.utmb.edu/cellbio/nucleus2.htm Wikipedia http://en.wikipedia.org/wiki/Epigenetics http://en.wikipedia.org/wiki/Karyotype http://en.wikipedia.org/wiki/Chromatin http://en.wikipedia.org/wiki/Histone

  6. EPIGENETIKA B150P85 • Úvod • koncept epigenetické modifikace, diverzita mechanismů a efektů, přehled přednášky • Histones I • - koncept struktury chromatinu. Heterochromatin a euchromatin. Core histones, linker histones, replacement histones, protamines. Metody studia chromatinu. • Histones II • - modifikace histonů, polycomb proteins, acetylace, fosforylace a methylace histonů, efekt na expresi genů. • DNA methylation I • - molekulární základy DNA methylace. CpG a non-CpG methylace. Methylace adenosinu. Metody studia DNA methylace. Bisulfite sequencing. • DNA methylation II • - efekt methylace na expresi genů, Methyl-binding proteins a mechanismy inhibice exprese, struktura methylace genů a savčího genomu. 9.10. 2007 23.10. 2007

  7. Imprinting - koncept imprintingu, imprinting u savců. Molekulární mechanismy regulující imprinting. Role imprintingu, válka pohlaví. X-inactivation - koncept a různé strategie dosage compensation. Kontrola X-inaktivace u savců. Epigenetické reprogramování v životním cyklu savců - integrace epigenetických modifikací v životním cyklu savců. Reprogramování genové exprese. Aktivace genomu zygoty. Epigenetické efekty u jiných organismů (rostliny, kvasinky, bezobratlí) - vybrané epigenetické mechanismy zajišťující funkci integritu genomu RNA silencing I - molekulární stroje pro RNA silencing - historický úvod do RNA silencing. Posttranskripční efekty. Role dsRNA. Proteiny a komplexy v RNA silecning. RNA silencing II - role of miRNA and RNAi pathways - RNAi a miRNA. Role, RNAi technologie - experimentální a terapeutické využití. RNA silencing III - chromatin connection - inhibice transkripce prostřednictvím mechanismů souvisejících s RNA silencing. 6.11. 2007 20.11. 2007 4.12. 2007 18.12. 2007

  8. EPIGENETIKA Epigenetika se zabývá přenosem vlastností (informací), které nejsou uložené v sekvenci DNA. • Tyto informace jsou přenášené především v: • struktuře a modifikacích chromatinu • chemických modifikacích DNA • RNAmolekulách

  9. HISTONES I(CHROMATIN STRUCTURE AND FUNCTION)

  10. STRUKTURA GENETICKÉ INFORMACE U EUKARYOT

  11. 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

  12. 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

  13. 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

  14. 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

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

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

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

  18. 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

  19. 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&

  20. 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

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

  22. Metody studia chromatinu – Immunofluorescence I • - malé rozlišení na savčích chromosomech • vhodné pro analýzu velkých domém (centomery, rDNA arrays …) a globální distribuci proteinů • kombinace IF a FISH - kolokalizace B C A 349 350 HA 349 CENP-A UBF UBF 349 Merge Merge Merge HEK293

  23. Metody studia chromatinu – Immunofluorescence I - větší rozlišení u polyténních chromozomů Drosophily Polytene chromosomes (blue) stained for Hairy (green) and Groucho (red) binding

  24. Metody studia chromatinu - Chromatin IP - dobré rozlišení (obvykle cca 0.5 - 1.0 kb) - vhodné pro analýzu jednotlivých genů, promotorů atp. - relativně drahé

  25. Metody studia chromatinu - 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

  26. 349 WT 349 KO unspecific ab WT unspecific ab KO Metody studia chromatinu - Chromatin IP Figure 4 A mouse rDNA repeat 10kb 5kb 15kb 20kb 0kb 25kb 30kb 35kb 40kb 45kb 5´ETS 18S 5.8S 28S 1kb 4kb 17kb 21kb 43kb B 2 * ** 1,5 * 1 % of1:100 input 0,5 0 GAPDH 1kb 4kb 17kb 21kb 43kb

  27. HISTONES II(MODIFICATIONS AND THEIR INTERPRETATION)

  28. 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

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

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

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

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

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

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

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

  36. Bannister 2005

  37. http://www.abcam.com/index.html?pageconfig=resource&rid=10583&pid=5http://www.abcam.com/index.html?pageconfig=resource&rid=10583&pid=5

  38. Shi 2007 mono di tri and back

  39. Arney 2007 … BACK TO HETEROCHROMATIN vs. EUCHROMATIN

  40. Martens 2005 … BACK TO HETEROCHROMATIN vs. EUCHROMATIN DISTINCT REGIONS WITHIN CHROMATIN

  41. Histone code and its interpretation Bannister 2002

  42. Li 2002

  43. Schwartz 2007 Polycombs

  44. Schwartz 2007 Polycombs H3K27

  45. Histone phosphorylation It has been estimated that a typical human cell must repair over 10,000 DNA lesions per day (Lindahl, T. Nature, 1993). http://www.case.edu/med/sanders/sanderslab/Research.html Fission yeast The modification itself does not specifically mark sites of DNA damage. Rather H4-K20 methylation appears to be present throughout the genome even in the absence of DNA damage but is inaccessible to Crb2 in "undamaged" chromatin.

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