1. Epigenetics of Human Marie Černá Lecture No 426-H

2. Interaction of environment and genotype gene pool 1 gene pool 2 gene pool 3 gene pool 4 gene pool 5 external factor 1 external factor 2 external factor 3 external factor 4 external factor 5 epigenetic modifications

3. Epigenetic modifications - allow interaction of environment with identical genotypes and by the way formation of their various phenotypes - modifications of gene expression which do not involve nucleotide changes - can be heritable

4. Metastable epialleles - identical alleles that are variably expressed due to epigenetic modifications that are established during life - can be hereditary

5. Epigenetic modifications change level of gene expression Influence of environment: - In utero - Ex utero

6. Epigenetic modifications change level of gene expression They depend on - environment (internal + external) - age - sex

7. Epigenetic modifications - genome is an open system with biological clock period of early pregnancy period of medium pregnancy period of late pregnancy early postnatal period (until 3 years) period of puberty (in ♀ 2 years early) period of mode exchange (30-40 years)

8. Reik W, Dean W, Walter J, 2001, Science, 293, 1089-1093. Epigenetic reprogramming in mammalian development

9. Epigenetic modifications in monozygotic twins Fraga et al., 2005, PNAS, 102, 10604-10609 in 65% pairs identical in 35% pairs different correlation with age and with time spending together

10. DNA methylation Histone modifications RNA interference Epigenetic modifications

11. Chromosomal position effects Polycomb and Trithorax complexes Prions and prion-like phenomena

13. Inactivation of gene expression heterochromatin – closed chromatin DNA methylation of CpG islands Histone modifications - deacetylation (H3K9) - methylation (H3K9, H3K27, H4K20) - sumoylation - ubiquitination

14. Activation of gene expression euchromatin – open chromatin DNA hypomethylation of CpG islands Histone modifications - acetylation (H3K9) - methylation (H3K4, H3K36, H3K79) - phosphorylation - ubiquitination

15. Mechanism of DNA methylation Participation of protein complexes: DNA methyltransferases (DNMT) - DNMT 1 – maintenance methylation - DNMT 3A+3B – de novo methylation Methyl-CpG-binding protein (MeCP)

16. DNA methylation of CpG islands

17. Mechanism of histone modification Participation of protein complexes: histone acetyltransferases (HAT) histone deacetylases (HDAC) histone methyltransferases (HMT) histone demethylases (LSD)

18. DNA-histone modifications

19. Dietary methyl supplementation

20. RNA interference a natural process of gene expression silencing, at the stage of translation or transcription, by non-coding RNA: 1)short RNAs – obtained from digestion of double-stranded RNAs (dsRNAs) 2)long RNAs (> 200 nucleotides)

21. Targets of RNA interference short RNAs: inactivation of RNA from viruses inactivation of RNA from mobile elements (transposons) long RNAs: inactivation of X chromosome

22. Mechanism of RNA interference Participation of protein complexes: Dicer with RNase activity RISC (RNA-induced silencing complex) RITS (RNA-induced transcriptional silencing) PIWI proteins

23. short RNAs 1)micro RNA (miRNA) 2)short interfering RNA (siRNA) 3)Piwi-interacting RNA (piRNA) short RNAs (in cytoplasm) x small RNAs (in nucleus et nucleolus)

24. micro RNA (miRNA) 21-23 nucleotides Endogenic Binding to RISC Partial recognition of complementary sequence in 3’-UTR (nontranslated region) Inhibition of translation process by binding to target mRNA

25. short interfering RNA (siRNA) 20-25 nucleotides Exogenic Binding to RISC or RITS (chromatin modifications) Full recognition of complementary sequence in coding region Initiation of mRNA digestion/degradation

26. Piwi-interacting RNA (piRNA) 27-30 nucleotides Exogenic Binding to PIWI proteins Influence of epigenetic regulation Control of transposons silencing

27. Significance for diagnosis Searching specific miRNAs in blood and urine of patients with a goal to obtain biomarkers for diagnostics and progress evaluation of disease (tumors) In man there was described ≈ 2 500 miRNA.

28. Chromosomal position effects Insulators – sequences along the DNA that attract particular proteins (CTCF protein) for the purpose of preventing distal enhancers from activating or silencing genes insulator functions: - establish the boundaries of regulatory domains - modify chromatin structure - affect nuclear organization

29. Polycomb and Trithorax complexes maintain and propagate expression states (active or silent) of homeotic genes, HOX genes, throughout development by the counteracting activities: - silencing through trimethylation of H3K27 by Polycomb complexes - activation through trimethylation of H3K4 by Trithorax complexes

30. Prions and prion-like phenomena An auto-regulatory loop – capable of maintaining persistent cellular characteristics the structural inheritance – the faithful reproduction of a structure made of proteins the regulatory inheritance – transgenerational continuing of established function of proteins, greatly influenced by the environment

31. Significance in etiology of tumors

32. Epigenetics Environment Expression Genome Physiology Disease Therapy Significance in therapy

33. Definition of live system and life Life is specific organization of matter, energy and information very complicated open system with high level of orderliness with capability of reproduction with capability of development (evolution)