From genetics to epigenetics The world beyond Mendel s laws

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1 Sapienza University of Rome Master in Medicine and Surgery From genetics to epigenetics The world beyond Mendel s laws What about epigenetics?..the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being. Conrad Hal Waddington, 1942 Epigenetics : different interactions between different genes and between genes and environment result in distinct phenotypes from the same genetic material Dr. Cecilia Battistelli, PhD What about epigenetics? What about epigenetics? The study of the mechanisms of temporal and spatial control of gene activity during the development of complex organisms. Holliday R., This definition means that epigenetics is a phenomena or a modification, other than DNA sequence, that influences organisms development. Geneticists study the gene; however, for epigeneticists, there is no obvious epigene. Nevertheless, during the past year, more than 2,500 articles, numerous scientific meetings and a new journal were devoted to the subject of epigenetics. It encompasses some of the most exciting contemporary biology and is portrayed by the popular press as a revolutionary new science an antidote to the idea that we are hard-wired by our genes. Adrian Bird (2007) Epigenetics is the study of inheritable changes of gene expression that are not caused by changes in DNA sequence. (Waterland & Michels, 2007) Our body contains hundreds of different kind of cells, although each one derives from the same starting point. As cells develop, their state is governed by the selective silencing of genes. This silencing is mainly regulated by epigenetic factors. Failure to silences genes can produce a hazardous cacophony. Epigenetic factors govern the interpretation of DNA within each cell. The difference between genetics and epigenetics is as the difference between reading and writing a book. Once the book has been written its text (DNA sequence) is the same in all the copies of the book. Each reader can give a different interpretation to the content of the book. In a similar way epigenetics allows different interpretations of the genetic code that depends on the different conditions of the biological model. Thomas Jenuwein. 1

2 Epigenetics Development There might be a place for a view of epigenetics that keeps the sense of the prevailing usages but avoids the constraints imposed by stringently requiring heritability. The following could be a unifying definition of epigenetic events: THE STRUCTURAL ADAPTATION OF CHROMOSOMAL REGIONS SO AS TO REGISTER, SIGNAL OR PERPETUATE ALTERED ACTIVITY STATES. Adrian Bird, Perceptions of epigenetics, NATURE Vol447, (2007) Epigenetics at work: Development Parental Imprinting Differentiation X-chromosome inactivation in mammals Variegation, variable expressivity Cancer Methylation is regulated during development Methylation is critical during differentiation 2

3 Central dogma of molecular biology Genetic Vs Epigenetic Genetic mutation Epigenetic mutation Neurogenin Myogenin Neurons Myotubes Same genome different phenotype distinct expression profiles loss or alteration of protein function loss or alteration of expression IRREVERSIBLE Inheritable If they occur in germ cells they are passed to the progeny according to Mendel s lows Cancer Evolution alteration of expression REVERSIBLE Stable during mitosis Inheritable In a specific cell type they are the base for the epigenetic memory of a specific cell lineage Tissue specific gene expression depends on epigenetic modifications that happen during development and differentiation and not on genetic modifications involving nucleotides sequence. Epigenome Different levels of epigenetic control Epigenome Transcriptional control: trancription factors binding tissue-specific, direct binding of hormons, growth factors or intermediate elements DNA Primary transcript (precursor) Epigenetic mechanisms: long-range control through chromatin structure remodelling It decides wich gene muste be «ON» or «OFF» in a single cell, determining a specific pattern of gene expression It can be inherited throwout different cell generations, saving the same genic program or changing it (epigenome plasticity) Nucleus Cytoplasm Translational control Translation Post-translational control PROTEIN Active Vs Inactive PROTEIN mrna mrna Post-transcriptional: alternative splicing alternativo, alternative polya, RNA editing tessue-specific Exportation control Stability control Degradation 3

4 Role of chromatin Mechanisms of action of epigenetics Epigenetics Heritable changes in gene function that occur without a change in the DNA sequence a) Housekeeping genes b) Tissue-specific genes FACOLTATIVE Inactive when it is condensed. COSTITUTIVE Inactive. Localized in centromeric and pericentromeric regions Mechanisms: DNA methylation and hydrossimethylation Histone modifications (acetylation, methylation, phosphorylation ) microrna and ncrna Histone variants Chromatin remodelling Mechanisms of action of epigenetics Octamer core in association with 146 bp DNA Histone modifications Histones: Eukaryotes aa Lys and arg -> charge + Interaction with phosphate of DNA Nucleosomes Chromatin condensation Different kinds of modifications: Acetylation and/or de-acetylation (lys) Methylation (lys, arg) Phosphorylation (his, ser, thr) Ubiquitination (lys) Histone Code : hypothesis Histone tails modifications act as a signal for proteins that control gene expression and DNA replication 4

5 H3 H4 modifications Histone acetylation Histone K Ac Me Active chromatin Inactive chromatin Acetylatio n Histone tail HATs It is associated with : chromatin de-condensation transcription factors recruitment transcriptionally active DNA Repressors and activators can direct histone deacetylation and hyperacetylation at specific genes Histone acetylation Vs deacetylation Histone deacetylases inhibitors Trichostatin A Suberoylanilide hydroxamic acid (SAHA) Sodium butyrate / phenylbutyrate Valproic acid Acetylation and deacetylation modifies the activity of proteins involved in transcription, nuclear transport, and the cytoskeleton as well as histones The balance between distinct acetylated states determines the activity of a specific DNA sequence 5

6 Histone methylation How to study histone modifications Cultured cells or tissue Purification of nuclei Chromatin purification Chromatin immunoprecipitation Histone demethylases: Arginine deiminase : Catalized by PAD (Protein Arginine Deiminases) that produces citrulline and methyl-ammonium Extraction of DNA from antibody-bound and unbound fractions Demethylation : Catalized by LSD (Lysine-specific Demethylases) Quantitative PCR ChIP on chip ChIP seq Nucleosome occupancy DNA methylation Nucleosomes distribution and the consequent DNA accessibility to transcription factors depend in part on histone modifications 6

7 DNA methylation DNA methylation It occurs especially at CpG level in mammals, in CpG-rich regions named CpG islands. Mechanism involved in: Gene expression regulation and transcriptional control Chromatin structure and heterochromatin formation and maintenance Cell differentiation (tissue specific genes are methylated in tissues where they are not expressed) Mechanism counteracting exogenous DNA, transposons and retro-transposable elements. After DNA replication DNMT restores methylation signature in order to give the epigenetic heredity to daughter cells. DNA methylation is the best characterized epigenetic mechanism. X chromosome inactivation Genomic imprinting Mutagenesis e carcinogenesis DNA methylation and gene silencing Methylation phases Differential DNA methylation (de novo) : DNMT3a and DNMT3b Maintenance of DNA methylation and transmission to the progeny : DNMT1 De-methylation (de-methylases? ) DNA methylation inhibits gene expression at transcriptional level. DNA methylation occurs at promoter regions. 7

8 Maintenance methylation DNA methylation inhibitors DNMT1 5-azacytidine 2-deoxy-5-azacytidine sirna (Dnmt1, 3a,3b) Decitabine(MDS) Zebularine (Cancer) Procainamide (Dnmt1) Hydralazine (PKCδ) Diet UV light 5-Azacytidine Inhibits methylation of newly synthesized DNA Induces gene expression Alters differentiation Requisite transcription factors must be present and activated! DNA methylation analysis Methylation sensitive restriction endonuclease digestion Methylation sensitive restriction endonuclease digestion Bisulfite sequencing Methylation specific PCR Pyrosequencing Isoschizomeric Restriction Endonuclease Analysis Hpa HpaII II cleaves C C-C-G-G but not C C-mC-G-G Msp MspI I cleaves C C-C-G-G and C-mC-G-G 8

9 Bisulfite sequencing Methylation specific PCR CH 3 CH 3 Genomic DNA 5 -ACGATCGCGACGATCGA-3 5 -AUGATCGUGAUGATCGA-3 5 -ATGATCGTGATGATCGA-3 3 -TACTAGCACTACTAGCT-5 Bisulfite treatment PCR amplification Cloning and sequencing CH 3 CH 3 5 -ACGATCGCGACGATCGA-3 DNA methylation enzymes DNA methylation enzymes DNMT1 Expressed in all tissues Responsible for the maintenance of methylation pattern during replication Localized on replication foci k.o. mice dead at 10 days post fertilization It could have a de novo methyl-transferase activity In tumours it is frequently over-expressed and methylates un-methylated regions, silencing cell cycle regulating genes. DNMT3a, 3b Involved in de novo and maintenance methylation They mediate global re-methylation after embryo implantation k.o. mice for DNMT3a dead at 4 weeks after birth k.o. mice for DNMT3b dead during gastrulation DNMT3b is involved in ICF syndrome Highly expressed in ES cells Down regulated after differentiation Low expression in adult somatic tissues 9

10 Methyl binding proteins Cross-talk between DNA methylation and histone tails modification Ubiquitously expressed in somatic tissues MBD domain in common MeCP2: It binds symmetrically to methylated CpG dinucleotide No sequence-specificity Co-localization with heterochromatic and highly methylated DNA Interaction with histone de-acetylation complexes Cross-talk between DNA methylation and histone tails modification micrornas RNA polymerase transcribes - mrnas - non coding RNAs snorna snrna hnrna lincrna microrna 10

11 micrornas Genomic localization mir-10 Single strand non coding RNA, 22nt long Involved in gene expression regulation through a repressive post- Exons and introns of transcripts non protein-coding (81%) HOX4B mir-155 transcriptional mechanism BIC They control development, cell proliferation, apoptosis, organogenesis and cancer genesis Introns of protein-coding transcripts (18%) DLEU2 mir-15a mir-16-1 Intergenic regions Translational repression Mechanism of action 5 3 Cytoplasm mrna 5 cap Ago Ago AAAAn 3 gene mirna 5 cap Ago Ago AAAAn 3 pri-mirna Endonucleolitic cleavage of target mrna Ago Mature mirna RISC complex pre-mirna mrna 5 cap Ago AAAAn 3 Exp-5 5 cap P AAAAn 3 Nucleus Ago 11

12 Genomic imprinting Male and female genome in gametes have to be considered equivalents (same genes, exception for sex chromosomes). Gene are usually expressed from both the alleles. Genomic imprinting They control gene expression of other genes, they are involved in development of embryonic and extra-embryonic tissues. A limited number of genes are expressed only from one of the two alleles (IMPRINTED). Maternal imprinting (maternal allele silenced) or paternal imprinting (paternal allele silenced). Embryos with only maternal chromosomes: no development of extra embryonic tissues; embryos with only paternal chromosomes: no development of embryo body. Phenomena due to different epigenetic modifications, especially DNA methylation and histone tail modifications, introduced in imprinted genes during gametes formation, with the formation of DMR (differentially methylated regions). Basis of cellular memory. Imprinted genes are not modified during nuclear reprogramming happening after fertilization but maintain epigenetic modifications introduced during gametogenesis. Imprinted genes : 80% in cluster Co-ordinately regulated in chromosomal domains ICR control imprinting regionally Balance between genes of paternal or maternal origin: Father: promotes foetal growth Mather: controls foetal growth Pathologies of imprinting Prader-Willi and Angelman are excellent examples of imprinting as pathology They are the result of a deletion in the same section of chromosome 15. Prader-Willi is the syndrome caused by epigenetic modification inherited from the father; Angelman is due to epigenetic mutations inherited from the mother Pathologies of imprinting Beckwith- Widemann Syndrome 1/ born. Prenatal overgrowth High mass of abdominal organs Asymmetry of the limbs Macroglossia Hypo- glycaemia Heart troubles Predisposition to select cancers (Wilms tumor of the kidney, HCC, neuroblastoma) 12

13 Pathologies of imprinting Beckwith- Widemann Syndrome: the chromosomal region X chromosome inactivation Inactive X chromosome is silenced by hyper-methylated DNA and hypo-acetylated histone tails. Essential for dosage compensation of gene expression From epigenetic mechanism to epigenetic pathology Epigenetic pathologies Tumours Neurodegenerative diseases Psichiatric disorders Autism Chron disease Alzheimer, Parkinson, Schizofrenia, eating disorders, drug use and abuse 13

14 Epigenetic pathologies ICF Syndrome: Immunodeficiency, centromere instability, facial anomalies Autosomal recessive, 50 cases in the world Hypertelorism, Low ears, Macroglossia, Development delay Immunoglobulin reduction (lower number of timocytes caused by a high level of apoptosis), Dead caused by infection, Neurological disorders Chromosomal fusions, Peri-centromeric chromatin extension, Chromosomes with long arms Lymphocytes have centromeric regions (satellite DNA, constitutive heterochromatin) completely de-methylated and fragile (breaks and re-unions with chromosomal rearrangements), mitotic lymphocytes have multi-radial chromosomes Bi-allelic mutations in the catalytic domain of DNMT3b (7% 5meC) Rett syndrome MeCP2 gene mutation Males dead at birth Alteration of neuronal development Loss of language and of hands use Epigenetic pathologies Microcefalia, convulsions, autism and atassia Due to a wrong silencing of specific genes II cause of mental delay in females (1/10000) Normal phenotype for 6-18 months than loss of the capacities (hand washing, clapping, mouthing), apraxia ( loss of co-ordinated movements), breathing abnormalities, epilepsia, sleeping irregularities, agitation. APL acute pro-myelocytic leukemia Epigenetic pathologies APL acute pro-myelocitic leukemia Epigenetic pathologies Pro-myelocytes differentiation granulocytes Clonal expansion of immature pro-myelocytes in bone narrow and in peripheral blood Retinoic Acid Receptor (RARa) Hormones receptor HDAC-regulated It forms heterodimers RAR/RXR Absence of RA histone de-acetylation differentiation genes repression Presence of RA loss of repressive complex histone acetylation differentiation of myeloid cells Translocation involving genes RARα (Ch.17) e PML (Ch.15) 14

15 Epigenetic pathologies Epigenetic pathologies Cancer Cancer Self sufficiency in growth signals Insensibility to anti-growth signals Evading apoptosis Limitless replicative potential Sustained angiogenesis Tissue invasion and metastasis Oncogene ras activation Loss of tumour suppressor e.g. Rb Production of IGF Telomerase activation VEGF production vascular endothelialgrowth factor E caderin inactivation Cellular adesion Chromatin modification can repress gene expression in tumor HDAC inhibitors have anti-tumor properties because they activate tumor-suppression genes (e.g. p21). HAT: Genes coding for HAT are trans located, amplified, over-expressed and/or mutated in different tumours HDAC: involved in lymphoma and leukaemia Epigenetic pathologies Epigenetic pathologies Cancer HAT: Missense mutations or associated with non-functional p300 are associated with colon tumours. Mutations inactivating HAT activity of CBP higher risk of tumours Loss of heterozygosis in CBP locus is associated with HCC p300 and CBP translocation resulting in fusions in frame with different genes haematological tumours Cancer Normal cells transcription me AC AC AC Me TF Tumour cells HKMT HDAC DNMT me Me HDAC: Non-Hodgkin lymphoma : LAZ3 (transcriptional repressor) is over-expressed and causes an aberrant transcriptional silencing leaving to tumours Acute myeloid leukemia : M2 is associated with t(8;21) that generates a fusion protein AML1/ETO acting as a potent transcriptional repressor through the recruitment of HDAC. me me Me Me Me Me Me TF me me Genes involved in the control of cell cycle, differentiation, apoptosis 15

16 Epigenetic therapies Epigenetic therapies HDAC inhibitors: a new anti-cancer therapy promoter Loss of function of tumour suppressor genes Tumour suppressor genes Cell cycle inhibitors genes HDAC inhibitors promoter Target genes TSA and butyrate Cell growth arrest Differentiation Apoptosis (CDKN1A, TRAIL/TNFR10, CDKN2A) Co-operates with 5-aza-2 -deoxicitidine To hypo-methylate and reactivate tumoursuppressor genes Re-expression of Tumour suppressor genes Epigenetic therapies Epigenetics Genetics Mutation Epigenetics Chromatinremodelling Epigenetics regulates gene expression without modifying DNA sequence Deletion promoter DNA methylation There are different levels of regulation: DNA methylation Genomic imprinting Histone modifications Methylated DNA promoter Epigenetic alteration mechanisms are the basis for different pathologies including tumours Gene therapy Epigenetic therapy We can reprogramming cellular epigenome through epigenetic modulators 16

17 Reprogramming Regenerative medicine Dolly demonstrated that also in mammals: - Genome remains pluripotent - Chromatin is plastic and can be pluripotent also in adult cells How to obtain autologous ES cells? Dolly opened the door to the regenerative medicine Nuclear reprogramming Nuclear transplantation oocyte enucleation Somatic cell Problems: 1) This process is very inefficient 1/400 (a high number of oocytes is required) 2) New potential individuals are generated (ethical problems) Therapeutic cloning Incomplete DNA de-methylation is the main cause of cloning problems through nuclear transfer in mammals oocytes. DNA de-methylation is necessary for Oct4 transcription (essential for blastocyst) 17

18 Which is the conclusion? Genetic cause Epigenetic cause Dr. Cecilia Battistelli, PhD 5 Clinica Medica 2 floor battistelli@bce.uniroma1.it 18