HGD 5502 EPIGENETICS. Dr. Abhi Veerakumarasivam (2011)

Transcription

1 HGD 5502 EPIGENETICS Dr. Abhi Veerakumarasivam (2011)

2 Outline History Definition Mechanisms of Epigenetics Epigenetic Processes Review

3 History Conrad Hall Waddington ( ) Developmental biologist, paleontologist, geneticist, embryologist and philosopher. Foundations for systems biology. Educated and Lectured at Cambridge University. Early 1930s: Waddington & embryologists probed the development of the amphibian neural tube. Paradigm at that time : Genes were unimportant and just played a role in minor phenomena such as eye colour. Waddington s view: embryology lies in genetics. Drosophila laboratory in California, 1935: how gene regulatory products could generate developmental phenotypes (mutation analysis) Canalisation: the ability of an organism to produce the same phenotype in various different environments. Genetic assimilation: animal s response to an environmental stress or change to become a fixed part of its inherited repertoire

4 Definition Epigenesis + genetics = Epigenetics Epigenesis: differentiation of cells from their totipotent state in embryonic development. Epigenetics: Waddington : Holliday: physical nature of genes (genes role in heredity was unknown then) Model of genes interacting with their environment to produce a phenotype. the study of mechanisms of temporal & spatial control of gene activity during the development of complex organisms. so any aspect apart from DNA sequences that influence the development of an organism. Modern: refers to heritable traits (cell cycles/transgenerations) that do not involve changes to the DNA sequence. Greek epi means on top of or in addition. Epigenetics traits exist in addition to the traditional molecular basis of inheritance.

5 Epigenetic landscape Metaphor for how gene regulation modulates development Imagine a marble rolling down a hill towards a wall. The marbles will compete for the grooves on the slope, and come to rest at the lowest points Marble = cell Grooves on the slope = represents the 'chreodes' or bundles of trajectories in state space Marble is rolled along the slope into different grooves = Irreversibility of cell type differentiation increases Marble rolls down to the point of lowest local elevation = represent the eventual cell fates, that is, tissue types Idea was actually based on experiment: Waddington found that one effect of mutation (which could modulate the epigenetic landscape) was to affect how cells differentiated

6 Landscape Expansion Epigenetic landscape: Behind the scenes The complex features of the epigenetic surface is a product of the expression of complex network of interactions underlying it Each valley in the landscape is formed by tension on ropes that are attached to pegs on the ground that represent complexes of genes The ropes are attached not only to random points on the overhead surface, but to points on the other ropes as well, and to pegs in the lower surface that themselves represent only semi-stabilised forms. Exponential multiplication of the non-linearities flowing through the system No change in any single parameter can fail to be relayed throughout the system and to affect, in turn, conditions all across the event surface

7 Modern Epigenetics Today, refers to the combined epigenetic modifications of a given domain of DNA sequence Molecular basis is complex Modifications of the activation of certain genes and or chromatin proteins but not DNA structure Muscle and iris cells contain DNA instructions how to make eye colour pigment but in the muscle cells, these genes are switched off Everyone is sitting in my class paying attention to different things exam fate will be different!! Epigenetic changes are preserved when cells divide (not cancer cells) Epigenetic changes occur within the course of an organism s lifetime (some inherited transgeneration)

8 Mechanisms of Epigenetics Mechanisms: Chromatin Remodelling & Histone Modification DNA Methylation Non-coding RNA mediated pathway Prion Epigenetic code: - defining code in every eukaryotic cell - consisting of the specific and combination of epigenetic modifications in each cell Genetic code in each cell is the same Epigenetic code is tissue- and cell-specific "The genetic code is the piano, the epigenetic code the tune."

9 Chromatin Remodelling & Histone Modification

10 Chromosome Set 23 pairs: 44 autosomes 2 sex chromosomes Each chromosome type contains different info

11 Chromosome specific traits

12 DNA Packaging Human cell has 2m of DNA & Nucleus is mm diameter DNA compacted 10,000X Chromatin = DNA + protein that comprises chromosomes

13 Chromatin Chromatin structure is dynamic and changes in chromatin structure adds another level of regulation to eukaryotic gene expression HISTONES form the NUCLEOSOME, which DNA loops around. Chromatin in eukaryotes is not uniform: EUCHROMATIN less condensed; actively transcribed HETEROCHROMATIN more condensed; transcriptionally inactive; either constitutive/facultative

14 Euchromatin Vs. Heterochromatin G-banding : Dark bands, stain strongly A,T rich (gene poor).

15 Nucleosome Eukaryotic DNA is packed into a nucleoprotein complex called chromatin The fundamental repeating unit of chromatin is the nucleosome Nucleosome contains 147 bp of genomic DNA wrapped around an octamer of histone proteins approximately 1.75 times The histone core complex includes 2 molecules of each of the following histones H2A, H2B, H3, H4 [variant H2A.Z also included in core complex under special circumstances]

16 Chromatin Remodeling Remember that chromatin structure is dynamic. Many proteins have been identified in a range of eukaryotic species that are involved in chromatin remodeling. Many of these proteins are highly conserved. Chromatin remodeling is initiated can be initiated via 2 ways: Post Translational Modification of the amino acids that make up Histone proteins Methylation of the DNA

17 Histone Modification Histones contain: a globular domain that interacts with other histone proteins and DNA flexible tail regions (N-term and C-term) that project laterally from the core Histone Tails: highly modified a substrate for various post-translational modifications Most common modifications include: acetylation, methylation, phosphorylation, and ubiquitination on the protruding N-terminal tail distinct modifications correlate with specific transcriptional states supports the histone-code hypothesis: patterns of modifications are read like a molecular bar-code for recruiting nuclear machinery that controls chromatin state

18 Histone Modifications Histone tails = N-termini of Histones e.g. Histones H3 and H4 N-termini e.g. Histones H2A, H2B and H1 N- and C- termini Post-translational modifications: Acetylation Lys Methylation Lys and Arg Phosphorylation Ser and Thr Ubiquitination Lys Sumoylation (Lys); ADP-ribosylation; glycosylation; biotinylation; carbonylation

19 Exp: Identification of modifications ChIP: Chromatin Immunoprecipitation to identify histone modifications

20 Histone Acetylation Histones in transcriptionally active genes are often acetylated Acetylation is the modification of lysine residues in histones Histone acetylation Histone acetyl transferases (HATs) Adds acetyl groups to histone tails Reduces positive charge and weakens interaction of histones with DNA Facilitates transcription by making DNA more accessible to RNA polymerase II Histone deacetylation Histone deacetylases (HDACs) Removes acetyl groups from histone tails Increases interaction of DNA and histones Represses transcription (usually) May involve the same Lys residues as targeted for methylation

21 Histone acetylation Histone acetyltransferase Histone deacetylase Hypoacetylation Strong internucleosomal interactions: Acetylation has two functions: Neutralise the positive charge on the lysine residues Destabilise interactions between histone tails and structural proteins Hyperacetylation (Yellow) Weak internucleosomal interactions: histone tails do not constrain DNA, which is accessible to transcription factors

22 Histone acetylation Mitosis does not erase acetylation, but merely distributes histones, between the daughter chromosomes. Specific acetyltransferases (red) end up distributed between the daughter chromosomes, too. Once segregated, an acetyltransferase would acetylate the adjacent nucleosomes (yellow) and thereby spread over the entire chromatin domain.

23 Histone Methylation Histone methylation Histone methyl transferases (HMTs): Histone lysine methyl transferases (HKMTs) Methylate lys (K) residues Protein arginine methyl transferases (PRMTs) Methylate arg (R) residues Varying number of methyl groups: Lys mono- di- or tri-methylated (on e-amino group) Arg mono- or di-methylated (symmetric or asymmetric) (on guanidine-e-amino groups) Methylation can result in repression or activation of expression Not permanent: LSD1 Jmj histone demethylases

24 Histone Phosphorylation Histone phosphorylation - kinases E.g. by aurora AIR2 Ipl1 kinase family Required for chromosome condensation and cell cycle progression E.g. by MSK1 and 2 or IKKa kinase Required for signal transduction leading to gene activation Can prevent nearby histone methylation due to steric hindrance and/or facilitation of competing acetylation Reversed by phosphatases like PP1 or PP2 Alters recruitment of binding proteins; e.g.- If phospho-acceptor precedes methylated residue activates transcription If phospho-acceptor follows methylated residue silences transcription

25 Histone ubiquitination Histone Ubiquitination -Mono-ubiquitination (by Rad6) and recruitment of proteasomal ATPases (Rpt4 and Rpt6) alters chromatin structure regulates H3 methylation De-ubiquitination (by SAGA-associated Ubp8) regulates mono- vs tri-methylation

26 Histone Code Hypothesis The histone code hypothesis: - A particular modification on a specific histone residue may regulate modification of the same or different residues within the same or a different histone - Different types or combinations of modifications are read by chromatinmodulating proteins, resulting in regulation of chromatin structure and hence, transcription Inactive X high H3 K9 Methylation no H4 Acetylation Active X high H3 K4 Methylation increased H3 and H4 Acetylation

27 Nucleosome Remodeling Complexes Chromatin remodeling influences the level of gene expression The positioning of histones along DNA is mediated by ATP-dependent nucleosome - remodeling complexes

28 SWI/SNF-Mediated Chromatin Remodeling BAF170 BAF57 BAF53 hsnf5 BAF155 BRG1 BAF60 BAF250 c-myc p53

29 SWI/SNF-Mediated Chromatin Remodeling c-myc p53 BAF57 BAF53 hsnf5 BAF155 BAF170 BRG1 BAF60 BAF250

30 SWI/SNF-Mediated Chromatin Remodeling c-myc p53 BAF57 BAF53 hsnf5 BAF155 BAF170 BRG1 BAF60 BAF250 ATP ADP+P i

31 SWI/SNF-Mediated Chromatin Remodeling c-myc p53 BAF57 BAF53 hsnf5 BAF155 BAF170 BRG1 BAF60 BAF250

32 Bulge Propogation: Repositioning This complexes use the energy of ATP hydrolysis to noncovalently reposition histone octamers and generate nucleosome free or dense chromatin.

33 Genes encoding ATP-dependent nucleosome - remodeling complexes Genes Mechanism involved Disease In ATRX cells, the DNA repeats are hypomethylated. SIOD - Schimle immuno-osseous dysplasia COFS - cerebro-oculo-facio-skeletal syndrome CBS - Cockayne syndrome type B RTS - Rubinstein Taybi syndrome

34 DNA Methylation Chemical modification of DNA. Can be inherited and subsequently removed without changing the original DNA sequence (reversible). Most well characterised epigenetic mechanism. Involves the addition of a methyl group to DNA Usually to the number 5 carbon of the cytosine pyrimidine ring Effect: reducing gene expression. DNA methylation at the 5 position of cytosine has been found in every vertebrate examined.

35 CpG Along the linear DNA chain, there are sites of DNA where a cytosine is followed by and linked via phospate to guanine. These sites are called CpG sites = phospate between cytosine and guanine. Cytosines in CpG dinucleotides are methylated by DNA methyltransferases in many eukaryotic organisms to form 5-methylcytosine. In mammals: 70%-80% of CpG cytosines methylated. In adult somatic tissues: DNA methylation typically occurs in a CpG dinucleotide context; non-cpg methylation is prevalent in embryonic stem cells. In plants, cytosines are methylated in CpG, CpNpG and CpNpNp, where N can be any nucleotide. Drosophila: virtually no DNA methylation.

36 CpG Islands CpG dinucleotides occur at lower frequency. Human genome with 42% GC content: The probability of CG = 0.21 * 0.21 = 4.41% of the time. But actual frequency of CpG dinucleotides in human genome is 1% Hypothesis: CpG deficiency is due to an increased vulnerability of methylcytosines to transition mutation in genomes with CpG cytosine methylation. CpG islands: Regions of DNA that have high density of CpG. Methylation is most active in CpG islands Many genes in mammalian genomes have CpG islands associated with the start of the gene. Presence of a CpG island used in gene prediction and annotation. These increased concentrations of CpGs might be associated with the decreased methylation of cytosines often observed in CpG islands this could result in a reduced vulnerability to transition mutations and, as a consequence, a higher equilibrium density of CpGs surviving.

37 DNA Methylation Enzymes Four active DNA methyltransferases have been identified in mammals. They are named DNMT1, DNMT2 (TRDMT1), DNMT3A and DNMT3B. DNMT3L is a protein that is closely related to DNMT3A and DNMT3B structurally and that is critical for DNA methylation, but appears to be inactive on its own. DNMT1: - most abundant DNA methyltransferase in mammalian cells, localises at the replication foci and interactes with the proliferating cell nuclear antigen. - key maintenance methyltransferase in mammals. - Predominantly methylates hemimethylated CpG dinucleotides fold more active on hemimethylated DNA as compared with unmethylated substrate in vitro, but it is still more active at de novo methylation than other DNMTs. - About 1620 amino acids long. - First 1100 amino acids constitute the regulatory domain of the enzyme, and the remaining residues constitute the catalytic domain. These are joined by Gly-Lys repeats. - Both domains are required for the catalytic function of DNMT1. - Several isoforms, the somatic DNMT1, a splice variant (DNMT1b) and an oocyte specific isoform (DNMT1o).

38 DNA Methylation Enzymes DNMT1o is synthesized and stored in the cytoplasm of the oocyte and translocated to the cell nucleus during early embryonic development, while the somatic DNMT1 is always found in the nucleus of somatic tissue. DNMT1 null mutant embryonic stem cells were viable and contained a small percentage of methylated DNA and methyltransferase activity. Mouse embryos homozygous for a deletion in Dnmt1 die at days gestation. DNMT 2 - Methylate position 38 in Aspartic acid transfer RNA and does not methylate DNA. - Known as TRDMT1 (trna aspartic acid methyltransferase 1) DNMT 3 - Methylate hemimethylated and unmethylated CpG at the same rate. - Similar architecture to DNMT1 with regulatory region attached to a catalytic domain. - There are three known members of the DNMT3 family: DNMT3a, 3b and 3L. - DNMT3a can co-localize with heterochromatin protein (HP1 ) and methyl-cpg binding protein (MeCBP). Interact with DNMT1 co-operatively.

39 DNA Methylation Enzymes - DNMT3a prefers CpG methylation to CpA, CpT, and CpC methylation - DNMT3a methylates CpG sites at a rate much slower than DNMT1, but greater than DNMT3b. - DNMT3L contains DNA methyltransferase motifs and is required for establishing maternal genomic imprints, despite being catalytically inactive. - DNMT3L is expressed during gametogenesis when genomic imprinting takes place. - The loss of DNMT3L lead to bi-allelic expression of genes normally not expressed by the maternal allele. - DNMT3L interacts with DNMT3a and DNMT3b and co-localized in the nucleus. Though DNMT3L appears incapable of methylation, it may participate in transcriptional repression.

40 DNA Methylation Mechanism Mitosis erases methylation only temporarily Cytosine methyl-transferases of vertebrates have preference for hemi-methylated targets Because of that newly synthetized DNA strand will receive same methylation pattern as parental strand DNMT1 enzyme = maintain methylation DNMTs coordinate transcriptional repression with histone deacetylases (HDACs) and methyl-cpg binding proteins (MBDs)

41 DNA Methylation Mechanism Regulates cell differentiation and function Too much / little : effect on gene function and result in alteration in the cell and even disease..how? Silencing: Methylation of CpG sites within the promoters of genes can lead to their silencing, a feature found in a number of human cancers (eg. silencing of tumor suppressor genes) Activation: In contrast, the hypomethylation of CpG sites has been associated with the overexpression of oncogenes within cancer cells.

42 Changes in DNA Methylation during Mammalian Development

43 DNA methylation and histone acetylation can be maintained through replication This allows the packing of chromatin to be passed on - just like a gene sequence. However, differences in chromatin packing are not as stable as gene sequences. Heritable but potentially reversible changes in gene expression are called EPIGENETIC phenomena Vertebrates use these differences in chromatin packing to IMPRINT certain patterns of gene regulation. Some genes show MATERNAL IMPRINTING while other show PATERNAL IMPRINTING. The alleles of some genes that are inherited from the relevant parent are methylated, and therefore are not expressed.

44 Functions of DNA Methylation Functions of DNA methylation in mammals: Transcriptional gene silencing Chromatin compaction Genome stability Suppression of homologous recombination between repeats Genome defence X chromosome inactivation (females)

45 Growing Epigenetic Mechanism: Non-coding RNAs

46 Epigenetic Processes Bookmarking X chromosome inactivation Position effect Carcinogenesis Imprinting Disorders Aging Reprogramming Transvection Maternal effects

47 X Chromosome Inactivation Lyon: random inactivation of one female X chromosome. Explain the mottled phenotype of female mice heterozygous for coat color genes. Lyon hypothesis: one copy of the X chromosome in female cells was highly condensed. Mice with only one copy of the X chromosome developed as fertile females. Beutler: in heterozygous women for G6PD deficiency, two red cell populations of erythrocytes: deficient & normal cells.

48 X Chromosome Inactivation Epigenetics?? Normal females possess two X chromosomes: one chromosome will be active (Xa) one will be inactive (Xi). However, in individuals with more than 2 X chromosomes, still only one Xa; and all the remaining X chromosomes are inactivated. Hence, default state of the X chromosome in females is inactivation, but one X chromosome is always selected to remain active. Sequences at the X inactivation center (XIC), present on the X chromosome, control the silencing of the X chromosome. Hypothesis: limited autosomally-encoded 'blocking factor' binds to the XIC of one X chromosome and prevents inactivation; the others then are not protected and get inactivaed. The XIC contains two non-translated RNA genes, Xist and Tsix, which are involved in X- inactivation.

49 X Chromosome Inactivation The X-inactive specific transcript, Xist RNA is the major effector of X-inactivation. Xi is coated by Xist RNA, whereas the Xa is not. Xist The Xist gene is the only gene which is expressed from the Xi but not from the Xa. X chromosomes which lack the Xist gene cannot be inactivated. Artificially placing and expressing the Xist gene on another chromosome leads to silencing of that chromosome. Prior to inactivation, both X chromosomes weakly express Xist RNA from the Xist gene. During the inactivation process, the future Xa ceases to express Xist, whereas the future Xi dramatically increases Xist RNA production, coasts the chromosome. The silencing of genes along the Xi occurs soon after coating by Xist RNA. Tsix gene is a negative regulator of Xist. X chromosomes lacking Tsix expression are inactivated much more frequently.

50 X Chromosome Inactivation Initial imprinting paternal X inactivation then reversed and then inner cell mass cells of blastocyst randomly inactivates 1 X chromosome. This inactivation is irreversible. So all daughter cells will also inactivate that same chromosome. This leads to mosaicism if a female is heterozygous for an X- linked gene, e.g. coloration of calico cats. X-inactivation is reversed in the female germline, so that all ova contain an active X chromosome

51 X Chromosome Inactivation Xi Silencing of Xi by repressive heterochromatin, which coats the Xi DNA and prevents the expression of most genes Barr Bodies. Xi has - high levels of DNA methylation, histone H3 lysine-9 methylation - low levels of histone acetylation, histone H3 lysine-4 methylation - a histone variant called macroh2a is exclusively found on Xi nucleosomes Effects of Xist expression through the presence of the blocking factor is passed through generation of cells is by a model of Polycomb group (PcG) protein action to maintain repressed states hence the irreversible epigenetic fate of the cell.

52 Bookmarking Mechanism for transmitting cellular memory of the pattern of gene expression in a cell, throughout mitosis, to its daughter cells. Vital for phenotype maintainence in a lineage of cells. Why? Characterised by non-compaction of some gene promoters during mitosis. Mechanism: prior to mitosis onset, gene promoters that exist in a transcription-competent state are bookmarked in some way. Bookmark persist during and after mitosis : transmits gene expression memory by: - preventing the mitotic compaction of DNA at this locus - facilitating reassembly of transcription complexes on the promoter Mediated by: - by binding of specific factors to the promoter prior to onset of mitosis - presence of histone variants that are characteristic of active genes persisting throughout mitosis. Eg. Hsp70, stress-inducible gene 1. Boomarking ensures that gene in transcribed in early G1 phase. 2. So if stress occurs in G1, no waste of time for decompacting. 3. Less vulnerable to stress-induced cell death 4. Integral for cell survival

53 Position Effect Variegation Epigenetic signals can spread Evidence that chromatin structure can regulate gene expression

54 Imprinting In diploid organisms somatic cells possess two copies of the genome. Each autosomal gene is therefore represented by two copies, or alleles, with one copy inherited from each parent at fertilisation. For the vast majority of autosomal genes, expression occurs from both alleles simultaneously. In mammals however, a small proportion (<1%) of genes are imprinted, meaning that gene expression occurs from only one allele. The expressed allele is dependent upon its parental origin. Parent Offspring Conflict Hypothesis: Conflict between male and female over allocation of maternal resources to offspring Dad uses imprinting to direct all resources to immediate offspring (not future litters) Mom uses imprinting to allocate resources to multiple litters Thus, predict paternally expressed genes would promote growth, maternally expressed genes should slow it down Imprinting mechanism: Methylation and Chromatin Remodeling

55 Genomic Imprinting Process ON E OFF E maternal allele paternal allele The process begins during gamete formation when in males certain genes are imprinted in developing sperm and in females, others are imprinted in the developing egg. All the cells in a resulting child will have the same set of imprinted genes from both its father and its mother EXCEPT for those cells ("germplasm") that are destined to go on to make gametes. All imprints both maternal and paternal are erased in them.

56 IGF2 H19 E E Insulator functions as enhancer blocker on maternal allele IGF2 H19 E E Insulator function is silenced by heterochromatin formation and DNA methylation which spreads to the H19 promoter

57 Cancer Epigenetics Epigenetic alterations are found in almost all types of cancers Large number of epigenetic alterations found in cancer cells due to: Stochastic occurrences that accumulate with age, selected for during tumour formation Caused by defects in components of the epigenetic machinery Changes in 5-me-C distribution in DNA Hypermethylation of promoter CpG islands (found in ~50% genes) associated with TSGs Decreased expression levels/silencing of TSGs Increased mutations Global hypomethylation Chromosome instability (particularly pericentromeric repeats) Activation of viruses Activation of proto-oncogene Changes in chromatin structure Histone modifications: Histone deacetylation Histone methylation (H3-K9); demethylation (H3-K4) Histone sumoylation Heterochromatin-associated proteins

58 DNA Hypermethylation Silencing may occur early, even in non- or precancerous cells E.g. Promoter methylation of CDKN2A/p16INK4a is detectable in preinvasive bronchial lesions, in pituitaries of Cushing s patients and in normal breast tissue Many other examples in other tissues Changes may be age-related or pre-malignant, or may be associated with environmental exposures and/or diet

59 DNA Methylases in Cancer Cells mrna of DNMT1 (maintenance methylation) mrna of DNMT-3b (de novo methylation of CpG islands) That explains the paradox: Global hypomethylation Hypermethylation of CpG islands DNMT-3b is a molecular target for cancer treatment ( to restore function of TSGs)

60 Inactivation of TSGs

61 DNA Methylation

62 Inactivation of TSGs (MMR gene) (DNA repair gene)

63 Hypermethylation of RASSF1A

64 Hypermethylation of CDKN2A

65 Hypermethylation of MLH1 Nature Rev 5:223, 2005

66 TP73 Inactivation of TSGs MLH1 RASSF1 CDKN2A p16 INK4A + p14 ARF HIC1 TP53 BRCA2 cadherin BRCA1 Red = only hypermethylation identified Green = only genetic mutations identified Purple = both mechanisms found HIC1 = hypermethylated in cancer 1 (frequent LOH)

67 Genes Frequently Silenced in Tumors by Methylation Cell cycle Signal transduction Apoptosis DNA repair Carcinogen metabolism Hormonal response Metastasis RB1, INK 4a, INK4b, p14 ARF APC, LKB1/STK11, RASSF1 DAPK, caspase-8 MGMT, BRCA1, MLH1 GSTP1 ER, PR, RAR E-cadherin, VHL Only methylation in or near promoter region results in gene silencing

68 Hypomethylation of proto-oncogene Eg: MAGE = melanoma Ag SERPINB5 = Ser protease inhibitor SNCG = synuclein g (Br/Ov onc) Biochem (Moscow) 70:533, 2005

69 DNA Methylation Influences Cancer Processes By influencing global gene expression DNA Repair Carcinogen Metabolism Cell Cycle DNA Methylation Hormonal Regulation Differentiation Apoptosis

70 Exp: Analysis of methylation status Gene expression microarray analysis Identifies non-transcribed genes as candidates for promoter hypermethylation Re-expression of silenced genes after HDAC/DMT blocks Promoter DNA methylation status Bisulfite genomic sequencing (known TSGs) Converts unmethylated C to U Bisulfite-based methylation-specific PCR (known TSGs) Bisulfite-based methylation-specific microarray and probes Two spots (sequence +/- bisulfite); bisulfite treated PCR probes (multiple known TSGs) Methylation-sensitive RE digests (RLGS, MS-RDA/PCR) RLGS = restriction landmark genome scanning eg NotI for total genomic digests look for missing spot on 2D gel MS-RDA = methylation sensitive representational difference analysis Eg HpaII, SacII ligate adaptors as primer sites; look for bands diffs MS-PCR = methylation sensitive PCR eg HpaII (sens) vs MseI (insens) PCR look for bands or hybridize to CpG island microarrays (random or known TSGs)

71 Conversion of unmethylated cystosines to uracil using sodium bisulfite Sequencing: unemethylated cytosines read as thymidine in sense strand; adenine in the anti-sense strand. Other technologies evolved from here.

72 Aging & Other Diseases DNA methylation decreases as cells age. Identical twins are epigenetically indistinguishable early in life. Substantial differences in epigenetics markers with age. Hence, environment plays an important role in shaping the epigenome. Aging process involves similar epigenetic pathways as cancers (inverse..e.g. telomere shortening) Increasing evidence that epigenetics play a critical role in the development of certain human diseases such as type-2 diabetes, cardiovascular diseases, obesity and infertility.

73 Epigenetics & Environment Enviromental & Dietary factors linked to changes in abnormal epigenetic pathways. Changes are subtle and cumulative and manifest over a long time so difficult to establish exact causal relationship. Eg. of environmental factors that are related to epigenetic changes: Heavy metals (cadmium) disrupts DNA methylation Vinclozin (pesticide) alters DNA methylation and effects persist in unexposed offspring through several generations (mouse model) Deficiency in folate and methionine which are involved in cellular processes that supply methyl groups for DNA methylation,, can change the expression of growth factor genes (IGF12) imprinting!! Cigarette smoke stimulates the demethylation of metastatic genes in lung cancer cells.

74 Drugs Since many tumor suppressor genes are silenced by DNA methylation during carcinogenesis. Attempts to re-express these genes by inhibiting the DNMTs. 5-aza-2'-deoxycytidine (decitabine) - nucleoside analog that inhibits DNMTs by trapping them in a covalent complex on DNA by preventing the β-elimination step of catalysis, resulting in enzyme degradation. However, for decitabine to be active, it must be incorporated into the genome of the cell, but this can cause mutations in the daughter cells if the cell does not die. Toxic to the bone marrow - limits the size of its therapeutic window. Development of antisense RNA therapies that target the DNMTs by degrading their mrnas and preventing their translation. However, it is currently unclear if targeting DNMT1 alone is sufficient to reactivate tumor suppressor genes silenced by DNA methylation DNMT3b!!!

75 Review In lecture, we developed the following ideas: Epigenetics means control of gene expression (specifically transcription), not dependent on DNA base sequence. Two organisms can have the identical DNA base sequence, and not express the same genes!! Epigenetics explains why identical twins are not entirely identical, and why cloned animals are not identical and are abnormal. Epigenetic control is due to modulation of chromatin structure, and whether genes are accessible for transcription. Which genes are modified during development. DNA of an embryo has a different methylation pattern than that present in each adult cell type. DNA methylation is also different in male and female germ cells, and in genes derived from the paternal or maternal genome later in development. This is referred to as IMPRINTING. A normal embryonic DNA methylation pattern is required for normal development.

76 Review Strategies of gene expression control form a hierarchy: Level 1: DNA base sequence Mutations in genes can render them non-functional or incorrectly functional. Mutations can be in the promoter, coding sequence, affect splicing or translation of any gene. Level 2: EPIGENETIC CONTROL. Chromatin (DNA packaged with histone proteins) must be opened in order to allow transcription of the DNA. This opening includes whether transcription factors can bind their target sequence, whether they can direct transcription initiation and elongation, and/or whether the DNA double helix can be unwound. Chromatin structure is largely dependent on methylation of cytosine in nuclear DNA. DNA methylation inhibits transcription, because it prevents modification of histones that would allow opening of the chromatin and transcription. Level 3: Third in importance: all the other mechanisms Transcriptional controls that regulate whether a specific transcription factor is present in a specific cell type. Post-transcriptional controls regulate whether the final gene product (usually a protein) is made from a transcript, e.g. protein transport/modification