Epigenetics of health and disease Lecture plan

Epigenetics of health and disease

Lecture plan 1. Epigenetics – above genetics 2. Chromatin structure 3. Histone modifications 4. DNA methylation 5. nc. RNAs 6. Epigenetic of germline and epigenetic memory 7. Epigenetics of disease 8. Transgenerational response 9. Epigenetics in neuroscience

Organisms have at least two levels of inheritance: genetic and epigenetic • Genetic – mechanisms of inheritance that mostly follow Mendelian laws of segregation. Genetic changes are based on stable changes in DNA sequence (mutations). • Alleles • Epigenetic – mechanisms of inheritance that frequently don’t follow Mendelian laws of segregation and are based on potentially reversible changes in cytosine methylation and histone modifications (epimutations). • Epialleles

Effect on environment on twins

What does “epigenetics” mean? Literally, epigenetics means above, or on top of, genetics. Transcription Usually this means information coded beyond the DNA sequence, such as in covalent modifications to the DNA or modifications to the chromatin structure. Practically, epigenetics describes phenomena in which genetically identical cells or organisms express their genomes differently, causing phenotypic differences. Epigenetic Silencing Different epigenetic modifications leading to different expression patterns Genetically identical cells or individuals Different phenotypes © 2013 American Society of Plant Biologists

In plants, the activity of many developmental genes and environmentallyregulated genes are epigenetically regulated, to be stably maintained in an ON or OFF position Holec, S. and Berger, F. (2012). Polycomb group complexes mediate developmental transitions in plants. Plant Physiol. 158: 35 -43. © 2013 American Society of Plant Biologists

The two major phases of genome-wide erasure of DNA methylation in the early embryo and in primordial germ cells (PGCs) of the mouse. S Feng et al. Science 2010; 330: 622 -627 Published by AAAS

Epigenetic marks: DNA methylation and histone modification e as r sfe an ltr thy e M cytosine 5 -methylcytosine The histone proteins that DNA is wrapped around can be covalently modified, affecting chromatin structure Histone octamer DNA can be covalently modified by cytosine methylation. Methylcytosine TTCGCCGACTAA NUCLEOSOME Epigenetic Silencing Histone modifications © 2013 American Society of Plant Biologists

Epigenetic marks contribute to largescale chromatin domains The centromere and regions around it are usually densely packaged with few protein-coding genes Densely packaged heterochromatin CENTROMERE Euchromatin DAPI DNA stain Less densely packaged, generich euchromatin Merged Centromeric heterochromatin Deal, R. B. , Topp, C. N. , Mc. Kinney, E. C. , and Meagher, R. B. (2007) Repression of flowering in Arabidopsis requires activation of FLOWERING LOCUS C expression by the histone variant H 2 A. Z. Plant Cell 19: 74 -83. © 2013 American Society of Plant Biologists

Chromatin structure Chromatin is a complex structure consisting of DNA and associated histones and non-histone proteins. Chromatin is not a rigid structure, and dynamic changes are crucial for most DNA-dependent processes such as replication, transcription and DNA repair. The chromatin status is defined by complex interactions of modifications of many specialized sets of nuclear proteins. Chromatin structure is regulated through: - DNA methylation; - ATP-dependent remodeling of nucleosome cores; - covalent modifications of histone tails; - the replacement of core histones by their variants; - nucleosome eviction.

Chromosome territories During the interphase, chromosomes in the nucleus of a cell are located in specific regions called chromosome territories (CTs), with the regions around them called interchromatin compartments (ICs). The nuclear architecture of the majority of eukaryotic cells is defined by gene-rich CTs that occupy the interior regions of the nucleus, whereas gene-poor CTs are located at the periphery of the nucleus.

Models of CT and IC organization Interchromatin compartment model (CT-IC) Interchromosome domain model (ICD) Gene-rich chromosome territory (CT) Gene-poor chromosome territory (CT) Interchromatin compartment (IC)

Chromatin-modifying and chromatin remodeling proteins Three major groups of proteins are involved in chromatin modifications: - chromatin remodeling complexes, - effector proteins, and - insulator proteins Chromatin remodeling complexes are energy-driven, multi-protein machinery that allows an access to specific DNA regions or histones by altering nucleosomal positions, histone-DNA interactions, and histone octamer positions.

Chromatin-modifying and chromatin remodeling proteins In chromatin remodeling complexes, ATPases are grouped into four subfamilies: -the SWI/SNF (SWITCH/ SUCROSE NONFERMENTING) ATPases, -the imitation switch (ISWI) ATPases, -the chromodomain and helicase-like domain (CHD) ATPases and -INO 80 ATPases.

Antagonistic functions of Trx. G and Pc. G proteins The antagonistic function of trithorax (trx. G) and polycomb (Pc. G) proteins was first described in Drosophila melanogaster. Whereas the polycomb complexes function as repressors of target genes, the trithorax proteins work as activators targeting the identical DNA regulatory elements - the Pc. G or trx. G response elements (PREs/TREs).

The role of chromatin remodeling complexes in the maintenance of pluripotency Pluripotency of embryonic stem cells (ESCs) is maintained by the action of es. BAF and TIP 60 -p 400 complexes that repress inappropriate gene expression as well as by the CHD 1 activity that apparently prevents chromatin compaction.

Effector proteins are proteins that read and implement modificationencoded biological messages. Effector proteins contain specific binding modules such as chromodomains, bromodomains, Tudor domains, PHD (Plant Homeodomain) domains and others. This allows effector proteins to associate with one or more specific histone modifications or DNA methylation.

HP 1 effector protein The mammalian HP 1 protein is a non-histone protein that regulates chromatin remodeling and transcription via the interaction with other histone and non-histone proteins. There are three isoforms of HP 1, HP 1α, HP 1β, and HP 1γ. Trimethylation of histone 3 at lysine 9 serves as a signal and the attachment site for HP 1 binding. Functions: - gene repression by heterochromatin formation, regulation of binding of cohesin complexes to centromere, sequesteration of genes to nuclear periphery, transcriptional arrest, maintenance of heterochromatin integrity, gene repression at single nucleosome level and gene repression by heterochromatization of euchromatin.

HP 1 effector protein

Effector proteins binding methylated DNA Other group of effector proteins is represented by proteins that bind to methylated DNA. These include: Me. CP 1, Me. CP 2, MBD 1, MBD 2, MBD 4 and Kaiso. These proteins have one common ability – the ability to bind methylated DNA and interpret DNA methylation marks in different biological contexts. It is speculated that the lack of expression of Me. CP 2 results in the overexpression of four neural development-related genes.

The role of protein insulators Insulators are DNA elements that can protect a gene from neighboring transcriptional influences to prevent inappropriate activation or repression of the gene. Insulators have two well-known functions that are represented by enhancer blocking and barrier insulators activities, so that they can either prevent distal enhancers from activating a promoter or block heterochromatin spreading that may lead to silencing of neighboring genes. A single insulator element can control the expression of a single gene or several genes. Likewise, the expression of a single gene can be controlled by several distinct regulatory elements.

Two major functions of insulators A. Preventing the regulation of promoter activity by an enhancer (ENH) element. B. Preventing spreading of repressive chromatin into active chromatin. d. CTCF – an insulator protein commonly found in animals.

Matrix attachments DNA observed in 10 -100 kb loops anchored to a chromosome scaffold or nuclear matrix made up of nonhistone proteins Major components of this matrix are lamins, topoisomerase II, and components of centromeres and telomeres Evidence for attachment to matrix = Extensive digestion with DNases still leaves some small pieces of DNA intact that are not released to a soluble fraction in nucleus Evidence for size of loops = Even the most limited DNase digestion does not produce pieces of DNA with an average length of > ~100 kb

Matrix attachments Binding sites for DNA and topoisomerase II known as scaffoldassociated regions (SARs) (MARs - nuclear matrix present in interphase) SARs (MARs) may be between transcription units and although they may serve to hold specific chromosomal domains in isolated loops MARs or SARs may function as: - attachment sites, - boundaries of replication, - transcription domains, - areas where topological changes in the DNA are undertaken

Heterochromatin and Euchromatin EM of nucleus indicates some areas remain condensed and darkstaining throughout cell cycle = heterochromatin Typically represent 5 -10 x increased compaction Euchromatin is less condensed and lighter staining regions within the interphase nucleus Typically heterochromatin has been observed at centromeres and telomeres and thought historically to represent sites of inactive genes since they are condensed throughout the cell cycle • These regions of chromosomes areas known to contain highly repeated DNA that are rarely transcribed • This is known as constitutive heterochromatin • Heterochromatin may also occur in entire chromosomes inactive in a particular lineage such as the mammalian X • This is known as facultative heterochromatin

Histones

Histones • Histones are a family of small, basic proteins found in all eukaryotic nuclei • They are very rich in basic amino acids (rich in lysine and arginine >20% of AA) facilitating interaction with the negative charge of phosphate groups (common pattern for DNA binding proteins) Six major histone classes are known: H 1 (the linker histone) H 2 A H 2 B H 3 H 4 Archaeal histones (form tetrasomes, like in eukaryotes) Two each of the class H 2 A, H 2 B, H 3 and H 4, so-called core histones, assemble to form one octameric nucleosome core particle by wrapping 146 -147 base pairs of DNA around the protein spool in 1. 65 left-handed super-helical turn.

Nucleosome structure The nucleosome core is formed of two H 2 A-H 2 B dimers and a H 3 -H 4 tetramer, forming two nearly symmetrical halves by tertiary structure.

Chaperones The assembly of mature nucleosomes requires the activity of histone chaperones, proteins that specialize in incorporating either histones H 2 A and H 2 B or histones H 3 and H 4 into nucleosomes.

Chromatin compaction The most basic such formation is the 10 -11 nm fiber or beads on a string conformation. This involves the wrapping of DNA around nucleosomes with approximately 50 -55 base pairs of DNA spaced between each nucleosome (also referred to as linker DNA).

Structure Histones are subject to posttranslational modification by enzymes primarily on their N-terminal tails, but also in their globular domains. Such modifications include methylation, citrullination, acetylation, phosphorylation, sumoylation, ubiquitination, and ADP-ribosylation. In general, genes that are active have less bound histones, while inactive genes are highly associated with histones during interphase.

Chromatin compaction Acetylation of H 3 and H 4 histones One of the H in the e-free amino group of internal lysine substituted with acetyl (addition of acetyl group = CH 3 CO-) Addition of acetyl group removes positive charge from NH 3+ group on lysine Methylation One of the H in the e-free amino group of internal lysine substituted with methyl group Also occurs on arginine and histidine Phosphorylation Addition of P on hydroxyl of serine and histidine -- this introduces negative charge Ubiquitination 76 AA protein which can be conjugated with histone H 2 A in which C-terminal carboxyl group of ubiquitin is joined to an internal lysine residue at the amino group

POST-TRANSLATIONAL MODIFICATIONS ON THE HISTONE TAILS CHROMATIN Yellow – phosphorilation Red – methylation Blue - acetylation POST-TRANSLATIONAL HISTONE MODIFICATIONS NUCLEOSOME

Effect of histone (de)acetylation Histone acetylation is performed by the activity of HISTONE ACETYL TRANSFERASE (HAT) enzymes, whereas the deacetylation is promoted by HISTONE DEACETYLASE (HDAC) enzymes. Whereas the former promotes transcription, the latter represses transcription. Histone acetylation is normally associated with promoters and the 5ʹend of transcribed sequences of regulated genes. Histone acetylation acts directly by decreasing the positive histone charge and loosening the association of histone with DNA, which leads to transcriptional activation.

Histone acetylation and methylation are the major histone modification marks Histone acetylation and methylation are the most common histone modifications in animals. An open chromatin configuration is associated with high levels of histone acetylation and trimethylation at H 3 K 4, H 3 K 36, or H 3 K 79. In contrast, condensed heterochromatin is associated with deacetylated histones and histones enriched in H 3 K 9, K 3 K 27, and H 4 K 20 trimethylation.

Effect of histone methylation Histone methyl transferases (KMTases) are the enzymes that add 1, 2 or 3 methyl groups to lysines (K) primarily Histone methylation regulates binding of other effector proteins and their complexes and thus regulates transcription either in a positive or negative way.

Histone methylation/demethylation Compared to acetylation, methylation is a more complex modification. 1. There are three different methylation states (mono-, di- and trimethylation). 2. Methylation can target lysine and arginine and 3. Methylation can activate or repress transcription depending on the position of modifications and the number of attached methyl groups.

Permissive, restrictive and bivalent states of gene promoters All gene promoters are found in three fundamental states of gene expression activity: - restrictive or inactive, - permissive or active, or - both restrictive and permissive, known as bivalent state.

Permissive, restrictive and bivalent states of gene promoters Gene expression states are determined by histone modification marks: - restrictive marks are mainly associated with trimethylated H 3 K 9 and/or H 3 K 27), - permissive marks are associated with trimethylated H 3 K 4 and acetylated H 3 K 9, - bivalent ones – with trimethylated H 3 K 27 and trimethylated H 3 K 4. Non-expressed genes and some genes that are expressed at low levels are often found in undifferentiated cells such as human embryonic stem cells.

Permissive, restrictive and bivalent states of gene promoters Upon differentiation, many promoters in bivalent state undergo twostep “conversion” into permissive or restrictive. 1. The promoters of these genes are primed prior to the entry into a specific differentiation pathway. 2. These promoters are placed in either an active state via the recruitment of permissive marks such as H 3 K 27 me 2 or a silent state through the recruitment of H 3 K 4 me 2 marks.

Regulation of transcription through histone modifications Transcriptionally active chromatin is associated with H 3 K 4 me and H 3 K 9 ac, established by K 4 -HMTs such as h. SET 1 A and HATs such as GNAT and MIST, respectively. Transcriptionally inactive chromatin is achieved through the removal of H 3 K 4 me by histone demethylase LSD 1, and H 3 K 9 ac by HDACs of classes I-III, followed by establishment of H 3 K 9 me by K 9 HMTase Suv 39 H 1. H 3 K 9 me is recognized by HP 1 protein resulting in further spreading of inactive chromatin. Chromatin inactivation at euchromatic loci is also associated with binding of retinoblastoma p. Rb protein. This chromatin state recruits DNMTs that result in dramatic increase in DNA methylation. Me. CP 2 proteins bind methylated DNA and HDACs remove remaining acetyl groups from histones.

Histone modifications and their impact on transcription in humans Modificat Histone ion type H 3 K 4 H 3 K 9 me 1 + + - - +/- me 2 me 3 ac + + H 3 K 14 H 3 K 27 H 3 K 79 H 4 K 20 + H 2 BK 5 + - + “+” – activation of transcription; “-“ – repression of transcription. The data are collected from Barski et al. (2007).

Histone variants: H 2 A. Z The H 2 A. Z histone variant is believed to primarily associate with transcriptionally active genomic regions and is mainly deposited at promoter regions. H 2 A. Z marks are deposited by the ATP-dependant chromatinremodeling complex SWR 1 at the 5′ end of genes in many eukaryotes. H 2 A. Z is mutually exclusive with DNA methylation and mainly associates with methylated and acetylated histone isoforms. Some evidence exists that H 2 A. Z is removed from nucleosomes during the process of transcription.

Histone variants: H 2 AX is a histone variant found in its phosphorylated form in the regions of DNA strand breaks. It has been named "the histone guardian of the genome", and its primary role may be the recruitment of the DNA damage repair machinery to DNA damage sites. The recognition of strand breaks in the genome involves phosphorylation of the histone variant H 2 AX in the position of Ser 139 producing γH 2 AX is believed to be required for: -the assembly of DNA repair proteins at sites of damaged chromatin and - the activation of checkpoints proteins which trigger cell cycle arrest.

Model of Mediator of DNA damage checkpoint 1 regulated phosphorylation of histone H 2 AX (1) The MRE 11, RAD 50 and NBS 1 (MRN) complex binds DNA ends at sites of DNA damage and recruits ataxia telangiectasia mutated (ATM), which phosphorylates proximal H 2 AX. (2, 3) MDC 1 binds phosphorylated proximal H 2 AX and recruits more MRN –ATM. The new pool of ATM phosphorylates more distal H 2 AX. These events could contribute to the ‘spreading’ of H 2 AX phosphorylation to more distal chromatin regions.

DNA methylation

DNA methylation definition DNA methylation is a covalent modification of DNA in which the methyl group is added to a cytosine residue at position C-5 or N-4 or to an adenine residue at position N-6. Whereas methylation of cytosine at C-5 and adenine at N-6 are common to many organisms, methylation of cytosine at N-4 occurs in bacteria only.

Cytosine DNA methylation in animals Cytosine DNA methylation is a covalent modification of DNA in which methyl group is transferred from S-adenosylmethionine to the 5 position of cytosine by a family of cytosine (DNA-5)-methyltransferases DNMT 1 DNMT 3 a DNMT 3 b SAM SAH DNMT 3 DNMT 1 - methylated; - unmethylated

Cp. G methylation In animals, cytosine methylation occurs predominantly symmetrically at Cp. G dinucleotide pairs. It is estimated that in the animal genome, about 80% of all cytosines in Cp. Gs are methylated, with the remaining 20% of unmethylated Cp. G dinucleotides located predominantly in promoter regions. The GC content of the human genome is approximately 42%; thus, it is expected that Cp. G dinucleotides occur with a frequency of 0. 21 × 0. 21 = 4. 41%. Instead, Cp. Gs are severely underrepresented in the genome, occurring at a frequency of less than 1%.

Cp. G methylation However, they are frequently found in clusters termed Cp. G islands which are defined by over 50% content of GC, and the ratio of observed to expected Cp. G occurrence is higher than 60% in a stretch of 200 nt. Cp. G islands play a crucial role in the regulation of gene expression and transcriptional silencing. Only about 10% of all cytosines in the animal genome are methylated. Although it is not common in animals, low levels of non-Cp. G methylation occur in embryonic stem cells.

De novo and maintenance DNA MTases De novo methyltransferases recognize something in the DNA that allows them to methylate cytosines de novo. These are expressed mainly in early embryo development and they set up the pattern of methylation. Maintenance methyltransferases add methylation to DNA when one strand is already methylated. These work throughout the life of the organism to maintain the methylation pattern that had been established by the de novo methyltransferases. In mammals, DNA methylation patterns are established by a family of de novo methyltransferases, DNA methyltransferase 3 (DNMT 3), and maintained by the maintenance methyltransferase, DNMT 1.

Mammalian DNA methyltransferase (DNMT) There are three families DNA MTases in animals: DNMT 1, DNMT 2 and DNMT 3. The DNMT 1 family consists of DNMT 1 s, DNMT 1 o, DNMT 1 b, DNMT 1ΔE 3— 6 and DNMT 1 p genes. The DNMT 3 family has three members including DNMT 3 a, DNMT 3 b, and DNMT 3 L; DNMT 3 a has four isoforms (DNMT 3 a 1 to DNMT 3 a 4); and DNMT 3 b has eight isoforms (DNMT 3 b 1 to DNMT 3 b 8). Five active DNA methyltransferases have been identified in mammals: DNMT 1, DNMT 2, DNMT 3 A, DNMT 3 B and DNMT 3 L.

De novo DNA methylation - DNMT 3 is a family of DNA methyltransferases that could methylate hemimethylated and unmethylated Cp. G at the same rate – de novo methyltransferase. DNMT 3 a can co-localize with heterochromatin protein (HP 1 ) and methyl-Cp. G binding protein (Me. CBP).

Dnmt 3 L-assisted de novo methylation of DNA by Dnmt 3 a DNMT 3 L interacts with DNMT 3 a and DNMT 3 b and co-localize in the nucleus. Though DNMT 3 L appears incapable of methylation, it may participate in transcriptional repression. The dashed line indicates possible process of oligomerization of the Dnmt 3 L/Dnmt 3 a complex.

Maintenance DNA methylation - DNMT 1 is the most abundant DNA methyltransferase in mammalian cells, and considered to be the key maintenance methyltransferase in mammals. It predominantly methylates hemimethylated Cp. G di-nucleotides in the mammalian genome. This enzyme is 7– 20 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.

Active loss of methylation in animals Cell undergoing active demethylation express two classes of enzymes: Activation-induced cytosine deaminase (AID) and apolipoprotein B RNA editing catalytic component 1 (APOBEC 1). AID catalyzes deamination of 5 -methylcytosine, resulting in T: G mismatches. Deamination may be the main process of active demethylation.

Passive loss of methylation in animals Passive loss of methylation occurs during replication and DNA repair (that involves the resynthesis step). Passive loss is most important during gamete and embryo development. It may involve suppression of DNMT 1 activity by retinoblastoma (Rb) pathway during gametogenesis. Petra Hajkova: Epigenetic reprogramming in mouse germ cells

nc. RNAs

Imprinting Xist/Tsix RNAs Stability Telomerase RNA DNA Telomere Transcription regulation Long nc. RNA, si. RNA, pi. RNA, 75 K RNA Editing g. RNA pre-r. RNA pre-m. RNA Modification sno. RNA Ψ Ψ Nucleus Splicing sn. RNA m. RNA Ψ sn. RNA Nucleolus NA sno. RNA sca. R r. RNA m. RNA Cytoplasm ER Modification RNase. P, sno. RNA Posttranscriptional regulation mi. RNA, si. RNA Transport SRP RNA Translation r. RNA, t. RF Figure 1. The main groups of nc. RNAs and their possible role in a human cell. The shaded boxes represent different functional groups of nc. RNAs, each of them combines several groups from NONCODE classification. sno. RNA and RNase. P are non-coding RNAs that modify other nc. RNAs.

Nucleus NA D HEN 1 DCL 1 Cleavage pri-mi. RNA Drosha. DGCR 8 pre-mi. RNA pri-mi. RNA * * Drosha. DGCR 8 * * Ex ADAR po rt in 5 AGO 1 Dicer/ ds. RBD AGO 1 Nuclear migration pri-mi. RNA AGO 1 Cytoplasm P body mi. RNA biogenesis mi. RISC AGO 1

Mirtrons as a novel concept of mi. RNA biogenesis DNA pre-m. RNA Exon Intron RNA folding Splicing Drosha Lariats Processing by Drosha phosphor-diester bond h. DBR 1 RNA re-folding Degraded -5 rtin po x E Dicer RISC Dicer

Mechanism – m. RNA cleavage Micro. RNAs can direct the RISC to downregulate gene expression by either of two posttranscriptional mechanisms: m. RNA cleavage or translational repression. The choice of posttranscriptional mechanisms is not determined by whether the small silencing RNA originated as an si. RNA or a mi. RNA but instead is determined by the identity of the target: once incorporated into a cytoplasmic RISC, the mi. RNA will either cleave when the homology is high or will repress productive translation if the homology is not sufficient.

DNA Nucleus Transcription inhibition Cytoplasm Transcription activation ? Miwi/Mili? ? ? ? Cleavage Ago 3 Piwi Rec. Q 1 pi. RCs The mechanism of biogenesis and function of pi. RNAs Translation activation Piwi Aub 5ʹU 3ʹ 5ʹ A Ago 3 3ʹ 3ʹ U 5ʹ A 3ʹ Piwi Aub 5ʹ U 3ʹ 3ʹ 5ʹ Ago 3 U 3ʹ 5ʹ A 5ʹ 3ʹ Ago 3 A 5ʹ

Epigenetics of germline and epigenetic memory

Epigenetics memory in animals The first wave of epigenetic reprogramming takes place during gametogenesis, whereas the second one occurs early during embryogenesis. Reprogramming includes passive and active losses and the reestablishment of epigenetic marks. Similar to genetic memory, epigenetic memory requires stable inheritance and should be passed onto the progeny through meiosis.

De novo DNA methylation – reprogramming Second round of methylation

De novo DNA methylation – reprogramming

De novo DNA methylation occurs twice during organismal development following waves of demethylation, which erase the DNA methylation imprints established in the previous generation. The first round of de novo DNA methylation occurs early during embryonic development, probably before or immediately after implantation.

De novo DNA methylation – reprogramming Following the first round of reprogramming, in female PGCs (primordial germ cells) the level of methylation drops by 70%, whereas in male PGCs it drops by 60%. De novo methylation of DNA in the male germ line occurs several days after the first round of reprogramming, between E 15 and E 16, whereas in the female germ line, the process takes place only postnatally during the oocyte growth phase.

De novo DNA methylation – reprogramming The second wave of de novo DNA methylation occurs later during post-implantation development in PGCs. After this second wave of de novo methylation, the methylation state of PGCs is similar to that of somatic cells, although substantial differences in methylation patterns can be observed in the parental imprinting control regions (ICRs).

Methylation levels Fertilization Zygote 2 -cell Female methylation Male methylation Time postfertilization 4 -cell Morula Blastocyst Inner cell mass Trophoblastic cells

Epigenetic inheritance – escaping reprogramming

Epigenetic Inheritance mitotic stability/heritability meiotic stability/ transgenerational inheritance Persistence of epigenetic marks. Alterations that last less than one cell cycle (green asterisk, a) do not qualify as epigenetic under the definition that strictly requires heritability, whereas non-mutational changes that are transmitted from one cell to its daughters (red asterisk, b) or between generations of an organism (blue asterisk, c) do qualify.

Inheritance – effects on 2 nd or 3 rd generation When exposure occurs during pregnancy, only changes in the F 3 generation can be considered as a result of truly heritable transgenerational effects © 2013 American Society of Plant Biologists

Genomic imprinting is regulated by epigenetic processes • The zygote receives two copies of each gene, one from the mother’s genome and one from the father’s. • At most loci, both copies are active. • Some loci, imprinted loci, show a “parent of origin effect”. • Expression of these loci is controlled by epigenetic factors. © 2013 American Society of Plant Biologists

Imprinting – nuclear transplant experiment Placing a sperm and an egg nucleus into an enucleated fertilized cell leads to a normal embryo. Zygotes that receive only maternal or only paternal nuclei do not survive. The two parental genomes are not equivalent © 2013 American Society of Plant Biologists

The MEDEA (MEA) gene is imprinted MEA/mea x MEA/MEA All seeds viable MEA/MEA x MEA/mea 50% of seeds abort In the second cross, 50% of the seeds receive the mutant mea allele from their mother. These seed abort, even though they also have a wild-type MEA allele inherited from their father; the paternal allele is epigenetically silenced and inactive. From: Grossniklaus, U. , Vielle-Calzada, J. -P. , Hoeppner, M. A. , Gagliano, W. B. (1998) Maternal control of embryogenesis by MEDEA, a Polycomb Group gene in Arabidopsis. Science 280: 446 -450. Reprinted with permission from AAAS. © 2013 American Society of Plant Biologists

Epimutations Hypermethylation of the cryptic IAP promoter leads to silencing of the Avy epiallele and reversion to a black coat color. The Avy/A epiallele phenotype is thus converted to a A/A wildtype phenotype. The progeny of Avy/A mothers have varying degrees of a yellow coat color, suggesting that the epigenetic mark is not completely erased in the female germline. +++ Avy/A --Avy/A A/A

DNA methylation-based inheritance Effect of environment – bisphenol A Methylation

Epigenetics of disease

Aging There is a global loss of DNA methylation during aging. But some genes become hypermethylated with age, including genes for the estrogen receptor, p 16, and insulin -like growth factor 2. The decreased expression of histone genes and the reduction of heterochromatin marks, such as DNA methylation and repressive histone marks, such as H 3 K 9 me 3, H 3 K 27 me 3 and H 4 K 20 me 3, during senescence and aging suggest that aging is associated with the loss of heterochromatin.

Model for the role of SUV 39 H 1 downregulation in the establishment of senescence The green ellipses represent the amount of SUV 39 H 1 present in a cell, indicating the reduction that is observed during senescence. The black lines represent DNA and the red circles represent H 3 K 9 me 3. Bent arrows indicate transcriptional activity.

Cancer In general, cancer cells have aberrant DNA methylation patterns – global genome hypomethylation and locus-specific hypermethylation. Hypermethylation typically occurs at Cp. G islands in the promoter region and is associated with gene inactivation. Global hypomethylation leads to activation of aberrant gene expression, including activation of transposon elements. Typically, there is hypermethylation of tumor suppressor genes and hypomethylation of oncogenes.

Atherosclerosis In animal models of atherosclerosis, vascular tissue as well as blood cells such as mononuclear blood cells exhibit global hypomethylation with gene-specific areas of hypermethylation. Monocytes and lymphocytes experience hypomethylation, likely due to elevated homocysteine levels that inhibit DNA methyltransferases. Hypomethylation of DNA affects gene that alter smooth muscle cell proliferation, cause endothelial cell dysfunction, and increase inflammatory mediators, leading to formation of atherosclerotic lesions.

Diabetes (type 2) Type 2 diabetes is characterized by chronic hyperglycaemia as a result of impaired pancreatic beta cell function and insulin resistance in peripheral tissues. The PPARGC 1 A gene regulates genes involved in energy metabolism and is hypermethylated and downregulated in type 2 diabetes patients. Promoter of Pdx 1 gene encoding transcription factor that regulates beta-cell differentiation and insulin gene expression is also hypermethylated in islets that dysfunction in diabetes in rats. Pancreatic islet-specific mi. R-375 inhibits insulin secretion in mouse pancreatic β-cells by inhibiting the expression of the protein myotrophin. An overexpression of mi. R-375 can completely suppress glucose-induced insulin secretion.

Epigenetics of the Nervous System

DNA methylation and brain During brain development, DNA methylation is believed to be important in regulating the proliferation of neural stem cells and their differentiation into neurons and glial cells (Mattson 2003). DNMTs are expressed throughout neural development and promote neuronal survival and plasticity (Mehler 2008). MBDs were shown to play an important role in brain development and cognitive functions, such as learning and memory (Chahrour et al. 2008, Mehler 2008). DNA methylation was shown to be important in synaptic plasticity, learning and memory in adult CNS neurons (Feng et al. 2010) and CNS repair.

Researchers mapped methylation sites in genomes of neurons and glia in the frontal cortex. m. CH methyl tags, or non-CG methylation (purple stars), were absent at birth, but were added rapidly during the first few years of life and then more slowly until about age 30. After age 50, the number of m. CH tags declined.

Chromatin remodeling, histone modifications and brain Changes in the chromatin structure occur not only during development, but also in mature neurons. Neuronal signaling appears to be strongly regulated by addition and removal of histone acetylation, histone and DNA methylation. The neuronal levels of monoacetylated H 4 decrease progressively during aging. Chromatin remodeling via the post-translational modification of histone proteins (primarily histone H 3 phosphorylation and acetylation) is important for the formation of long-term memory.

Chromatin remodeling, histone modifications and brain Histone methylation is actively regulated in the hippocampus and facilitates the formation of long-term memory, while the deregulation of H 4 K 12 acetylation may cause memory impairments in the aging mouse brain. The alteration in balance between HDAC and HAT functions can lead to neurodegenerative disease. HDAC inhibitors have shown a therapeutic efficacy in animal models of neurodegenerative diseases, suggesting their neuroprotective role.

nc. RNAs and brain mi. RNAs are particularly abundantly expressed in the brain. Many mi. RNAs are expressed in a spatially and temporally controlled manner in the nervous system, suggesting that their regulation may be important in neural development and function. In the past years, a growing body of scientific evidence has indicated that mi. RNAs may be a contributing factor to aging-related neurodegenerative diseases. Schaefer et al. (2007), for instance, demonstrated that a substantial loss of mature mi. RNA in the cerebellum of mice with the knockedout Dicer gene causes progressive neurodegeneration in a mouse model.

Prenatal stress Altered DNA methylation machinery and mi. RNA expression in placenta Pregnancy complications (preeclampsia, fetal growth restriction, preterm birth) Epigenetic changes in the fetal brain High risks of mental illness later in life Low infant birth weight Figure. Proper functioning of mi. RNA and DNA methylation machineries are required for normal placental and brain development. Human studies showed that alteration in these epigenetic mechanisms are associated with low infant birth weight and pregnancy complication. Animal studies demonstrated that stress can induce epigenetic changes in placenta and brain. Whether stress -induced epigenetic changes in the placenta and brain cause higher risk of mental illness later in life is yet to be verified

Prenatal stress Traumatic war experiences, death of father Repeated variable stressors n? sio ? es s xpr ange e h A c i. RN etic m n red epige e Alt her Ot Human evidence High risk of developing schizophrenia children Animal studies Molecular changes in the brain Schizophrenia-like phenotype in the offspring: cognitive - deficits, disrupted social behaviour, hyperactivity - Altered DNA methylation in prefrontal cortex es Disrupted maturation ng tic ha e r c igen ere of prefrontal cortex la cu y ep at w le Impaired HPA axis mo ed b s, th uring e regulation Ar ulat nism d d fe? reg cha rupte tal li Impaired synaptic me dis rena plasticity p

Michael Meaney and Moshe Szyf experiments Michael Meaney and Moshe Szyf and their colleagues demonstrated a direct relation between variations in maternal care and the phenotype of the offspring. They showed that variations in maternal licking/grooming (LG) behavior influence behavioral and hypothalamic-pituitary-adrenal (HPA) responses to stress in adult offspring through epigenetic mechanisms. LG is a source of tactile stimulation that triggers important endocrine and metabolic responses and regulates somatic growth (Kappeler and Meaney 2010).

It was shown that the progeny of the dams that display high LG behavior had decreased methylation of glucocorticoid receptors (GR) in the hippocampus, which leads to increased GR expression and results in a decreased stress response. This early-life experience has a long-lasting stable effect on the progeny, but it can be reversed by cross-fostering experiments. Michael Meaney and Moshe Szyf experiments

Pups born to low LG mothers but reared by high LG mothers show stress responses that are similar to those of the normal offspring of high LG mothers and vice versa. Even more intriguing is the evidence that these effects of maternal care can be transmitted to the next generations. Michael Meaney and Moshe Szyf experiments

Changes in the expression pattern of some genes in the brain that regulate the stress response GR in hippocampus, a central benzodiazepine receptor (CBR) in amygdala, and a corticotropin-releasing factor (CRF) in hypothalamus) were transmitted from one generation to the next. Michael Meaney and Moshe Szyf experiments

Is everything wrong what can be wrong – is wrong due to epigenetics?

Potential mechanism of genome evolution – when epigenetics precedes genetics Boyko and Kovalchuk, Curr Opin Plant Biol, 2011

References/reading pages Kovalchuk and Kovalchuk “Epigenetics in health and diseases” Ch. 2. pp. 19 -31; 36 -46. Ch. 5. pp. 119 -123; 125 -130; 133 -134; 143 -144 Ch. 4. pp 75 -83; 92 -117; Ch. 6 pp. 147 -161; Ch: 7 pp. 177 -195; Ch: 12 http: //books. google. ru WIKIPEDIA Sam Griffiths-Jones The Wellcome Trust Sanger Institute http: //www. sanger. ac. uk/Software/Rfam/ http: //www. stats. ox. ac. uk/~hein/Human. Genome/