Epigenetics and Epigenomics PDF

Epigenetics and Epigenomics Professor Rebecca Oakey Department of Medical and Molecular Genetics [email protected] The Central Dogma in Molecular Biology Kuang, Jujiao et al. “An overview of technical considerations when...

Epigenetics and Epigenomics Professor Rebecca Oakey Department of Medical and Molecular Genetics [email protected] The Central Dogma in Molecular Biology Kuang, Jujiao et al. “An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research.” PloS one vol. 13,5 e0196438. 10 May. 2018  The DNA does not work alone, it needs the epigenome to function along side Genetic information encoded in DNA (A,T,G,C) Epigenetic information includes DNA methylation of C, post-translational modifications to histone proteins & non-coding RNAs Epigenetic factors help regulate gene expression These mechanisms interact with transcription factors to regulate gene- expression patterns inherited from cell to cell. The patterns underlie embryonic development, differentiation and cell identity  Classic genetic paradigms X-inactivation Sex determination Genomic Imprinting How does a multicellular organism develop from one genome? Epigenetic factors provide information for tissue specific gene expression Stable in somatic cells (cell division) Reprogrammed on a genome wide level in germ cells & early embryos Pluripotency Differentiation Imprinting X inactivation Transposon control DNA methylation of cytosine DNA methylation is the addition of a methyl group to cytosine CpG dinucleotides and DNA methylation CpGs are found in clusters called CpG islands CpG islands are unmethylated at promoters of genes Germline genes have methylated CpG island promoters In the rest of the genome CpGs are depleted in frequency and usually methylated Histone modifications- including acetylation, methylation, phosphorylation, & ubiquitylation Each nucleosome is made of two identical subunits, each of which contains four histones: H2A, H2B, H3, and H4 Histone proteins undergo post-translational modification Examining histone modifications can reveal gene activation states ‘Histone code’ for activating or shutting down regions of the genome and their genes  Epigenetics in mammals: X-inactivation Dosage compensation of sex chromosomes between females & males is achieved through X- chromosome inactivation Xist long noncoding RNA Xist initiates DNA methylation & repressive chromatin process early in development which spreads across the X chromosome from which it is transcribed Loda A, Heard E (2019) Xist RNA in action: Past, present, and future. PLOS Genetics 15(9): e1008333. https://doi.org/10.1371/journal.pgen.1008333 The study of imprinting provided the basis for understanding epigenetics Most genes are expressed from both parental alleles of a gene BUT a few hundred genes are expressed from either the maternal or the paternal allele, but NOT both and are known as imprinted genes We inherit one copy of each gene from each parent Imprinted genes are dangerous X This plane can land with 2 This plane can still land working engines with 1 working engine Loss of heterozygosity X X A gene with no working alleles cannot function If you start off with one silenced copy, you are at a greater risk of having no working copies, so there must be a compelling reason to have imprinted genes in mammals!  Parent-specific expression of imprinted genes Paternal allele Same sequence Same environment One ACTIVE & one SILENCED We can study the epigenetic factors on the two parental alleles of a gene in the same cell Nuclear transplantation studies formed some of the first imprinting experiments Nuclear Transplantation Genomic imprinting imposes biparental reproduction in mammals Mouse uniparental conceptuses by nuclear transfer Fertilized Parthenogenote Androgenote Biparental Embryonic lethality < 9dpc Barton et al., McGrath et al., 1984 Naturally occurring situations in humans Parthenogenetic conceptuses Androgenetic conceptuses Ovarian teratoma (dermoid cyst) Hydatidiform mole Hyperdifferentiation Trophoblastic hyperplasia Naturally occurring situations in humans 10 week twins, one normal and one hydatidiform mole in one gestational sac “Take a break” Ovarian teratoma Imprinting is involved with... Development and growth of the Reproductive Birth defects & technologies that involve embryo and EET cancer cloning Rare genetic disorders Silver Russell Beckwith Wiedemann syndrome Transient Neonatal Diabetes Mellitus syndrome Neurological and Psychiatric Autoimmune disease (RA, SLE) Many cancer types Common disorders Heart disease Epigenetic defects in rare imprinting disorders, cancer & common diseases  Uniparental Disomies Normal-one each parent Paternal Maternal UPD syndromes Maternal UPD14 syndrome (growth failure) Paternal UPD14 syndrome (dwarfism) Maternal UPD2 (growth failure) Maternal UPD16 (growth failure) Paternal UPD6 (neonatal diabetes)  Clinical phenotype of imprinted disorders (a)Angelman (b) Prader–Willi syndrome. syndrome. Happy disposition, Typical central obesity, inappropriate laughter, short stature, small hands widely spaced teeth & wide and feet, and mild facial mouth, stiff, upheld arms dysmorphism, PWS patients and broad stance and are always hungry mental retardation AS is associated with R. D. Nicholls, mutations S. Saitoh in the TrendsPWS and B. Horsthemke involves in Genetics loss of function of 1998, 14:194-200 imprinted gene, UBE3A multiple paternally expressed genes PWS & AS Shared cytogenetic deletion of 15q11-13 AS mutations are transmitted from the mother AS mutations from the father are asymptomatic compared to PWS deletions are transmitted from the father PWS deletions from the mother are asymptomatic AS is inherited through the maternal line AS results from mutations in a maternally expressed gene called UBE3A PWS is is inherited through the paternal line PWS results from deletions or mutations of paternally expressed genes  Molecular classes of PWS and AS Deletions and imprinting mutations occur with equal frequency in both syndromes UPD is more common in PWS than AS because of higher rates of maternal nondisjunction. The gene mutation class in AS is lacking in PWS, because PWS is a contiguous gene syndrome. M, maternal (red); M(P), maternal inheritance of imprinting center (IC) mutations with a fixed paternal epigenotype (horizontal lines); P, paternal (blue); P(M), paternal inheritance of IC mutations with a fixed maternal epigenotype; UPD, uniparental disomy R.D. Nicholls, S. Saitoh and B. Horsthemke Trends in Genetics 1998, 14:194-200  Angelman syndrome No cure but: Mouse model in which scientists have: Activated the silenced pat allele of Ube3a Results show that this ameliorates many disease-related symptoms including motor coordination defects & cognitive deficit in mice Potential for human therapy? Meng et al, PLoS Genetics, 2013 Beckwith-Wiedemann Syndrome BWS is a human disease with parent-of-origin-associated prenatal overgrowth and cancer pre-disposition, located on human chromosome 11p15.5 BWS occurs in around one in 14,000 births  BWS is characterized by a large tongue (macroglossia), large organs (visceromegaly), large body size (macrosomia), hernia of the navel (omphalocele) and small head (microcephaly) Prognosis: Neonatal mortality rate is approximately 21% mainly by congestive heart failure. De-regulation of imprinted genes Most cases are sporadic & result from epimutations at either of the two 11p15.5 imprinting centres (IC1 and IC2) Germline mutations in NLRP2 gene in a familial form of this imprinting disorder BWS incidence elevated in IVF Reports in animals and humans suggest BWS is increased after ART Imprinting defects in IVF/BWS are higher than in BWS conceived naturally Further research into embryo culturing and imprinting mechanisms  Silver-Russell Syndrome IUGR & low birth weight Decreased birth length Triangular shaped face Postnatal growth retardation Poor appetite / Reflux Fifth finger clinodactyly Normal head size appearing large because of reduced body length & weight 40-60% of cases due to an epimutation (DNA methylation defect at Igf2/H19 genes) remainder unknown  Characteristic features of the Mendelian disorders of the epigenetic machinery Hans Tomas Bjornsson Genome Res. 2015;25:1473-1481 © 2015 Bjornsson; Published by Cold Spring Harbor Laboratory Press Epigenetic differences in identical twins Identical DNA sequence Effects of intrauterine environment Effects of post-natal environment (eg smoking) Epigenetics could explain twin discordance in disease eg. sickle cell anaemia, automimmune diseases and psychiatric disorders amongst others BWS and unaffected twins  Advent of Multi-’Omics Draft of the human genome ENCODE The Encyclopedia of DNA sequence published 2001 elements 2003 2021 2001 2012 Completed human genome 20th Birthday 2021 sequence in 2003 https://genome.ucsc.edu/ https://www.ensembl.org/index.html Multiomics https://www.youtube.com/watch?v=Xsyp0qqKzkY&t=261s Epigenetic dysregulation as a hallmark of cancer Stahl M, Kohrman N, Gore SD, Kim TK, Zeidan AM, et al. (2016) Epigenetics in Cancer: A Hematological Perspective. PLOS Genetics 12(10): e1006193. https://doi.org/10.1371/journal.pgen.1006193 https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006193 Phaeochromocytomas & Paragangliomas Germline Mutation Patients with PPGLs All tumours have a metastatic potential Operative management if possible Genetic screening If pathogenic variant identified Genetic counselling Screening for family members Follow up Annual review with bloods Full body MRI every 2-3 years Using multi-omics to characterize PPGLS Genomic analysis Whole exome sequencing DNA copy number analysis Transcriptomic analysis mRNA seq miRNA seq Epigenomic analysis CpG DNA methylation Proteomic analysis Reverse phase protein array  Hypermethylation is a hallmark of PPGLs Genomic analysis Methylomic analysis Transcriptomic analysis Personalised medicine for PPGLs Question Is there a difference in gene expression between metastatic and non- metastatic SDHx related PPGLs? Single cell transcriptomics 1. Fresh tumour collection from operating theatre (following enrolment and consent) 2. Immediate transfer to our lab for… 3. Single cell dissociation and single cell transcriptomics  An analysis pipeline is being developed to examine metastatic or aggressive tumour behaviour Non-aggressive Establish baseline disease course All patient ‘omics Aggressive or What are the metastatic tumour differences to disease course above? similarities differences The genes and (epi)genomes identified through multi-’omic analysis are now being interrogated for their functional roles and their use as diagnostic predictive markers Take-away messages Epigenetic mechanisms direct the genome Epigenetic mechanisms are essential in mammalian development Epimutations are involved in rare disorders Epimutations are found in cancer Appreciate how epigenetic mechanisms can go awry aids diagnostics and treatment regimens [email protected]