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EPIGENETICS
OUTLINES
• Introduction and Definition• Factor affecting epigenetics and Inheritance• Mechanism• Epigenetic phenomenon• Cancer and epigenetics• Diagnosis • Therapies targeting epigenetic modification• Future
Introduction • Genetics explains phenotypic trait in
organisms through presence or absence of specific nucleotide sequence of DNA.
• However, that does not adequately explain the expression or lack of expression of all genes.
• That indicates, there are other control mechanism; such as DNA methylation and chromatin modification
Definition • C.H. Waddington coined the term
epigenetics to mean above or in addition to genetics to explain differentiation
• Epigenetics refers to the study of changes in the regulation of gene activity and expression that are not dependent on gene DNA sequence.
• While epigenetics often refers to the study of single genes or sets of genes, epigenomics refers to more global analyses of epigenetic changes across the entire genome
• Disruption of such control mechanism is associated with a variety of disease
– With behavioral/neurological manifestation
– Disorder of tissue growth
– Endocrine disorder
– Neoplasia
Inheritance
• parent’s experiences are passed on to offspring through epigenetic tags
• When the zygote is formed many epigenetic tags are removed from the chromosomes of the parents, but some remain
• Identical twins– from same zygote– same genetic information
(including epigenetic tags)
• While infants – similar environments, – so little variation in
epigenome
• Difference in the twins’ epigenomes makes them become different when they are older
• One can develop a disease while the other is fine
Methylation of Agouti Genes in Mice
Environment can Influence Epigenetic Changes
Emma Whitelaw, Henry Stewart Talks
Factors during pregnancy affecting the transmission of epigenome
• nutrition of mother
• Stress, social interactions, physical activity as Stress hormones can travel from the mother to a fetus
• exposure to toxins
GRANDMOTHER’S CURSE!!!!!
Some of the effects ofsmoking may be passed from grandmother to
grandchild
Epigenetics Mechanisms
Gene Expression
RNA Interference
Histone Modifications DNA Methylation
• Epigenetic alteration are outside the primary sequence, but nonetheless affect the ability of gene to be expressed.
• DNA in most cells packaged with histone to form nucleosome in ‘beads on a string’ structure.
• Chromatin regulation involves high-order conformational changes– Relaxation or tightening of this thread of DNA-
histone complex
• Further regulation is by assembling promoter-enhancer complexes via long-range DNA looping– Can be blocked by specific DNA-sequence called
insulator
• The core histones are subject to diverse post-translational modifications, including methylation and acetylation– That project from tightly structured nucleosome core
DNA methylation
Histone
MethylatedDNA
Effect of methylation
Methylation turns off genes.
Acetylation turn genes on.
5-Methyl Cytosine is Found in Heterochromatic Regions
Natural Roles of DNA Methylation in Mammalian System
Imprinting
X chromosome inactivation
Heterochromatin maintenance
Developmental controls
Tissue specific expression controls
DNA Methylation and Other Human Diseases
• -- Imprinting Disorder:• Beckwith-Wiedemann syndrom (BWS)• Prader-Willi syndrome (PWS)• Transient neonatal diabetes mellitus (TNDM)
• -- Repeat-instability diseases• Fragile X syndrome (FRAXA)• Facioscapulohumeral muscular dystroph
• -- Defects of the methylation machinery• Systemic lupus erythemtosus (SLE)• Immunodeficiency, centromeric instability and facial
anomalies (ICF) syndrome
Histone Modifications
Histone modificationthe histone code
– Acetylation– Methylation– Phosphorylation– Ubiquitylation– sumoylation
• Enzymes catalyzing– Histone
acetyltransferase– Histone deacetylase– Histone
methyltransferase– Histone kinase
Histone Modification Status Correlates with Transcriptional Activity
• Gene activation correlated with H3-K9 acetylation • Gene silencing associated with H3-K9 methylation
Role of histone modification
• DNA transcription• DNA repair• DNA replication
Histone Modifications and Human Diseases
Coffin-Lowry syndrome is a rare genetic disorder characterized by mental retardation and abnormalities of the head and facial and other areas. It is caused by mutations in the RSK2 gene (histone phosphorylation) and is inherited as an X-linked dominant genetic trait. Males are usually more severely affected than females.
Rubinstein-Taybi syndrome is characterized by short stature, moderate to severe intellectual disability, distinctive facial features, and broad thumbs and first toes. It is caused by mutations in CREB-binding protein (histone acetylation)
RNA interference
siRNA Mediated Heterochromatin Maintenance
X-inactivation
• One X-chromosome in female cells is condensed and located on nuclear periphery
• Complete silencing of one entire chromosome is certainly one of the most dramatic epigenetic phenomenon
• Key-transcript, X-inactivation specific transport lack protein-coding function
• Production of Xist rapidly attract histone-modifying enzymes, followed in turn by DNA-methylation
• Inactive x-chromosome is evidently ‘locked in’ by chromatin modification-– Methylation of Histone H3 at lysine K27– Hypoacetylation of histone at H3 ande H4– Dense CpG methylation of CpG islands
Genomic imprinting
• Most autosomal genes in mammalian have biallelic expression
• But a small number of genes are expressed from only one parental allele – Imprinting –become essential for viability
• Maternally derived genes are growth-limiting• Paternally derived genes are growth-
enhancing
Androgenetic and gynogenetic tumor
• Benign overian teratoma (bimaternal origin)
• Hydatidiform mole (biparenatal origin)
• Usually benign
• Diagnosis: histopathology + immunostaining of protein product of imprinted gene– Neoplastic trophoblast of complete
hydatidiform mole fails to express imprinted gene (CDKN1C and PHLDA2)- those normally imprinted from maternal allele
Prader-willi and Angelman syndrome
• Chromosomal band 15q11-q13 is a megabase scale chromosomal domain including multiple imprinted gene
– One portion of gene is maternally expressed/paternally silenced
– Adjacent portion oppositely imprinted
– Deletion of paternal homologue – PWS – Deletion of maternal homologue - AS
• Whether these diseases are due to large deletion, microdeletion, epimutation majority can be diagnosed by simple Southern blot or PCR-based assays for DNA mutation
• Gain of methylation in PWS
• Loss of mutation kin AS
Cancer epigenetics
• A common paradigm of cancer epigenetics is hypermethylation of CpG island of tumor suppressor gene promoter
• Hypermethylated promoter DNA is associated with virtually every type of human tumor– With each type of tumor having own signature of
methylated genes
Cancer Methylated genes
Prostate GSTP1
Renal VHL
Colon and endometrial MLH1 (mismatch repair gene)
Esophageal APC
• Global hypomethylation: overall in 5-methylcytosine content in the genome– Found in premalignant and early stages of some
neoplasm– Important in tumor progression
• Gene-specific hypomethylation: – Often affect promoter region of proto-oncogene
and oncogene which are normally highly methylated
DNA Methylation and Cancer
Technologies for Studying Epigenetics/Epigenomics
DNA Methylation
Irizarry et. Al. (2008) Genome Research 18(5):780
Microarray or deep sequencing
Bisulfite Sequencing
5’3’
Expression array
Exon array
Splicing array
Promoter array
Tiling array
exon intron
Global Interrogation of DNA Methylation using Microarrays
promoter
• Earliest method, still the most direct and least subject to artifact is southern blotting of genomic DNA with methylation-sensitive restriction enzyme.
• Restriction enzymes ( e.g. HpaІІ, SmaІ, NotІ) with CpG as part of their recognition sequence do not cut that sequence when the C is methylated
Technologies for Interrogating Epigenetics/Epigenomics
http://www.nature.com/jid/journal/v125/n2/extref/5603467x1.jpg
ChIP-chip
Antibody specific to one type of histone modification
Histone Modifications
ChIP-seq
Deep sequencing
Demethylation in cancer therapy
HDAC inhibitors
Interchromosome regulation of genes
Scaffold proteinSATB1
• More higher structure than chromatin
• Play important role in T-cell development