Introduction to Epigenetics Manoj Kannan BITS-Pilani & NCI-Frederick

Recap of some familiar terms in genetics Allele Genotype Phenotype Gene expression Gene silencing Genome

Genome expression Well-established paradigm of how genetic information is transcribed and translated Human genome project has given plenty of data, which is still being mined for useful information An estimated 140,000 proteins in the human body Different cells express a different subset of proteins Yet almost all cells have the same genomic sequence comprised of just under 25,000 genes 25,000 genes  140,000 proteins?? Simple math doesn’t explain! Hence, it’s not just the genes, but how they are regulated that explains the phenotype variations

Here is a phenotype variation… What do you think is the basis? But epigenetically different! ALL THE MICE ARE GENETICALLY IDENTICAL! May be from different parents? Well, age difference… Oh, okay - mutation in the pigment causing gene?

What is Epigenetics? Study of heritable changes in gene function that do not involve changes to the nucleotide sequence of DNA When a cell undergoes mitosis or meiosis, the epigenetic information is stably transmitted to the subsequent generation Epigenetic controls add an ‘extra layer’ of transcriptional control

Three major epigenetic processes we will discuss today… DNA Methylation Histone modifications RNA-mediated phenomena

DNA Methylation Most well-studied epigenetic tag/mark; best understood epigenetic cause of disease Conserved across various kingdoms of life SAM – S-adenosylmethionine SAH – S-adenosylhomocystine So, G, A, T, C…. and the fifth base, mC in mammalian genome

Distribution of DNA methylation In mammals, in the context of CpG dinucleotides (plants have other types too) Methylated CpGs are associated with silenced DNA, eg. Transposons, inactive X chromosome, imprinted genes “CpG islands”, associated with promoters of 40% of mammalian genes, are generally free of methylation eg. housekeeping genes, tissue-specific genes

DNA methyltransferases (DNMTs) 2 major classes of enzymes in mammalian systems De novo methylases Maintenance methylase Mouse knockouts of these genes tell us they are necessary for the survival and proper development of the organism.

How does DNA methylation affect gene transcription? Unmethylated (or hypomethylated) promoter allows gene transcription Methylated CpGs block binding of TFs; hence, transcription blocked Me-CpG binding proteins also preclude TF binding to the promoter region Other ways too…

Role of DNA methylation Tight control for maintaining gene silencing (vertebrate genes are less “leaky” compared to bacterial) Transcriptional silencing of transposons (‘genome defense’ model) Genomic imprinting – one of the alleles of a gene is silenced, depending on the parent of origin X inactivation – all but one of the X chromosomes in female is inactivated – methylation of the inactive X copy

Three major epigenetic processes DNA Methylation Histone modifications RNA-mediated phenomena

Structural organization of the genome Unless the genome is accessible by the transcription machinery of the cell, the genome cannot be functional! Hence, the utilization of the biological information in the genome is dependent on the chromatin organization.

Structure of a nucleosome Histone octamer core ~146 bp DNA

Post-translational histone modifications A = acetylation M = methylation P = phosphorylation U = ubiquitination

Consequences of tail modifications Higher order chromatin structure is affected eg. Addition of acetyl groups (-ve) neutralizes the positive charge on lysine => affinity of the histone to bind tightly to DNA is reduced => chromatin becomes less compact => transcription of the associated gene is favored Vice versa for deacetylation (the gene is repressed) Other proteins are attracted to these sites of modifications….which, in turn, affect gene expression

Enzymes catalyze these covalent tail modifications Histone Acetyl Transferases (HATs) function as large, multiprotein complexes, eg. SAGA, ADA complexes (yeast), TFTC complexes (humans); associated with transciptional activation. Histone Deacetylases (HDACs) part of multiprotein complexes, eg.Sin3, NuRD; associated with transcriptional repression. Histone Methyl Transferases (HMTs) Histone Demethylases

Comparing chromatin types Transcriptionally active chromatin/euchromatin Transcriptionally inactive chromatin/ heterochromatin Chromatin conformation Open, extended conformation Highly condensed conformation DNA CpG methylation Relatively unmethylated, especially at promoter regions Methylated, including at promoter regions Histone acetylation Histone methylation Acetylated histones H3-K4me3, R17me2 Deacetylated histones H3-K9me

Crosstalk between DNA methylation and chromatin modification Histone deacetylation Histone H3-K9 methylation Self-reinforcing repressive cycle

Three major epigenetic processes DNA Methylation Histone modifications RNA-mediated phenomena

RNA interference (RNAi) causes gene silencing RNAi initiates heterochromatin formation in fission yeast and DNA methylation in plants.

Epigenetics in human disease Association with various cancers – stomach, kidney, colon, pancreas, liver, uterus, lung and cervix ICF syndrome Fragile X syndrome Angelman’s syndrome Rett Syndrome Coffin-Lowry Syndrome HUMAN “EPIGENOME” PROJECT

Epigenetics….Environment

Epigenetics….Environment

Epigenetics….Environment And Consciousness! “The Biology of Belief: Unleashing the Power of Consciousness, Matter and Miracles” is a recent book in the market on epigenetics! Disclaimer: I haven’t read the book yet!

References Genomes (3/E) – T.A. Brown Molecular Biology of the Cell (4/E) – Bruce Alberts, et al. Human Molecular Genetics (2/E) – Strachan & Read Developmental Biology (7/E) - Gilbert NCBI Bookshelf - free online books! (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books)

Thank you! Questions? Discussion?

If you want to read more about Epigenetics… Chapter 10 of T.A. Brown’s Genomes (3/E), entitled “Accessing the Genome” is an good place to start Special issue of Science 10 Aug. 2001 has a bunch of excellent articles written by pioneers in the field. (slightly dated, but still relevant) Science Functional Genomics Resources: Epigenetics (portal hosted by the Americal journal, Science) – gives a series of articles published in the field, and also a list of useful websites http://www.sciencemag.org/feature/plus/sfg/resources/res_epigenetics.dtl