2016 Complex Disease talk - Galton Institute

2016 Complex Disease talk - Galton Institute

Epigenetics and gene regulation Andrew P Read Centre for Genomic Medicine St Marys Hospital, Manchester http://www.galtoninstitute.org.uk/wp-content/uploads/2018/10/BN180916_Genetics-in-Medicine-41.pdf-web-1.pdf Galton institute Teachers Day Manchester, June 2019 Definitions of epigenetics The narrow view: Epigenetic changes are changes in gene expression that do not depend on changes in

DNA sequence, and that are transmitted through mitosis from mother cell to daughter cell. (but not through meiosis from parent to child) X-inactivation is the classic example: NCG3 Fig 7.7 Why does epigenetics matter? We have no more protein-coding genes than the 1mm-long nematode worm Caenorhabditis elegans 1 mm

Thousands of phenotypically different cells in the human body, all with the same genome GIM4 p.6 20,338 genes 20,362 genes so our greater complexity must be due to more complex gene regulation Regulatory elements

Cell cycle controls Epigenetic regulation MicroRNAs etc Modification & location Regulation of transcription is regulation of promoters Promoters: A few hundred bp upstream of the 5 end of a gene

Site of assembly of the transcription initiation complex RNA polymerase + a variety of other proteins (transcription factors) Enhancers Regulatory sequences that are some way away from the gene they control. May be upstream or downstream, and up to 1 megabase away Like promoters, they bind transcription factors. They loop round to bring their bound proteins into proximity to the promoter HMG5 Fig 10.24

Genes can have multiple enhancers Tissue-specific gene expression largely depends on enhancers HMG5 Fig 10.25 Transcription factors DNA-binding proteins that control transcription General (basal) transcription factors are always present and necessary for all gene expression: TFIIA, TFIIB etc Tissue-specific transcription factors are present only in some cells and under some conditions; they determine which genes are expressed in that cell and at that time.

Transcription factors bind to short (typically 4-12) DNA sequence motifs but real binding sites are often much weaker and more variable than the optimal motifs defined by in vitro experiments At a given promoter or enhancer multiple TFs often operate in a combinatorial way HMG5 Fig 10.6 The three pillars of gene regulation Chromatin conformation

DNA methylation Histone modification DNA methylation DNA methyltransferase Cytosine 5-methyl cytosine deoxyribose

but the methyl group acts as a signal to recruit methylDNA-binding proteins Methylation doesnt alter the genetic code: 5mC basepairs with G just like C does DNA methylation patterns are transmitted from mother cell to daughter cells Two DNA methyltransferases: DNMT3 methylates cytosines that are next to guanines CpG sequences DNMT1 is the maintenance methylase Patterns of DNA methylation characterise different types of cell

Studying DNA methylation In the lab (as distinct from in cells with DNA methyltransferases etc) all methylation is lost when you make copies of the DNA. PCR and standard sequencing methods use copies of the original DNA so you cant study DNA methylation by standard laboratory methods. The standard method for methylation uses sodium bisulphite Na2SO3 or Na2S2O5. This converts C to U, but leaves 5meC unchanged. Sequence the sample twice, with and without bisulphite treatment and compare.

The three pillars of gene regulation Chromatin conformation DNA methylation Histone modification Nucleosomes DNA in the cell nucleus exists as chromatin - a complex with proteins and some RNA species In the most basic level of organisation chromatin consists of nucleosomes A nucleosome is a ball of 8 molecules of histone with 146bp of DNA wrapped round it.

Histones Small basic proteins (ca. 130 amino acids) that have an affinity for negatively-charged DNA 4 types in nucleosomes: H2A, H2B, H3 & H4; 2 molecules of each. A fifth type, H1, where the linker DNA exits. N-terminal tails of the four core histones protrude from the nucleosome and are subject to extensive covalent modifications. These modifications play an essential role in epigenetics HMG5 Fig 2.18

Histone modifications Methylation of lysines and arginines esp. H3K4, H3K9, H3K27, H4K20 Acetylation of lysines esp. H3K9, H3K27 Lysines can be mono-, di- or trimethylated e.g. H3K4me1, H3K4me2, H3K4me3 Many other modifications e.g. phosphorylation of serines H3S10, H3S28; mono-, di- & trimethylation of arginines H3R2, H3R8, H3R17, H4R3; phosphorylation of threonines H3T3, H3T6, H3T11; ubiquitylation of lysines

H2AK119, H2BK120 Studying histone modifications ChIP-seq: chromatin immunoprecipitation followed by sequencing. Use antibodies against specific histone modifications e.g. H3K27ac, H3K4me1 etc DNA sequencing reveals which DNA sequences are associated with which specific histone modifications Can identify histone modifications specific to promoters, enhancers etc Can then use this knowledge to identify novel enhancers etc. HMG5 Box 9.3 The three pillars of gene regulation

Chromatin conformation DNA methylation Histone modification How long is the human genome? 3,200,000,000 bp Base pairs are 0.34 nm apart

Length of human genome = 3.2 x 109 x 0.34 x 10-9 m = 1 metre Length of DNA in a diploid cell = 2 metres13 Ca. 10 cells in your body 2 x 1013 m of DNA in your body = 2 x 1010 km (20,000,000,000 km) 130 x distance from us to the sun 2 x diameter of solar system out to Neptune

Packaging the DNA Cell nucleus Need to pack 2m of DNA into a 10m cell nucleus Chromosome territories A&B compartments Topologically-associating Euchromatin (open) domains (TADs)

Bare DNA Nucleosom es Heterochromatin (closed) Packaging the genome Chromatin Conformation Capture HMG5 Box 10.1 Dekker J et al.(2013) Exploring the three-dimensional organization of genomes: interpreting

chromatin interaction data. Nat. Rev. Genet. 14: 390403. Packaging the DNA - TADs Revealed by chromatin conformation capture Typically 100-500 kb A constant feature of chromatin organisation, conserved across tissues and species DNA loops Chromosome

territories Enhancer TADs promoter loops Enhancers can regulate genes within the same TAD, but not genes in different TADs Smaller loops within TADs may control enhancer promoter interactions Wikipedia

Chromatin remodelling complexes Large ATP-powered multiprotein machines Able to shuffle nucleosomes along the DNA, e.g. to create nucleosome-free stretches of DNA accessible to regulatory proteins. Create areas of accessible (nucleosome-free) DNA where transcription factors etc can bind. Can also swap individual histone molecules into and out of nucleosomes Involved whenever DNA activity

changes e.g. replication, differentiation, repair Clinical epigenet ics (A quick overview; lots more detail in GIM4) Imprinting A few genes carry a mark of their parental origin Paternal and maternal copies are expressed differently Imprintin

g is epigeneti c and reversibl e Total of ca. 90 genes are imprinted Parent-specific epigenetic marks imposed on gamete In imprinted genes mark persists in somatic cells Marks erased in germ cells Imprinting:

Prader-Willi & Angelman syndromes Two very different conditions: Prader-Willi Hypotonia, moderate intellectual syndrome: disability, later uncontrollable eating Angelman syndrome: Developmental delay, severe intellectual disability, no speech, jerky movements

Both caused by the same deletion on chromosome 15 See GIM4 for Prader-Willi syndrome: deletion of more details and paternal copy Angelman syndrome: deletion of further examples

maternal copy Why imprinting?? Think selfish genes Fathers interest: As big and strong a baby as possible, even at the expense of the mother Mothers interest: Limit depredations of the parasitic fetus to preserve her for further childbearing

www.parents.com Epigenetic diseases Failures of writers, readers or editors Writers impose epigenetic marks: DNA methyltransferases Histone methyltransferases Histone acetyltransferases Histone kinases Readers react to epigenetic

etc etc Many different proteins marks: Editors remove epigenetic marks: TET enzymes Histone demethylases Histone deacetylases Histone phosphatases etc etc Check the OMIM database for examples https://www.ncbi.nlm.nih.gov/omim

Failures of writers: Some examples of epigenetic disease (NCG3 Box table 7.1) Failures of editors: Some examples of epigenetic disease Failures of readers: Rett syndrome

X-linked dominant condition, OMIM 312750 Failures of chromatin remodelling: Reacting to the environment Short term reacting to changing conditions Long term Barker hypothesis: intrauterine and neonatal environment set future metabolism Evidence: epidemiology; Dutch hunger winter; many animal experiments Transgenerational definitely happens but controversial how important Evidence: well documented in plants; many animal experiments In humans: grandpaternal effects in verkalix region of Sweden, etc.

Inheritance of acquired characters!

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