Functional characterisation

Transcription

1 Me Me Ac Ac Functional characterisation How can we know if measured changes in DNA methylation and function (phenotype) and linked, and in what way? DNMT Dianne Ford Professor of Molecular Nutritional Institute for Cell and Molecular Biosciences/Human Nutrition Research Centre Newcastle University Signal of gene expression (e.g. northern blot (or RT-PCR); western blot)? Epigenetic Epidemiology Short Course 11 th -15 th June 2012 Institute of Genetic Medicine, Newcastle University, UK

2 Possible relationships between measured changes in DNA methylation and measured functional/phenotypic changes Intervention/ exposure DNA methylation function/ phenotype Dietary resveratrol supplement Reduced DNA methylation of genes in insulin signalling pathway Increased expression of affected genes in muscle and adipose tissue Enhanced muscle and adipose sensitivity to insulin Improved glucose tolerance

3 Possible relationships between measured changes in DNA methylation and measured functional/phenotypic changes Intervention/ exposure DNA methylation function/ phenotype Intervention/ exposure Dietary resveratrol supplement function/ phenotype Improved glucose tolerance DNA methylation Reduced DNA methylation of genes in insulin signalling pathway Reduced net exposure of tissues to glucose Effects on DNMT expression

4 Possible relationships between measured changes in DNA methylation and measured functional/phenotypic changes Intervention/ exposure DNA methylation function/ phenotype Intervention/ exposure function/ phenotype DNA methylation Intervention/ exposure DNA methylation Dietary resveratrol supplement function/ phenotype Reduced DNA methylation of genes in insulin signalling pathway Effects on DNMT expression DNA methylationindependent effects on expression of genes in insulin signalling pathways Improved glucose tolerance

5 Approaches to uncovering causal relationships between DNA methylation and function/phenotype We need to create the observed change/difference in DNA methylation and determine if this then causes the functional effect; i.e. manipulate DNA methylation and measure function/phenotype Cell line models Reduce/inhibit DNA methylation Chemical/pharmaceutical inhibition» 5-aza-2 -deoxycytidine Transgenic approaches» DNMT knockout/sirna Increase DNA methylation Transgenic approaches» DNMT overexpression Promoter-reporter constructs Animal models (mice?; honeybees!) Reduce/inhibit DNA methylation Chemical/pharmaceutical inhibition» 5-aza-2 -deoxycytidine Transgenic approaches» DNMT knockout/sirna Increase DNA methylation Transgenic approaches» DNMT overexpression Biochemical measurements

6 Cell line models; reduce DNA methylation chemically/pharmaceutically 5-aza-2 -deoxycytidine Mechanism of action of nucleoside analogue inhibitors Deoxynucleoside analogues such as 5-aza-2-deoxycytidine (depicted by Z) are converted into the triphosphate inside S-phase cells and are incorporated in place of cytosine into DNA. Once in DNA, the fraudulent bases form covalent bonds with DNA methyltransferases (DNMTs), resulting in the depletion of active enzymes and the demethylation of DNA. Pink circles, methylated CpG; cream circles, unmethylated CpG. Egger G et al. (2004) Epigenetics in human disease and prospects for epigenetic therapy. Nature 429:

7 Cell line models; reduce DNA methylation chemically/pharmaceutically 5-aza-2 -deoxycytidine Western blot of proteins extracted from cell line in which Fas is methylated No expression of Fas detected Fas detected after treating cells with 5-Aza; shows expression was suppressed by DNA methylation Spannhoff A et al (2011) Histone deacetylase inhibitor activity in royal jelly might facilitate caste switching in bees. EMBO reports 12:

8 Cell line models; reduce DNA methylation through transgenic approaches - sirna

9 Cell line models; reduce DNA methylation through transgenic approaches - sirna

10 Cell line models; reduce DNA methylation through transgenic approaches - sirna DNMT Signal of gene expression (e.g. northern blot (or RT-PCR); western blot)?

11 Cell line models; reduce DNA methylation through transgenic approaches DNMT knockout by homologous recombination sirna achieves only partial knockout A gene can be inactivated completely by using the process of homologous recombination to replace it (or a section) with non-functional DNA (if in mouse embryonic stem (ES) cells, these can then be used to generated transgenic mice) Targeting construct DNMT gene sequence Replacement sequence (e.g. Neo R ) DNMT gene on chromosome DNMT gene disrupted by sequence replacement

12 Cell line models; reduce DNA methylation through transgenic approaches DNMT knockout by homologous recombination sirna achieves only partial knockout A gene can be inactivated completely by using the process of homologous recombination to replace it (or a section) with non-functional DNA (if in mouse embryonic stem (ES) cells, these can then be used to generated transgenic mice) Targeting construct DNMT gene sequence Replacement sequence (e.g. Neo R ) DNMT gene on chromosome DNMT gene disrupted by sequence replacement

13 Cell line models; reduce DNA methylation through transgenic approaches DNMT knockout by homologous recombination sirna achieves only partial knockout A gene can be inactivated completely by using the process of homologous recombination to replace it (or a section) with non-functional DNA (if in mouse embryonic stem (ES) cells, these can then be used to generated transgenic mice) DNMT Targeting construct DNMT gene sequence Replacement sequence (e.g. Neo R ) DNMT gene on chromosome DNMT gene disrupted by sequence replacement Signal of gene expression (e.g. northern blot (or RT-PCR); western blot)?

14 Cell line models; increase DNA methylation; transgenic approaches; DNMT overexpression strong promoter (e.g. CMV) DNMT1 gene (cdna) Deliver vector to cells (transfection) Expression vector High level of DNMT1 expression Increase DNA methylation Signal of gene expression (e.g. northern blot (or RT-PCR); western blot)?

15 Cell line models; increase DNA methylation promoter-reporter constructs GENE X Gene promoter to test for effects of methylation Reporter gene (e.g. β-galactosidase or luciferase) Deliver vector to cells (transfection) Promoterreporter plasmid Methylate promoter-reporter plasmid in vitro (SssI DNA methylase) Promoterreporter plasmid Prepare cell extract and measure reporter gene activity Colour change (βgalactosidase) or light emission (luciferase) indicates promoter active Prepare cell extract and measure reporter gene activity If no activity is detected conclude that promoter is inactivated by DNA methylation

16 Use of animal models Mouse DNMT knockout embryo lethal (use homologous recombination in embryonic stem (ES) cells to inactivate gene; for viable knockouts can then generate animals from these ES cells) Technology to achieve conditional knockout (i.e. time or tissue-specific) is welladvanced; reports where DNMTs targeted seem very limited e.g. Endres, M., G. Fan, et al. (2001). Effects of cerebral ischemia in mice lacking DNA methyltransferase 1 in post-mitotic neurons. Neuroreports 12(17): Can reduce DNMT expression but retain sufficient to produce viable mice hypomorphic alleles e.g. Oghamian, S., N. M. Sodir, et al. (2011). Reduction of pancreatic acinar cell tumor multiplicity in Dnmt1 hypomorphic mice. Carcinogenesis 32(6): Administration of 5-aza-2-deoxycytidine to mice is a rather crude approach, and used mainly in context of its use as an anti-tumour agent Transgenic mice that overexpress DNMTs do not appear to have been used extensively. e.g. Samuel, M.S., Lundgren-May T. & Ernst, M. (2007). Identification of putative targets of DNA (cytosine-5) methylation-mediated transcriptional silencing using a novel conditionally active form of DNA methyltransferase 3a. Growth Factors 25(6): Do we need better animal models? e.g. honeybees?

17 Diet-induced epigenetic changes and lifespan in the honey bee Lifespan of a queen bee is 20X longer than that of a worker. All bees are born equal (genetically indistinct). DNA methylation plays an important role in development into the queen. Royal jelly suppresses Dnmt3 expression leading to altered DNA methylation patterns that induce the queen bee phenotype. RNAi-mediated silencing of Dnmt3 induces queen-like traits in workerdestined larvae.

18 Diet-induced epigenetic changes and lifespan in the honey bee

19 Diet-induced epigenetic changes and lifespan in the honey bee

20 Is the honeybee a good model to investigate functional effects of changes in DNA methylation? Advantages? Easy manipulation of DNMT expression by administration of sirna in food DNMT homologues to mammalian enzymes with CpG methylation contrast with flies and worms DNA methylation appears to have same function as in mammals; clustered in promoter regions of silenced genes and at splice sites Advantages for specific areas of research e.g. relationship between DNA methylation and longevity Cheaper than mice Simplified (smaller) genome with fewer sites of DNA methylation Disadvantages? Simplified (smaller) genome with fewer sites of DNA methylation May not present relevant phenotype Relationship between phenotypes/functions in bees and mammals questionable

21 A more targeted approach to manipulating DNA methylation? None of the approaches to manipulating DNA methylation considered can target only specific, defined CpGs within the genome all affect multiple CpGs Biochemical approaches may complement observation of associations between specific DNA methylation differences and phenotypic/functional outcomes and/or manipulation of DNA methylation in cell and animal models to provide evidence of functional effects on gene expression of methylation of specific, individual CpGs e.g. electrophoretic mobility shift analysis (EMSA) A Oligo ZTRE CpG metylated Oligo All CpGs metylated

22 A more targeted approach to manipulating DNA methylation? None of the approaches to manipulating DNA methylation considered can target only specific, defined CpGs within the genome all affect multiple CpGs Biochemical approaches may complement observation of associations between specific DNA methylation differences and phenotypic/functional outcomes and/or manipulation of DNA methylation in cell and animal models to provide evidence of functional effects on gene expression of methylation of specific, individual CpGs e.g. electrophoretic mobility shift analysis (EMSA) B A Oligo ZTRE CpG metylated Oligo All CpGs metylated Caco-2 extract, 150 µm Zn Oligo Oligo ZTRE CpG metylated Oligo All CpGs metylated

23 References Spannhoff A, Kim YK, Raynal NJ, Gharibyan V, Su MB, Zhou YY, Li J, Castellano S, Sbardella G, Issa JP, Bedford MT (2012) Histone deacetylase inhibitor activity in royal jelly might facilitate caste switching in bees. EMBO Reports 12: Egger G, Liang G, Aparicio A, Jones PA (2004) Epigenetics in human disease and prospects for epigenetic therapy. Nature 429: Endres, M., G. Fan, et al. (2001) Effects of cerebral ischemia in mice lacking DNA methyltransferase 1 in post-mitotic neurons. Neuroreports 12(17): Oghamian, S., N. M. Sodir, et al. (2011). Reduction of pancreatic acinar cell tumor multiplicity in Dnmt1 hypomorphic mice. Carcinogenesis 32(6): Samuel, M.S., Lundgren-May T. & Ernst, M. (2007). Identification of putative targets of DNA (cytosine-5) methylation-mediated transcriptional silencing using a novel conditionally active form of DNA methyltransferase 3a. Growth Factors 25(6): Ford D (2012) Honeybees and cell lines as models of DNA methylation and ageing in response to diet. Experimental Gerontology (publication late 2012)