* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download How to measure chromatin modifications
Butyric acid wikipedia , lookup
Exome sequencing wikipedia , lookup
Molecular Inversion Probe wikipedia , lookup
Point mutation wikipedia , lookup
Transformation (genetics) wikipedia , lookup
DNA sequencing wikipedia , lookup
SNP genotyping wikipedia , lookup
Whole genome sequencing wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Molecular cloning wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
DNA supercoil wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Non-coding DNA wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Community fingerprinting wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Genomic library wikipedia , lookup
How to measure chromatin modifications Dr Jelena Mann chromosome What is chromatin? Chromatin DNA Histones Structural RNA This structure is called chromatin Smallest unit of chromatin is a nucleosome 146bp of DNA sequence is wrapped around an octamer of proteins called histones- two copies of H2A, H2B, H3 and H4 and a single linker histone H1 Cross section • Histone tails are subject to a vast number of post-translational modifications, including methylation, acetylation, phosphorylation, ubiquitination, all involved in gene-specific regulation • Furthermore, lysine residues can also be mono-, di- and tri-methylated, representing the stability of the modification • Histone acetylases (HATs), histone deacetylases (HDACs) and histone methyltransferases (HMTs) Histone tail sequences H3 H2A H2B H4 Number of histone post translational modifications is vast Histone methylation MLL1/2, Ash1, CARM1 PRMT1/5 Suv39H1/2, Set1, Smyd3 G9a,Clr4, Riz1 R26 K4 R2 R8 G9a K9 EZH2 R17 R3 Set2 K27 Dot1 K36 K79 Histone ADPribosylation PARP1/2 E3 E115 E3 E16 Histone acetylation CBP,p300,P/CAF,SRC-1,ACTR,Tip60 etc K12 K20 K15 K5 K13 K5 K8 K16 K20 K12 K15 K9 K219 K27 K23 K9 K14 K18 K79 K120 K119 K4 K125 K129 K12 K9 K127 K8 S1 S1 S32 S36 Biotinidase Histone biotinylation Rad6/Bre1 PRC1 Histone ubiqitination S14 S28 S10 T11 Histone phosphorylation T3 Histone methylation MLL1/2, Ash1, CARM1 PRMT1/5 Suv39H1/2, Set1, Smyd3 G9a,Clr4, Riz1 R26 K4 R2 R8 G9a K9 EZH2 R17 R3 Set2 K27 Dot1 K36 K79 Histone ADPribosylation PARP1/2 E3 E115 E3 E16 Histone acetylation CBP,p300,P/CAF,SRC-1,ACTR,Tip60 etc K12 K20 K15 K5 K13 K5 K8 K16 K20 K12 K15 K9 K219 K27 K23 K9 K14 K18 K79 K120 K119 K4 K125 K129 K12 K9 K127 K8 S1 S1 S32 S36 Biotinidase Histone biotinylation Rad6/Bre1 PRC1 Histone ubiqitination S14 S28 S10 T11 Histone phosphorylation T3 What do these histone modifications do? Best studied is histone lysine/arginine methylation Combinations of histone modifications can signal to induce or close down transcription thus achieving cell type and signal specific gene expression How can we find out where particular histone modifications are in the genome? Chromatin immunoprecipitation assay (ChIP) Native ChIP Monococcal nuclease digest Mononucleosomes contain 146bp of DNA sequence This is the limit of resolution Crosslinked ChIP Sonication Average fragment size is ~500bp of DNA sequence Limit of resolution is therefore 1kb Chromatin Immunoprecipitation Assay (ChIP) 1. Crosslink Protein-DNA complexes in situ 2. Isolate nuclei and fragment DNA (sonication or digestion) 3. Immunoprecipitate with antibody 4a. Identify against target nuclear protein and reverse crosslinks protein components of isolated complexes 4b. Identify DNA sequence by PCR, cloning and sequencing Can histone modifications be measured in human studies? Yes, basic method is the same (ChIP), then associated DNA can Be investigated in several ways Isolate cells or sample the tissue Make chromatin Carry out ChIP assay ChIP on chip method The ChIP–chip method can be used to study many of the epigenomic phenomena. The example presented here shows how ChIP–chip can be used to study histone modifications. Modified chromatin is first purified by immunoprecipitating crosslinked chromatin using an antibody that is specific to a particular histone modification (shown in green). DNA is then amplified to obtain sufficient DNA. The colour-labelled ChIP DNA, together with the control DNA prepared from input chromatin and labelled with a different colour, is hybridized to a DNA microarray. The microarray probes can then be mapped to the genome to yield genomic coordinates. Chromatin immunoprecipitation combined with serial analysis of gene expression (ChIP–SAGE). The combination of ChIP experiments with SAGE can be used to profile histone modifications at a genomic scale. The ChIP–SAGE procedure begins with a ChIP step to purify chromatin regions that are associated with a specific histone modification (shown in green), and proceeds as follows. First, crosslinks are reversed, a biotinylated universal linker (UL) is ligated to DNA ends and DNA is bound to streptavidin beads. Then NlaIII, which recognizes CATG, is used to digest DNA and a linker containing the recognition sequence of MmeI is ligated to the cleaved DNA ends. MmeI digestion produces 21–22 bp sequence tags from the immunoprecipitated fragments; the sequence tags are concatenated, cloned into a sequencing vector and sequenced. About 20 to 30 short sequence tags of 21 bp can be generated from each sequencing reaction. The sequence tags can then be mapped to the genome to identify modified regions. Chromatin immunoprecipitation combined with high-throughput sequencing techniques (ChIP–Seq). One of the most exciting recent advances in technologies for studying epigenetic phenomena at a genomic scale relies on the combination of ChIP experiments with highthroughput sequencing. The procedure that is outlined here is specific to the Illumina Genome Analyzer using Solexa technology, although other high-throughput sequencing techniques would also work in principle. The first step is the purification of modified chromatin by immunoprecipitation using an antibody that is specific to a particular histone modification (shown in green). The ChIP DNA ends are repaired and ligated to a pair of adaptors, followed by limited PCR amplification. The DNA molecules are bound to the surface of a flow cell that contains covalently bound oligonucleotides that recognize the adaptor sequences. Clusters of individual DNA molecules are generated by solid-phase PCR and sequencing by synthesis is performed. The resulting sequence reads are mapped to a reference genome to obtain genomic coordinates that correspond to the immunoprecipitated fragments. Comparison of ChIP–chip, ChIP–SAGE and ChIP–Seq Resolution The resolution of ChIP–Seq depends on the size of the chromatin fragments that are used for ChIP (chromatin immunoprecipitation), as well as the depth of sequencing. Using mononucleosomes generated by micrococcal nuclease digestion, the histone modification signals that are detected by ChIP–Seq can be assigned to individual nucleosomes in the genome. The resolution of ChIP–chip depends on both the size of the chromatin fragments that are used for ChIP and the probes on the array. The resolution of ChIP-SAGE (serial analysis of gene expression) depends on how frequently restriction enzyme sites occur in the DNA that has been subject to ChIP. Quantification For ChIP–chip, the quantification depends on the hybridization efficiency of the ChIP DNA molecules to the probes on the array, which can vary dramatically depending on the sequence. No hybridization is required for ChIP–Seq and the ChIP DNA is minimally amplified to generate clusters of molecules that are directly counted by the sequencing procedure. Similar to ChIP–Seq, no hybridization is required for ChIP–SAGE. Much less PCR amplification of the ChIP DNA is required for ChIP–Seq than for ChIP–chip; therefore, ChIP–Seq and ChIP–SAGE are probably more quantitative that ChIP–chip. Cost To achieve nucleosome resolution in mammalian genomes, ChIP–Seq is less expensive than ChIP–chip given the current cost of whole-genome tiling arrays. ChIP–chip might be more cost-effective for profiling of subgenomic regions. ChIP–SAGE is also more expensive than ChIP–Seq because it uses the more expensive traditional sequencing methods. Options ChIP–Seq does not require pre-selection of genomic regions whereas ChIP–chip can only analyse the portion of the genome on a microarray. Although ChIP–SAGE can be used to study entire genomes, it is limited to regions that have recognition sites for the restriction enzyme that is used to cleave the ChIP DNA. If you collect numerous samples of tissues, cells chromatin or even gDNA post ChIP, all of those can be kept long term in -800C freezer in siliconized tubes. Why would you study histones? What evidence is there that histone modifications change with environmental exposure/pressure? Sits in balance between activities of HDACs and HATs Substances that alter HDACs and HATs activities also alter the histone acetylation and with it gene expression Natural inhibitor References HDAC Allyl mercaptan Nian et al. (2008) Amamistatin Fennell and Miller (2007) Apicidin Darkin-Rattray et al. (1996) Azumamide E Maulucci et al. (2007) Caffeic acid Waldecker et al. (2008) Chlamydocin Brosch et al. (1995) Chlorogenic acid Bora-Tatar et al. (2009) Cinnamic acid Bora-Tatar et al. (2009) Coumaric/ hydroxycinnamic acid Waldecker et al. (2008) Curcumin Bora-Tatar et al. (2009) Depudecin Kwon et al. (1998) Diallyl disulfide Lea et al. (1999) Equol Hong et al. (2004) Flavone Bontempo et al. (2007) Genistein Kikuno et al. (2008) Histacin Haggarty et al. (2003) Isothiocyanates Ma et al. (2006) Largazole Ying et al. (2008) Pomiferin Son et al. (2007) Psammaplin Pina et al. (2003) SAHA (Vorinostat) Richon et al. (1998) S-allylmercaptocysteine Lea et al. (2002) Sulforaphane Myzak et al. (2004) Trapoxin Kijima et al. (1993) Ursolic acid Chen et al. (2009) Zerumbone Chung et al. (2008) Natural inhibitor References HAT Allspice Anarcardic acid EGCG Curcumin Gallic acid Garcinol Quercetin Anguinarine Plumbagin Lee et al. (2007) Balasubramanyam et al. (2003), Ghizzoni et al. (2010) Choi et al. (2009a) Balasubramanyam et al. (2004), Marcu et al. (2006) Choi et al. (2009b) Balasubramanyam et al. (2004) Ruiz et al. (2007) Selvi et al. (2009) Ravindra et al. (2009) References Simone Reuter, Subash C. Gupta, Byoungduck Park, Ajay Goel, and Bharat B. Aggarwal “Epigenetic changes induced by curcumin and other natural compounds” Genes Nutr. 2011 May; 6(2): 93–108. Cheung P, Lau P. “Epigenetic regulation by histone methylation and histone variants” Mol Endocrinol. 2005 Mar;19(3):563-73. Epub 2005 Jan 27. Review. Schones DE, Zhao K. “Genome-wide approaches to studying chromatin modifications” Nat Rev Genet. 2008 Mar;9(3):179-91. Kouzarides T. “Chromatin modifications and their function” Cell. 2007 Feb 23;128(4):693705. Review. Turner BM. “Defining an epigenetic code” Nat Cell Biol. 2007 Jan;9(1):2-6.