1.EUKARYOTIC GENE REGULATION II Jill Howlin PhD Canceromics Branch Department of Oncology, Lund University Medicon Village http://www.med.lu.se/english/klinvetlund/canceromics/research

2. Lecture structure Eukaryotic Gene Regulation I A Revision of the basics What is a gene, chromosome etc. Transcription mRNA processing & Splicing Translation DNA mutations Eukaryotic Gene Regulation II B Gene Structure and function Structure of typical gene in the genome and its regulatory units How gene structure influences regulation.. A Control of gene expression Producing specificity: Transcription factors Other forms of regulation of gene expression, non-coding RNA, epigenetic regulation: imprinting & methylation etc. B Analysis of Gene Expression & Regulation Analysis of gene expression in the genomics era: transcriptomics RNA-seq Studying gene regulation, DNA-protein interaction 3. Source material: Molecular Biology of the Cell. 5th edition. Alberts et al. The Cell: A Molecular Approach. 2nd edition. Cooper GM. Human Molecular Genetics. 4th edition. Strachan and Read Virtual Cell Animation Collection http://vcell.ndsu.nodak.edu/animations/ (also available as free iphone/ipad app from itunes) Wikipedia http://en.wikipedia.org/ & YouTube http://www.youtube.com ENCODE explorer http://www.nature.com/encode/#/threads 4. Summaryof EukaryoticGeneRegulationI 5. PART IIA control of gene expression 6. Gene Regulation Why do we need gene regulation? All cells carry All the genetic information! - how is cell specificity determined? DNA = = = 7. Gene Expression Specificity of function is determined by regulation of gene expression Gene products will be produced only when & where they are needed A specific transcription factor is the when and where determinant Mediate temporal and spatial control of gene expression Control the rate of gene transcription by influencing RNA polymerase binding to DNA >2000 proteins with DNA binding domains in the genome (10%) protein coding transcripts 8. Transcription Factors General and sequence-specific Transcribed like any other protein coding gene Intracellular & extracellular stimuli (growth factors, hormones, cytokines) Developmental programs & signaling cascades Feedback loop Nuclear localization Protein-protein interaction (co-regulations, chromatin remodelers etc.) 9. Transcriptional Regulation upregulation activation or promotion of the rate of transcription downregulation repression or suppression of the rate of transcription co-activator a protein that works with transcription factors to increase the rate of gene transcription co-repressor a protein that works with transcription factors to decrease the rate of gene transcription co-regulators 10. Regulated transcription 11. Classes of Transcription Factors All have at least two functional domains or regions: Transactivation domain interacts with other proteins DNA-binding domain binds a specific DNA sequence several common protein structural motifs that TF use -Zinc finger proteins -Helix-turn-helix -Leucine zipper proteins -Helix-loop-helix proteins -Nuclear Receptors 12. Nuclear Receptors e.g.:ERalpha,AR nuclear/steroid receptors have a third steroid-binding domain bind ligand Zinc finger proteins HRE 13. HRE -hormone response element short sequence in/near promoter region, 5 to TSS a pair of inverted repeats separated by 3 nucleotides HRE Transcription Factor Consensus Sequence ERE Estrogen receptor AGGTCANNNTGACCT GRE Glucocorticoid receptor AGAACANNNTGTTCT Estrogen receptor Estrogen AGGTCANNNTGACCT TSS 14. Position weight matrices used to depict the DNA binding preferences of transcription factors e.g.: Always G at 3rd position 15. Transcriptional Co-regulators interact with transcription factors to either activate or repress the transcription of specific genes They can be referred to as coactivators or corepressors. They dont bind DNA themselves Co-regulators connect transcription factors with the complex of proteins that carry out transcription Many co-regulators are proteins that modify histones (or recruit proteins that can) to allow the transcription factors access to the underlying DNA sequence 16. Accessingthe DNA roleofchromatininregulatinggeneexpression The structure and function of chromatin is influenced by: 1. Histone modifications 2. DNA modifications 17. Heterochromatin & Euchromatin Packaged chromatin has 2 basic variants: 1. Heterochromatin closed & inactive either constitutive or facultative 2. Euchromatin - open & accessible XX-example in females Specific histone modifications differentiate the main types of chromatin (e.g.: H3k9me2/3) Heterochromatin Feature Transcriptionally active chromatin Transcriptionally inactive chromatin Chromatin conformation Open, extended conformation Highly condensed conformation; particularly apparent in heterochromatin (c&f) 18. Nucleosome structure 147bp DNA wrapped around a complex of histones "beads on a string Histone N- and -C terminal tails protrude from the nucleosome Core of 8 histones (H2A, H2B, H3, H4) X 2 Histone H1 Linker DNA Core DNA Histone tails 19. Histones small (130 aa) basic proteins, positively charged Enzymatic modifications of specific amino acid in the tails can determine nucleosome behavior DNA accessibility Modification Effecting enzymes Reversing enzymes Acetylation (HATs) histone acetyltransferases (HDACs) histone deacetylases Methylation (HMTs) histone methyltransferases Histone demethylases Phosphorylation Histone kinases Histone phosphatases Large families of enzymes Writers Erasers 20. Histone modifying enzymes http://www.actrec.gov.in/histome/The histone Infobase Examples of Writers Examples of Erasers Amino acid Possible modifications Lysine Methylation, Acetylation, Ubiquitination, Arginine Methylation Serine Phosphorylation Threonine Phosphorylation Lysine methyltransferases EZH1/2, SETD1A/B Lysine acetyltransferases NCOA1/3, CBP/P300 Lysine deacetylases HDAC1/2/3..; SIRT1/2/3.. Lysine demethylases KDM1A/2A, JHDM1D, JMJD5 21. Histone deacetylases (HDACs) are negative regulators of transcription Histone acetylases (HATs) are positive regulators of transcription In general + - e.g.: , CBP/P300 e.g.: , HDAC1/2 22. The Histone Code Specific histone modifications determine function H3K4me3 Histone 3 Lysine Position 4 from the N-terminal Modification type Tri-methylated ac: acetylation ph: phosphorylation ub: ubiqutination Reading the notation.. Amino acid Possible modifications Lysine Methylation, Acetylation, Ubiquitination, Arginine Methylation Serine Phosphorylation Threonine Phosphorylation 23. Barth and Imhof ; Trends in Biochemical Sciences Volume 35, Issue 11 2010 618 626c Active gene TSS gene body H3K36me3 is found in actively transcribed gene bodies H3K4me3 found in actively transcribed promoters, particularly just after the transcription start site 24. Barth and Imhof ; Trends in Biochemical Sciences Volume 35, Issue 11 2010 618 - 626 Inactive gene even distribution of silencing modifications H3K9ME2/3, H4K20ME3 H3K27me3 is enriched in the promoter gene body TSS 25. Reading the Histone Code Specific histone modifications act as binding sites for other proteins Readers Families of proteins with the right type of domain recognize particular modifications Bromodomain containing proteins (BRD4, BRD2) recognize acetylated lysines Chromodomain proteins (CHD1, CDY) recognizes methylated residues Examples of Readers 26. UCSC Genome Browser encodehistonetracks 27. DNA modifications DNA can be modified by methylation of adenine and cytosine bases Methylated Base Methylation Sequence C5-methylcytosine (5-mC) CpG C5-hydroxymethylcytosine (5- hmC) CpG, CpHpG1, CpHpH1 H = Adenine, Cytosine, or Thymine "p" in CpG refers to the phosphodiester bond 28. DNA methylation Histonemodifications ++++transcription acetylated Histones/unmethylated cytosines -transcription deacetylated Histones/methylated cytosines 29. Epigenetics Epigenetics - regulation of gene expression that is not at the level of the DNA sequence The so called Histone Code can be considered part of a wider Epigenetic Code, as can DNA methylation 30. Imprinting expressed differently according to the parent of origin always either the version from the mother or father Prader-Willi syndrome : missing gene activity that normally comes from only from the paternal copy Angelman syndrome missing gene activity that normally comes from only the maternal copy Igf2 is imprinted on the chromosome inherited from the mother H19 is imprinted on the paternal chromosome. genes that escape epigenetic reprogramming chromosome 15q partial deletion, 31. Genetic conflict hypothesis Beckwith-Wiedemann Syndrome http://learn.genetics.utah.edu/content/epigenetics/imprinting/index.html 32. Epigenetic Inheritance transmittance of information from one generation to the next without alteration of the primary DNA sequence Various complex cellular mechanisms allow for retention of epigenetic marks Retention of epigenetic marks is seen during chromosomal replication where modifications of new histones and DNA are patterned on those of the old histones and DNA 33. Transgenerational Epigenetic Inheritance From organism to offspring not just cell to cell Can be hard to prove true transgenerational inheritance Intergenerational effect Examples: male-line transgenerational effects Swedish verkalix Study variation in the food supply during the early life of paternal grandparents was related to variation in mortality rate specifically diabetic deaths in their grandchildren Rat liver fibrosis study Liver injury to male rats led to epigenetic changes in their sperm, which transmit increased protection from fibrosis into their male offspring Mother -1st Fetus -2nd Offspring's reproductive cells -3rd Simultaneous exposure to environmental agent Nature Reviews Genetics 14, 228-235 (March 2013) 34. END OF PART IIA 35. PART IIB Analysis of Gene Expression & Regulation 36. Evolution of Genomics Technologies Kahvejian et al., (2007) Nat Biotechnology 26:1125 37. Studying Gene Regulation.inthepost-genomicsera Gene expression profiling /transcriptomics DNA/oligonucleotide microarray based assays RNA sequencing analysis Mapping DNA-protein interactions Chromatin IP followed by microarray based analysis or sequencing ChIP-Chip ChIP-seq 38. Gene expression profiling..microarraytechnology Microarray hybridization technology developed in the late 1990s Hybridization is the process of establishing a non-covalent, sequence-specific interaction two perfectly complementary strands will bind to each other readily Hybridization strong weak Relative gene expression high low upregulation downregulation 39. Results of GEX 1000s Individual genes Hierarchical clustering Control Experimental Red genes are upregulated in the experimental samples green downregulated in the control 40. Gene expression profiling using microarrays has been successfully used to subtype breast tumors based on their gene expression patterns Sorlie et al., 2001, PNAS Individual genes Individual tumor samples 41. Sequencing Sanger (First Generation) Developed by Frederick Sanger and colleagues in 1977 Days to weeks per genome..costly Remain useful for small scale projects & longer reads Method: chain-termination incorporation of radioactive or dye-labeled nucleotides Next-Generation Sequencing (costs less by four orders of magnitude) Hours for a genome @ $10-20k Method: reversible chain-termination - Ilumina system 42. Sanger Sequencing chaintermination Readout capillary gel + A G C TPCR measure the length and color of the resulting individual fragments 43. Next Generation Sequencing www.genomesunzipped.org No individual fragments to measure because the dye termination is reversible DNA is immobilized in dense clusters Massively parallel, sequencing by synthesis: Illumina video clusters of library to be seq 44. Types of NG Sequencing experiments RNA whole transcriptome -total RNA seq mRNA seq (poly-A tailed RNA) specific target-enriched DNA whole genome whole exome specific target-enriched -normal cellular nucleic acid, disease tissue, tumor tissue 45. mRNA-Seq Poly-AdependantRNAanalysisthroughcDNAsequencing TSS, alternative splicing, novel transcripts & gene fusion events 46. total RNA-Seq wholetranscriptome NotPoly-Abasedcapture,rRNA depletedtotalRNA both coding and non- coding RNA species 47. NGS target enrichment (RNA & DNA) DNA : selectively sequence particular coding regions of the genome e.g.: X chromosome, custom selection of oncogenes & tumor suppressors RNA: particular RNA species, e.g.: Kinome all kinases in the genome, custom selection of genes 48. DNA-protein interactions where in the genome does a particular TF bind? or where is a particular histone modification located? ChIP Chromatin Immuoprecipitation 1. DNA & protein complex cross-linked using formaldehyde 2. Bound chromatin is sheared by sonication 3. Antibodies bind either TF or Histone modification & immunoprecipitate the DNA protein complex 4. Cross-linking is reveresed & DNA asssoc. with the TF/histone modification is purified transcription factor X histone modification anti-H3K36me3 antibody anti X antibody identify DNA fragments 49. ChIP-Chip Determine genome-wide DNA binding sequence for a specific TF ChIP-seq DNA fragments identified by hybridization to a microarray oligonucleotide probes from entire genome or selected regions e.g.: promoter regions, specific chromosomes Like GEX, limited by sequences on the microarray DNA fragments of interest are sequenced directly Not limited by known sequences on an array 50. Results of ChIP-seq 51. UCSC genome browser specificTFtracks 52. END