1. EUKARYOTIC GENE REGULATION I 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 Collectio http://vcell.ndsu.nodak.edu/animations/ (also available as free iphone/ipad app from itunes) Wikipedia http://en.wikipedia.org/ ENCODE explorer http://www.nature.com/encode/#/threads 4. BIM12 5. PART IA revision 6. the basics.. What is a gene? How many genes do we have? Cell Nucleus and the Chromosome What is DNA? The double helix DNA-RNA-PROTEIN -the central dogma What is a mutation? DNA replication and inheritance 7. What is a GENE? discrete segments of DNA (or RNA) that comprise a functional unit of hereditary material make a complementary RNA molecule that serves as a template for making a protein the total complement of genes in an organism or cell is known as its Genome the human genome approx. 3Gbp DNA and around 20,000 protein coding genes 8. A brief History of Genetics Gregor Mendel (father of modern genetics) published his Experiments in Plant Hybridization in 1866 Charles Darwin published Origin of the Species in 1859 the theories of both men were understood together and merged in early 20th century Around 1910 Thomas hunt Morgan genes are on chromosomes and are the mechanical basis of heredity The HersheyChase experiments 1952 by Alfred Hershey and Martha Chase confirmed that DNA was the genetic material 9. The Double Helix the double helix allowed scientists to understand how DNA was replicated 1962 Nobel Prize James Watson, Francis Crick & Maurice Wilkins. Rosalind Franklin generated the X-ray diffraction images used to formulate Watson and Cricks hypothesis Photo 51 10. DNAdeoxyribonucleicacid Sugar-phosphate polymer/backbone Base pairs A - T C - G 11. DNA Replication DNA polymerase can only extend an existing DNA strand, it cannot begin the synthesis helicase unwinds DNA at the replication fork, the DNA ahead is forced to rotate enzymes that solve the physical problems of twisted & coiling DNA 100 to 200 nucleotides long on the lagging strand the new DNA is made in installments 12. The Cell Nucleus DNA (and RNA) is referred to as nucleic acid because its found in the nucleus of a cell Site of protein synthesis Where ribosomes are produced 13. DNA Packaging 30nm 1nm per turn, 3.6nm pitch Nucleosome 147bp DNA wrapped around a complex of 8 core histones (H2A, H2B, H3, H4) Histone H1 The nucleosomes fold to a 30nm fiber.. that form loops 300nm in length ..the 300nm fiber is compressed and folded further to produce a 250nm wide fiber 700nm Tight coiling of the 250nm fiber produces the chromatid of a chromosome 1400nm Chromsome 14. Humans have 23 pair of chromosomes and (22 autosomes and one pair of sex chromosomes) Chromosomes 15. Cell Division & Genetic Inheritance 16. Chromosome disorders Abnormal number aneuploidy Down Syndrome 3 x chromosome 21 Klinefelter syndrome 46/47, XXY, or XXY syndrome Abnormal structure deletions, duplications, translocations Jacobsen Syndrome 11q deletion disorder Most chromosome abnormalities occur as an accident in the egg or sperm 17. Somatic error Gross chromosomal abnormalities thousands of clustered chromosomal rearrangements single catastrophic event of fragmentation and reassembly cancer BIMA30 2011-09-14/20 Chromothripsis shattering 18. Expression of Genetic Information Chromosomal DNA functions in two ways: - to allow replication of the genetic material (cell division & inheritance) - allow expression of genetic information in an organism the central dogma 19. Double stranded DNA Single stranded RNA Amino acids (protein) The Central dogma 20. RNA ribonucleicacid Similar to DNA but contains the sugar ribose instead of deoxyribose RNA also contains the base uracil (U) in place of thymine (T) RNA molecules are less stable than DNA and are typically single-stranded 21. The Genetic Code DNA and RNA is organized in sets of three nucleotides, known as codons DNA (base pairing A-T,G-C) while RNA (A-U,G-C) Each codon correspond to a specific amino acid or to a signal (e.g.: START, STOP) AUG, or the amino acid Met is the typical START signal Three STOP codons tell the translation machinery that the end of the gene has been reached. 64 (43) possible codons (4,4,4) & 20 standard amino acids genetic code is redundant 22. Deciphering the code.. 5 3 N C coding strand /sense strand/Crick strand template strand/antisense strand / Watson strand -NH2 positively charged amino group -COOH Negatively charged carboxylgroup 23. RNA codons 24. transcription DNA to RNA basic mechanism- DNA helix unwinds Direction of transcription RNA polymerase RNA strand Template DNA strand 25. cis-acting factors onthesamemoleculee.g.:promoter RNA Polymerase II is the enzyme that synthesizes RNA from genes encoding proteins It recognizes and requires certain regulatory sequences in a genes promoter such as: -TATA box -GC box -CAAT box 26. RNA Polymerase II requires the assembly of the basal transcription apparatus to initiate transcription This consists of general transcription factors: -TFIIA -TFIIB -TFIID -TFIIF -TFIIH trans-acting factors producedelsewhere 27. transcription animation 28. mRNA processing 29. Capping & Poly-A-tail RNA capping, 5 methyl cap, is the first modification of the mRNA as soon as it emerges from the polymerase In the nucleus, the mRNA cap binds a protein complex, the CBC (cap-binding complex) Following splicing and cleavage of the mRNA, RNA binding proteins and processing enzymes generate the 3 poly-adenylation signal, Poly-A tail Poly-A-binding proteins 30. splicing following the 5 capping, the processed mRNA must still undergo splicing before translation into protein can occur removal of the intronic regions cleavage of the exons mature spliced mRNA also has 5 and 3 UTRs , untranslated regions 31. Splice junctions & the Spliceosome splicing machinery or Spliceosome large ProteinRNA complex (snRNA snRNP) made up of small nuclear RNAs and >50 proteins 3 consensus sequence sites: Most introns start with a GT (GU for RNA) and end with AG (always the same) An A forms the branch point of the lariat produced by splicing (can vary) lariat 32. splicing animation 33. translation mRNA to protein mature mRNA is DECODED by the ribosome to produce a specific polypeptide (amino acid chain) proceeds through initiation, elongation and termination tRNAs, transfer RNAs: function as adaptors between the mRNA template and the amino acids 34. tRNA 70 to 80 nucleotide long Always CCA at the 3 end Binds to the codon in the mRNA sequence 35. translation animation 36. Wobble hypothesis There are 64 = 43 possible codons 49 different tRNAs In 1966, Francis Crick proposed the Wobble hypothesis the 5' base on the anticodon, which binds to the 3' base on the mRNA, is not as spatially confined as the other two bases, and can have non-standard base pairing 37. Mutations Permanent changes in the DNA sequence Mutations can be : spontaneously occurring (mistakes in replication, failure of DNA repair) or induced (by radiation such as UV, or by chemical exposure to agents that bind or react with DNA) Types of mutations include: point mutations insertions deletions translocation 38. The effect on the resulting protein can vary and and includes the following consequences: Frame shift mutation : a disruption in the reading frame The sun was hot but the old man did not get his hat T hes unw ash otb utt heo ldm and idn otg eth ish at Or Th esu nwa sho tbu tth eol dma ndi dno tge thi sha t Nonsence mutation premature STOP or truncated protein Missence Single point mutation resulting in different amino acid Neutral mutation e.g.: AAA to AGA, lysine to argine Silent mutation No effect on final protein 39. Mutations causing disease BIMA30 2011-09-14/20 heritable genetic disorders (single mutation) SCD sickle cell anaemia: a point mutation in the -globin chain of haemoglobin, causing the hydrophilic amino acid glutamic acid to be replaced with the hydrophobic amino acid valine, GAG to GTG (Malaria resistance) Cystic Fibrosis: most commonly (F508) a deletion that results in a loss of phenylalanine in the protein encoded by the CFTR gene. 40. SNPsnaturalvariation => 1% of population accounts for 90% of all human genetic variation every 100 to 300 bases coding (gene) and noncoding regions many SNPs have no effect on cell function GWAS BIMA30 2011-09-14/20 41. GWAS genome-wideassociationstudies SNP arrays, NG sequencing focus on the associations between SNP and a particular disease influences the risk of disease occurring in an individual BIMA30 2011-09-14/20 42. BIMA30 2011-09-14/20 Types of SNPsassociatedwiththediseasephenotype SNPs usually occur in non-coding regions more frequently than in coding regions SNPs rather than cause disease may confer differential susceptibility e.g.: Alzheimer's disease and ApoE SNPs 43. END OF PART IA 44. PART IB gene structure and function 45. Common misconceptions: The only purpose of a gene is to encode a protein One gene encodes one mRNA and gives rise to one specific protein DNA that is not a gene is considered junk DNA "Biologists should not deceive themselves with the thought that some new class of biological molecules, of comparable importance to proteins, remains to be discovered. This seems highly unlikely." (Francis Crick, 1958) 46. 2003 (14th April) completion of Human Genome Project (99% /99.99% accuracy) 2012 (5th September) The ENCODE project regions of transcription, transcription factor association, chromatin structure and histone modification in the entire genome 80% of the components of the human genome now have at least one biochemical function associated with them! BIMA30 2011-09-14/20 Human Genome annotation 47. Size approx. 3Gbp GENCODE consortium aims to identify all protein-coding, long non-coding RNA and short RNA genes: >51,096 genes: 20,026 protein-coding and 31,070 non- coding (GENCODE version 8, March 2011) A lot fewer genes than originally thought! ~20,000 proteincoding ( 1 promoter & > 1 splicing pattern At least half of all genes have 2 or more promoters Alternative splicing, as well as creating different protein isoforms, can also generate different 5 and 3 UTRs 59. Transcript variation manyproteinisoforms Equally however the same final protein sequence can come from slightly different transcripts! As long as CDS not affected 60. Roleof mRNA processingin generegulation? seems wasteful exon-intron arrangement facilitates new genetic recombination evidence in protein domains variation - several protein variants can be produced from one gene The 5, 3 modifications of mRNA also have a role in stability, transport and recognition 61. Influenceof mRNA structurein generegulation Export ready (correctly spliced and polyadenylated) mRNA is assembled in nucleus It moves through the nuclear pore complex as a curved fiber, 5 cap first Final mRNA checks Translation Initiation factors 5 cap distinguish mRNA from other types (pol I and III) 5 cap binds CBC (cap-binding complex) the Poly-A-tail is bound by Poly-A binding proteins splicing junctions are marked by EJCs exon junction complexes processing, exp ort & initiation of translation 62. Quality control In the cytosol, the 5cap and Poly-A-tail are recognized by the translation initiation machinery EJCs serve to label the mRNA Nonsense-mediated decay (NMD) rids cells of mRNAs with premature termination codons hUpf complex triggers NMD in the cytoplasm when recognized downstream Exon junction complexes Correct stop codon Premature stop codon Upf proteins Upf triggers mRNA degradation 63. mRNA stability & degradation The stability of mRNAs is very variable A deadenylase enzyme acts to shorten the Poly-A tail in the cytoplasm Decapping enzymes - uncapped mRNA is rapidly degraded by exonucleases 3 binding of miRNA mediated degradation RNAi mediated degradation AREs can stimulate Poly-A tail removal AU-rich elements 50150 nt (rich in adenosine and uridine) 3-UTRs of many mRNAs with a short half- life 100o genes RNA editing Adenosine deaminases acting on RNA (ADARs) C-U deamination of cytosine to produce uracil Apoplipoprotein B: each isoform has a different role in lipid metabolism 65. Control at the protein level ..lostaftertranslation In principle, every step required for the process of gene expression can be regulated The steps following translation are no different Protein folding is initiated as synthesis proceeds, often with the help of molecular chaperones (such as Hsp70) The ubiquitin-proteasome system efficiently destroys incorrectly or incompletely folded proteins and acts to limit the life of normal or correctly folded proteins Abnormally folded proteins can form disease causing protein aggregates 66. Summary 67. END OF PART IB