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![Control
of
transcription
promoters,
enhancers,
repressors..
• Complex
control
• -‐cis
and
-‐trans
factors
including:
regulatory
&
promoter
sequences,
RNA
polymerase,
the
general
transcription
factors
and
gene
specific
activators
and
repressors
• RNA
pol
II:
-‐protein-‐coding
genes
[Pol
I
(rRNA)
and
III
(tRNA
and
some
small
RNAs)]](https://image.slidesharecdn.com/eukaryoticgeneregulationi2014slides-150705174934-lva1-app6891/75/Eukaryotic-Gene-Regulation-I-2014-slides-40-2048.jpg)
Uploaded byJill Howlin
Eukaryotic Gene Regulation I 2014 slides
This document provides an overview of a lecture on eukaryotic gene regulation given by Jill Howlin PhD. The lecture covers topics such as transcription factors, epigenetic regulation through imprinting and methylation, and analysis of gene expression through techniques like transcriptomics and RNA sequencing. Reference materials for the lecture include textbooks on molecular biology, genetics, and human molecular genetics, as well as online resources like the Virtual Cell Animation Collection and PubMed.
Eukaryotic Gene Regulation I 2014 slides
- 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. Lectures • Eukaryotic Gene Regulation I • Eukaryotic Gene Regulation II The Gene, the Genome & Gene Expression Some revision, some new concepts Control of gene expression: Transcription factors Epigenetic regulation: imprinting & methylation Analysis of Gene Expression & Regulation: transcriptomics RNA-‐seq 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 • PubMed http://www.ncbi.nlm.nih.gov/pubmed
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- 5. What is a GENE? discrete segments of DNA (or RNA) that comprise a functional unit of hereditary material • complementary RNA molecule that serves as a template for protein • Genome : 3Gbp DNA, < 20,000 protein coding genes
- 6. Genetics Gregor Mendel published his ‘Experiments in Plant Hybridization’ in 1866 Charles Darwin published ‘Origin of the Species’ in 1859 merged in early 20th century Around 1910 Thomas hunt Morgan demonstrated genes are on chromosomes and are the mechanical basis of heredity The Hershey–Chase experiments 1952 by Alfred Hershey and Martha Chase confirmed that DNA was the genetic material
- 7. DNA deoxyribonucleicacid Sugar-‐phosphate polymer/backbone Base pairs A -‐ T C -‐ G
- 8. 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 Photo 51
- 9. 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
- 10. 23 pairs -‐ 11 autosomes and -‐ sex chromosomes Chromosomes
- 11. Cell Division & Genetic Inheritance NB: Somatic mutations, Germ-‐line mutations
- 12. 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 -‐ Germ line -‐
- 16. transcription • DNA to RNA basic mechanism-‐ DNA helix unwinds Direction of transcription RNA polymerase RNA strand Template DNA strand
- 17. cis-‐acting factors on the same molecule e.g.: promoter RNA Polymerase II synthesizes RNA from genes encoding proteins promoters sequences: -‐TATA box, -‐GC box, -‐CAAT box RNAPOLII requires basal transcription apparatus General transcription factors: -‐TFIIA, TFIIB, TFIID, TFIIF, TFIIH trans-‐acting factors produced elsewhere
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- 20. Capping & Poly-‐A-‐tail 5’ methyl cap: first modification of the mRNA as soon as it emerges • In the nucleus, it binds a protein complex, the CBC (cap-‐binding complex) • Binding proteins and processing enzymes generate the 3’ poly-‐adenylation signal: Poly-‐A tail -‐ Following splicing and cleavage • Poly-‐A-‐binding proteins
- 21. splicing • processed mRNA undergos splicing before translation • removal of the intronic regions • cleavage of the exons • Mature mRNA has 5’ and 3’ UTRs , untranslated regions
- 22. Splice junctions & the Spliceosome Spliceosome Protein–RNA complex (snRNA –snRNP) -‐ small nuclear RNAs -‐ >50 proteins 3 consensus sites: • introns start /end • GT (GU for RNA) • AG (always the same) • An A forms the branch point (can vary) lariat
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- 24. translation • mRNA to protein • DECODED by the ribosome to produce specific polypeptides • initiation, elongation and termination • tRNAs, transfer RNAs: function as adaptors Binds to the codon in the mRNA sequence Always CCA at the 3’ end 70 to 80 nucleotide long tRNA
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- 26. Wobble hypothesis 64 = 43 possible codons 49 different tRNAs 1966 Francis Crick Less spatially confined = non-‐standard base pairing
- 27. Mutations Permanent changes in the DNA sequence spontaneously occurring (mistakes in replication, failure of DNA repair) or induced (radiation, UV, chemical) • point mutations • insertions • deletions • translocation
- 28. • 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/ truncated protein • Missence Single point mutation = different amino acid • Neutral mutation e.g.: AAA to AGA, lysine to argine • Silent mutation No effect
- 29. Mutations & SNPs Heritable disorders e.g.: single mutation , germ cell (rare) SCD sickle cell anaemia: haemoglobin (β-‐globin ) GAG to GTG point mutation (Malaria resistance) Cystic Fibrosis: ΔF508 deletion (phenylalanine) the CFTR gene SNPs…natural variation => 1% of population (common) ~ 90% of variation Coding/noncoding, 100 to 300 bases no effect on cell function
- 30. GWAS genome-‐wide association studies • SNP arrays, NG sequencing • SNPs & Risk of disease
- 31. Types of SNPs…associated with the disease phenotype non-‐coding regions > coding SNPs rather than cause disease may confer differential susceptibility e.g.: Alzheimer's disease and ApoE SNPs
- 32. 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) The ‘gene’ today has an identity crisis old!
- 33. • 2003 (April) completion of Human Genome Project (99% /99.99% accuracy) • 2012 (September) The ENCODE project , based on GENCODE regions of transcription, transcription factor association, chromatin structure and histone modification in the entire genome • 2014 (August) GENCODE v20 built on Grch38 (December 2013) Human Genome assembly & annotation
- 34. A lot fewer genes than originally thought! -‐ no. of protein coding genes continues to fall /lncRNA rises <20,000 protein–coding (<1.5%) 20,026 protein-‐coding (GENCODE v8, March 2011) Non-‐protein-‐coding sequences makes up majority ¾ capable of being transcribed 80% of the components of the human genome now have at least one biochemical function associated with them!?? Human Genome conclusions
- 35. Gene Structure and Function REFERENCE GENOME: GRCH38ASSEMBLY ANNOTATION 5’ – 3’ ANNOTATION 3’ – 5’ 1. 2. What’s a ‘gene’?? 1. Protein-‐coding genes 2. Pseudogenes 3. RNA genes (Sebastian) Non-‐protein coding
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- 37. Pseudogenes They are dysfunctional relatives of known genes in the genome that never become proteins
- 38. Pseudogenes Gene of origin 1 .Reterotransposition mRNA spontaneously reverse transcribed back into DNA and inserted into chromosomal DNA (looks like cDNA structure, exons, no promoter) 2. Gene duplication with subsequent acquisition of deleterious mutations (has introns, exon structure and promoter) 3. Disabled/Unitary Genes A gene disabled by a mutation that does not have a deleterious effect on the organism and gets fixed in the population L-‐gulono-‐γ-‐lactone oxidase (GULO)-‐ biosynthesis of vitamin C (GULOP gene in humans)
- 39. Pseudogenes in Gene Regulation • Pseudogene-‐derived small interfering RNAs (siRNA) Some pseudogenes actually encode siRNAs that regulate the expression of protein-‐coding mRNAs-‐ ‘parent’ genes • An miRNA decoy function e.g.: PTEN • -‐PBL case
- 40. Control of transcription promoters, enhancers, repressors.. • Complex control • -‐cis and -‐trans factors including: regulatory & promoter sequences, RNA polymerase, the general transcription factors and gene specific activators and repressors • RNA pol II: -‐protein-‐coding genes [Pol I (rRNA) and III (tRNA and some small RNAs)]
- 41. • Pol II requires assembly of the general TFs on the promoter in order to initiate transcription • specific gene transcription may also be regulated by activator and repressor proteins binding at near OR distant sites RNA Pol II promoters vary but often include: TATA box GC box CAAT box
- 42. Transcriptional repressors compete with transcriptional activators
- 43. Transcript variation one gene = many mRNAs Mainly due to the fact that: 1 Gene > 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
- 44. Transcript variation …many protein isoforms • Equally however the same final protein sequence can come from slightly different transcripts! As long as CDS not affected
- 45. Role of mRNA processing in gene regulation? • 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
- 46. Influence of mRNA structure Final mRNA checks Translation Initiation factors Poly-‐A binding proteins 5’ cap & CBC Assembly > > export > > translation
- 47. Quality control in cytosol • 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 of STOP Exon junction complexes Correct stop codon Premature stop codon Upf proteins Upf triggers mRNA degradation
- 48. mRNA stability • varies (mins-‐hrs) • Exo/endonuclease, other decay mechanisms • A deadenylase shortens 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 50–150 nt (rich in adenosine and uridine) 3-‐UTRs of mRNAs with a short half-‐life <10% genes, e.g: growth factors
- 49. Final aa differs from the genomic DNA A-‐I deamination adenine > inosine: >100o genes RNA editing Adenosine deaminases acting on RNA (ADARs) C-‐U deamination cytosine>uracil By cytidine deaminase Apoplipoprotein B: lipid metabolism
- 50. Control at the protein level ..lost after translation • Every step required for the process of gene expression can be regulated • Translation is no different • Protein folding is initiated as synthesis proceeds, (Hsp70) • Ubiquitin-‐proteasome system • Abnormally folded proteins can form disease causing protein aggregates
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