Regulation of Eukaryotic Gene Expression

1. Regulation of Eukaryotic Gene Expression Chapter 18 What regulates the precise pattern of gene expression in the developing wing of a fly embryo?

2. Concept 18.2: Eukaryotic gene expression is regulated at many stages • All organisms must regulate which genes are expressed at any given time • In multicellular organisms regulation of gene expression is essential for cell specialization

3. Differential Gene Expression • Almost all the cells in an organism are genetically identical with the exception of gametes • Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome • RBC from an epithelial cell to a nerve cells • Abnormalities in gene expression can lead to diseases including cancer • Gene expression is regulated at many stages

4. Signal Figure 18.6 NUCLEUS Chromatin Chromatin modification:DNA unpacking involvinghistone acetylation andDNA demethylation DNA Gene availablefor transcription Gene Transcription Exon RNA Primary transcript Intron RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Translation Degradationof mRNA Polypeptide Protein processing, suchas cleavage and chemical modification Active protein Degradationof protein Transport to cellulardestination Cellular function (suchas enzymatic activity,structural support)

5. Signal Figure 18.6a NUCLEUS Chromatin Chromatin modification:DNA unpacking involvinghistone acetylation andDNA demethylation DNA Gene availablefor transcription Gene Transcription Exon RNA Primary transcript Intron RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm CYTOPLASM

6. CYTOPLASM Figure 18.6b mRNA in cytoplasm Translation Degradationof mRNA Polypeptide Protein processing, suchas cleavage and chemical modification Active protein Degradationof protein Transport to cellulardestination Cellular function (suchas enzymatic activity,structural support)

7. Regulation of Chromatin Structure • The genes within highly packed heterochromatin (DNA + proteins) are usually not expressed • Euchromatin is found in prokaryotes. No coiling of DNA involved • Chemical modifications to histonesand DNA of chromatin influence both chromatin structure and gene expression • Acetylation • Methylation • Phosporylation

8. Histone Modifications For the Transcription of a gene on DNA to occur, histones must be modified to uncoil the DNA to allow for transcription • In histoneacetylation, acetyl groups (-COCH3) are attached to positively charged lysinesin histone tails • Histone tails no longer will bind to adjacent nucleosomes • This loosens chromatin structure, thereby promotes the initiation of transcription • Histoneacetylation enzymes may promote the initiation of transcription. Kosigrin animation Mc Graw Hill

9. Figure 18.7 Histone tails DNA double helix Amino acidsavailablefor chemicalmodification Nucleosome(end view) (a) Histone tails protrude outward from a nucleosome Acetylated histones Unacetylated histones (b) Acetylation of histone tails promotes loose chromatinstructure that permits transcription

10. The addition of methyl groups – CH3(methylation) to histone tails promotes condensing of chromatin. • So this turns off genes • The addition of phosphate groups (phosphorylation) next to a methylated amino acid canloosen chromatin • Turns on genes

11. The histone code hypothesisproposes that specific combinations of modifications, as well as the order in which they occur, help determine chromatin configuration and influence transcription

12. DNA Methylation • DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species • DNA methylation can cause long-term inactivation of genes in cellular differentiation • Such as in X inactivation when the X chromosomes become methylated • Unexpressed genes are more heavily methylated than active genes. • In Genomic Imprinting, methylation regulates expression of either the maternalor paternal alleles of certain genes at the start of development • PraderWilli and Angelman syndrome is determine by which parent donates the gene

13. Epigenetic Inheritance • Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells • The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance • Ghosts in Your Genes

15. Control & Organization of a Typical Eukaryotic Gene • Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery • Associated with most eukaryotic genes are multiple control elements, segments of noncoding DNA that serve as binding sites for transcription factors that help regulate transcription • Control elements and the transcription factors they bind are critical to the precise regulation of gene expression in different cell types

16. Figure 18.8-1 Proximalcontrolelements Enhancer(distal controlelements) Poly-Asignalsequence Transcriptionterminationregion Transcriptionstart site Exon Exon Intron Intron Exon DNA Upstream Downstream Promoter

17. Figure 18.8-2 Enhancer(distal controlelements) Poly-Asignalsequence Proximalcontrolelements Transcriptionterminationregion Transcriptionstart site Exon Intron Intron Exon Exon DNA Upstream Downstream Promoter Poly-Asignal Transcription Exon Intron Intron Exon Exon Primary RNAtranscript(pre-mRNA) Cleaved3 end ofprimarytranscript 5

18. Figure 18.8-3 Enhancer(distal controlelements) Proximalcontrolelements Poly-Asignalsequence Transcriptionterminationregion Transcriptionstart site Exon Intron Intron Exon Exon DNA Upstream Downstream Promoter Poly-Asignal Transcription Exon Intron Intron Exon Exon Primary RNAtranscript(pre-mRNA) Cleaved3 end ofprimarytranscript 5 RNA processing Intron RNA Coding segment mRNA 3 G P P P AAA AAA Startcodon Stopcodon Poly-Atail 5 UTR 5 Cap 3 UTR

19. The Roles of Transcription Factors • To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors • General transcription factors are essential for the transcription of all protein-coding genes • In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors

20. Enhancers and Specific Transcription Factors • Proximal control elements are located close to the promoter • Distal control elements, groupings of which are called enhancers, may be far away from a gene or even located in an intron

21. An activator is a protein that binds to an enhancerand stimulates transcription of a gene • Activators have two domains, one that binds to DNA and a second that activates transcription • Bound activators facilitate a sequence of protein-protein interactions that result in transcription of a given gene McGraw Hill animation

22. Some transcription factors function as repressors, inhibiting expression of a particular geneby a variety of methods • Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription

23. Figure 18.10-1 Promoter Activators Gene DNA Distal controlelement Enhancer TATA box

24. Figure 18.10-2 Promoter Activators Gene DNA Distal controlelement TATA box Enhancer Generaltranscriptionfactors DNA-bendingprotein Group of mediator proteins

25. Figure 18.10-3 Promoter Activators Gene DNA Distal controlelement TATA box Enhancer Generaltranscriptionfactors DNA-bendingprotein Group of mediator proteins RNApolymerase II RNApolymerase II Transcriptioninitiation complex RNA synthesis

26. Enhancer Promoter Figure 18.11 Controlelements Albumin gene Crystallingene LENS CELLNUCLEUS LIVER CELLNUCLEUS Availableactivators Availableactivators Albumin genenot expressed Albumin geneexpressed Crystallin genenot expressed Crystallin geneexpressed (a) Liver cell (b) Lens cell

27. Figure 18.11a Enhancer Promoter LIVER CELLNUCLEUS Controlelements Albumin gene Availableactivators Crystallingene Albumin geneexpressed Crystallin genenot expressed (a) Liver cell

28. Figure 18.11b Enhancer Promoter LENS CELLNUCLEUS Controlelements Albumin gene Availableactivators Crystallingene Albumin genenot expressed Crystallin geneexpressed (b) Lens cell

29. Coordinately Controlled Genes in Eukaryotes • Unlike the genes of a prokaryotic operon, each of the co-expressed eukaryotic genes has a promoter and control elements • These genes can be scattered over different chromosomes, but each has the same combination of control elements • Copies of the activators recognize specific control elements and promote simultaneous transcription of the genes

30. Mechanisms of Post-Transcriptional Regulation • Transcription alone does not account for gene expression • Regulatory mechanisms can operate at various stages after transcription • Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes

31. RNA Processing • In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

32. Figure 18.13 Exons DNA 4 1 3 5 2 Troponin T gene PrimaryRNAtranscript 3 5 2 4 1 RNA splicing or mRNA 3 5 5 2 2 4 1 1

33. Initiation of Translation • The initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to sequences or structures of the mRNA • Alternatively, translation of all mRNAs in a cell may be regulated simultaneously • For example, translation initiation factors are simultaneously activated in an egg following fertilization

34. Protein Processing and Degradation • After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control (phosporylation) • Proteasomesare giant protein complexes that bind protein molecules and degrade them

35. Figure 18.14 Proteasomeand ubiquitinto be recycled Ubiquitin Proteasome Proteinfragments(peptides) Ubiquitinatedprotein Protein tobe degraded Protein enteringa proteasome

36. Figure 18.UN04a Chromatin modification Transcription • Regulation of transcription initiation:DNA control elements in enhancers bindspecific transcription factors. • Genes in highly compactedchromatin are generally nottranscribed. • Histoneacetylation seemsto loosen chromatin structure,enhancing transcription. Bending of the DNA enables activators tocontact proteins at the promoter, initiatingtranscription. • DNA methylation generallyreduces transcription. • Coordinate regulation: Enhancer forlens-specific genes Enhancer forliver-specific genes Chromatin modification Transcription RNA processing • Alternative RNA splicing: RNA processing Primary RNAtranscript Translation mRNAdegradation mRNA or Protein processingand degradation

37. Figure 18.UN04b Chromatin modification Transcription RNA processing Translation mRNAdegradation Protein processingand degradation Translation • Initiation of translation can be controlledvia regulation of initiation factors. mRNA degradation • Each mRNA has a characteristic life span,determined in part bysequences in the 5 and3 UTRs. Protein processing and degradation • Protein processing anddegradation by proteasomesare subject to regulation.

38. Cancer results from genetic changes that affect cell cycle control • The gene regulation systems that go wrong during cancer are the very same systems involved in embryonic development • Types of Genes Associated with Cancer • Cancer can be caused by mutations to genes that regulate cell growth and division • Tumor viruses can cause cancer in animals including humans

39. Oncogenesare cancer-causing genes • Proto-oncogenesare the corresponding normal cellular genes that are responsible for normal cell growth and division • Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle

40. Figure 18.23 Proto-oncogene DNA Translocation ortransposition: genemoved to new locus,under new controls Point mutation: Gene amplification:multiple copies ofthe gene within a controlelement withinthe gene New promoter Oncogene Oncogene Normal growth-stimulatingprotein in excess Normal growth-stimulatingprotein in excess Normal growth-stimulatingprotein inexcess Hyperactive ordegradation-resistantprotein

41. Proto-oncogenes can be converted to oncogenes by • Movement of DNA within the genome: if it ends up near an active promoter, transcription may increase • Amplification of a proto-oncogene: increases the number of copies of the gene • Point mutations in the proto-oncogene or its control elements: cause an increase in gene expression

42. Tumor-Suppressor Genes • Tumor-suppressor genes help prevent uncontrolled cell growth • Mutations that decrease protein products of tumor-suppressor genes may contribute to cancer onset • Tumor-suppressor proteins • Repair damaged DNA • Control cell adhesion • Inhibit the cell cycle in the cell-signaling pathway

43. Interference with Normal Cell-Signaling Pathways • Mutations in the rasproto-oncogene and p53 tumor-suppressor geneare common in human cancers • Mutations in the rasgene can lead to production of a hyperactive Ras protein and increased cell division

44. 3 2 1 2 3 4 5 1 Figure 18.24 MUTATION Growthfactor Protein kinases Hyperactive Ras protein(product of oncogene)issues signals on itsown. Ras MUTATION GTP G protein Defective or missingtranscription factor,such asp53, cannotactivatetranscription. Ras P P GTP Activeformof p53 UVlight P P P P Protein kinases(phosphorylation cascade) Receptor DNA damagein genome DNA NUCLEUS Transcriptionfactor (activator) Protein thatinhibitsthe cell cycle DNA Gene expression (b) Cell cycle–inhibiting pathway Protein that stimulatesthe cell cycle EFFECTS OF MUTATIONS Proteinoverexpressed Protein absent (a) Cell cycle–stimulating pathway Cell cycleoverstimulated Increased celldivision Cell cycle notinhibited (c) Effects of mutations

45. 1 5 4 3 2 MUTATION Growthfactor Figure 18.24a Hyperactive Ras protein(product of oncogene)issues signals on itsown. Ras G protein GTP Ras P P GTP P P P P Protein kinases(phosphorylation cascade) Receptor NUCLEUS Transcriptionfactor (activator) DNA Gene expression Protein that stimulatesthe cell cycle (a) Cell cycle–stimulating pathway

46. 2 1 3 Figure 18.24b Protein kinases MUTATION Defective or missingtranscription factor,such asp53, cannotactivatetranscription. Activeformof p53 UVlight DNA damagein genome DNA Protein thatinhibitsthe cell cycle (b) Cell cycle–inhibiting pathway

47. Suppression of the cell cycle can be important in the case of damage to a cell’s DNA; p53 prevents a cell from passing on mutations due to DNA damage • Mutations in the p53 gene prevent suppression of the cell cycle

48. Figure 18.24c EFFECTS OF MUTATIONS Proteinoverexpressed Protein absent Cell cycleoverstimulated Increased celldivision Cell cycle notinhibited (c) Effects of mutations

49. The Multistep Model of Cancer Development • Multiple mutations are generally needed for full-fledged cancer; thus the incidence increases with age • At the DNA level, a cancerous cell is usually characterized by at least one active oncogene and the mutation of several tumor-suppressor genes

50. 1 2 4 3 5 Figure 18.25 Colon Lossof tumor-suppressorgene APC(or other) Lossof tumor-suppressorgene p53 Activationof rasoncogene Additionalmutations Lossof tumor-suppressorgene DCC Colon wall Small benigngrowth(polyp) Largerbenign growth(adenoma) Normal colonepithelial cells Malignanttumor(carcinoma)