1. Gene Regulation in Eukaryotes

2. Outline of Chapter 17 How we use genetics to study gene regulation How we use genetics to study gene regulation Using mutations to identify cis-acting elements and trans-acting proteins Using mutations to identify cis-acting elements and trans-acting proteins How genes are regulated at the initiation of transcription How genes are regulated at the initiation of transcription Three polymerases recognize three classes of promoters Three polymerases recognize three classes of promoters Trans-acting proteins control class II promoters Trans-acting proteins control class II promoters Chromatin structure affects gene expression Chromatin structure affects gene expression Signal transduction systems Signal transduction systems DNA methylation regulates gene expression DNA methylation regulates gene expression How genes are regulated after transcription How genes are regulated after transcription RNA splicing RNA splicing RNA stability RNA stability mRNA editing mRNA editing Translation Translation Posttranslational modification Posttranslational modification A comprehensive example of sex determination in Drosophila A comprehensive example of sex determination in Drosophila

3. Regulatory elements that map near a gene are cis-acting DNA sequences cis-acting elements cis-acting elements Promoter – very close to gene’s initiation site Promoter – very close to gene’s initiation site Enhancer Enhancer can lie far way from gene can lie far way from gene Can be reversed Can be reversed Augment or repress basal levels of transcription Augment or repress basal levels of transcription Fig. 17.1 a

4. Reporter constructs are a tool for studying gene regulation Sequence of DNA containing gene’s postulated regulatory region, but not coding region Sequence of DNA containing gene’s postulated regulatory region, but not coding region Coding region replaced with easily identifiable product such as β-galactosidase (Lac Z) or green fluorescent protein (GFP) Coding region replaced with easily identifiable product such as β-galactosidase (Lac Z) or green fluorescent protein (GFP) Reporter constructs can help identify promoters and enhancers by using in vitro mutagenesis to systematically alter the presumptive regulatory region Reporter constructs can help identify promoters and enhancers by using in vitro mutagenesis to systematically alter the presumptive regulatory region

5. Regulatory elements that map far from a gene are trans-acting DNA sequences because they encode transcription factors Genes that encode proteins that interact directly or indirectly with target genes cis- acting elements Genes that encode proteins that interact directly or indirectly with target genes cis- acting elements Known genetically as transcription factors Known genetically as transcription factors Identified by: Identified by: Mapping Mapping Biochemical studies to identify proteins that bind in vitro to cis- acting elements Biochemical studies to identify proteins that bind in vitro to cis- acting elements Fig. 17.1 b

6. In eukaryotes three RNA polymerases transcribe different sets of genes RNA polymerase I transcribes rRNA RNA polymerase I transcribes rRNA rRNAs are made of tandem repeats on one or more chromosomes rRNAs are made of tandem repeats on one or more chromosomes RNA polymerase I transcribes one primary transcript which is broken down into 28S, 5.8S, and 18S by processing RNA polymerase I transcribes one primary transcript which is broken down into 28S, 5.8S, and 18S by processing Fig. 17.2 a

7. RNA polymerase III transcribes tRNAs and other small RNAs (5S rRNA, snRNAs) RNA polymerase III transcribes tRNAs and other small RNAs (5S rRNA, snRNAs) Fig. 17.2 b

8. RNA polymerase II recognizes cis-acting regulatory regions composed of one promoter and one or more enhancers RNA polymerase II recognizes cis-acting regulatory regions composed of one promoter and one or more enhancers Promoter contains initiation site and TATA box Promoter contains initiation site and TATA box Enhancers are distant from target gene Enhancers are distant from target gene Sometimes called upstream activation sites Sometimes called upstream activation sites Fig. 17.2 c

9. RNA polymerase II transcribes all protein coding genes RNA polymerase II transcribes all protein coding genes Primary transcripts are processed by splicing, a poly A tail is added to the 3’ end, and a 5’ GTP cap is added Primary transcripts are processed by splicing, a poly A tail is added to the 3’ end, and a 5’ GTP cap is added

10. Large enhancer region of Drosophila string gene Fourteenth cell cycle of the fruit fly embryo A variety of enhancer regions ensure that string is turned on at the right time in each mitotic domain and tissue type Fig. 17.3

11. trans-acting proteins control transcription from class II promoters Basal factors bind to the promoter Basal factors bind to the promoter TBP – TATA box binding protein TBP – TATA box binding protein TAF – TBP associated factors TAF – TBP associated factors RNA polymerase II binds to basal factors RNA polymerase II binds to basal factors Fig. 17.4 a

12. Activator proteins Also called transcription factors Also called transcription factors Bind to enhancer DNA in specific ways Bind to enhancer DNA in specific ways Interact with other proteins to activate and increase transcription as much as 100-fold above basal levels Interact with other proteins to activate and increase transcription as much as 100-fold above basal levels Two structural domains mediate these functions Two structural domains mediate these functions DNA-binding domain DNA-binding domain Transcription-activator domain Transcription-activator domain

13. Transcriptional activators bind to specific enhancers at specific times to increase transcriptional levels Transcriptional activators bind to specific enhancers at specific times to increase transcriptional levels Fig. 17.5 a

14. zinc-finger proteins and helix-loop- helix proteins bind to the DNA binding domains of enhancer elements zinc-finger proteins and helix-loop- helix proteins bind to the DNA binding domains of enhancer elements Examples of common transcription factors Fig. 17.5 b

15. Some proteins affect transcription with out binding to DNA Coactivator – binds to and affects activator protein which binds to DNA Coactivator – binds to and affects activator protein which binds to DNA Enhancerosome – multimeric complex of proteins Enhancerosome – multimeric complex of proteins Activators Activators Coactivators Coactivators Repressors Repressors Corepressors Corepressors

16. Localization of activator domains using recombinant DNA constructs Fusion constructs from three parts of gene encoding an activator protein Fusion constructs from three parts of gene encoding an activator protein Reporter gene can only be transcribed if activator domain is present in the fusion construct Reporter gene can only be transcribed if activator domain is present in the fusion construct Part B contains activation domain, but not part A or C Part B contains activation domain, but not part A or C Fig. 17.6

17. Most eukaryotic activators must form dimers to function Eukaryotic transcription factor protein structure Eukaryotic transcription factor protein structure Homomers – multimeric proteins composed of identical subunits Homomers – multimeric proteins composed of identical subunits Heteromers – multimeric proteins composed of nonidentical subunits Heteromers – multimeric proteins composed of nonidentical subunits Fig. 17.7 a

18. Leucine zipper – a common activator protein with dimerization domains Fig. 17.7 b

19. Repressors diminish transcriptional activity Fig. 17.8

20. Repressors Reduction of transcriptional activation but do not affect basal level of transcription Reduction of transcriptional activation but do not affect basal level of transcription Activator-repressor competition Activator-repressor competition Quenching (corepressors) Quenching (corepressors) Some repressors stop basal level of transcription Some repressors stop basal level of transcription Binding directly to promoter Binding directly to promoter Bind to DNA sequences farther from promoter, contact basal factor complex at promoter by bending DNA causing a loop where RNA polymerase can not access the promoter Bind to DNA sequences farther from promoter, contact basal factor complex at promoter by bending DNA causing a loop where RNA polymerase can not access the promoter

21. Transcription factors may act as activators or repressors or have no affect Action of transcription factor depends on Action of transcription factor depends on Cell type Cell type Gene it is regulating Gene it is regulating

22. Specificity of transcription factor can be altered by other molecules in cell yeast  2 repressor – determines mating type yeast  2 repressor – determines mating type Haploid –  2 factor silences the set of “a” genes Haploid –  2 factor silences the set of “a” genes Diploid –  2 factor dimerizes with a1 factor and silences haploid-specific genes Diploid –  2 factor dimerizes with a1 factor and silences haploid-specific genes Fig. 17.9

23. Myc-Max system is a regulatory mechanism for switching between activation and repression Myc polypeptide has an activation domain Myc polypeptide has an activation domain Max polypeptide does not have an activation domain Max polypeptide does not have an activation domain Fig. 17.10

24. Myc-Max system is a regulatory mechanism for switching between activation and repression As soon as a cell expresses the myc gene, the Max-Max homodimers convert to Myc-Max heterodimers that bind to the enhancers As soon as a cell expresses the myc gene, the Max-Max homodimers convert to Myc-Max heterodimers that bind to the enhancers Induction of genes required for cell proliferation Induction of genes required for cell proliferation Fig. 17.10

25. Gene repression results only when the Max polypeptide is made in the cell max gene Fig. 17.10 b

26. Gene activation occurs when both Myc and Max are made in cell Fig. 17.10

27. The locus control region is a cis-acting regulatory sequence that operates sequentially Human  -globin gene cluster contains five genes that can all be regulated by a distant LCR (locus control region) Human  -globin gene cluster contains five genes that can all be regulated by a distant LCR (locus control region) Fig. 17.12 a

28. Proof that cis-acting factor such as LCR is needed for activation of  -globin gene Fig. 17.12 b

29. One mechanism of activation that brings LCR into contact with distant globin genes may be DNA looping Fig. 17.12 c

30. Other mechanisms of gene regulation Chromatin structure Chromatin structure Slows transcription Slows transcription Hypercondensation stops transcription Hypercondensation stops transcription Genomic imprinting Genomic imprinting Silences transcription selectively if inherited from one parent Silences transcription selectively if inherited from one parent Some genes are regulated after transcription Some genes are regulated after transcription RNA splicing can regulate expression RNA splicing can regulate expression RNA stability controls amount of gene product RNA stability controls amount of gene product mRNA editing can affect biological properties of protein mRNA editing can affect biological properties of protein Noncoding sequences in mRNA can modulate translation Noncoding sequences in mRNA can modulate translation Protein modification after translation can control gene function Protein modification after translation can control gene function

31. Normal chromatin structure slows transcription Fig. 17.13

32. Remodeling of chromatin mediates the activation of transcription Fig. 17.13

33. Hypercondensation over chromatin domains causes transcriptional silencing. This is achieved by the methylation of cytosine residues Fig. 17.14

34. In mammals hypercondensation is often associated with methylation It is possible to determine the methylation state of DNA using restriction enzymes that recognize the same sequence, but are differentially sensitive to methylation It is possible to determine the methylation state of DNA using restriction enzymes that recognize the same sequence, but are differentially sensitive to methylation Fig. 17.14

35. Genomic imprinting results from chromosomal events that selectively silence genes inherited from one parent 1980s, in vitro fertilization experiments in mice demonstrated pronuclei from two females could not produce a viable embryos 1980s, in vitro fertilization experiments in mice demonstrated pronuclei from two females could not produce a viable embryos

36. Experiments with transmission of Ig f 2 deletion showed mice inheriting deletion from male were small. Mice inheriting deletion from female were normal. Experiments with transmission of Ig f 2 deletion showed mice inheriting deletion from male were small. Mice inheriting deletion from female were normal. Figure 15.15 a

37. H19 promoter is methylated during spermatogenesis and thus the H19 promoter is not available to the enhancer and is not expressed H19 promoter is methylated during spermatogenesis and thus the H19 promoter is not available to the enhancer and is not expressed

38. Epigenetic effect – whatever silences the maternal or paternal gene is not encoded in the DNA. The factor is outside the gene, but is heritable Epigenetic effect – whatever silences the maternal or paternal gene is not encoded in the DNA. The factor is outside the gene, but is heritable Methylation can be maintained across generations by methylases that recognize methyl groups on one strand and respond by methylating the opposite strand Methylation can be maintained across generations by methylases that recognize methyl groups on one strand and respond by methylating the opposite strand Fig. 15.15 c

39. RNA splicing helps regulate gene expression Fig. 17.16

40. Fig. 17.16 b

41. RNA stability provides a mechanism for controlling the amount of gene product Cellular enzymes slowly shorten the poly-A tail. mRNA then degrades. Cellular enzymes slowly shorten the poly-A tail. mRNA then degrades. Length of poly-A tails of mRNAs affects the speed at which mRNAs are degraded after they leave the nucleus. Length of poly-A tails of mRNAs affects the speed at which mRNAs are degraded after they leave the nucleus. Histone transcripts receive no poly-A tail Histone transcripts receive no poly-A tail mRNA quickly degrades after S phase of cell cycle mRNA quickly degrades after S phase of cell cycle

42. Specialized example of regulation through RNA stability Note also the untranslated sequences that help modulate their translation Fig. 17.17

43. mRNA editing can regulate the function of protein products – e.g., AMPA receptor gene in mammals Fig. 17.18

44. Protein modifications after translation provide a final level of control over gene function Ubiquitination targets proteins for degredation Ubiquitination targets proteins for degredation Ubiquitin – small, highly conserved protein. Ubiquitin – small, highly conserved protein. Covalently attaches to other proteins Covalently attaches to other proteins Ubiquitinized proteins are marked for degredation by proteosomes Ubiquitinized proteins are marked for degredation by proteosomes Fig. 17.19 a

45. Sex determination in Drosophila A comprehensive example of gene regulation Sex specific traits in Drosophila Fig. 17.20

48. The X:A ratio regulates expression of the Sex lethal (sxl) gene Key factors of sex determination Key factors of sex determination Helix-loop-helix proteins encoded by genes on the autosomes Helix-loop-helix proteins encoded by genes on the autosomes Denominator elements Denominator elements Helix-loop-helix proteins encoded by genes on the X chromosome Helix-loop-helix proteins encoded by genes on the X chromosome Numerator elements – monitor the X:A ratio through formation of homodimers or heterodimers Numerator elements – monitor the X:A ratio through formation of homodimers or heterodimers Sisterless-A and sisterless-B Sisterless-A and sisterless-B

49. Fig. 17.21

50. Hypothesis to explain why flies with more numerator homodimers transcribe Sxl early in development Numerator subunit homodimers may function as transcription factors that turn on Sxl Numerator subunit homodimers may function as transcription factors that turn on Sxl Females Females Some numerator subunits remain unbound by denominator elements Some numerator subunits remain unbound by denominator elements Free numerator elements act as transcription factors at P e promoter early in development Free numerator elements act as transcription factors at P e promoter early in development Males Males Carry half as many X-encoded numerator subunits Carry half as many X-encoded numerator subunits All numerator proteins are bound by abundant denominator elements All numerator proteins are bound by abundant denominator elements Pe promoter is not turned on Pe promoter is not turned on The Sxl protein expressed early in development in females regulates its own later expression through RNA splicing The Sxl protein expressed early in development in females regulates its own later expression through RNA splicing Females Females Sxl protein produced early in development catalyzes the synthesis of more of itself through RNA splicing of the P L transcript Sxl protein produced early in development catalyzes the synthesis of more of itself through RNA splicing of the P L transcript Males Males No Sxl transcript in early development results in a unproductive transcript in later development from the P L promoter with a stop codon near the beginning of the transcript No Sxl transcript in early development results in a unproductive transcript in later development from the P L promoter with a stop codon near the beginning of the transcript

51. Effects of Sxl mutations Recessive Sxl mutations making gene nonfunctional Recessive Sxl mutations making gene nonfunctional Females – lethal Females – lethal Absence of Sxl allows expression of dosage compensation genes on X chromosome Absence of Sxl allows expression of dosage compensation genes on X chromosome Increase transcription of X-linked genes is lethal Increase transcription of X-linked genes is lethal Males Males No Sxl expression No Sxl expression No affect on phenotype No affect on phenotype Dominant Sxl mutations that allow expression even in XY embryos Dominant Sxl mutations that allow expression even in XY embryos Females Females No affect because they normally produce the protein No affect because they normally produce the protein Males Males Repression of genes used in dosage compensation Repression of genes used in dosage compensation No hypertranscription of X-linked genes and do not have enough X-linked gene product to survive No hypertranscription of X-linked genes and do not have enough X-linked gene product to survive

52. Sxl triggers a cascade of splicing Sxl influences splicing of RNAs in other genes Sxl influences splicing of RNAs in other genes e.g., transformer (tra) e.g., transformer (tra) Presence of Sxl produces functional protein Presence of Sxl produces functional protein Absence of Sxl results in nonfunctional protein Absence of Sxl results in nonfunctional protein Fig. 17.22

53. Cascade of splicing continues Cascade of splicing continues e.g., doublesex (dsx) e.g., doublesex (dsx) Tra protein synthesized in females along with Tra2 protein (produced in males and females) influences splicing of dsx Tra protein synthesized in females along with Tra2 protein (produced in males and females) influences splicing of dsx Females - Produces female specific Dsx-F protein Females - Produces female specific Dsx-F protein Males – No Tra protein and splicing of Dsx produces Dsx-M protein Males – No Tra protein and splicing of Dsx produces Dsx-M protein Fig. 17.22

54. Dsx-F and Dsx-M are transcription factors that determine somatic sexual characteristics Alternative forms of Dsx bind to YP1 enhancer, but have opposite effects of expression on YP1 gene Alternative forms of Dsx bind to YP1 enhancer, but have opposite effects of expression on YP1 gene Dsx-F is a transcriptional activator Dsx-F is a transcriptional activator Dsx-M is a transcriptional repressor Dsx-M is a transcriptional repressor Fig. 17.23

55. Tra and Tra-2 proteins also help regulate the expression of Fruitless Primary fru mRNA transcript made in both sexes Primary fru mRNA transcript made in both sexes Presence of tra protein in females causes alternative splicing encoding fru-F Presence of tra protein in females causes alternative splicing encoding fru-F Absence of tra protein in males produces fru-M Absence of tra protein in males produces fru-M Fig. 17.24