1. Genetics: From Genes to Genomes
Powerpoint to accompany
Genetics: From Genes to Genomes
Third Edition
Hartwell ● Hood ● Goldberg ● Reynolds ● Silver ● Veres
Chapter 18
Prepared by Malcolm Schug
University of North Carolina Greensboro
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2. Gene Regulation in Eukaryotes
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3. Outline of Chapter 18 How we use genetics to study gene regulation
Using mutations to identify cis-acting elements and trans-acting proteins
How genes are regulated at the initiation of transcription
Three polymerases recognize three classes of promoters.
Trans-acting proteins control class II promoters.
Chromatin structure affects gene expression.
Signal transduction systems
DNA methylation regulates gene expression.
How genes are regulated after transcription
RNA splicing
RNA stability
mRNA editing
Translation
Posttranslational modification
A comprehensive example of sex determination in Drosophila
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4. Regulatory elements that map near a gene are cis-acting DNA sequences.
cis-acting elements
Promoter – very close to gene’s initiation site
Enhancer
Can lie far way from gene
Can be reversed
Augment or repress basel levels of transcription
Figure 18.1 a
Fig a
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5. Reporter constructs are a tool for studying gene regulation.
Sequence of DNA containing gene’s postulated regulatory region, but not coding region
Coding region replaced with easily identifiable product such as b-galactosidase or green fluorescent protein
Reporter constructs can help identify promoters and enhancers by using invitro mutatgenesis to systematically alter the presumptive regulatory region.
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6. Regulatory elements that map far from a gene are trans-acting DNA sequences.
Genes that encode proteins that interact directly or indirectly with target genes cis-acting elements
Known genetically as transcription factors
Identified by:
Mapping
Biochemical studies to identify proteins that bind invitro to cis-acting elements
Figure 18.1 b
Fig b
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7. In eukaryotes three RNA polymerases transcribe different sets of genes.
RNA polymerase I transcribes rRNA.
rRNAs are made of tandem repeats on one or more chromosomes.
RNA polymerase I transcribes one primary transcript which is broken down into 28s and 5.8s by processing.
Figure 18.2 a
Fig a
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8. RNA polymerase III transcribes tRNAs and other small RNAs (5s rRNA, snRNAs).
Figure 18.2 b
Fig b
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9. RNA polymerase II recognizes cis-acting regulatory regions composed of one promoter and one or more enhancers.
Promoter contains initiation site and TATA box.
Enhancers are distant from target gene.
Sometimes called upstream activation sites
Figure 18.2 c
Fig c
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10. RNA polymerase II transcribes all protein coding genes.
Figure 18.2 c
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.
Fig c
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11. 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.
Figure 18.3
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Fig. 18.3

12. Trans-acting proteins control transcription from class II promoters.
Basal factors bind to the promoter.
TBP – TATA box binding protein
TAF – TBP associated factors
RNA polymerase II binds to basal factors.
Figure 18.4 a
Fig a
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13. Activator proteins Also called transcription factors
Bind to enhancer DNA in specific ways
Interact with other proteins to activate and increase transcription as much as 100-fold above basal levels
Two structural domains mediate these functions:
DNA-binding domain
Transcription-activator domain
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14. Transcriptional activators bind to specific enhancers at specific times to increase transcriptional levels.
Figure 18.5 a
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Fig a

15. Examples of common transcription factors
Zinc-finger proteins and helix-loop-helix proteins bind to the DNA binding domains of enhancer elements.
Figure 18.5 b
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Fig b

16. Some proteins affect transcription without binding to DNA.
Coactivator – binds to and affects activator protein which binds to DNA
Enhancerosome – multimeric complex of proteins
Activators
Coactivators
Repressors
Corepressors
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17. Localization of activator domains using recombinant DNA constructs
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.
Part B contains activation domain, but not part A or C.
Figure 18.6
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Fig. 18.6

18. Most eukaryotic activators must form dimers to function.
Eukaryotic transcription factor protein structure
Homomers – multimeric proteins composed of identical subunits
Heteromers – multimeric proteins composed of nonidentical subunits
Figure 18.7 a
Fig a
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19. Leucine zipper – a common activator protein with dimerization domains
Figure 18.7 b
Fig b
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20. Repressors diminish transcriptional activity
Figure 18.8 a.b
Fig. 18.8
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21. Repressors
Reduces transcriptional activation but does not affect basal level of transcription
Activator-repressor competition
Quenching (corepressors)
Some repressors stop basal level of transcription.
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
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22. Transcription factors may act as activators or repressors or have no affect.
Action of transcription factor depends on:
Cell type
Gene it is regulating
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23. Specificity of transcription factor can be altered by other molecules in the cell.
yeast a2 repressor – determines mating type
Haploid – a2 factor silences the set of “a” genes
Diploid – a2 factor dimerizes with a1 factor and silences haploid-specific genes
Figure 18.9
Fig. 18.9
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24. Myc-Max system is a regulatory mechanism for switching between activation and repression.
Figure 18.10
Myc polypeptide has an activation domain.
Max polypeptide does not have an activation domain.
Fig a
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25. Myc-Max system is a regulatory mechanism for switching between activation and repression.
As soon as a cell expresses the myc gene, the Myc-Myc homodimers convert to Myc-Max heterodymers that bind to the enhancers.
Induction of genes required for cell proliferation
Figure 18.10
Fig b
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26. Gene expression results only when the Max polypeptide is made in the cell.
max gene
Figure 17
Figure b
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Fig c

27. Gene activation occurs when both Myc and Max are made in the cell.
Figure 18.10
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Fig d

28. The locus control region is a cis-acting regulatory sequence that operates sequentially.
Human b-globin gene cluster contains five genes that can all be regulated by a distant LCR (locus control region).
Figure 18.12
Fig a
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29. Proof that cis-acting factor such as LCR is needed for activation of b-globin gene
Figure b
Fig b
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30. One mechanism of activation that brings LCR into contact with distant globin genes may be DNA looping.
Figure c
Fig c
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31. Other mechanisms of gene regulation
Chromatin structure
Slows transcription
Hypercondensation stops transcription.
Genomic imprinting
Silences transcription selectively if inherited from one parent
Some genes are regulated after transcription.
RNA splicing can regulate expression.
RNA stability controls amount of gene product.
mRNA editing can affect biological properties of protein.
Noncoding sequences in mRNA can modulate translation.
Protein modification after translation can control gene function.
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32. Normal chromatin structure slows transcription
/figure a,b
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Fig a-b

33. Remodeling of chromatin mediates the activation of transcription.
Figure c,d
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Fig c-d

34. Hypercondensation over chromatin domains causes transcriptional silencing.
Figure 18.14
Fig a
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35. In mammals hypercondensation is often associated with methylation.
Figure 18.14
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 b
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36. Genomic imprinting results from chromosomal events that selectively silence genes inherited from one parent.
1980s, invitro fertilization experiments demonstrated pronuclei from two females could not produce viable embryos.
Fig a
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37. Figure a
Experiments with transmission of Ig f 2 deletion showed mice inheriting deletion from male were small. Mice inheriting deletion from female were normal.
Fig a
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38. Figure d
H19 promoter is methylated during spermatogenesis and thus the H19 promoter is not available to the enhancer and is not expressed.
Fig d
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39. Figure c
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.
Fig c
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40. RNA splicing helps regulate gene expression.
Figure 18.16
Fig a
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41. Figure b
Fig b
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42. Micro-RNAs mediate RNA interference.
– micro-RNAs identified and characterized
RNA interference – trans-acting single stranded micro-RNAs that regulate eukaryotic gene expression
¼ from introns of protein coding transcripts
¾ from products of primary transcripts devoid of ORFs
Number of miRNAs may exceed the number of protein coding genes.
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43. Micro-RNAs mediate RNA interference.
miRNAs distinguished by ribonucleotide-long segments of reverse sequence complimentarity – will snap back spontaneously into hairpin stem-loop structures.
Involved in posttranscriptional regulation and trans acting transcription factors
Provides a method for development of powerful new RNA-based therapies for treatment of diseases.
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44. Examples of primary transcripts from micro-RNA-containing genes
Figure 18.17
Fig
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45. Micro-RNA processing and modes of action
Immediately after transcription, pri-miRNAs are recognized by Drosha which crops
out pre-miRNA stem loops from larger RNA.
Pre-miRNAs undergo active transport from nucleus to cytoplasm where they are recognized by Dicer.
Dicer reduces the pre-miRNA into a short-lived miRNA:miRNA duplex which is released and picked up by RISC.
Figure 18.18
Fig a
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46. Two modes of RNA interference
If miRNA and its target mRNA contain perfectly complementary sequences, miRISC cleaves the mRNA. RNase rapidly degrades cleavage product.
If miRNA and its target mRNA have only partial complementarity, cleavage does not occur. miRISC remains bound to its target and represses its movement across ribosomes.
Figure 18.8b
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47. Protein modifications after translation provide a final level of control over gene function.
Ubiquitination targets proteins for degradation.
Ubiquitin – small, highly conserved protein
Covalently attaches to other proteins
Ubiquitinized proteins are marked for degradation by proteosomes
Figure 18.19
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Fig a

48. Sex specific traits in Drosophila
Sex determination in Drosophila A comprehensive example of gene regulation
Sex specific traits in Drosophila
Figure 18.20
Fig
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49. Table 18.2
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50. Table 18.3
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51. The X:A ratio regulates expression of the Sex lethal (sxl) gene.
Key factors of sex determination:
Helix-loop-helix proteins encoded by genes on the autosomes
Denominator elements
Helix-loop-helix proteins encoded by genes on the X chromosome
Numerator elements – monitor the X:A ratio through formation of homodimers or heterodimers
sisterless-A and sisterless-B
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52. Figure 18.21
Fig
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53. 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.
Females
Some numerator subunits remain unbound by denominator elements.
Free numerator elements act as transcription factors at Pe promoter early in development.
Males
Carry half as many X-encoded numerator subunits
All numerator proteins are bound by abundant denominator elements.
Pe promoter is not turned on.
The Sxl protein expressed early in development in females regulates its own later expression through RNA splicing.
Sxl protein produced early in development catalyzes the synthesis of more of itself through RNA splicing of the PL transcript.
No Sxl transcript in early development results in an unproductive transcript in later development from the PL promoter with a stop codon near the beginning of the transcript.
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54. Effects of Sxl mutations
Recessive Sxl mutations making gene nonfunctional
Females – lethal
Absence of Sxl allows expression of dosage compensation genes on X chromosome.
Increase transcription of X-linked genes is lethal.
Males
No Sxl expression
No affect on phenotype
Dominant Sxl mutations that allow expression even in XY embryos
Females
No affect because they normally produce the protein
Repression of genes used in dosage compensation
No hypertranscription of X-linked genes and do not have enough X-linked gene product to survive
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55. Sxl triggers a cascade of splicing.
Sxl influences splicing of RNAs in other genes.
e.g., transformer (tra)
Presence of Sxl produces functional protein.
Absence of Sxl results in nonfunctional protein.
Figure a
Fig a
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56. Cascade of splicing continues
e.g., doublesex (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
Males – No Tra protein and splicing of Dsx produces Dsx-M protein
Figure b
Fig b
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57. Dsx-F and Dsx-M are transcription factors that determine somatic sexual characteristics.
Figure 18.23
Alternative forms of Dsx bind to YP1 enhancer, but have opposite effects of expression on YP1 gene.
Dsx-F is a transcriptional activator.
Dsx-M is a transcriptional repressor.
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Fig

58. Tra and Tra-2 proteins also help regulate the expression of Fruitless.
Figure 18.24
Primary fru mRNA transcript made in both sexes
Presence of tra protein in females causes alternative splicing encoding fru-F.
Absence of tra protein in males produces fru-M.
Fig
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