1. Gene Regulation in Eukaryotes

2. Outline of Chapter 17 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

3. 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 17.1 a
Fig a

4. 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 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

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
Known genetically as transcription factors
Identified by:
Mapping
Biochemical studies to identify proteins that bind in vitro to cis-acting elements
Figure 17.1 b
Fig b

6. 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, 5.8S, and 18S by processing
Figure 17.2 a
Fig a

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

8. 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 17.2 c
Fig c

9. 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
Figure 17.2 c

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
Figure 17.3
Fig. 17.3

11. 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 17.4 a
Fig a

12. 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

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

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

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

16. 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 17.6
Fig. 17.6

17. 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
Fig a
Figure 17.7 a

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

19. Repressors diminish transcriptional activity
Figure 17.8 a.b
Fig. 17.8

20. Repressors
Reduction of transcriptional activation but do 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

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

22. Specificity of transcription factor can be altered by other molecules in 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 17.9
Fig. 17.9

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

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
Induction of genes required for cell proliferation
Figure 17.10
Fig

25. Gene repression results only when the Max polypeptide is made in the cell
max gene
Fig b
Figure 17
Figure b

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

27. 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 17.12
Fig a

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

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

30. 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

31. Normal chromatin structure slows transcription
/figure a,b
Fig

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

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

34. In mammals hypercondensation is often associated with methylation
Figure 17.14
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

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

36. 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.
Figure a

37. Figure d
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
Methylation can be maintained across generations by methylases that recognize methyl groups on one strand and respond by methylating the opposite strand
Figure c
Fig c

39. RNA splicing helps regulate gene expression
Figure 17.16
Fig

40. Figure b
Fig b

41. Cellular enzymes slowly shorten the poly-A tail. mRNA then degrades.
RNA stability provides a mechanism for controlling the amount of gene product
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.
Histone transcripts receive no poly-A tail
mRNA quickly degrades after S phase of cell cycle

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

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

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

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

46. Table 17.2

47. Table 17.3

48. 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

49. Figure 17.21
Fig

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
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 a unproductive transcript in later development from the PL promoter with a stop codon near the beginning of the transcript

51. 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

52. 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

53. 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

54. Dsx-F and Dsx-M are transcription factors that determine somatic sexual characteristics
Figure 17.23
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
Fig

55. Tra and Tra-2 proteins also help regulate the expression of Fruitless
Figure 17.24
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