1. How Genes Are Regulated
Gene Regulation in Eukaryotes
Overview of Eukaryotic Gene Regulation
Control of Transcription Initiation
Regulation After Transcription
A Comprehensive Example: Sex Determination in Drosophila
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2. Overview of eukaryotic gene regulation
Eukaryotes use complex sets of interactions
Regulated interactions of large networks of genes
Each gene has multiple points of regulation
Themes of gene regulation in eukaryotes:
Environmental adaptation in unicellular eukaryotes
Maintenance of homeostasis in multicellular eukaryotes
Genes are turned on or off in the right place and time
Differentiation and precise positioning of tissues and organs during embryonic development

3. Compared to prokaryotes, eukaryotes have additional levels of complexity for controlling gene expression
Eukaryotic genomes are larger than prokaryotic genomes
Chromatin structure in eukaryotes makes DNA unavailable to transcription machinery
Additional RNA processing events occur in eukaryotes
In eukaryotes, transcription takes place in the nucleus and translation takes place in the cytoplasm

4. Key regulatory differences between eukaryotes and prokaryotes
Attenuation is a regulatory feature found throughout Archaea and Bacteria causing premature termination of transcription.[1] Attenuators are 5'-cis acting regulatory regions which fold into one of two alternative RNA structures which determine the success of transcription.[2] The folding is modulated by a sensing mechanism producing either a Rho-independent terminator, resulting in interrupted transcription and a non-functional RNA product; or an anti-terminator structure, resulting in a functional RNA transcript. 
Table 16.1

5. Multiple steps where production of the final gene product can be regulated in eukaryotes
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16
there are many more steps
in the process leading to an active product beyond transcription initiation.
Fig. 16.1

6. Control of transcription initiation
Three types of RNA polymerases in eukaryotes
RNA pol I – transcribes rRNA genes
RNA pol II – transcribes all protein-coding genes (mRNAs) and micro- RNAs
RNA pol III – transcribes tRNA genes and some small regulatory RNAs

7. RNA polymerase II transcription
RNA pol II catalyzes synthesis of the primary transcript, which is complementary to the template strand of the gene
Most RNA pol II transcripts undergo further processing to generate mature mRNA
RNA splicing – removes introns
Addition of 5' GTP cap – protects RNA from degradation
Cleavage of 3' end and addition of 3' polyA tail

8. cis-acting elements: promoters and enhancers
Promoters – usually directly adjacent to the gene
Include transcription initiation site
Often have TATA box:
Allow basal level of transcription
Enhancers – can be far away from gene
Augment or repress the basal level of transcription
Fig. 16.2

9. Use of reporter genes to identify promoters and enhancers in eukaryotes
Examples of reporter genes used in eukaryotes
lacZ gene (Ch 15), blue color when X-gal used
Gene for "green fluorescent protein" (GFP)
Mutations in regulatory regions can be engineered and tested for effects on expression
Fig. 16.3

10. trans-acting factors interact with cis-acting elements to control transcription initiation
Activators and repressors bind to enhancers
Direct effects of transcription factors:
Through binding to DNA
Indirect effect of transcription factors:
Through protein-protein interactions
Basal Factors bind to promoter
Fig. 16.4

11. Use of reporter genes to identify trans-acting factors in transcriptional regulation
Identifying trans-acting factors.
(a) Trans-acting factors bind to regulatory regions (enhancers) to increase
transcription.
(b) A trans-acting mutation that reduces transcription identifies a regulatory protein.
Fig. 16.5

12. Basal transcription factors
Basal transcription factors assist the binding of RNA pol II to promoters (see Fig 16.6)
Key components of basal factor complex:
TATA box-binding protein (TBP)
Bind to TATA box
First of several proteins to assemble at promoter
TBP-associated factors (TAFs)
Bind to TBP assembled at TATA box
RNA pol II associates with basal complex and initiates basal level of transcription

13. Basal factors bind to promoters of all protein-encoding genes
Ordered pathway of assembly at promoter: 1. TBP binds to TATA box 2. TAFs bind to TBP 3. RNA pol II binds to TAFs
Fig. 16.6

14. Activators are transcription factors that bind to enhancers
Activators are responsible for much of the variation in levels of transcription of different genes
Increase levels of transcription by interacting directly or indirectly with basal factors at the promoter
3-dimensional complex of proteins and DNA (Fig. 16.7)
Mechanisms of activator effects on transcription
Stimulate recruitment of basal factors and RNA pol II to promoters
Stimulate activity of basal factors already assembled on promoters
Facilitate changes in chromatin structure
Although similar sets of basal factors bind to all the promoters of the tens of thousands of genes in the eukaryotic
genome, a cell can transcribe different genes into widely varying amounts of mRNA. This enormous range of transcriptional
regulation occurs through the binding of different transcription factors to enhancer elements associated
with different genes.

15. Binding of activators to enhancers increases transcriptional levels
Low level transcription occurs when only basal factors are bound to promoter
When basal factors and activators are bound to DNA, rate of transcription increases
Fig. 16.7

16. Domains within activators
Activator proteins have two functional domains
Sequence-specific DNA binding domain (Fig 16.8)
Binds to enhancer
Transcription-activator domain
Interacts with other transcriptional regulatory proteins
Some activators have a third domain (Fig 16.9)
Responds to environmental signals
Example - steroid hormone receptors

17. DNA-binding domains of activator proteins
Interacts with major groove of DNA
Specific amino acids have high-affinity binding to specific nucleotide sequence
The three best-characterized motifs:
Helix-loop-helix (HLH)
Helix-turn-helix (HTH)
Zinc finger
Fig. 16.8

18. Steroid hormone receptors are activators only in the presence of specific hormones
Steroid hormones don't bind to DNA but are coactivators of steroid hormone receptors
In the absence of hormone, these receptors cannot bind to DNA and so cannot activate transcription
In the presence of hormone, these receptors bind to enhancers for specific genes and activate their expression
Fig. 16.9

19. Coactivators
Proteins and other molecules that play a role in transcriptional activation without binding directly to DNA are called coactivators.
The hormone component of a DNA bound hormone- receptor activation complex is one example of a coactivator.

20. Many activators must form dimers to function
Homodimers: multimeric proteins made of identical subunits
Heterodimers: multimeric proteins made of nonidentical subunits
Examples - Fos and Jun, both have leucine zippers
Fos forms heterodimers with Jun, but cannot form homodimers
Jun can form homodimers
Jun-Jun and Jun-Fos dimers recognizes different sequences
Jun-Jun and Jun-Fos dimers bind to the same enhancer sequences, but have different affinities
Fig

21. Dimerization domains make up another class of transcription factor domains
Dimerization domains are specialized for polypeptide-polypeptide interactions
Leucine zippers are a common dimerization motif in eukaryotes
Amino acid sequence twirls into an a helix with leucines protruding at regular intervals
Heterodimers greatly increases the
number of potential regulatory transcription complexes
Fig

22. Repressor proteins suppress transcription initiation through different mechanisms
Some repressors have no effect on basal transcription but suppress the action of activators
Compete with activator for the same enhancer (Fig 16.12a) OR
Block access of activator to an enhancer, quenching, (Fig 16.12b)
Some repressors eliminate virtually all basal transcription from a promoter
Block RNA pol II access to promoter
Bind to sequences close to promoter or distant from promoter

23. Repressor proteins that act through competition with an activator protein
Repressor binds to the same enhancer sequence as the activator
Has no effect on the basal transcription level
Fig a

24. Repressor proteins that act through quenching an activator protein
Quenchers bind to the activator but do not bind to DNA
Type I: Repressor blocks the DNA-binding domain
Type II: Repressor blocks the activation domain
Fig b

25. Corepressors
These blocked activators still bind to their enhancers, but once bound, they are unable to carry out activation.
Quenching polypeptides that operate in this manner are termed corepressors.
Just like coactivators, corepressors associate indirectly with enhancers through their interaction with DNA- binding proteins.

26. Some repressors eliminate virtually all basal transcription
by binding to DNA sequences very close to the promoter and thereby blocking RNA polymerase’s access to the promoter. OR
bind to DNA sequences farther from the promoter and then reach over and contact the basal factor complex at the promoter,
allow contact between the repressor and the basal factor complex.

27. The same transcription factor can play different roles in different cells
Example: a2 repressor in yeast
Determines mating type by binding to enhancers of certain genes
In a haploids and a/a diploids, a2 binds to enhancers of a- specific genes but not to enhancers of haploid-specific genes
In a/a diploids, binding specificity of a2 is altered so that it can bind to enhancers of haploid-specific genes
Fig

28. The Myc-Max mechanism can activate or repress transcription
Myc − identified as an oncogene
Regulates transcription of genes involved in cell proliferation
Doesn't have DNA binding activity on its own
Max − identified through its binding to Myc
Max/Max homodimers and Myc/Max heterodimers bind to the same enhancers
Max/Max represses transcription
Myc/Max activates transcription

29. Comparative structures of Myc and Max
Myc/Myc homodimers cannot form
Max/Max homodimers and Myc/Max heterodimers can form
Max/Max homodimers bind DNA but cannot act as activators
Fig

30. Dimer structure and subunit concentrations of Myc and Max affect activation or repression
Max is constitutively expressed but Myc expression is tightly regulated
Max/Max acts as a repressor when Myc is not expressed
Fig a

31. Dimer structure and subunit concentrations of Myc and Max affect activation or repression
When Myc is present, Myc/Max heterodimers form and activate transcription
Myc - Max affinity is higher than Max - Max affinity
Fig b

32. Complex regulatory regions enable fine-tuning of gene expression
Each gene can have many regulatory proteins
In humans, ~2000 genes encode transcriptional regulatory proteins
Each regulatory protein can act on many genes
Each regulatory region can have dozens of enhancers

33. Not just a matter of turning genes on and off
It also entails fine-tuning the precise level of transcription
higher or lower in different cells,
higher or lower in cells of the same tissue but at different
stages of development.
It also includes mechanisms that allow each cell to modify its program of gene activity in response to constantly changing signals from its neighbors.

34. Complex regulatory regions enable fine-tuning of gene expression
Enhanceosome – multimeric complex of proteins and other small molecules that associate with an enhancer
Enhancers can be bound by activators and repressors with varying affinities
Different sets of cofactors and corepressors compete for binding to activators and repressors
These changes lead to the assembly of an altered
enhanceosome, which recalibrates gene activity.
In short, a large, exquisitely controlled machinery determines the level of primary transcript produced.

35. Control of the string gene in drosophila
The enhancer regions of some genes are very large, containing multiple elements that make possible the fine-tuned regulation of a gene.
Those genes in multicellular eukaryotes that must be expressed in many different tissues.

36. Different enhancers for the string gene in Drosophila are used in different cell types
Cells in different parts of Drosophila embryos go through the 14th mitosis at different times
The string gene product activates the 14th mitosis
Different activators for string are expressed at different times in different tissues
Fig

37. Chromatin structure and epigenetic effects
Chromatin structure can affect transcription
Nucleosomes can sequester promoters and make them inaccessible to RNA polymerase and transcription factors
Histone modification and DNA methylation
Chromatin remodeling and hypercondensation
Epigenetic changes – changes in chromatin structure that are inherited from one generation to the next
Altered chromatin structure can cause changes in gene expression and therefore phenotypic changes in a cell or an organism.
DNA sequence is not altered

38. Chromatin reduces transcription
(a) DNA molecules containing a promoter and an associated gene can be purified away from chromatin proteins in vitro. The addition of basal factors and RNA polymerase to this purifi ed DNA induces high levels of transcription.
(b) Within the eukaryotic nucleus, DNA is present
within chromatin. Promoter regions are generally sequestered within the nucleosome and only rarely bind to basal factors and RNA
polymerase. Thus, the chromatin structure maintains basal transcription at very low levels.
Fig

39. Effects of chromatin structure on transcription: Histone modification and DNA methylation
N-terminal tails of histones H3 and H4 can be modified
Methylation, acetylation, phosphorylation, and ubiquitination
Can affect nucleosome interaction with other nucleosomes and with regulatory proteins
Can affect higher-order chromatin structure
the differentiation of pluripotent stem cells into specific tissue lineages, development
DNA methylation occurs at C5 of cytosine in a CpG ? dinucleotide
Associated with gene silencing
CpG is shorthand for 5'—C—phosphate—G—3' , that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides together in DNA.

40. Determining methylation state of DNA using two restriction enzymes and Southern blotting
HpaII and MspI have the same recognition sequence (CCGG), but different sensitivity to DNA methylation
HpaII doesn't cut if 2nd C is methylated
Methylation doesn't affect MspI cutting
A determination of the methylation status of a DNA region can be made using a pair of restriction enzymes that both recognize the same base
sequence, with one being able to digest methylated DNA, while the other can’t.
In this example, the restriction site is CCGG, and the enzymes are MspI, which can digest both methylated and unmethylated sites, and HpaII, which cannot digest methylated sites. If a methylated site is present between two unmethylated sites, HpaII digestion will leave a larger fragment than MspI. After electrophoresis and Southern blot analysis with a probe that hybridizes to a sequence on one side of the methylated site, there will be a clearly observable difference in band size.
Fig

41. Chromatin remodeling can expose the promoter
Nucleosomes can be repositioned of removed by chromatin remodeling complexes
After remodeling, DNA at promoters and enhancers becomes more accessible to transcription factors
Can be assayed using DNase digestion (DNase hypersensitive sites, DH sites)
DH sites show up at the 5’ ends of genes that are either undergoing transcription or are being prepared for transcription in a later step of cellular differentiation
Fig

42. The SWl-SNF remodeling complex
One of best-studied remodeling complexes in yeast
Uses energy from ATP hydrolysis to alter nucleosome positioning
chromatin decompaction gives basal factors much greater access to promoter regions, and consequently, transcription rapidly accelerates
In Drosophila
Human cells
Fig

43. Hypercondensation of chromatin results in silencing of transcription
Examples of heterochromatin – inactive X chromosome, centromeres, telomeres
Heterochromatin has methylation of CpG dinucleotides and methylation of lysine in histone H3 tails
Fig

44. Gene silencing by the SIR complex in yeast
Gene for a2 repressor is at the MAT (mating type) locus
HML and HMR are copies of MAT locus but are silenced by SIR complex
SIR mutations cause sterility because HML and HMR genes not silenced and both a- and a- specific genes are expressed
They allow the simultaneous expression of a and α information. The resulting cells, which behave as diploids, do not mate.
Fig

45. Genomic imprinting results from transcriptional silencing
Mendal states that the parental origin of an allele not affect its function in the F 1 generation ???? NOT CASE ALWAYS!!
Genomic imprinting – expression pattern of a gene depends on whether it was inherited from the mother or father
Occurs with some genes of mammals
Epigenetic effect (no change in DNA sequence)
Paternally imprinted gene is transcriptionally silenced if it was transmitted from the father
Maternal allele is expressed
Maternally imprinted gene is transcriptionally silenced if it was transmitted from the mother
Paternal allele is expressed

46. For most genes, we inherit two working copies -- one from mom and one from dad.
But with imprinted genes, we inherit only one working copy. Depending on the gene, either the copy from mom or the copy from dad is epigenetically silenced.
they are reset during egg and sperm formation. Regardless of whether they came from mom or dad, certain genes are always silenced in the egg, and others are always silenced in the sperm.

47. In mice, deletion of Igf2 causes a mutant phenotype only when transmitted by father
Maternal imprinting of Igf2 gene
Maternally-inherited Igf2 allele is silenced
Igf2 deletion heterozygotes have normal phenotype if the mutant allele was inherited from the mother
The phenotypic effect of an Igf2 deletion is determined by the parent transmitting the mutant locus.
This parent-of-origin effect can be demonstrated in the two-generation cross illustrated
here.
Fig a

48. Methylation of complementary strands of DNA in genomic imprinting
Epigenetic state can be maintained across cell generations through the action of DNA methylases
One important component is the methylation of cytosines in CG dinucleotides within the imprinted region
Fig b

49. H19, found just 70 kb downstream of Igf 2,
Imprinted but in opposite way
With H19, the copy inherited from the father is silenced and the copy inherited from the mother is active in normal mice.
identified an enhancer region downstream of the H19 gene that can interact with promoters for both genes ( Fig c ).
In the region between the two genes lies another type of transcriptional regulation element called an insulator
an insulator becomes functional, it stops communication between enhancers on one side of it and promoters on the other side.

50. Insulators limit the chromatin region over which an enhancer can operate
An insulator that binds CTCF is involved in reciprocal imprinting of the Igf2 and H19 genes
Methylation of the H19 promoter and the insulator occurs in spermatogenesis
In the Igf2-H19 region, the insulator DNA becomes functional by binding a protein called CTCF. The binding
normally occurs on the maternal chromosome.
As a result, the enhancer element on the maternal chromosome can interact only with the promoter of H19; this interaction, of course, turns on the H19 gene. In such a situation, the
Ig f2 gene remains unexpressed.
On the paternal chromosome, by contrast, both the insulator and the H19 promoter are methylated. Because methylation of the
insulator prevents the binding of CTCF, the insulator is not functional; and without a functional insulator, the enhancer downstream of H19 can reach over a great distance
to activate transcription from the Igf2 promoter.
In addition, methylation of the H19 promoter suppresses transcription of the paternal H19 gene.
Imprinting of the paternal chromosome by methylation thus turns on transcription of Ig f2 and prevents transcription of H19.
A methylase is essential for imprinting of the H19 gene
Fig.16.23c

51. The resetting of genomic imprints during meiosis
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Epigenetic imprints remain throughout the lifespan of the mammal
In germ cells, epigenetic imprints are reset at each generation
During meiosis, imprints are erased and new ones are set
Fig d

52. With imprinting, it is the sex of the parent carrying a mutant allele that counts, and not the sex of the individual inheriting the mutation
An inactivating mutation in a maternally imprinted gene could pass unnoticed from mother to daughter for many generations (because the maternally derived gene copy is inactive due to imprinting).
If, however, the mutation passed from mother to son, the son would have a normal phenotype (having received an active wild type allele from his father), but the son’s children, both boys and girls, would each have a 50% chance of receiving a mutant paternal allele.

53. Genomic imprinting and human disease
Examples: two syndromes associated with small deletions in chromosome 15
At least two genes within this region are differently imprinted
Praeder-Willi syndrome occurs when the deletion is inherited from the father
Angelman syndrome occurs when the deletion is inherited from the mother
Affected individuals have mental retardation and development disorders

54. Inheritance patterns of disorders resulting from mutations in imprinted genes
These pedigrees may appear to be instances of incomplete penetrance, but are distinctly different
In each pedigree, affected individuals (represented by filled in, orange circles and squares) are heterozygotes for a deletion removing a gene that has either a paternal or a maternal
imprint.
In these pedigrees, a dotted symbol indicates individuals carrying a deleted chromosome but not displaying the mutant phenotype.
Fig e

55. Regulation after transcription
Posttranscriptional regulation can occur at any step
At the level of RNA
Splicing, stability, and localization
Example – alternative splicing of mRNA
Generates more diversity of proteins
Common feature in eukaryotes
At the level of protein
Synthesis, stability, and localization

56. Expression of the Sxl mRNA in early Drosophila development
Sex lethal (Sxl) gene encodes a protein required for female-specific development (see Comprehensive example)
In early embryos, Sxl is transcribed only in females
Fig a

57. Differential RNA splicing of Sxl mRNA during Drosophila development
Later in development, Sxl gene is transcribed in both sexes from different promoter Sxl protein regulates alternative splicing of its own mRNA
Later in development, the transcription factors activating
the Sxl early promoter in females disappear;
but to develop as females, these animals still need the Sxl
protein.
How can they still make the Sxl protein they need?
Fig b

58. Some small RNAs are responsible for RNA interference (RNAi)
Specialized RNAs that prevent expression of specific genes through complementary base pairing
Small (21 – 30 nt) RNAs
Micro-RNAs (miRNAs) and small interfering RNAs (siRNAs)
First miRNAs (lin-4 and let-7) identified in C. elegans
Nobel prize to A. Fire and C. Mello in 2006
Posttranscriptional mechanisms for gene regulation
mRNA stability and translation
May also affect chromatin structure

59. Primary transcripts containing miRNA
Most miRNAs are transcribed by RNA polymerase II from noncoding DNA regions that generate short dsRNA hairpins
Fig a

60. miRNA processing
Drosha excises stem-loop from primary miRNA (pri-miRNA) to generate pre-miRNA of ~ 70 nt
Dicer processes pre-miRNA to a mature duplex miRNA
One strand is incorporated into miRNA-induced silencing complex (RISC)
Fig

61. Two ways that miRNAs can down-regulate expression of target genes
When complementarity is perfect:
Target mRNA is degraded
When complementarity is imperfect:
Translation of mRNA target is repressed
Fig

62. siRNAs detect and destroy foreign dsRNAs
Two biological sources of dsRNAs that are precursors of siRNAs (pri- RNAs)
Transcription of both strands of an endogenous genomic sequence
Arise from exogenous virus
pri-RNAs are processed by Dicer
siRNA pathway targets dsRNAs for degradation
siRNAs are very useful experimental tools to selectively knock down expression of target genes
To study function of a gene, dsRNAs for that gene can be introduced into cells

63. Posttranslational modifications of proteins
Ubiquitination – covalent attachment of ubiquitin to other proteins targets those proteins for degradation by the proteosome
Cascades of phosphorylation and dephosphorylation
Transmission of signals across the cell membrane to the nucleus (discussed more in Chapter 17)
Sensitization – tissues exposed to hormones for long periods of time lose ability to respond to the hormone
Example: binding of epinephrine to β-adrenergic receptors on surface of heart muscle cells

64. Phosphorylation and desensitization of β-adrenergic receptor
Phosphorylation of receptor has no effect on its binding to epinephrine, but blocks its downstream functions
Fig

65. A comprehensive example of gene regulation: Sex determination in Drosophila
In Drosophila, ratio of X chromosomes to autosomal chromosomes (X:A ratio) determines sex, fertility, and viability
X:A ratio influences sex through three independent pathways:
Male vs female appearance and behavior
Development of germ cells as eggs or sperm
Dosage compensation – males have increased transcription of X- linked genes

66. Sex-specific traits in Drosophila
Fig

67. How chromosomal constitution affects phenotype in Drosophila
Table 16.2

68. Drosophila mutations that affect the two sexes differently
Table 16.3
*Sxfl is a recessive mutation of Sex lethal **SxlML is a dominant mutation of Sex lethal

69. The X:A ratio regulates transcription of the Sxl gene
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Several HLH transcription factors are key regulators of sex determination
X-linked genes are "numerator elements"
Autosomal genes are "denominator elements"
Numerator/denominator determines ratio of HLH homodimers and heterodimers
Only the homodimer activates transcription of Sxl
Fig

70. Sxl protein regulates splicing of tra mRNA
The Sxl protein triggers a cascade of splicing of transformer (tra) and double-sex (dsx) mRNAs
Sxl protein regulates splicing of tra mRNA
Tra protein regulates splicing of dsx mRNA
Fig

71. Dsx-F is a transcriptional activator and Dsx-M is a transcriptional repressor
Fig

72. The Tra protein regulates splicing of fruitless (fru) mRNA
Fig