1. Eukaryotic gene expression
Bacterial genes have a ground state that permits transcription
Without CAP site or operator, the sigma subunit will locate a gene
Eukaryotic genes require complex systems to turn them on
Chromatin structure must be relaxed in order for RNA polymerase to gain access to DNA sequence information
Eukaryotic genes are positively regulated. They are not transcribed in the absence of active mechanisms.
The regulatory components and systems are more complex than bacteria
Transcription is removed from translation
There are no systems equivalent to attenuation in eukaryotes

2. The evidence for alteration of chromosomal structure during transcription
DNase I cleaves chromatin at the linker junctions between nucleosomes
When run on an agarose gel, the DNA resulting from cleavage forms ladders reflecting discrete units increasing in size by 200 nucleotides
This means that nucleosomes cover the bulk of DNA and protect from Dnase digestion
But this procedure reveals the structure of DNA, and not any specific genes

3. DNAse digestion of heat shock genes
When genes are identified within these ladders, they are found in two forms
Genes that are not transcribed are also found to form ladders in response to DNAse I
Genes that are transcribed are fragmented into smaller pieces
The nucleosomes are gone in the upstream regions of genes undergoing transcription
A heat shock gene was digested over time following heat shock and the upstream region identified with a specific probe
Following heat shock, the control regions of the gene become hypersensitive to digestion

4. Where there are no nucleosomes
The nucleosome free sites are not throughout the entire gene, but in certain places called hypersensitive sites
Hypersensitive sites correspond to regions of DNA that bind transcription factors
Thus hypersensitive sites are found upstream of the coding region of genes
They also may be found wherever transcription factors bind
For eukaryotic genes, that is not always just 5’ to the transcriptional start

5. Other alterations to transcriptionally active DNA
Loss of histone H1
This is the histone that exists between the DNA/histone octomer coils
Loss of methyl group from 5 methyl cytosine in CpG islands
Transcriptionally silent DNA tends to have more 5 methyl cytosine than active DNA
Other alterations to transcriptionally active DNA

6. A clinical example - thalassemia
The shutdown of g globin is due to methylation of the upstream region of the genes before and after birth
In the human disease thalassemia, b and d globin chains are lost due to mutation of the globin genes.
One therapy involves administration of 5 azacytidine
Incorporation of this nucleotide results in a loss of methylation
This results in the activation of fetal globin genes which assume some of the oxygen carrying capacity of the mutant globin

7. Histone acetylation Histones have two functional domains
One for binding other histones and wrapping DNA around the nucleosome core
The other is a modification site for control of histone assembly
Multiple lysine residues are presented to the exterior of the histone
Histones are acetylated prior to import into the nucleus following their synthesis on ribosomes
They are actively assembled on DNA by an enzymatic mechanism
Acetylation sites
Of Histone H4

8. Acetylation near transcriptionally active genes
A nuclear histone acetylase further acts on histones H3 and H4
Increasing acetylation decreases the affinity of the histone octomer for DNA
This makes the DNA more available for binding interactions with other proteins
Histones are moved out of the way by an ATP driven process involving a multiprotein complex
Repressors may stimulate deacetylation
Activators may stimulate acetylation
Acetylation near transcriptionally active genes

9. Eukaryotic promoters Why are they subject to positive regulation?
The genes within are sequestered because of chromatin structure
The size of the genome favors non-specific binding of regulatory proteins at random
In a diploid genome of 6 billion nucleotide pairs, a short sequence capable of binding regulatory proteins would occur many times by chance
So regulatory systems demand that multi-protein complexes form before a gene is transcribed
It is more efficient to negatively regulate the entire genome with a single mechanism (chromatin structure) and then specifically turn on the set of genes needed by the cell than to specifically negatively regulate every gene of a eukaryote
That would mean tens of thousands of repressors for each cell type

10. Promoters and Enhancers
Promoters include, for example, TATAA boxes, GC boxes and CAAT boxes that are responsible for positioning RNA polymerase II at the beginning of a gene
Polymerase II has no affinity for the TATAA box on its own.
Assembly of a transcriptional complex depends on the sequence around the 5’ end of the gene
Enhancers are sequences that are distant from the promoter but positively affect its function
They may be pointed in either orientation

11. Three classes of transcription factors
Basal (general) transcription factors
These interact directly with RNA polymerase II or with each other in building a complex around the promoter
They also recognize the promoter sequences
The TATA box is highly conserved
The TATA binding protein + transcription factors for polymerase II (TF II) assemble and provide the minimal assembly for transcription
But transcription still requires a positive signal
This complex marks the spot where RNA polymerase is to bind and begin transcription

12. Enhancer binding proteins
Also known as DNA binding transactivators
These bind enhancers that are far away from the promoter
They recognize the specific enhancer sequence
Some enhancer binding proteins work on a large number of genes, permitting coordinate control of transcription
Others are specific to a single gene
They then loop inward toward the promoter so that the enhancer binding protein can interact with the basal transcription factors at the promoter site
Protein-protein interactions are mediated through motifs such as the leucine zipper and the helix loop helix
Enhancer binding proteins

13. Coactivator proteins
These bind RNA polymerase II complexes and enhancer binding proteins and mediate the signaling between them
RNA polymerase II may carry the coactivator proteins with it as it transcribes
Coactivators are necessary for transcription

14. The process of transcriptional activation
Remodeling chromatin
May involve
Demethylation of 5 methyl C
Acetylation of histones
Binding of basal transcription factors
Transactivator binding enhances the remodeling of chromatin and facilitates opening up chromatin structure
This helps other enhancer binding proteins to interact with exposed DNA sequence
Transactivators interact with coactivators and help RNA polymerase position itself on the transcription complex at the TATA box
The process of transcriptional activation

15. Induction and repression
Inducibility and especially repression is not as common a phenomenon in eukaryotic cells
Especially higher eukaryotic cells
The larger the organism, the more stable the environment a cell experiences
So it needn’t respond to radical changes in the environment
However some transcriptional regulation is still necessary
Transactivators can serve the function of inducers or repressors
A repressor generally inhibits the function of an inducer by some mechanism
Competitive binding
To DNA
To basal transcription factors
Directly binding the activator
Induction and repression

16. Induction and repression
Binding to a small molecule can result in an increase or decrease in the ability of a transactivator to work
Steroid hormone receptor becomes a functional DNA binding transactivator in response to binding its ligand
Binding to a ligand displaces HSP90 (a heat shock protein) and permits translocation to the nucleus and subsequent DNA binding
In the absence of ligand, it interferes with transcription and thus becomes a repressor
AD: activator domain
DBD: DNA binding domain
LBD: ligand binding domain

17. A specific example The GAL genes of yeast
These are a set of individual genes under coordinate control
Eukaryotes don’t have operons
Each gene has a promoter and set of enhancers (called UAS)
Turning on one of the GAL genes means activating a set of enhancer binding proteins and coactivators that turn on all of the other GAL genes
The products of the genes are needed for importation of galactose and its metabolism

18. The GAL genes can be repressed
The logic is the same as with bacteria
When glucose is present, there is no necessity to make galactose importation and metabolizing enzymes, so the genes are shut down
This repression overrides induction

19. Induction
Gal4p is a transactivator that induces transcription at a GAL locus by interacting with the coactivator assembly at promoter
In the absence of galactose, Gal4p is sequestered by another regulatory protein Gal80
Gal4p is displaced from Gal80 by Gal3p when Gal3p binds galactose
Thus in contrast to bacterial inducers, the ligand binding and the DNA binding proteins are not the same

20. Minimum structure of enhancer binding proteins
Each must
Bind its target DNA
Bind the promoter complex and/or activating proteins
The ability of a protein to perform each function is due to functional domains in the protein
The domains permit interaction with other proteins and specific recognition of DNA sequence
In addition to DNA and protein interaction domains, there are 3 common types of activator domains
Acidic – Gal 4p
Glutamine rich – SP1
Proline rich - CFT1

21. GAL 4p This has a domain resembling a zinc finger
Instead of two cys and two his coordinating a Zn , it has 6 cys residues
It is a homodimer, bound by interactions of two coiled coils
The two zinc fingers interact with a palindromic sequence
The protein is controlled by another domain that is rich in aspartic and glutamic acid residues
This was identified by constructing mutants of the Gal4p gene that substituted other amino acids in this domain
The mutants lost function

22. SP1 and CTF1 SP1 binds the GC box CTF1 binds the CAAT box
GC boxes are located close to the TATA sequence
SP1 is a very common enhancer binding protein
Many genes lack a GC box
There are 3 Zn fingers for DNA binding
Two glutamine rich activator domains
CTF1 binds the CAAT box
The DNA binding domain is unique and is neither helix turn helix or a zinc finger
The activation domain is proline rich
SP1 and CTF1

23. Domain swapping
Since the domains for DNA binding and activation are distinct, their domains may be separated on the level of DNA
By taking a domain for DNA binding and adding it to a domain for activation, a new protein may be engineered
This binds the DNA sequence specified by one gene, and responds to the signals of another
Such experiments permit the manufacture of proteins with unique control abilities
Although not therapeutically useful right now, they are important experimental tools in defining the way that genes respond to external signals.
Questives
What form of transcriptional regulation is used in eukaryotic cells?
What problem does RNA polymerase encounter when attempting to gain access to a eukaryotic promoter?
What does Dnase I do to isolated chromatin?
When the DNA fragments from Dnase I treatment of chromatin are visualized by agarose gele electrophoresis, what is seen?
Why is a ladder produced?
What does the size of the bands reflect?
Would you expect to see this pattern in all DNA?
Which part of a transcriptionally active gene would you expect to lose the ladder?
So what does Dnase hypersensitivity reflect?
What happens to histone H1 during transcription of a gene?
How does methylation of cytosine in CpG islands affect transcription?
Is there a drug that can affect methylation?
What are two domains of histones?
How does acetylation of histones affect
Nucleosome assembly?
Nucleosome structure?
Transcription?
Where is a TATA box located?
What distinguishes an enhancer from a promoter?
What are 3 classes of eukaryotic transcription factors?
What do basal transcription factors do?
What does a DNA binding transactivator do?
What DNA sequence does it bind?
What protein(s) does it interact with?
What do coactivators do?
What are three ways in which a repressor interacts with an inducer in eukaryotes?
Give an example of a DNA binding transactivator that binds to hormones?
What three domains are necessary for a Ligand binding/DNA binding transactivator to work?
What type of control is exerted over the GAL genes of yeast?
Why are these genes coordinately controlled?
How does the induction/repression system in GAL work?
What is the function of
Gal4p?
Gal3p
Gal80
What is SP1? What does it bind?
What is CFT1? What does it bind?

24. Regulated gene expression
Gene expression in multicellular organisms is often controlled by intercellular signaling
Some genes are directly responsive to environmental stimulus however
UV induction of DNA repair enzymes
Stress response (heat shock) genes
Signaling takes two forms
Hormones may be bound by
Membrane bound receptors
Diffusible receptors
Regulated gene expression

25. Diffusible receptors
Diffusible receptors act by directly binding a hormone and then moving into the nucleus
Hormone binding induces a conformational change that permits the receptor to act as a transcriptional activator
Diffusible molecules can be transactivators
“trans” means something that acts on a gene that originates from another site. In this fashion they resemble the activators of bacteria
However hormones are made by one cell in order to command a transcriptional response in another cell
Bacterial effectors are nutrients or their metabolites or analogs

26. Steroid hormones These are
endocrine hormones
hydrophobic molecules that are synthesized using cholesterol as a precursor
made by certain cell types and secreted in response to biochemical, developmental or neurological signals
carried by the blood from their cell of origin to target cells either dissolved or by a protein carrier
Many are too hydrophobic to dissolve directly in blood
They enter a cell by dissolving in the plasma membrane and diffusing to their receptor
Steroid hormones

27. The steroid hormone receptor
This is a DNA binding protein with a hormone binding domain at the carboxyterminal end of the protein
There are several related types
Each receptor has a specific complement of transcription factors it must interact with which vary from one receptor to another and one cell type to another
They are all related in structure
The DNA binding domain contains two Zn fingers and is in the middle of the protein
The domain that interacts with transcription factors is amino terminal and varies in structure
The hormone binding region is highly variable in structure
Each must recognize and bind its cognate ligand
The steroid hormone receptor

28. Loss of responsiveness to a hormone can be caused by changes in any of the three domains
Hormone-ligand complexes may serve either positive or negative regulatory functions
Mutations prevent transcriptional activation or repression in response to hormone binding
Mutation in the androgen binding domain of the androgen receptor creates androgen unresponsiveness
Mutation in the DNA binding or transcription activation domains would mean the protein could bind androgen, but nothing would happen
This results in developmental abnormalities such as XY females
To the left are four XY siblings suffering from androgen insensitivity
MUTATIONS

29. The cis elements Cis refers to sequence involved in gene expression
Trans elements interact with cis elements but arise from other genes
The glucocorticoid responsive element (GRE) and estrogen responsive element (ERE) share sequence homology
The cis elements that are important in hormone responsiveness are the binding sites for the hormone-receptor complexes
Hormone responsive elements: HRE
These are direct repeats that interact with the Zn finger domains
The consensus sequences for these receptors are very similar
This reflects the similarity in the Zn finger domains among the various receptors
The cis elements

30. Following binding of a hormone, the receptor diffuses to the nucleus and binds the HRE
The receptor is a dimer
Each subunit of the dimer binds to one of the two repeat elements
The strength of binding is determined by the variation of the HRE away from the consensus sequence
The stronger the binding between the receptor and HRE, the longer the receptor will remain bound and the longer transcription will be activated
Binding is an all or none event
If bound, activation due to the receptor is full
Receptor – DNA binding

31. Phosphorylation of transcription factors
Transcription factors are subject to phosphorylation on serine and threonine residues
This is the result of second messenger activation of serine-threonine kinases or ras activation
In abnormal, though common, conditions, such as mutations or viral infections, gene expression is deregulated and genes are inappropriately expressed because of deregulated phosphorylation mechanisms
This is because second messengers activate a complex cascade of enzymatic steps that can be perturbed at many different points
Here the transcription factor Elk-1 is activated through phosphorylation
Phosphorylation of transcription factors

32. Repression This occurs at the transcriptional and translational level
Genes are usually turned off as a default at the transcriptional level
But this does not mean the mRNA is gone
It could have been stabilized through sequestration
Translational regulation permits rapid responsiveness
The primary transcript of a gene may take several minutes to synthesize because of its size
It also must be spliced and transported to the ribosomes
A sequestered transcript that is released in response to a signal is faster

33. Translational repression affects more than just ribosomal proteins
The distribution of mRNA within some cells creates a distribution of protein inside a cell
This results in intracellular protein gradients that are important in development
Translational repression affects more than just ribosomal proteins

34. Regulatory mechanisms of translational initiation
Inhibition of initiation factors through phosphorylation
eIF phosphorylation inhibits its function and can be reversed through dephosphorylation
Inhibition of initiation factors by binding to specific factors
Interference with eIF4E and eIF-4G activity by 4E-BP’s.
Inhibition of specific mRNA by binding of inhibitory proteins to sequences in the 3’untranslated region

35. Phosphorylation of eIF-2 inhibits its activity
Maturation of red blood cells involves a stage in which reticulocytes translate mRNA left behind after the loss of the nucleus
Reticulocytes regulate the amount of globin synthesized by phosphorylating eIF-2
When there is heme deficiency globin synthesis is wasteful since hemoglobin cannot be synthesized
Low heme activates HCI which phosphorylates eIF-2
Phosphorylated eIF2 binds eIF2 binding protein and is unavailable for translational initiation

36. eIF4E inhibition
eIF4E is necessary to bind the 5’ CAP in order to from an initiation complex for translation
Normally it binds eIF4G
Maskin binds eIF4E (preventing it from binding eIF4G) when it is bound to an mRNA through interaction with CEPB

37. Developmental control of gene expression
The study of fruit fly development resulted in the discovery of a number of genes involved in human disease
Although fruit fly development is greatly different in the processes leading to the final form, the activation of genes and the structure of gene products and their participation in the formation of patterns and structures have parallels in human gene regulation and the structure of human regulatory gene products

38. Fly development is controlled by gene expression
The conceptually difficult part of this is to understand how a single cell can create multiple, morphologically different structures starting from a seemingly symmetrical, undifferentiated state merely by dividing.
It is easier to think of the process in parts and then add up the whole than to see a cell turn into a fly and attempt to understand the entire process at once

39. Three gene families are responsible for early development
Maternal genes
Made by the female and exist within the egg at the time of fertilization
Responsible for establishing the polarity of the early embryo
Zygotically acting genes
Segmentation genes
These establish a repeating pattern of body segments
Homeotic genes
These establish the identity of the segments
Three gene families are responsible for early development

40. This is a distinction in structure established between two poles.
The distinction needn’t be great, only a morphological difference is enough to create polarity
Without polarity, further structures would have no way of organizing themselves
Segments would be repeating structures that are all the same
Establishing polarity is thus the earliest developmental event
Polarity is actually established by the assymetry of the egg
This yields pole cells on one end of the zygote
Polarity

41. Segmentation This is obvious in the formation of the fly abdomen
Repeating abdominal segments are very similar in appearance
However this patterning extends from end to end of the fly
The patterns are given different identities by homeotic genes
Thus the head and abdomen begin as segments similar to abdominal segments
But polarity makes them different, and therefore the genes that are expressed within each segment differs
Segments are created and further divided into smaller segments
Gap genes create the largest segments
Pair rule and segment polarity genes subdivide the largest segments
Segmentation

42. Homeotic genes These give rise to the dramatic mutants of Drosophila
Once segments are established with the proper polarity, homeotic genes create unique structures
Mutation of a particular homeotic gene results in the formation of a structure that is due to the action of another homeotic gene
The homeotic genes are controlled by their position within a gradient of polarity
So if one segment was destined to give rise to antennae, but lacked the homeotic gene due to mutation, it would create the next most available structure
Homeotic genes

43. Antennaepedia
Antennaepedia represents the mutation of a gene that would create an antennae
Antennapedia is a transcription factor that coordinately controls expression of genes, that when expressed result in an antennae
In its absence, the next most similar structure is a leg
The normal leg is also formed in the next segment
The gene encoding the protein controlling leg development is expressed at lower concentrations in the head segment than antennapedia gene, but in the absence of antennapedia, it is the most highly expressed protein capable of activating genes that result in a structure

44. Key genes are expressed early
Maternal genes establish polarity due to formation of gradients
Front to back and top to bottom gradients establish anterior posterior and dorsal ventral gradients
When cells are formed in the blastoderm, they form within an environment in which the concentration of transcription factors will vary along one axis
This varies the type and numbers of genes that are expressed within any cell
To the left is bicoid RNA (upper) and bicoid protein (lower) in the early embryo
The RNA gradient is present in the egg and establishes the protein gradient

45. A few examples of developmental genes
Bicoid is a maternal gene that controls expression of segmentation genes
It is a transcription factor that activates segmentation genes
And a translational repressor
It appears in the anterior of an egg, and its concentration falls of towards the posterior
The gradient is maintained during formation of the larvae
Experiments with bcd mutants
(a) A failure of bicoid to be expressed means a fly develops with two posteriors rather than an anterior and a posterior
(b)injecting cytoplasm from a normal embryo rescues the embryo (makes an anterior)
( c) injecting bicoid mRNA also rescues

46. It represses translation of caudal in the anterior of the fly larvae
Caudal is a transcription factor found uniformly throughout the larvae, and it creates the posterior end
And activates expression of hunchback
Hunchback is a transcription factor that creates the anterior end
The bicoid gradient means it has these effects only in the anterior end
Without bicoid, caudal is not repressed in the anterior end and hunchback is not transcribed
What bicoid does

47. Nanos
This is a translational repressor that is found at highest concentrations in the posteror of a fly larvae
It acts in concert with the uniformly distributed pumilio gene product to translationally repress hunchback
This results in establishment of high caudal gene product and inactivated hunchback mRNA, meaning the posteriorizing effect of caudal dominates

48. Some Gap genes
Gap genes create a gross form of segmentation in the early embryo
They are overlayed onto the pair-rule gene expression to create complex transcriptional signals
These are the expression patterns of the hb-z, Kr and kni genes

49. fushi tarazu and eve
These are two segmentation genes known as pair rule genes that split a segment in two
Ftz establishes the “pair rule”
Two segments form out of one
Without it the fly forms 7 rather than 14 segments
Ftz (blue) is expressed in each segment, in the anterior half of the segment
This expression pattern is again the result of the action of an anterior posterior gradient, but now within each segment
Eve (brown) for even-skipped are expressed in the posterior half of each segment
Both ftz and eve are homeodomain transcription factors that control expression of genes expressed in the segments
Quiztiff
What two types of hormone receptors are there?
Where are diffusible receptors located prior to binding hormone?
What happens on binding hormone?
What type of transcription factor is a diffusible receptor?
Name a few steroid hormones.
How does a steroid reach its target tissue?
Why do some require carriers?
How do they cross the membrane barrier of a cell?
Draw a typical steroid hormone receptor
Which region is most variable in sequence?
What is the defect in testicular feminization?
What is the difference between cis and trans elements?
What is the HRE?
What modification of a transcription factor commonly affects its activity?
What advantage does translational regulation of gene expression through sequestration of an mRNA confer on gene expression as opposed to regulation at the level of transcriptional initiation?
What are three mechanisms of translational repression in eukaryotes?
In what cell does eIF2 inhibition act to interfere with globin synthesis?
What protein regulates this translational repression?
What structure does EIF4G bind and what does this result in?
How does maskin interfere with this interaction?
What three gene families are responsible for fly development?
Where are Maternal effect genes active?
What doe maternal effect genes establish?
What do segmentation genes do?
What do homeotic genes do?
In what structure is polarity established?
What is the main difference between the effect of gap genes and pair rule genes?
A segment originally destined to give rise to a haltere in a fly has given rise to a set of wings. Which gene has been mutated?
What type of gene is antennaepedia?
What creates polarity?
How is the initial gradient within an egg established?
What does the gradient consist of?
How does bicoid regulate caudal?
How does bicoid regulate hunchback?
What does hunchback do?
What is the result of a mutation in bicoid?
What type of gene is fushi tarazu?
What does fushi tarazu do?