1. Control of Gene Expression Department of Physiology
Shaw-Jenq Tsai
Department of Physiology

2. Thinking about Gene Regulation
Humans begin life from a single cell; all the genetic information needed to create an adult is in our genome.
Embryonic cells undergo differentiation to produce specific cell types such as muscle, nerve, and blood cells.
Different cell types are the consequence of differential gene expression.

3. A typical differentiated mammalian cell makes about 100,000 proteins from approximately 35,000 genes.
Most of these are housekeeping proteins needed to maintain all cell types.
Certain proteins can only be detected in specific cell types.
How is gene expression regulated?
Regulation of gene expression is very complex
Presently – we have a superficial understanding

4. Control of Gene Expression
Synthesis of a protein involves discrete steps
Several levels at which control mechanisms work
Transcriptional control
RNA processing control
Translation control
Protein activity control

5. Transcriptional level control
Differential gene transcription – the major mechanism of selective protein synthesis
Governed by a large number of proteins known as transcription factors
Two functional classes of transcription factors
General transcription factors
Specific transcription factors

6. Specific transcription factors
A single gene controlled by many regulatory sites – bind different regulatory proteins
A single regulatory protein may become attached to numerous sites on the genome
Cells respond to environmental stimuli by synthesizing different transcription factors
Bind to different sites on DNA

7. Specific Transcription Factors
PEPCK
A key enzyme of gluconeogenesis (conversion of pyruvate to glucose)
Synthesized in liver in response to low glucose
Synthesis drops sharply after a meal
Level of synthesis of PEPCK controlled by different transcription factors
e.g. receptors for hormones involved in regulating carbohydrate metabolism

8. Promoter Structure Closest upstream sequence
TATA box – major element of the gene’s promoter
Region from TATA box to start of transcription site is the core promoter
Site of assembly of preinitiation complex – RNA polymerase II and general transcription factors
Two other promoter sequences
CAAT box
GC box
Core
promoter

9. Control of PEPCK Gene Expression
TATA box determines the site of initiation of transcription
CAAT and GC boxes regulate the frequency of transcription
All located within 100 to 150 base pairs upstream of the transcription start site – proximal promoter elements

10. Activation of Transcription
Hormones which affect transcription of PEPCK include insulin, thyroid hormone, glucagon and glucocorticoids
All affect transcription factors which bind DNA
DNA sites bound by transcription factors are termed – response elements
Glucocorticoids stimulate PEPCK expression by binding to a specific DNA sequence termed – a glucocorticoid response element (GRE)

12. Activation of Transcription
Same GRE is located upstream from different genes on different chromosomes
Thus – a single stimulus – elevated glucocorticoid concentrations – simultaneously activates a range of genes needed in a comprehensive response to stress

13. Enhancers Activation of Transcription
Expression of genes also regulated by more distant DNA elements termed enhancers
Can be experimentally moved without affecting their ability to enhance gene expression
May be 1000s or 10000s base pairs upstream or downstream from the gene
How??
Brought into close proximity to the gene as DNA can form loops
Promoters and enhancers cordoned off from other genes by sequences called insulators

14. Control of Gene Expression
Activation of Transcription
Transcription factor

15. Control of Gene Expression
Activation of Transcription
Enhancers

18. Action of an Insulator

19. Figure 12.34
Two hypotheses for the mechanism of insulator activity.

22. Action of Transcription Factor
A transcription factor bound to an enhancer may act via the following mechanisms:
Recruit general transcription factors and DNA polymerase II to the core promoter
Stabilize the transcription machinery located in the core promoter
Via an intermediary termed a coactivator
Coactivators are large complexes with 15 to 20 subunits
Do not directly bind DNA
Interact with a range of transcription factors

23. Structure of Transcription Factors
Contain different domains which mediate the different functions – at least two domains
DNA-binding domain
Activation domain
Commonly form dimers
Example
Glucocorticoid receptor
Binds DNA at the glucocorticoid response element (GRE)
Ligand-binding domain / DNA-binding domain / Activation domain

24. Transcription Factors Binding Element
GRE
A palindrome
Two-fold nature is important
Pairs of GR polypeptides bind to DNA forming dimers
5’-AGAACAnnnTGTTCT-3’
3’-TCTTGTnnnACAAGA-5’

25. Transcription Factor Motifs
Transcription factors belong to each of several classes based upon specific types of binding domains or motifs
Many contain an a-helix which is inserted into the DNA major groove
Recognizes the particular nucleotide sequence lining the groove
Binding between aa and DNA (including DNA backbone) via:
Van der Waals (hydrophobic) forces
Ionic bonds
And hydrogen bonds

26. Control of Gene Expression
Transcription Factor Motifs
Common transcription factor motifs
Zinc finger
Helix-loop-helix
Leucine zipper
HMG box
Shared feature
Structurally stable framework
Specific DNA recognizing sequences are correctly positioned

27. Types of DNA binding proteins DNA and RNA polymerase repair enzymes
structural proteins
transcription factors
DNA binding motifs
zinc fingers
leucine zippers
helix-turn-helix
helix-loop-helix

28. Zinc finger Zn ion coordinated to two cysteines and two histidines
Each contains multiple zinc finger domains

29. Helix-loop-helix (HLH)
Two a helices separated by a loop
Often preceded by a stretch of basic aa which interact with a specific nucleotide string
Always occur as dimers
homodimers
heterodimers

30. Leucine zipper motif Leucines every seven aa along an a-helix
All leucines face the same direction
Two a-helices can zip together forming a coiled coil
Basic aa on opposite side of coils

32. Repression of Transcription
Cells also possess negative regulatory elements
Mechanisms:
Binding to promoter elements
Blocking assembly of the preinitiation complex
Inhibiting binding or functioning of transcriptional activators
Modifying DNA and its interaction with nucleosomes
Some transcription factors activate some genes and repress others

33. Mechanisms of Transcription Repression
Binding to promoter elements
Blocking assembly of the preinitiation complex

34. Mechanisms of Transcription Repression
Inhibiting binding or functioning of transcriptional activators

35. Repression of Transcription
DNA Methylation
Methyl groups may be attached to cytosine (C5 position)
Methyltransferases
Methyl groups provide a tag
In mammals always part of a symmetrical sequence
Concentrated in CG-rich domains
Often in promoter regions
Methylation of promoter DNA highly correlated with gene repression

36. DNA Methylation
Maintains a gene in inactive state rather than initiating gene repression – Example:
Inactivation of genes of one X chromosome in female mammals occurs prior to a wave of methylation
Shifts throughout life in DNA-methylation levels
Early Zygote – most methylation tags removed
Implantation – a new wave of methylation occurs
Important example – Genomic Imprinting

37. DNA Methylation Genomic Imprinting
Certain genes are active or inactive during early development
Depending on whether they are paternal or maternal genes
e. g.– IGF-2 is only active in the gene from the male parent
The gene is imprinted according to parental origin
Mammalian genome has > 100 imprinted genes in clusters
Imprinted due to selective methylation of one of the alleles

38. DNA Methylation Genomic Imprinting
In the early embryo the waves of demethylation and new methylation do not affect the methylation of imprinted genes
Thus the same alleles are affected in the zygote through to the adult stage in the individual

39. Chromatin structure and transcription
DNA is not naked – but wrapped around histone complexes to form nucleosomes
How are transcription factors and RNA polymerases able to interact with DNA tightly associated with histones?
Apparently nucleosome structure does inhibit initiation of transcription
Initiation of transcription requires assembly of large complexes and nucleosomes block assembly at the core promoter

40. Role of Acetylation
Genes which are actively transcribed are bound by histones which are acetylated
Each of the histones has a flexible N-terminal tail
Extends outside the core particle and the DNA helix
Acetyl groups are added to lysine residues by enzymes
Histone acetyl transferases (HATs)
Acetylation has two functions
Neutralize the positive charge on the lysine residues
Destabilize interactions between histone tails and structural proteins

41. Role of Acetylation Some coactivators have HAT activity
Links histone acetylation, chromatin structure and gene activation
HAT activity of coactivator acetylates core histones bound to promoter DNA causing
release of nucleosome core particles or loosening of histone-DNA interaction
Subsequent binding of transcription factors and RNA polymerase
Once transcription is initiated – RNA polymerase is able to transcribe DNA packaged into nucleosomes
Acetylation is dynamic – enzymes also remove acetyl groups

42. Role of Deacetylation Removal of acetyl groups
Histone deacetylases (HDACs)
HDACs associated with transcriptional repression
HDACs are subunits of larger complexes – corepressors
HDACs guided to regions of DNA by methylation patterns

43. Role of Deacetylation Example: Inactive X chromosome of female
Largely deacetylated histones
Active X chromosome has a normal level of histone acetylation

44. Control of Acetylation / Deacetylation

45. Control of Acetylation / Deacetylation

48. Processing-Level Control
Recall that the formation of multigene families is a mechanism that generates protein diversity
Protein diversity also generated via alternate splicing
Regulates gene expression at the level of RNA processing
A mechanism by which a single gene can encode two or more related proteins
Most genes (and their primary transcripts) contain multiple introns and exons
Often – more than one pathway for processing of primary transcript

49. Processing-Level Control
Transcripts from approx 35% of human genes may be subjected to alternate splicing
Simplest case – a specific segment either spliced out or retained – Example:
Fibronectin:
Synthesized by fibroblasts – two additional peptides compared to that synthesized by liver
Extra peptides encoded by pre-mRNA retained in fibroblast

50. Translational-Level Control
Wide variety of mechanisms – affecting mRNA previously transported from the nucleus
Subjects include:
Localization of mRNA in the cell
mRNA translation
Half-life of mRNA
Mediated via interactions between mRNA and cytosolic proteins

51. Translational-Level Control
mRNA noncoding segments – untranslated regions (UTRs)
5’ – UTR – from methylguanosine cap to AUG initiation codon
3’ – UTR – from termination codon to end of poly(A) tail
UTRs contain nucleotide sequences which mediate translational-level control

52. Translational-Level Control
Cytoplasmic localization of mRNAs – Example ferritin
Translation regulated by iron regulatory protein (IRP)
Activity of IRP dependent on cellular iron concentration
At low iron concentration – IRP binds the 5’ UTR
Bound IRP interferes physically with the binding of a ribosome to the 5’ end of the mRNA
At high iron concentration the IRP changes conformation and looses affinity for the 5’ UTR

53. Control of mRNA stability
Half-life of mRNA is variable – 10 minutes to 24 hours
Specific mRNAs are recognized in the cytoplasm and treated differentially
mRNAs lacking the poly(A) tail are rapidly degraded
Poly(A) tail is not naked mRNA but bound by the poly(A) binding protein (PABP)
Each PABP bound to about 30 adenosine residues

54. Control of mRNA stability
PABP protects poly(A) tail from general nuclease activity
But – increases its sensitivity to poly(A) ribonuclease
mRNA in cytoplasm is gradually reduced in length by poly(A) ribonuclease
When the tail is reduced to approx 30 residues
mRNA is rapidly degraded
Degradation occurs from the 5’ end
Suggests two ends held in close proximity