1. Control of Eukaryotic Genes “Epigenetics”
CHAPTER 19
Control of Eukaryotic Genes
“Epigenetics”

2. The BIG Questions… How are genes turned on & off in eukaryotes?
How do cells with the same genes differentiate to perform completely different, specialized functions?

3. REVIEW Evolution of gene regulation
Prokaryotes
single-celled
evolved to grow & divide rapidly
must respond quickly to changes in external environment
exploit transient resources
Gene regulation = (?) Operons
turn genes on & off rapidly
flexibility & reversibility
adjust levels of enzymes for synthesis & digestion
prokaryotes use operons to regulate gene transcription, however eukaryotes do not.
since transcription & translation are fairly simultaneous there is little opportunity to regulate gene expression after transcription, so control of genes in prokaryotes really has to be done by turning transcription on or off.

4. Evolution of gene regulation
Eukaryotes
Multicellular = only expresses a fraction of its genes
evolved to maintain constant internal conditions even with changing conditions
(?) Homeostasis
must REGULATE the body as a whole rather than serve the needs of individual cells
Also need to regulate:
(?) growth & development
long term processes
(?) specialization
turn on & off large number of genes
Specialization
each cell of a multicellular eukaryote expresses only a small fraction of its genes
Development
different genes needed at different points in life cycle of an organism
afterwards need to be turned off permanently
Continually responding to organism’s needs
homeostasis
cells of multicellular organisms must continually turn certain genes on & off in response to signals from their external & internal environment

5. Points of control
The control of gene expression (?)can occur at any step in the pathway from gene to functional protein
1. packing/unpacking DNA
2. transcription
3. mRNA processing
4. mRNA transport
5. translation
6. protein processing
7. protein degradation

6. Structural Organization
Chromatin is packed into chromosomes
=ordered into higher structural levels compared to prokaryotes
Figure 19.1

7. 1. DNA packing as gene control
Unfolded chromatin has the appearance of beads on a string
Each “bead” is a (?) nucleosome
Made up of (?) histones
2 nm
10 nm
DNA double helix
Histone
tails
His-
tones
Linker DNA
(“string”)
Nucleosome
(“bead”)
Histone H1
(a) Nucleosomes (10-nm fiber)
Figure 19.2 a

8. 2. Where does transcription occur on chromosome
Degree of packing of DNA regulates transcription
If tightly wrapped around histones
= (?) no transcription
= (?) genes turned off
heterochromatin
darker DNA (H) = tightly packed
euchromatin
lighter DNA (E) = loosely packed H E

9. Higher Levels of DNA Packing
The next level of packing
Forms the 30-nm chromatin fiber
Nucleosome
30 nm
(b) 30-nm fiber
Figure 19.2 b

10. (c) Looped domains (300-nm fiber)
The 30-nm fiber, in turn
Forms looped domains, making up a 300-nm fiber
Protein scaffold
300 nm
(c) Looped domains (300-nm fiber)
Loops
Scaffold
Figure 19.2 c

11. (d) Metaphase chromosome
In a mitotic chromosome
The looped domains coil and fold
= metaphase chromosome
700 nm
1,400 nm
(d) Metaphase chromosome
Figure 19.2 d

12. What regulates Chromatin Structure ?
Acetylation of histones (?) unwinds DNA
(?) attachment of acetyl groups (–COCH3)
(?) enables transcription
(?) genes turned on
transcription factors have easier access to genes
Figure 19.4 b
(b) Acetylation of histone tails promotes loose chromatin structure that permits transcription
Unacetylated histones
Acetylated histones

13. DNA methylation Methylation of DNA (?) blocks transcription factors
(?) attachment of methyl groups (–CH3) to cytosine
 (?) genes turned off
nearly permanent inactivation of genes
ex. inactivated mammalian X chromosome = Barr body

14. Epigenetic Inheritence
=Terminology for gene expression
** DNA sequence NOT changed, just the expression of the gene (on or off)
Can Chromatin modifications be passed offspring?
(sometimes – poorly understood)
**** In a new embryo, all tags are removed except for “imprinted” tags for getting development started

16. Examples of “epigenetics”
Morphogenesis and specialization

17. Examples of “epigenetics”
FTO gene & obesity
Cytogenetic Location: 16q12.2
Molecular Location on chromosome 16: base pairs 53,703,962 to 54,114,466

18. Examples of “epigenetics”
Cancer

19. Examples of “epigenetics”
Twin Studies…”epigenetic drift”

20. III. PROCESS CONTROLS
(?) transcription controls seem to be the most “important” factor in gene expression

21. A. The Roles of Transcription Factors
To initiate transcription
(?) RNA polymerase requires transcription factors (proteins) to bind to the
(?) promotor region

22. B. Role of Enhancers enhancer DNA sequence (?) upstream from promotor
Activator protein – “enhance” (high level) or transcription

23. Model for Enhancer action
Enhancer DNA sequences
(?) distant control sequences
Activator proteins
(?) bind to enhancer sequence & stimulates transcription
Repressor proteins
bind to enhancer sequence & (?)block gene transcription
Much of molecular biology research is trying to understand this: the regulation of transcription.
Silencer proteins are, in essence, blocking the positive effect of activator proteins, preventing high level of transcription.

24. Transcription complex
Activator Proteins
• regulatory proteins bind to DNA at distant enhancer sites
• increase the rate of transcription
Enhancer Sites
regulatory sites on DNA distant from gene
Enhancer
Activator
Activator
Activator
Coactivator B F E
RNA polymerase II A
TFIID H
Coding region
T A T A
Core promoter
and initiation complex
Initiation Complex at Promoter Site binding site of RNA polymerase

25. C. Significance of protein-mediated bending
Activators BEND TOWARD transcription factors stimulating transcription (influence chromatin structure)
Distal control
element
Activators
Enhancer
Promoter
Gene
TATA
box
General
transcription
factors
DNA-bending
protein
Group of
Mediator proteins
RNA
Polymerase II
RNA synthesis
Transcription
Initiation complex
Chromatin changes
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
A DNA-bending protein
brings the bound activators
closer to the promoter.
Other transcription factors,
mediator proteins, and RNA
polymerase are nearby. 2
Activator proteins bind
to distal control elements
grouped as an enhancer in
the DNA. This enhancer has
three binding sites. 1
The activators bind to
certain general transcription
factors and mediator
proteins, helping them form
an active transcription
initiation complex on the promoter. 3
Figure 19.6

26. 2. Post-transcriptional controls
= RNA modification
* Gene expression can be blocked or stimulated during RNA modification

27. A. Role of RNA Processing
(?) exons joined after introns cut out
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Exons
DNA
Primary
RNA
transcript
RNA splicing or
Figure 19.8

28. B. Role of mRNA Degradation
The life span of mRNA molecules in the cytoplasm (hours to weeks)
Degradation of the leader (5’cap) and trailer regions (poly-A tail) by enzymes
Prokaryotes vs. Eukaryotes lifespan

29. (?) Regulatory proteins – attach to 5’ end of mRNA
3. Translation Control
(?) Regulatory proteins – attach to 5’ end of mRNA
Prevent attachment of ribosome
Block translation

30. B. Protein Processing After translation
(?)protein processing/modification are controlled by cellular events (Endo. System)
= folding, cleaving, adding sugar groups, targeting for transport

31. Protein degradation (Figure 19.10)
Ubiquitin tagging
Proteasomes
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Ubiquitin
Protein to
be degraded
Ubiquinated
protein
Proteasome
and ubiquitin
to be recycled
Protein
fragments
(peptides)
Protein entering a
proteasome
Enzymatic components of the
proteasome cut the protein into
small peptides, which can be
further degraded by other
enzymes in the cytosol. 3
The ubiquitin-tagged protein
is recognized by a proteasome,
which unfolds the protein and
sequesters it within a central cavity. 2
Multiple ubiquitin mol-
ecules are attached to a protein
by enzymes in the cytosol. 1
Figure 19.10

32. Ubiquitin 1980s | 2004 “Death tag” Nobel Prize 2004
mark unwanted proteins with a label
76 amino acid polypeptide = ubiquitin
labeled proteins are broken down rapidly in "waste disposers“ = proteasomes
Since the molecule was subsequently found in numerous different tissues and organisms – but not in bacteria – it was given the name ubiquitin (from Latin ubique, "everywhere")
Nobel Prize 2004
Aaron Ciechanover
Israel
Avram Hershko
Israel
Irwin Rose
UC Riverside

33. Proteasome (?) Protein-degrading “machine” cell’s waste disposer
breaks down any proteins into 7-9 amino acid fragments
cellular recycling
A human cell contains about 30,000 proteasomes: these barrel-formed structures can break down practically all proteins to 7-9-amino-acid-long peptides. The active surface of the proteasome is within the barrel where it is shielded from the rest of the cell. The only way in to the active surface is via the "lock", which recognises polyubiquitinated proteins, denatures them with ATP energy and admits them to the barrel for disassembly once the ubiquitin label has been removed. The peptides formed are released from the other end of the proteasome. Thus the proteasome itself cannot choose proteins; it is chiefly the E3 enzyme that does this by ubiquitin-labelling the right protein for breakdown
play Nobel animation

34. RNA interference Small interfering RNAs (siRNA)
short segments of RNA (21-28 bases)
bind to mRNA
create sections of double-stranded mRNA
“death” tag for mRNA
triggers degradation of mRNA
causes (?) gene “silencing”
post-transcriptional control
(?) turns off gene = no protein produced
Nobel Prize 2006
UMASS!!!
siRNA

35. Action of siRNA siRNA mRNA degraded functionally turns gene off
Hot…Hot new topic in biology
Action of siRNA
dicer enzyme
mRNA for translation
siRNA
double-stranded miRNA + siRNA
breakdown enzyme
(RISC)
mRNA degraded
functionally turns gene off

36. Now that the complete sequence of the human genome is available
98.5% does not code for proteins, rRNAs, or tRNAs
Exons (regions of genes coding
for protein, rRNA, tRNA) (1.5%)
Repetitive
DNA that
includes
transposable
elements
and related
sequences
(44%)
Introns and
regulatory
(24%)
Unique
noncoding
DNA (15%)
DNA
unrelated to
(about 15%)
Alu elements
(10%)
Simple sequence
DNA (3%)
Large-segment
duplications (5-6%)
Figure 19.14

37. Another post-transcriptional control….
RNAi RNA interference by microRNAs (miRNAs)
Can lead to degradation of an mRNA or block its translation
The micro-
RNA (miRNA)
precursor folds
back on itself,
held together
by hydrogen
bonds. 1 2
An enzyme
called Dicer moves
along the double-
stranded RNA,
cutting it into
shorter segments.
One strand of
each short double-
stranded RNA is
degraded; the other
strand (miRNA) then
associates with a
complex of proteins. 3
The bound
miRNA can base-pair
with any target
mRNA that contains
the complementary
sequence. 4
The miRNA-protein
complex prevents gene
expression either by
degrading the target
mRNA or by blocking
its translation. 5 5
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Degradation of mRNA OR
Blockage of translation
Target mRNA
miRNA
Protein
complex
Dicer
Hydrogen
bond
Figure 19.9