Odisha NET Academy – ONA

Regulation of Eukaryotic Gene Expression

Arun Sir………
 WHY REGULATE GENE
EXPRESSION??

 Development and differentiation

 Adaptation
GENE EXPRESSION IN DIFFERENT CELLS

Muscle cell Pancreatic cells Blood cells

Genes for alpha cells beta cells WBCs RBCs

Glycolysis ON ON ON ON ON

Insulin OFF OFF ON OFF OFF

Glucagon OFF ON OFF OFF OFF

Hemoglobin OFF OFF OFF OFF ON
 The Heat-Shock Genes (Proteins)
 When organisms are subjected to the stress of high
temperature, they synthesize a group of proteins (the heat-
shock proteins) that help to stabilize the internal cellular
environment.

 The expression of the heat-shock proteins is regulated at the

transcriptional level; transcription of the heat-shock genes is
induced by heat.

© John Wiley & Sons, Inc.

Induction of the Drosophila hsp70
Gene by Heat Shock
36.7 to 37ºC

41 to 42ºC

© John Wiley & Sons, Inc.

Eukaryotic
DNA Is
Organized
Into
Chromatin
 A. Gene Regulation at DNA Level
Chromatin Remodeling

1. Changes of DNA Topo structure

DNase I hypersensitive site

DNase I hypersensitive sites (DHSs) are regions of chromatin that
are sensitive to cleavage by the DNase I enzyme. In these specific
regions of the genome, chromatin has lost its condensed structure,
exposing the DNA and making it accessible.

This raises the availability of DNA to degradation by enzymes, such as

DNase I. These accessible chromatin zones are functionally related to
transcriptional activity, since this remodeled state is necessary for the
binding of proteins such as transcription factors.
These regions have been shown to map many
types of cis-regulatory elements including
promoters, enhancers, insulators, silencers and
locus control regions.
 Chromatin remodeling
In preparation for transcription, nucleosomes
are altered by multiprotein complexes in a
process called chromatin remodeling.

Remodeling complexes disrupt nucleosome

structure near a gene’s promoter by sliding or
transferring histone octamers to new locations (e.g.,
the yeast SWI/SNF complex)
DNA compaction and chromatin remodeling
Family ATP-chromatin remodeling factors

ISWI SWI/SNF

•RSF •hSWI/SNF(BRG1)
man
•WCRF/hACF •hSWI/SNF(BRM)
•NURF
•CHRAC fly •Brahma
•ACF

•ISWI1 yeast •SWI/SNF

•ISWI2 •RSC
 SWItch/Sucrose Non-Fermentable

It possesses a DNA-stimulated ATPase activity and can

destabilise histone-DNA interactions in reconstituted
nucleosomes in an ATP-dependent manner, though the
exact nature of this structural change is unknown.
SWI2/SNF2 subfamily of
chromatin-remodeling ATPases

Isolated as sucrose-non-fermenting mutants in yeast

snf2, snf5, snf6

Isolated as mating type switch deficient mutants in yeast

swi1, swi2, swi3

SNF2=SWI2
 LARGE COMPLEXES (generally)

Ca. 11 subunits, 2 MD in size Casten et al., Cell snapshot 2011

 SWI2/SNF2 complexes
Core complex
ATPase Snf2 (BRM/BRG1)
Snf5 (BAF47)
Swi3 (BAF155/BAF170)
other subunits
Swp73 or BAF60
actin related proteins (ARP)
BAF 57
Function of some subunits not yet understood
ATPase and core: sufficient in vitro,
other subunits required in vivo
 Chromatin remodeling

(SWItch/Sucrose Non-Fermentable)
 ROLE of ISWI domains
Imitation SWI (SWItch) of Drosophila melanogaster

SANT/HAND domain contacts histone tails

- charge: histone tail interaction
+ charge: DNA interaction

Slide domain
linker DNA contact, ’measures’ distance
equal spacing of nucleosomes

ATPase domain
near dyad, motor, translocation
Chromatin Remodeling by ISWI
2. DNA Methylation

DNA Methylation
 CpG islands
----- are genomic regions that contain a high
frequency of CG dinucleotides.

----- CpG islands particularly occur at or near

the transcription start site of genes.
 TF RNA pol

Active
transcription
Unmethylated CpG island

TF RNA pol

CH3 CH3 CH3 Repressed

transcription

Methylated CpG island

 Housekeeping gene -A gene involved in basic
functions is required for the sustenance of the cell.
Housekeeping genes are constitutively expressed

 Luxury gene - are those coding for specialized

functions synthesized (usually) in large amounts in
particular cell types.
3. Histone modification

 Methylation
 Acetylation
Histone Acetylation

HAT
acetyltransferases
deacetylases
 S-adenosyl-L-
S-adenosyl-L-methionine homocysteine

histone methyltransferases

histone demethyltransferases
&&methylation
 N-terminal tail modifications of H3 and H4.
M=methylated, A=acetylated,
P=phosphorylated.
 X-Inactivation
 Inactive X-Chromosome (Barr body)
 Underacetylated at H4
 Hypermethylated
Demonstration of X Inactivation:
The classic mosaic example is Calico cats. One of the genes for hair
color is found on the X chromosome. The gene makes either orange
or black (white is on a different chromosome). Each colored patch on
a calico has a different X turned on. If the patch is orange, then there
are a bunch of cells there with the orange X on. A black patch has a
bunch of cells with the black X on. This is why a male calico is so rare.
Most males only have a single X and so either the orange or the black
hair color gene, not both.
Calico Cat
 X-inactivation in humans
 Red-green color blindness
 Males = fully color blind
 Females = mosaic retinas

 Anhidrotic ectodermal dysplasia

 Males = absence of teeth, lack of sweat glands
 Females = random patterns of tissue with or
without sweat glands
The mosaic effect is seen in human
females affected by anhidrotic
ectodermal dysplasia in which a
mutant gene on one X chromosome
results in patches of skin with no
sweat glands.
4. Changes of nucleosome

High Mobility Group (HMG) Protein

 non-histone chromatin proteins

 high mobility in PAGE
 soluble in 2-5% TCA
 small < 30 kDa
 high content of charged amino acids: Asp, Glu
5. Other gene regulation at DNA Level
(1) Gene Deletion
(2) Gene Duplication
(3) DNA Rearrangement
(4) Gene Amplification
(5) Chemical Activator
(6) Environmental Activator
Arrangement of DNA
 Antibody Diversity
 Light chains:
 Up to 300 Variable, 4 Joining and 1 Constant region

 300 x 4 = 1, 200 light chains

 Heavy chains:
 Up to 500 Variable, 4 Joining and 12 Diversity
regions and 12 constant regions
 500 x 4 x 12 = 24, 000 heavy chains

1200 x 24,000 = 28,800,000 antibody

molecules
 B. Transcriptional Regulation

1. Cis-acting element and Trans factors

What is cis-acting element?

DNA sequences close to a gene that are required
for gene expression
Promoter, UAS, Blocker seq etc
Enhancer
A regulatory DNA sequence that greatly
enhances the transcription of a gene.

Silencer
A DNA sequence that helps to reduce
or shut off the expression of a nearby gene.
Mediator Complex
(5) Insulators
No transcription
2. What is trans-acting factor?
 Concept
trans-acting factors - usually they are
proteins, that bind to the cis-acting elements to
control gene expression.
 These trans-acting factors can control gene
expression in several ways:

 may be expressed in a specific tissue

 may be expressed at specific time in development
 may be required for protein modification
(1) RNA polymerase
 prokaryotic RNA Pol
 eukaryotic RNA Pol

(2) Transcription factors

 Basal/general TFs
 Specific TFs
(3) Domains of trans-acting factors
 DNA binding domain DBD
 transcription activating domain
(Protein interacting Domain (PID)
a. HTH (helix-turn-helix) (in DBD)

α-helix (N-terminus)----specific
α-helix (C-terminus)----non-specific
 Helix Turn Helix motif: binds to DNA
A major structural motif capable of binding DNA
 b. Leu zipper
Present in both PBD and DBD
 c. Zinc finger (Present in DBD)
X2-Cys-X2,4-Cys-X12-His-X3,4,5-His
 Transcription factor Sp1, also known as specificity protein 1 is a
protein that in humans is encoded by the SP1 gene.

The encoded protein is involved in many cellular processes,

including cell differentiation, cell growth, apoptosis, immune
responses, response to DNA damage, and chromatin
remodeling.
SP1
Post-translational modifications
such as phosphorylation, acetylation,
glycosylation, and proteolytic
processing significantly affect
the activity of this protein, which
can be an activator or a repressor.[
d. Helix-loop-helix (DBD + PBD)
A basic helix-loop-helix (bHLH) is a protein structural motif that characterizes a
family of transcription factors.
 7.4 Co / post-Transcriptional Regulation
1. Gene Regulation of mRNA Processing
Alternative gene splicing Discussed
Alternative Splicing
2. Gene Regulation of mRNA Editing
3. mRNA Longevity
4. mRNA Transport Control
5. RNA Interference (RNAi)
 miRNA
 siRNA Discussed
 RNA EDITING
- includes the insertion, deletion, and base
substitution of matured m RNA (processed/spliced)
- RNA editing has been observed in some tRNA, rRNA, mRNA or
miRNA molecules of eukaryotes and viruses, archaea and prokaryotes.

-RNA editing is relatively rare.

-The common forms of RNA processing are splicing, 5'-
capping and 3'-polyadenylation and these are not usually
included as editing.
RNA editing occurs in the cell nucleus and cytosol, as well as
within mitochondria and plastids.
 RNA Apo-B gene
GTT ATT
EDITING

apolipoprotein B protein
Translational and Post-translational Regulation

1. Translation Control
Blocking mRNA Attachment to Ribosomes

2. Regulation of Protein Processing

Protein Modification
  Amino acid residus phosphorylated
 Ser/Thr type
 Tyr type

 Catalysis Features of Actions

 Integrated signals from different pathways effectively
 The same kind kinase or phosphatase is multible-substrates.
 modified different amino acids, different influences
3. Regulation of Protein Stability
7.6 Gene Regulation & Cancer
1. Features of Cancer Cells
2. Causes of Cancer:
 Viruses
 Tobacco smoke
 Food
 Radiation
 Chemicals
 Pollution
3. Proto-oncogene & Oncogene

 Proto-oncogene - is a normal gene that can

become an oncogene due to mutations or
increased expression.
 Oncogene - is a protein encoding gene, which -
when deregulated - participates in the onset and
development of cancer.
 Tumour suppressor gene - or antioncogene is a
gene that protects a cell from being cancer.
4. Proto-oncogene activation
5. p53 and cancer
 It can activate DNA repair proteins when DNA has
sustained damage.

 It can also hold the cell cycle at the G1/S regulation point
on DNA damage recognition

 It can initiate apoptosis, the programmed cell death, if

the DNA damage proves to be irreparable.
Summary
1. Genomic structure of eukaryotes
2. Regulation levels of eukaryotic gene expression.
3. Regulation at DNA level
4. Regulation at transcritional level
5. The mechanism of RNAi
6. Translational and post-translational regulation: