GENE REGULATION IN EUKARYOTES

1. K. Narayanapura, Kothanur (PO), Bengaluru 560077
Tel+91 80 – 68737777 / 28465770 /28465353 Fax. 080- 68737799
e-mail:info@kristujayanti.com, www.kristujayanti.edu.in
Regulation of gene expression in
Eukaryotes
Dr. Manikandan Kathirvel
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
Bengaluru

2. Regulation of Gene Expression in
Eukaryotes
Gene expression is the combined process of :
1. the transcription of a gene into mRNA,
2. the processing of that mRNA, and
3. its translation into protein (for protein-encoding genes).
Levels of regulation of gene expression

3. Purpose of regulation of gene expression:
Regulated expression of genes is required for:
1)Adaptation- Cells of multicellular organisms respond to varying
conditions. Such cells exposed to hormones and growth factors
change substantially in – shape, growth rate and other characteristics
2) Tissue specific differentiation and development
•The genetic information present in each somatic cell of a organism is
practically identical.
•Cells from muscle and nerve tissue show strikingly different morphologies and
other properties, yet they contain exactly the same DNA.
•These diverse properties are the result of differences in gene
expression.
•Expression of the genetic information is regulated during
developmental stage of an organism and during the differentiation of
tissues and biologic processes in the multicellular organism.
•Transcription control can result in tissue-specific gene expression.

4. Differences between prokaryotic and eukaryotic gene expression:
1) Absence of operons: Prokaryote gene expression typically is
regulated by an operon, the collection of controlling sites adjacent
to polycistronic protein-coding sequences.
• Eukaryotic genes also are regulated in units of protein-coding
sequences and adjacent controlling sites, but operons are not
known to occur.
2.) Complexity: Eukaryotic gene regulation is more complex because
eukaryotes possess a nucleus.
3) Different cell types: Different cell types are present in most
eukaryotes. Liver and pancreatic cells, for example, differ dramatically
in the genes that are highly expressed. Different mechanisms are
involved in the regulation of such genes.
4.) Uncoupled transcription and translation processes: transcription and
translation are not coupled).
i. In prokaryotes, transcription and translation are coupled processes, the primary transcript is
immediately translated.
ii. The transcription and translation are uncoupled in eukaryotes, eliminating some potential
generegulatory mechanisms.
iii. The primary transcript in eukaryotes undergoes modifications to become a mature functional m RNA.

5. 5) Chromatin structure (in eukaryotes)
- The DNA in eukaryotic cells is extensively folded and packed into the
protein-DNA complex called chromatin.
Histones are an important part of this complex since they both form
the structures known as nucleosomes and also contribute significantly
into gene regulatory mechanisms.
- Heterochromatin (tanscriptionally inactive) and Euchromatin
(transcriptionally active)
Regulation of gene expression in eukaryotes:
Two “categories” of eukaryotic gene regulation exist:
 Levels of control of gene expression
Short term control:
Genes are quickly turned on or off in response to the
environment and demands of the cell.
(to meet the daily needs of the organism)
Long term control
(genes for development/differentiation)

6. Mechanism of regulation of gene expression- An
overview
•Gene activity is controlled first and foremost at the level of
transcription.
•Much of this control is achieved through the interplay
between proteins that bind to specific DNA sequences and
their DNA binding sites.
•This can have a positive or negative effect on transcription.
•Transcription control can result in tissue-specific gene
expression.
•In addition to transcription level controls, gene
expression can also be modulated by translational
level.
Eukaryotic gene expression is controlled by:
•Cis-Trans acting elements- Transcriptional activation/
repression
•Histone modifications
•Chromatin modifications
•Regulation by noncoding RNA

7. 1. Cis-Trans acting elements: Transcriptional activation/Repression:
Transcriptional control of gene regulation is controlled by:
a) Promoters
b) Enhancers
c) Silencers
d) Transcriptional factors-Activators
e) repressors

8. 1) Cis and Trans acting elements:
Promoters
1. Transcriptional activation:

9. Promoters

10. Promoters
• Occur upstream of the transcription start site.
• Some determine where transcription begins (e.g., TATA),
whereas others determine if transcription begins.
• Promoters are activated by specialized transcription factor (TF)
proteins (specific TFs bind specific promoters).
• Transcription of rRNA – Transcriptional factor- I
• Transcription of mRNA- Transcriptional factor- II
• Transcription of tRNA- Transcriptional factor -III
• One or many promoters (each with specific TF proteins) may
occur for any given gene.
• Promoters may be positively or negatively regulated.

11. Enhancers and Repressors
i. Enhancer elements are regulatory DNA sequences, although they have no promoter activity of
their own but they greatly increase the activities of many promoters in eukaryotes.
ii. Enhancers function by serving as binding sites for specific regulatory proteins such as
transcriptional factors., when bound by transcription factors, enhance the transcription of an
associated gene.
iii. An enhancer is effective only in the specific cell types in which appropriate regulatory proteins
are expressed.
iv. Enhancer elements can exert their positive influence on transcription even when separated by
thousands of base pairs from a promoter;
v. they work when oriented in either direction; and they can work upstream (5') or downstream (3')
from the promoter.
vi. Enhancers are promiscuous; they can stimulate any promoter in the vicinity and may act on more
than one promoter.
• Occur upstream or downstream of
the transcription start site or within
the coding sequence
• Regulatory proteins bind specific
enhancer sequences; binding is
determined by the DNA sequence.
• Interactions of regulatory proteins
determine if transcription is activated
or repressed (positively or negatively
regulated).

12. Enhancers and Transcription
In some eukaryotic genes, there are regions
that help increase or enhance transcription.
These regions, called enhancers, are not
necessarily close to the genes they enhance.
They can be located upstream of a gene, within
the coding region of the gene, downstream of a
gene, or may be thousands of nucleotides
away.
Enhancer regions are binding sequences, or
sites, for transcription factors.
When a DNA-bending protein binds to an
enhancer, the shape of the DNA changes. This
shape change allows the interaction between
the activators bound to the enhancers and the
transcription factors bound to the promoter
region and the RNA polymerase to occur.
Therefore, a nucleotide sequence thousands of
nucleotides away can fold over and interact
with a specific promoter.
An enhancer is a DNA sequence that promotes
transcription. Each enhancer is made up of
short DNA sequences called distal control
elements. Activators bound to the distal
control elements interact with mediator
proteins and transcription factors.

13. a) Change in topology. The enhancer (E, blue)
and promoter (P, orange) are both located
on a loop of DNA. Binding of a gene-specific
transcription factor (green) to the enhancer
causes supercoiling that facilitates binding
of general transcription factors (yellow) and
polymerase (red) to the promoter.
(b) Sliding. A transcription factor binds to the
enhancer and slides down the DNA to the
promoter, where it facilitates binding of general
transcription factors and polymerase.
(c) Looping. A transcription factor binds to the
enhancer and, by looping out the DNA in
between, binds to and facilitates the binding of
general transcription factors and polymerase to
the promoter.
(d) Facilitated tracking. A transcription factor
binds to the enhancer and causes a short DNA
segment to loop out downstream. Increasing
the size of this loop allows the factor to track
along the DNA until it reaches the promoter,
where it can facilitate the binding of general
transcription factors and RNA polymerase.
Four hypotheses of enhancer action-
Interaction of enhancer with promoter

14. Trans acting elements

16. Transcriptional Activators
Activators can either stimulate or inhibit transcription by RNA polymerase II,
and
they have structures composed of at least two functional domains:
a DNA-binding domain
and a transcription-activating domain.
Many also have a dimerization domain that allows the activators to bind to
each other, forming homodimers (two identical monomers bound together),
heterodimers (two different monomers bound together), or even higher
multimers such as tetramers.
Some even have binding sites for effector molecules like steroid hormones.

17. The GAL4 Protein
The GAL4 protein is a yeast activator that
controls a set of genes responsible for
metabolism of galactose.
Each of these GAL4-responsive genes contains
a GAL4 target site (enhancer) upstream of the
transcription start site.
These target sites are called upstream
activating sequences, or UASGs.
GAL4 binds to a UASG as a dimer.
Its DNA-binding motif is located in the first 40
amino acids of the protein, and its dimerization
motif is found in residues 50–94.
Conditions:
• When galactose is absent, the GAL4 product
(GAL4p) and another product (GAL80p) bind
the UASG sequence; transcription does not
occur.
Conditions:
• When galactose is added, a galactose
metabolite binds to GAL80p and GAL4p
amino acids and are phosphorylated.
• Galactose acts as an inducer by causing a
conformation change in GAL4p/GAL80p.
Transcriptional Activators

18. Dimerization of activators
• Dimerization is a great advantage to
an activator because it increases the
affinity between the activator and its
DNA target.
• Some activators form homodimers,
but others function as heterodimers.
• Example: GAL4 activator

19. Silencers:
•The elements that decrease or repress the expression of specific genes have
also been identified called Silencers.
•Silencers are control regions of DNA that, like enhancers, may be located
thousands of base pairs away from the gene they control.
•However, when transcription factors bind to them, expression of the gene they
control is repressed.

21. MYC gene encodes a multifunctional,
nuclear phosphoprotein that controls a
variety of cellular functions, including
cell cycle, cell growth, apoptosis,
cellular metabolism and biosynthesis,
adhesion, and mitochondrial
biogenesis.

22. 2) Chromatin Remodeling
• Chromatin structure provides an important level of control of gene
transcription.
• The development of specialized organs, tissues, and cells and their function in
the intact organism depend upon the differential expression of genes.
• Some of this differential expression is achieved by having different regions of
chromatin available for transcription in cells from various tissues.
Large regions of chromatin are
transcriptionally inactive in some cells,
while they are either active or
potentially active in other specialized
cells.
For example, the DNA containing the -
globin gene cluster is in "active"
chromatin in the reticulocytes but in
"inactive" chromatini n muscle cells.

23. MAJOR CLASSES OF CHROMATIN-REMODELING
COMPLEXES
Chromatin
remodeling
complex
ATP Independent
modeling complexes
ATP Dependent
modeling complexes
Histone modifications
The ATP dependent remodeling
complexes require ATP hydrolysis for
modification of architecture of
nucleosome which helps to expose
the required sequence in DNA for
gene expression.
The enzymes are as follows:
ISWI (imitation switch )
SWI/SNF (Switching of mating
types/ sucrose non fermenting)

24. EFFECTS OF HISTONES ON TRANSCRIPTION ACTIVATION
 Histone modification – post
translational modification includes
methylation, phosphorylation,
acetylation, ubiquitylation,
summoylation.
 DNA wrap around the histone
octamer in a structure like beads
on string ,which makes the basic
chromatin unit.
 Chromatin folds into higher level
structures, helps to determine the
DNA accessibility.
 The transcriptional machinery
cannot access the DNA and genes
remain inactive.

25. Histones
 Histones are a group of basic proteins that
associate with DNA to condense it into
chromatin.
 Histones contain a large proportion of the
positively charged (basic) amino acids ,
lysine and arginine in their structure.
 DNA is negatively charged due to the
phosphate groups on its backbone.
 The results of these attraction and therefore
high binding affinity between histones and
DNA structure called nucleosomes.
 DNA wraps around histones, they also
play a role in gene regulation.
 The basic unit of chromatin is the
nucleosome core particles, which contains
147 bp of DNA wrapped nearly twice around
an octamer of the core histones.
 Each nucleosome is separated by 10-60 bp
of “linker” DNA, and the resulting
nucleosomal array constitutes a chromatin
fiber of 10 nm in diameter.

26.  Two types of Histones:
1) Core Histones- H2A, H2B, H3,
H4
2) Linker Histones- H1
 The eight histones in the core are
arranged into a (H3)2(H4)2
tetramer and a pair of H2A-H2B
dimers.
 The tetramer and dimers come
together to form a left- handed
superhelical ramp around which
the DNA wraps.
 Hydrogen bonds between the
DNA backbone and the amide
group on the main chain of
histone proteins.

27. Formation and disruption of
nucleosome structure:
• The presence of nucleosomes
and of complexes of histones and
DNA provide a barrier against the
ready association of transcription
factors with specific DNA regions.
1. Chromatin composed of cells DNA and
associated proteins.
2. There are five histone proteins in the family
H1,H2A,H2B,H3,and H4.
3. Two H3 and two H4 proteins form a tetramer
which combines with two H2A,H2B dimers to
form the disk shaped histone core .
4. 150bp of DNA wrap around the protein about
twice making a nucleosome core particle with
linker histone and linker DNA.
5. Linker DNA varies in length ( 10 and 90bp).
6. Nucleosome repeats every 200bp and is close to
10nm diameter.

28. Histone modification
 N-terminal tails of histones are the most
accessible regions of these peptides as they
protrude from the nucleosome and possess
no specific structure.
The amino-terminal portion of the core histone proteins contains a flexible and
highly basic tail region, which is conserved across various species and is
subject to various PTM.
Chromatin can be highly packed or loosely packed, and correlated to the gene
expression levels.
Post-translational modifications(PTM) of histones is a crucial step in
epigenetic regulation of a gene.
Modifications in histone proteins affects the structure of chromatin.
Gene regulation
DNA damage and repair
Chromosome condensation

29. Types of histone modification
 N-terminal tails of all histones are particularly of interest since they
protrude out of the compact structure. These N-terminal tails are often
subjected to a variety of post-translational modifications such as,
a) Acetylation
b) Methylation
c) Phosphorylation
d) Ubiquitination
e) Sumoylation
f) ADP ribosylation

30. The disruption of nucleosome structure
is therefore an important part of
eukaryotic gene regulation and the
processes involved are as follows:
i) Histone acetylation and deacetylation
Acetylation is known to occur on lysine
residues in the amino terminal tails of
histone molecules.
This modification reduces the positive
charge of these tails and decreases the
binding affinity of histone for the
negatively charged DNA.
Accordingly, the acetylation of histones
could result in disruption of
nucleosomal structure and allow
readier access of transcription factors
to cognate regulatory DNA elements.

31. N-terminal tails are
reversible
acetylated in Lys,
particularly in
H3+H4.
 Acetyl group
addition to
lysine in histone
tails loosens
nucleosome
grip on DNA by
neutralizing
positive charge.
i) Histone acetylation and deacetylation

32. i) Histone acetylation and deacetylation

33. ATP INDEPENDENT REMODELING COMPLEX

34. ii) Methylation
 It is the introduction of an methyl functional group to only on Lysine or Arginine
of the histone tail.
 These reactions are catalysed by enzymes like histone methyltransferases
(HMTs).
 Histone lysine methyl transferases (HKMTs) methylate Lysine (K) residues.
 Protein argenine methyl transferases (PRMTs) methylate Arginine (R)
residues.
 A role in both activation and repression.
 Arginines can be mono or di methylated whereas lysines can be mono, di , tri
methylated.

35. ii) Methylation
Methylation of deoxycytidine residues in DNA may effect gross changes
in chromatin so as to preclude its active transcription.
Example: Acute demethylation of deoxycytidine residues in a specific region of
the tyrosine aminotransferase gene—in response to glucocorticoid hormones—has
been associated with an increased rate of transcription of the gene.

37. iii) Ubiquitination
 Ubiquitination (or ubiquitylation)
refers to the post translational
modification of the amino group of a
lysine residue by the covalent
attachment of one
(monoubiquitination) or more
(polyubiquitination) ubiquitin
monomers.
 Ubiquitin is a 76 amino acid protein
highly conserved in eukaryotes.
 Histone Ubiquitination alters
chromatin structure and allows the
access of enzymes involved in
transcription.
 Ubiquitination is carried out in three
steps: activation, conjugation and
ligation, performed by ubiquitin-
activating enzymes (E1s), ubiquitin-
conjugating enzymes (E2s) and
ubiquitin ligases (E3s), respectively.

38. v) Sumoylation:
 Small ubiquitin like modifier
(SUMO) proteins are a
family of small proteins that
are attached to and
detached from other
proteins in cell to modify
their function.
 Sumoylation consists in the
addition of a small ubiquitin
related modifier protein
(SUMO) of 100 amino acids.
 Histone Sumoylation has a
role in transcription
repression by opposing
other active marks such as
Acetylation, methylation,
Ubiquitination, etc.
iv) Phosphorylation
 Phosphorylation is the addition of a
phosphate group (PO43-) to a
molecule.
 Phosphorylation is catalyzed by
various specific protein kinases.
 Histones are phosphorylated and the
most studied sites of histone
Phosphorylation are the serine 10 of
histone H3 (H3S10)

39. vi) DNA binding proteins
•The binding of specific transcription factors to certain DNA elements may result
in disruption of nucleosomal structure.
•Many eukaryotic genes have multiple protein-binding DNA elements.
•The serial binding of transcription factors to these elements may either directly
disrupt the structure of the nucleosome or prevent its re-formation.
•These reactions result in chromatin-level structural changes that in the
end increase DNA accessibility to other factors and the transcription
machinery.

40. Studies postulate that SWI2/SNF2 and related proteins can function
to destabilize nucleosome structure and thereby to facilitate the
binding of transcription factors to chromatin.
SWI2/SNF2:
Genetic studies of transcriptional regulation in Saccharomyces cerevisiae led to
the identification of a number of SWI and SNF genes (SWI refers to yeast
mating type swi tching, while SNF is an abbreviation for s ucrose n on f
ermenting.
The gene encoding the first SNF2/SWI2 enzyme was discovered by the yeast
geneticists Ira Herskowitz and Marian Carlson in the 1980s.

41. SNF2 protein
A SNF2 protein is an enzyme that belongs to the SF2 helicase-
like superfamily, and it is the founding member of a subfamily of
enzymes called SNF2-like helicases, which all harbor a conserved
helicase-related motifs similar to SNF2.
The SNF2 family proteins have multiple members, which are
approximately 30 different enzymes in human cells and 17
different enzymes in budding yeast.
SNF2 enzymes can be further classified into six groups
based on the structure of the helicase domain. These groups
are Swi2/Snf2-like, Swr1-like, SS01653-like, Rad54-like, Rad5/6-
like, and distant (SMARCAL1) enzymes.
 Many of the SNF2 enzymes have been shown to remodel
chromatin in vitro in an ATP-dependent manner, and several
enzymes remain to be tested.

42. SWI2/SNF2 Complex:
Experiments revealed that the SWI/SNF complex possesses a DNA-
stimulated ATPase activity and can destabilize histone-DNA
interactions in reconstituted nucleosomes in an ATP-dependent
manner, though the exact nature of this structural change is not known.
In addition, this SWI2/SNF2-mediated destabilization of nucleosomes was
found to increase the binding of transcription factors, such as GAL4
derivatives or the TATA box-binding protein (TBP), to the histone-associated
DNA.
These results, combined with the genetic data, led to the hypothesis that the
SWI/SNF complex facilitates the binding of transcription factors to
chromatin.

44. A Simple Model Depicting a Suggested
Mechanism for the Destabilization of
Nucleosomes by SWI/SNF Complex
and Related Factors by ATP-Driven
Translocation of the Protein along
Nucleosomal DNA
ATP DEPENDENT REMODELING
COMPLEXES

45. Members of the SNF2-like family exhibit an impressive range of
biological functions.
These activities include
•gene-specific transcriptional activation,
•transcriptional repression,
•destabilization of reconstituted nucleosomes,
•transcription-coupled repair,
•nucleotide excision repair of nontranscribed regions of the
genome,
•recombination repair,
•and chromosome segregation.
SNF2-like family members are also involved in human disease.
•Mutations in the human ERCC6 gene can lead to Cockayne's syndrome,
which is characterized by progressive neurodegeneration, dwarfism,
photosensitivity, and developmental abnormalities.
• In addition, mutated forms of the human ATR-X gene (also known
as NUCPRO; tentatively assigned to the RAD54 subfamily) cause a
combined α-thalassemia and mental retardation syndrome

46. NURF—A Complex Containing ISWI, a
Member of the SNF2L Subfamily
The analysis of an ATP-dependent activity
that is required to alter nucleosome
structure upon binding of the GAGA
transcription factor (a sequence-specific
DNA-binding factor in Drosophila) has led
to the purification of a factor termed NURF
(Nucleosome Remodeling Factor) from
Drosophila embryos.
NURF is an ∼0.5 MDa complex that
contains four polypeptides, one of which is
the ISWI (imitation switch) protein.
ISWI is a member of the SNF2L subfamily,
which is closely related to the SNF2
subfamily. At present, downstream targets
of ISWI are not known.
ISWI, a Member of the SWI2/SNF2
ATPase Family, Encodes the 140 kDa
Subunit of the Nucleosome Remodeling
Factor