Gene Regulation in

Eukaryotes
 Elements involved

• Activators
• Repressors
• Mediator
• Insulators
• Molecular Mechanisms
 Activators – Positive control,
turns genes on.
Activators regulated by – Ligand binding ,
Phosphorylation , Dimerization ,Proteolysis etc.
Two domains – DNA binding domain & Activating
Domain.
DNA binding domain helps in binding to a
particular DNA sequence by interaction within the
major groove of DNA sequence, paricularly H-
Bonds.
Activating domain helps in binding of RNA
polymerase to promoter or change the
conformation from a bound form to active form.
 Function
• 1 Stimulation of the recruitment and binding of general
transcription factors and RNA pol II to the corepromoter
to form a preinitiation complex.

• 2 Induction of a conformational change or post-

translational modification (such as phosphorylation) that
stimulates the enzymatic activity of the general
transcription machinery.

• 3 Interaction with chromatin remodeling and modification

complexes to permit enhanced accessibility of the
template DNA to general transcription factors or specific
activators.
 Site of binding
• Upstream Activating Sequence
• Ex – In yeast GAL1 gene activating
protein.GAL4 binds to UAS 275 bp
upstream of GAL1.
 Enhancers
A typical protein-coding gene is likely to contain several
enhancers which act at a distance. These elements
are usually 700–1000 bp or more away from the start of
transcription. The hallmark of enhancers is that,
unlike promoter elements, they can be downstream,
upstream, or within an intron, and can function in
either orientation relative to the promoter . A typical
enhancer is around 500 bp in length
and contains in the order of 10 binding sites for several
different transcription factors. Each enhancer is
responsible for a subset of the total gene expression
pattern.
 Activators recruit Nucleosome modifiers that help in
binding of transcriptional machinery to bind at
promoter

 Histone acetyl transferases

 Chromatin remodelling complexes like
ATP dependent activity of SWI/SNF
 Locus Control region
• LCRs are DNA sequences that organize
and maintain a functional domain of active
chromatin and enhancethe transcription of
downstream genes. Although sometimes
referred to as “enhancers” of
transcription.LCRs, unlike classic
enhancer elements, operate in an
orientation-dependent manner.
• Found 30-50 kb upstream of the cluster of
globin genes.
• Made up of multiple sequence elements :
Enhancers, Insulators, promoters.
 Multiple Activators Work
Synergistically

 Cooperative Binding
 Binding to a common third protein
 First protein recruits a nucleosome
remodeller which creates a site for second
protein
 Binding of first protein unwinds DNA
pg 545
 Examples

• HO gene expression- SWI5 / SBF

SWI5 recruits nucelosome modifiers , SBF interacts with mediators.

• The human β-interferon gene is activated in cells upon

viral infection. Infection triggers three activators :
NFκB, IRF, and Jun/ATF. They bind cooperatively
to sites within an enhancer located 1 kb upstream of the
promoter, to form a structure called enhanceosome.
 Mediator

• 20-subunit complex, which transduces

regulatory information from activator and
repressor proteins to RNA pol II.
• serves as a molecular bridge that
connects transcriptional activators bound
at enhancers, or other long-range
regulatory elements, with RNA pol II
Transcriptional Repressors

• Bind to sites overlapping promoter hence

inhibit binding of RNA Polymerase
• Binds to sites adjacent promoter and
inhibit the enzyme
• Interfere with action of activators
 Mechanisms used:-

• Competetion
• Inhibiton
• Direct Repression
• Indirect Repression – Histone
Deacetylation & DNA Methylation.
Pg 550 -
Watson
 Example

Glucose +ve
 Mig 1 binds btw GAL 1 & UAS
Recruits Tup1
 Repress Expression of GAL1
 Insulators
 An insulator is a DNA sequence element,
typically 300 bp to 2 kb in length, that has two
distinct functions
 Chromatin boundary marker: an insulator marks
the border between regions of heterochromatin
and euchromatin.
 Enhancer blocking activity: an insulator
prevents inappropriate cross-activation or
repression of neighbouring genes by blocking
the action of enhancers and silencers.
• Insulators prevent transcriptional silencing.
 Specialized form of repression that can
spread along chromatin switching off
multiple genes, without the need for each
to bear binding sites for specific
repressors.
 Hence, prevents both indiscriminate
activation and repression.
 Molecular Mechanisms
• Gene Silencing

 Modification of Histones
 Modification of DNA
 Gene Silencing
• Positional effect- Gene silenced due to its
position.
• Heterochromatin – most common form of
silencing is associated with it.

• Transcription can be silenced by

mehtylation of DNA by DNA methylases.
 Control at DNA level by DNA
methylation
– Heterochromatin is the most tightly packaged form of
DNA.
transcriptionally silent, different from cell to cell

– Methylation is related to the Heterochromatin

formation

• Small percentages of newly synthesized DNAs (~3% in

mammals) are chemically modified by methylation.

• Methylation occurs most often in symmetrical CG

sequences.

• Transcriptionally active genes possess significantly lower

levels of methylated DNA than inactive genes..
 DNA methylation
• Methylation of DNA blocks transcription factors
– no transcription = genes turned off
– attachment of methyl groups (–CH3) to cytosine
• C = cytosine
– nearly permanent inactivation of genes
• ex. inactivated mammalian X chromosome
 Histone acetylation
• Acetylation of histones unwinds DNA
– loosely packed = transcription
– = genes turned on
• attachment of acetyl groups (–COCH3) to histones
– conformational change in histone proteins
– transcription factors have easier access to genes
 Control at DNA level by Histone
modifications(Chromatin Remodeling)

• Acetylation by HATs
and coactivators leads to
euchromatin formation

•Chromatin Remodelling
complexes like SWI/SNF
help activate
transcription.
 Switching a gene off

I. Methylation of DNA sequence

II. Recognized by proteins which
recognize methylated DNA, Ex- MeCP2
III. Recruitment of Histone Methylases/
Deacetylases.
IV. Gene completely switched off.