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Gene Regulatory
By
Fatchiyah
([email protected])
Dept. of Biology, Brawijaya University
5/23/2017
fatchiyah, JB-UB
Regulation of Genes
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What turns genes on (producing a
protein) and off?
Where (in which cells) is a gene
turned on?
When is a gene turned on or off?
How many copies of the gene product
are produced?
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Overview of Gene Control
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The mechanisms that control the
expression of genes operate at many levels.
source: Molecular Biology of the Cell (4th ed.), A. Johnson, et al.
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Multi-scale Model Problem
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Organizational Structure in Biology
– Single Cells (Building blocks)
– Tissues (organization of multiple cells)
– Organism
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Fundamental Cellular Processes
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Inputs (Signal Transduction)
Transcription
Translation
Outputs – adaptive processes
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DNA to chromosome
M ovie: DNA to Chromosome
The structure of DNA dictates the way it
encodes genetic information. A strand of
DNA consists of three types of fundamental
chemical units, repeated many times.
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Prokaryote regulation
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Lac operon
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Transcriptional Regulation
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The transcription of each gene is
controlled by a regulatory region of
DNA relatively near the transcription
start site (TSS).
two types of fundamental
components
– short DNA regulatory elements
– gene regulatory proteins that recognize
and bind to them.
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Gene Signals in DNA
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CpG islands; methylation patterns;
promotors
Polymerase binding site; cofactors
binding sites; enhancer; silencer
transcription start sites; introns
Donors; acceptors; branch point
Termination signals
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Regulation of Genes (1)
Transcription Factor
(Protein)
RNA polymerase
(Protein)
DNA
ATG
Regulatory Element
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Start site
Gene
Termination
site
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source: M. Tompa, U. of Washington
Regulation of Genes (2)
Transcription
ATG
DNA
Regulatory Element
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Gene
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source: M. Tompa, U. of Washington
Regulation of Genes (3)
ATG
DNA
mRNA
source: M. Tompa, U. of Washington
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Protein
Animation
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Transcription regulatory
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Post-Transcription regulation
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Post translation
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Cellular
Processes
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Alignment of CpG island
CpG island
mRNA
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TATA-box
Transcription start
(homology-based assignment)
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Gene Regulatory Motif
What is a motif?
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A subsequence (substring) that occurs in
multiple sequences with a biological
importance.
Motifs can be totally constant or have
variable elements.
Protein Motifs often result from structural
features.
DNA Motifs (regulatory elements)
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Binding sites for proteins
Short sequences (5-25)
Up to 1000 bp (or farther) from gene
Inexactly repeating patterns
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daf-19 Binding Sites in C.
elegans
-150
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source: Peter Swoboda
GTTGTCATGGTGAC
GTTTCCATGGAAAC
GCTACCATGGCAAC
GTTACCATAGTAAC
GTTTCCATGGTAAC
-1
che-2
daf-19
osm-1
osm-6
F02D8.3
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Splice Motifs in A/T rich
Organisms
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Motif Representing
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Consensus sequence: a single string with
the most likely sequence(+/- wildcards)
Regular expression: a string with wildcards,
constrained selection
Profile: a list of the letter frequencies at
each position
Sequence Logo:
– graphical depiction of a profile
– conservation of elements in a motif.
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Motif Logos: an Example
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(http://www-lmmb.ncifcrf.gov/~toms/sequencelogo.html)
Finding Regulatory Motifs
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Given a collection of genes with common
expression,
Find the (TF-binding) motif in common
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An Example: Hormonal Gene
Regulation
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To understand the cellular
mechanisms of hormonal (hydrophobic
and Hydrophilic) gene regulation
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Hormonal Gene regulation
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OBJECTIVES:
Describe basic mechanisms of gene regulation.
Describe gene regulation at transcriptional level.
Describe key elements present in the region of gene
involved in gene transcription.
Give a definition of Promoters, enhancers, Response
elements, Transcription factors and co-activators.
Aware that hydrophobic hormone receptor is a
transcription factor.
Describe basic organisation of steroid receptor molecule
Describe how hydrophobic hormones regulate gene
expression
Describe how hydrophilic
hormones
regulate gene
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JB-UB
expression via cellular signalling molecules.
Regulation at
Transcriptional level
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Transcription by tissue specific factors: muscle cell
specific transporter factor(myoD)- in myoblasts.
Ker1=keratinocyte differentiating factor-skin cells.
HNF-5=hepatic nuclear factor5- liver cells
Hormone, growth factor or cellular
messenger binding to response element:
glucocorticoid response element (GRE): binds
glucocorticoid receptor complex
Alternative promoter: dystrophin gene
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Promoter Regions
Many factor sites: c-fos, c-jun, c-myc/mac; Sp1, CREB, etc
CREB
C-myc/
max
Sp-1
C/EBP
RNA Pol II
tgacgca
cacgtg
gggcgg
ccatt
-100
tata
-35
Exon1
Exon2
Transcription ON
mRNA
TF II-B
TF II-A
TF II-D
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Protein
Transcriptional regulation
cis-acting transcriptional control
sequences: Promoter, Response elements;
Enhancers, silencer
 Transcription regulatory proteins
(Transcription
·
factors)
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Cis acting transcriptional
control sequences
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Promoters:
Located in the immediate upstream region, serves
to initiate transcription.
Consists of combination of short sequence
elements (ccaat box, tata box, gc box).
Recognised by ubiquitous transcription factors.
Promoters of gene that show tissue specific
expression pattern often include cis-acting
sequence elements that can be recognised by
tissue specific transcription factors.
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Cis acting transcriptional
control sequences
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Response elements:
Found in selected genes whose
expression is controlled by an external
factor. Located short distance
upstream of promoters.
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Cis acting transcriptional
control sequences
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Enhancers:
Positive regulatory elements, function
independent of both orientation and distance
from the genes they regulate. They contain
elements recognised by ubiquitous transcription
factors and tissue specific transcription factors.
Silencers:
negatively regulatory elements.
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Transcriptional regulatory
proteins (Transcription factors)
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Transcription is regulated by a large number of
DNA binding proteins.
These DNA binding proteins mostly bind the
response elements in a dimeric form, either
as homodimers or or as heterodimers.
The symmetry of these dimeric transcription factor
is matched by their response elements which
frequently posses an incomplete dyad symmetry.
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Structural motifs of the
regulatory DNA binding protein
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Helix-turn-helix (HTH) motif: mediated DNA binding by fitting
into major groove of the DNA helix, allowing precise
alignment of the factor in relation to DNA sequence
recognised
Helix –loop helix: promotes both DNA binding and
protein dimer formation.
Zinc Finger: loops or fingers of amino acids that have a zinc
ion at their core. The adjacent amino acid sequences form a
α-helices that makes contact with DNA in the major groove
Leucine Zipper: forms a dimer that grips the
DNAdouble helix like a peg, by inserting into the major
groove.
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DNA binding motifs in
Proteins
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Properties of dimeric
transcription factors.
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DNA binding module: Provides a non-selective
affinity for DNA and the ability to recognise the
specific base sequence of the response element.
A dimerisation module allows protein to form
an active dimeric state.
Transcription activation (or inhibition) region:
required for stimulation or inhibition of transcription
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Regulation of
transcription factors
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Level of synthesis
Covalent modification by phosphorylation
/desphosphorylation
Binding of small molecules can act as positive or
negative allosteric effectors
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Specific transcription
factors
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Tissue specific gene expression
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Cell growth and differentiation
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Response to lipid soluble hormones and second messenger
molecules
Cellular transcription factors required for the
transcription of viral gene.
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Specific Hormonal Gene
regulation
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Hormones regulate the function of their target cells by
receptor mediated pathways.
Water soluble hormones for which the lipid barrier of the
plasma membrane is impenetrable, interact with their
receptors at the cell surface
Hydrophobic hormones that are lipid soluble and therefore
can pass the cell membrane interact with another family of
receptors that are intracellular (cytosolic or nuclear). These
intracellular receptors function as hormone-regulated
transcription factors, controlling the expression of specific
target genes by interacting with regions known as hormone
response elements located close to the promoter in the
regulatory region
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Receptor-mediated induction of
gene transcription by steroid
hormones
Steroid ligands
Vacant Receptor
hsp90
Pre-initiation complex
RNA Pol-II + GTFs
Coactivator protein(s)
Activated
receptors
HRE
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TATA box
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Mechanism of gene activation by
the steroid hormone receptors
(transcription factors)
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The DNA binding domain is characterised by the
presence of two zinc fingers.
In the case of steroid hormones Zinc is coordinated by four cysteine residues.
In other Zinc fingers Zn can be co-ordinated by two
histidine and two cysteine residues.
Although these are referred to as fingers, the
tertiary structure is more like a fist made from beta
sheet and α helical structure. DNA interacting
residues are found in the α-helical part of the
molecule.
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Mechanism of gene regulation
by Hydrophilic hormones
Intracellular second messenger cAMP and
gene regulation.
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In the previous lecture you learnt that cAMP is produced as a
result of activation of adenylate cyclase by G protein which in
turn had been acivated by hormone which can not pass the
hydrophobic cell membrane.
This cAMP in turn acivates a cAMP depedent protein kinase
(PKA) which modulates multiple aspects of cell function. PKA
phosphorylates a transcription factor called CREB (cAMP
response element binding protein).
This is then translocated to the nucleus where it binds to a
short pallinodromic sequence in the promoter region of cyclic
AMP-regulated genes referred to as CRE.
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Thank You
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