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ADDITION BY ME

CHAPTER 20 (GENES X)

PROMOTERS AND ENHANCERS

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Eukaryotic gene control: purposes and general principles

• Unlike bacterial cells and most single cell eukaryotes, cells in multicellular organisms have relatively few genes that are reversibly regulated by environmental conditions

• However, gene control in multicellular organisms is important for development and differentiation, and is generally not reversible

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Regulatory elements in eukaryotic DNA often are many kilobases from start sites

• The basic principles that control transcription in bacteria also

apply to eukaryotic organisms: transcription is initiated at a

specific base pair and is controlled by the binding of trans-

acting proteins (transcription factors) to cis-acting regulatory

DNA sequences

• However, eukaryotic cis-acting elements are often much

further from the promoter they regulate, and transcription

from a single promoter may be regulated by binding of

multiple transcription factors to alternative control elements.

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Promoter sequence components can beidentified by:

Transcription control sequences can be identified by analysis of a 5’-deletion series.

Finding precise consensus sequence that is bound by a particular factor in multiple promoters.

Introduction of point mutations at particular base pairs and testing the promoter function.

Finding the proteins which bound to promoters by footprinting and mutation.

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Transcription control sequences can be identified by analysis of a 5’-deletion series.

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REPORTER GENES

The primary objective is to identify gene regulatory sequences by testing a large number of deletion and point mutants in an in vivo cell transfection assays.

Promoter mapping studies can be done by creating artificial “reporter gene” by fusing regulatory region of the test gene to a heterologous gene coding sequences that direct the synthesis of a readily detectable protein product.

The steady state level of the reporter protein in the transfected cell is directly correlated to the steady state level of reporter mRNA, then it is possible to use protein based reporter assays for promoter mapping experiments.

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Repoter gene constructs

When trying to determine what are the cis-acting regulatory regions

that control the expression (transcription) levels of a gene, is usually

routine to clone a large piece of DNA containing these sequences and

hook it to a downstream sequence that encodes an easy-to-measure

protein called a reporter.

This is a lot more convenient and accurate than trying to estimate the

absolute levels of mRNA.

In vivo transgene reporter expression driven by the promoter of

interest will reveal the temporal/spatial pattern of expression of the

gene, measured by an easier method than the alternative in situ

hybridization.

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Reporter genes:

1. must encode a protein activity that is

similar to one already present in the cell.

2. The protein assay should be sensitive

enough, reproducible and easy to perform.

3. The reporter protein function should not

interfere with host cellular processes in a way

that will alter intracellular signaling pathways

or metabolic rates.

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Repoter gene constructs:

The most used reporters are:bacterial -galactosidase,chloramfenicol acetyl transferase (CAT),fire-fly luciferaseand most recently, the green fluorescent protein (GFP) which

allows measurements in living cells or organisms.

Part of the whole of a protein rather than a promoter can also be fused in frame to this reporters to find out where it localizes. Reporter gene must not being expressed endogenously in the cell types that you are working on, so their background should be null.

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How does one identify transciptional control mechanisms?

promotertranscriptional unit

• promoter & transcriptional unit are independent

reporter gene (e.g. luciferase)

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Use reporter to identify promoter sequence

promoter?transcriptional unit

reporter gene (e.g. luciferase)

promoter?

promoter?

promoter?

promoter?

1

2

3

4

5

luciferaseactivity

1 2 3 4 5

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Use reporter to identify promoter sequence

promoter

luciferaseactivity

1 2 3 4 5

= AAAATCACCCCACTGCAA

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Purify Transcription Factor

AAAATCACCCCACTGCAA

Affinity chromatography

affinitycolumn

1) pour nuclear extract2) allow to bind3) elute transcription

factor

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Gene Promoter Reporter Gene

Start site of Transcription

Transcriptional Reporter Gene Assay

+1

Translational Start Site

ATG

-2000

Chloramphenicol Acetyltransferase

Luciferase

LDL Receptor Promoter-141-- +36

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Construction and analysis of a 5-deletion series

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Identification of transcription-control elements with linker mutants

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asUsing reporter protein to

determine the pattern of a gene’s expression.

A) In this example the coding sequence for protein X is replaced by the coding sequence for Y protein.

B) B) Various fragments of DNA containing candidate regulatory sequences are added in combinations. The recombinant DNA molecules are then tested for expression after their transfection into a variety of different cell types of mammalian cells, and the result is summarized in C.

For experiments in eukaryotic cells, two commonly used reporter proteins are enzymes b-

galactosidase (b-gal) and green fluorescent protein or GFP.

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METHODS FOR STUDYING DNA-PROTEIN

INTERACTIONS

A. DNA footprinting

B. Electrophoretic mobility shift assay

C. Chromatin immunoprecipitation assay

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DNA FOOTPRINTING

identify protein-DNA interactions

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DNase I footprinting assays identify protein-DNA interactions

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Electrophoretic mobility shift assay

identify protein-DNA interactions

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Gel-shift assays identify protein-DNA interactions

Fragment bound to proteins are retarded

The free DNA fragment migrate rapidly to the bottom of the gel

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ChIP (Chromatin ImmunoPrecipitation)

Chromatin immunoprecipitation, or ChIP, refers to

a procedure used to determine whether a given

protein binds to a specific DNA sequence in vivo

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DNA-binding proteins are crosslinked to DNA with formaldehyde in vivo

Isolate the chromatin. Shear DNA along with bound proteins into small fragments.

Bind antibodies specific to the DNA-binding protein to isolate the complex by precipitation. Reverse the cross-linking to release the DNA and digest the proteins.

Use PCR( Polymerase Chain Reaction ) to amplify specific DNA sequences to see if they were precipitated with the antibody

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Chromatin immunoprecipitation

This methodology allows the identification

of sites in a genome that are occupied in

vivo by a gene regulatory protein. The

identities of the precipitated, amplified

DNA fragments can be determined by

hybridizing the mixture of fragments to

DNA microarrays.

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ChIP: Chromatin immunoprecipitation

(formaldehyde)

Shearing of DNA

Antibody against acetyl’edH3 & H4

Gene promoter is amplified using primers specific for that region

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Genes can be expressed with different efficiencies.Gene A is transcribed and translated much more efficiently than gene B. This allows the amount of protein A in the cell to be much greater than that of protein B.

From DNA to RNA

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DNA is transcribed by the enzyme RNA polymerase. The RNA polymerase (pale blue) moves stepwise along the DNA, unwinding the DNA helix at its active site. As it progresses, the polymerase adds nucleotides (here, small “T” shapes) one by one to the RNA chain at the polymerization site using an exposed DNA strand as a template. The RNA transcript is thus a single-stranded complementary copy of one of the two DNA strands. The polymerase has a rudder that displaces the newly formed RNA, allowing the two strands of DNA behind the polymerase to rewind. A short region of DNA/RNA helix (approximately nine nucleotides in length) is therefore formed only transiently, and a “window” of DNA/RNA helix therefore moves along the DNA with the polymerase. The incoming nucleotides are in the form of ribonucleoside triphosphates (ATP, UTP, CTP, and GTP), and the energy stored in their phosphate–phosphate bonds provides the driving force for the polymerization reaction (see Figure 5–4).

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Cells produce several types of RNA

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RNA polymerase I rRNA transcriptionRNA Polymerase II mRNA transcriptionRNA Polymerase III tRNA and other small RNAs

Promoters

• RNA polymerase I and RNA Polymerase II are mostly upstream of the start point.

• RNA Polymerase III lie downstream of the start point.

• Each promoter contains characteristic sets of short conserved sequences that are recognized by the appropriate class of factors.

RNA pol I and III recognize restricted set of promoters, rely upon a small number of accessory factors.

RNA pol II show more variation in sequence, cis acting elements are spread out over a region of >200bp.

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Start TerminationExon ExonIntronPromoterEnhancer

Mammalian gene

Promoter: DNA sequence located 5’ to a gene that is the sites where transcription is initiated. Example: TATA box.

It is the binding sites for transcription factors.

Enhancer: Eukaryotic control element that can increase expression of a gene. Located some distance from the gene either up- or downstream.

Human gene number estimate: 28,000–34,000 genes.

Gene

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Figure 24.01: Transcription is controlled by a promoter and enhancer.

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Figure 24.02: RNA polymerase has >10 subunits.

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RNA POLYMERASE I PROMOTER

• It transcribes genes for rRNA from a single type of promoter• It consists of a bipartite sequence• The core promoter and upstream control element (UCE)

The core promoter:Surrounds the start point (-45 to +20)It is sufficient to start transcription

UCE: -180 to -170

Both regions are rich in G.C basesThey are ~85% identical

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RNA polymerase I requires two factors:

• The factor UBF1 wraps DNA around a protein structure to bring the core and UPE into proximity.

• SL1 includes the factor TBP that is involved in initiation by all three RNA polymerases.

• RNA polymerase binds to the UBF1-SL1 complex at the core promoter.

SL1 consists of 4 proteins:

3 TBP-associated factors (TAF1) + 1 TBP (TATA box binding protein)

TBP is necessary to start transcription for RNA polymerase II and III as well.

TBP is well conserved between species

TBP does not directly bind to G.C rich DNA. Other factors are also necessary.

47Figure 24.05: Pol I promoters have two sequence components.

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RNA Polymerase III promoter:

The promoters have two general classes

1) Promoters for 5S and tRNA genes are internal. They lie

downstream of the start point between positions +55

and +80 within the gene.

2) The promoters for snRNA genes lie upstream of the

start point.

3) There are two types of internal promoters: Type I and

Type II

Type I Box A and Box C

Type IIBox A and Box B

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There are three accessory factors:

TFIIIAZinc finger protein

TFIIIB TBP and two proteins (B” and BRF proteins)

TFIIIC contains 5 subunit, >500 kD

TFIIIA and TFIIIC do not fully affect the initiation

reaction (assembly factors), their role is to bind TFIIIB

at the right location.

TFIIIB is crucial. It allows RNA pol III to bind at the

start site point (initiation factor or positioning factor).

50Figure 24.06: Pol III promoters may be downstream of startpoint.

• Internal promoters:– have short consensus sequences

located within the transcription unit

– cause initiation to occur a fixed distance upstream

• Upstream promoters contain three short consensus sequences upstream of the startpoint that are bound by transcription factors.

51Figure 24.07: There are three types of pol III promoters.

5sRNA

tRNA

SnRNA

They increase the efficiency of transcription

52Figure 24.08: Type 2 internal promoters use TFIIIC.

tRNA

• TFIIIA and TFIIIC bind to the consensus sequences and enable TFIIIB to bind at the startpoint.

• TFIIIB has TBP as one subunit and enables RNA polymerase to bind.

TFIIIB Is the Commitment Factor for Pol III Promoters

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Figure 24.09: Type 1 pol III promoters use TFIIIA/C.

5sRNA

Assembly factor (TFIIIA) ensure that TFIIIB (which includes TBP is bound just upstream of

the start point, to provide the positioning information.

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The Startpoint for RNA Polymerase II

• RNA polymerase II requires general transcription factors (called TFIIX) to initiate transcription.

• RNA polymerase II promoters have a short conserved sequence Py2CAPy5 (the initiator InR) at the startpoint.

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• The TATA box is a common component of RNA polymerase II promoters – It consists of an A-T-rich octamer located ~25 bp upstream of the startpoint.

• The DPE is a common component of RNA polymerase II promoters that do not contain a TATA box.

• A core promoter for RNA polymerase II includes:– the InR– either a TATA box or a DPE

Figure 24.10

56Figure 24.11: Polymerases bind via commitment factors.

TBP Is a Universal Factor

• TBP is a component of the positioning factor that is required for each type of RNA polymerase to bind its promoter.

• The factor for RNA polymerase II is TFIID, which consists of:

• TBP

• 11 TAFs

– The total mass is ∼800 kD.

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TBP Binds DNA in an Unusual Way

• TBP binds to the TATA box in the minor groove of DNA.

• It forms a saddle around the DNA and bends it by ∼80°.

• Some of the TAFs resemble histones and may form a structure resembling a histone octamer.

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TBP binds in the narrow groove and bends the DNA.

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Start with binding of TFIID which binds a short DNA sequence with many A-Ts - TATA box

This binding causes a dramatic distortion in the DNA.

TBP is a subunit of TFIID which binds the TATA box.

Partially unwound areas of TATA-Box DNA

The conserved C-terminal domain of TBP binds to TATA-box DNA

TBP

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Most eukaryotic genes are regulated by multiple transcription control

mechanisms

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The basal apparatus assembles at the promoter

Binding of TFIID to the TATA box is the first step in initiation.

Other transcription factors bind to the complex in a defined order, extending the length of the protected region on DNA.

When RNA polymerase II binds to the complex, it initiates transcription.

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RNA polymerase II requires general transcription factors

Initiation of transcription of a eucaryotic gene by RNA polymerase II.

To begin transcription, RNA

polymerase requires a number of

general transcription factors (called

TFIIA, TFIIB, and so on). (A) The

promoter contains a DNA sequence

called the TATA box, which is

located 25 nucleotides away from the

site at which transcription is initiated.

(B) The TATA box is recognized and

bound by transcription factor TFIID,

which then enables the adjacent

binding of TFIIB (C).

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. (D) The rest of the general transcription factors, as well as the RNA polymerase itself, assemble at the promoter. (E) TFIIH then uses ATP to pry apart the DNA double helix at the transcription start point, allowing transcription to begin. TFIIH also phosphorylates RNA polymerase II, changing its conformation so that the polymerase is released from the general factors and can begin the elongation phase of transcription. As shown, the site of phosphorylation is a long C-terminal polypeptide tail that extends from the polymerase molecule. The assembly scheme shown in the figure was deduced from experiments performed in vitro, and the exact order in which the general transcription factors assemble on promoters in cells is not known with certainty. In some cases, the general factors are thought to first assemble with the polymerase, with the whole assembly subsequently binding to the DNA in a single step. The general transcription factors have been highly conserved in evolution; some of those from human cells can be replaced in biochemical experiments by the corresponding factors from simple yeasts.

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General transcription factors assemble on all promoters transcribed by RNA polymerase II

Start with binding of TFIID which binds a short DNA sequence with many A-Ts - TATA box - typically 25 nucleotides upstream from the transcription start site. This binding causes a dramatic distortion in the DNA.

Other factors assemble along with RNA polymerase = transcription initiation complex. TFIIH contains a protein kinase subunit which adds phosphates to RNA polymerase, releasing it from the complex to start transcription

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Figure 24.16: TFIIB helps position RNA polymerase II.

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The basal / general transcription factors (core promoter factors):

TFIID: It has many subunits. TBP and TAFs

TBP is the only general transcription factor makes sequence-specific contacts with

DNA.

Within TFIID as a free protein complex, the factor TAFII230 binds to TBP and

controls the ability of TBP to bind to DNA.

The N-terminal domain of TAFII230 must be displaced from the DNA-binding

surface of TBP in order for TFIID to bind to DNA.

TFIIA: binds to TBP and stablilizes the TBP-DNA interaction. It also antogonizes transcription repressors, it physically displaces or blocks several negative transcriptional regulators from TFIID complex.

TFIIB: it is involved in the selection of transcription start sites, possibly setting distances between promoters and transcription start sites. It interacts with diverse activators that may serve to recruit TFIIB to the promoters.

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TFIIF:

It binds tightly to RNA pol.II, suppresses nonspecific DNA

binding, and stabilizes the preinitiation complex.

It stimulates polymerase elongation rates by suppressing

transient pause during transcription.

Its subunit RAP74 makes contacts with DNA to position the

template during initiation. RAP74 has an ATP-dependent DNA

helicase activity. It affects the DNA topology, facilitates

promoter melting.

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TFIIE: Interacts with TFIIH; involved in recruitment, stimulation of TFIIH and

promoter opening.

TFIIH: Its subunits have at least three enzymatic activities: DNA-dependent

ATPase, ATP-dependent helicase, and CTD kinase.

Two of its subunits have helicase activity (5’ to 3’ DNA helicase activity),

essential promoter opening and promoter escape.

TFIIH kinase/cyclin subcomplex: Cdks phosphorylate serines in the RNA

polymerase II CTD.

TFIIH kinase also functions as a Cdk activating kinase (CAK) and regulates

cell cycle transitions.

Some of its subunits required for nucleotide excision repair (transcriptional

coupled repair function).

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The function of the CTD tail of RNA pol II

Most of the TFs are released before RNA polymerase II leaves the promoter.Phosphorylation of RNA pol II tail is needed to release RNA pol II from the TFs so it can make the transition to the elongation form.CTD may coordinate processing of RNA with transcription.

In mammals long polypeptide tail of RNA pol II contains 52 repeats of the amino acid sequence. The serine and threonin side chains are phosphorylated.

Figure 24.17: The CTD is phosphorylated at initiation.

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The RNA refractory concept for eucaryotic RNA polymerase II:

Not only does the polymerase transcribe DNA into RNA, but it also carries pre-mRNA-processing proteins on its tails, which are then transferred to the nascent RNA at the

appropriate time.

The capping enzyme (Guanlylyl transferase), which adds the G residues to the 5’ end of the newly synthesized mRNA, binds to phosphorylated CTD.

Some splicing factors bind to CTD and also some components of the cleavage/polyadenylation apparatus.

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Transcriptional coupled repair

The template strand of DNA in a transcribed gene is preferentially repaired when DNA is damaged.

What identifies the template strand of the DNA to repair apparatus?

RNA polymerase stalls when it encounters the damage in the template strand since it can not use the damaged strand to direct complementary base pairing.

Mfd gene has two roles:• Displacing RNA pol. from DNA• Causing the uvr ABC enzyme to bind to the damaged DNA.DNA is repaired by excision repair system.RNA polymerase binds and produces a normal transcript.

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Chemically modified bases, such as thymine-thymine dimers, are corrected by excision repair

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Dimeric bases andbulky lesions,eg. large chemical adducts,are repaired by Nucleotide Excision Repair (NER)

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Rereference: de Laat W.L., Jaspers N.J.,Hoeijmakers J.H.

Genes & Development 13 2000

Nucleotide ExcisionRepair in Eucaryotes

Damage removed in the

context of a 24 - 28nt

fragment

Two pathways:

Global Genome repair

(GG-NER)

Transcription coupled

repair (TC-NER)

(Helicases)

(SS-DNA binding protein)

(CSA and CSB)

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In eucaryotes The template strand of the transcribed gene is preferentially repaired after UV damage.TFIIH. TFIIH is found in alternative forms which consists of a core associated with other subunits. TFIIH core protein and subunits associated with other subunits that have kinase activity and also a repair subunit. TFIIH is required for initiation, but may be replaced by other forms in response to DNA damage. TFIIH dissociates from RNA polymerase, then reassociates at the site of DNA damage with additional coupling components (i.e. Rad26). The repair function may require modification or degradation of RNA pol.

TFIIH plays multiple roles.

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Short sequence elements bind activators

Short conserved sequence elements are

dispersed in the region preceding the startpoint.

The upstream elements increase the

frequency of initiation.

The factors that bind to them to stimulate

transcription are called activators.

77Figure 24.21: The ß-globin promoter has three short sequence elements.

Promoters of RNA pol. II have short sequence elements.TATA box -30 bpCAAT box -75 bpGC box -90 bp

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Figure 24.22: Promoters have mix and match modules.

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Enhancers contain bidirectional elements that assist initiation

An enhancer activates the nearest promoter to it, and can be any distance either

upstream or downstream of the promoter. Similar sequence elements are found in enhancers and promoters. Enhancers form complexes of activators that interact directly or indirectly with

the promoter.

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Figure 24.24: Essential elements are more concentrated in enhancers.

Enhancers Contain the Same Elements That Are Found at Promoters

• Enhancers are made of the same short sequence elements that are found in promoters.

• The density of sequence components is greater in the enhancer than in the promoter.

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The essential role of an enhancer is to increase the

concentration of transcription factors in the vicinity of the

promoter.• An enhancer could change the overall structure of the template by influencing the density of the supercoil.

• It could be responsible for locating the template at a particular place within the cell. i.e. attaching it to the nuclear matrix.

• It provides an “entry site”, a point which RNA pol. (or some other essential proteins) associate with chromatin.

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A model for gene activation from an enhancer

General transcription factors usually can not efficiently initiate transcription alone.

Enhancers can be thousands of base pairs away, either upstream or downstream and can either increase or decrease transcription.

“Action at a distance”

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Enhancers work by increasing the concentration of activators near the promoter

Enhancers usually work only in cis configuration with a target promoter.

Enhancers can be made to work in trans configuration by linking the DNA that contains the target promoter to the DNA that contains the enhancer via a protein bridge or by catenating the two molecules.

The principle is that an enhancer works in any situation in which it is constrained to be in the same proximity as the promoter.

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Figure 24.25: Enhancer activity requires proximity to the promoter.

• The principle is that an enhancer works in any situation in which it is constrained to be in the same proximity as the promoter.

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Gene Expression Is Associated with Demethylation

• Demethylation at the 5 end of the gene is necessary for transcription.

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CpG Islands Are Regulatory Targets

• CpG islands surround the promoters of constitutively expressed genes where they are unmethylated.

• CpG islands also are found at the promoters of some tissue-regulated genes.

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Gene expression is associated with demethylation

•The state of methylation of DNA is controlled by • methylases, which add methyl groups to the 5 position of cytosine• demethylases which remove the methyl group.

•There are two types of DNA methylase:

• De novo methylase• modifies the DNA at a new position

recognizes DNA by the presence of a specific sequence

Maintenance methylaseacts on hemimethylated sites to convert to fully methylated

sites. It is a ubiquitious enzyme.

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Formation of 5-methylcytosine occurs by methylation of a cytosine base in the DNA double helix. In vertebrates this event is confined to selected cytosine nucleotides located in the sequence CG.

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Methylation pattern during embryogenesis:

In males:

• spermatocytes display methylation pattern characteristic of mature sperm

• no further changes occur during spermatogenesis

• after fertilisation further changes occur

In females:

* oocytes mature through meiosis after birth and methylation pattern is established.

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Genes are inactive in gametesPrimordial germ cells develop in the embryo and lose all

their allelic differences irrespective of sex

(genome-wide demethylation)

Sex-specific pattern is imposed During somatic development individual sequence-specific

methylation events occur in particular for genes to

become inactive.

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How DNA methylation patterns are faithfully inherited:In vertebrate DNAs a large fraction of the cytosine nucleotides in the sequence CG are methylated. Because of the existence of a methly-directed methylating enzyme (the maintenance methlytransferase), once a pattern of DNA methylation is established, each site of methylation is inherited in the progeny DNA, as shown.

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Gene expression is associated with demethylation

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CpG islands are regulatory targets

CpG islands surround the promoters of

constitutively expressed genes where they are

unmethylated. They are also found at the promoters of some

tissue-regulated genes. There are ~29,000 CpG islands in the human

genome. Methylation of a CpG island prevents activation

of a promoter within it.

Repression is caused by proteins that bind to methylated CpG doublets.

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How DNA methylation may help to turn off genes:

The binding of gene regulatory

machinery near an active promoter may

prevent DNA methylation by excluding

de novo methyltransferases. If most of

these proteins dissociated from the

DNA, however, as generally occurs

when a cell no longer produces the

required activator proteins, the DNA

becomes methylated, which enables

other proteins to bind, and these shut

down the gene completely by further

altering chromatin structure.

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Figure 24.26: Restriction enzymes differ at methylated sites.

MspI cleaves CCGG sequences both methylated and unmethylated.

HpaII cleaves CCGG sequences only nonmethylated state.

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Restriction digests can analyze methylation

MspI cleaves CCGG sequences both methylated and unmethylated.

HpaII cleaves CCGG sequences only unmethylated state.

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The DNA sequence given below is methylated in one type of tissue but unmethylated in another type of tissue.What sort of gel electrophoresis pattern would you obtain if you cut the below DNA sequence isolated from different tissues with these two enzymes?

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Methylation status was checked by “methylation specific PCR (MSP)”

• All unmethylated cytosines are deaminated and sulfonated, converting them to

uracils, while 5-methylcytosines remain unaltered.

(Sodium bisulfite converts unmethylated cytosine to uracil).

• Thus, the sequence of the treated DNA will differ depending on whether the DNA

is originally methylated or unmethylated.

• Also, the initially complementary DNA strands will no longer be complementary

after cytosine conversion.

• Primers for use in MSP can be designed to specifically amplify either bisulfite-

sensitive, unmethylated strand or a bisulfite-resisant, methylated strand, based

upon these chemically-induced differences.

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Unmethylated DNA

ggg gcg gac cgc g

ggg gug gau ugu g

Methylated DNA

ggg gcmg gac cmgcm g

ggg gcmg gau cmgcm g

Sodium bisulfitemodification

Sodium bisulfitemodification

DNA treatment with Sodium Bisulfite

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Methylation specific PCR

The PCR primers are designed to specifically amplify the promoter regions of the gene of interest.

If the DNA samples was originally unmethylated, an MSP reaction product will be detectable when using primer set (labeled as “U”) designed to be complementary to the unmethylated DNA sequence.

No product will be generated using primer set (labeled as “M”) designed to be complementary to the derivative methylated DNA sequence.

Conversely, an MSP product will be generated only using the M primer set if the sample was originally methylated, and the U primers will not amplify such a template.

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• Methylation status was checked by “methylation specific PCR (MSP)”.

• Sodium bisulfite converts unmethylated cytosine to uracil

• 2 SETS OF PRIMERS are used in 2 SEPERATE PCR REACTIONS

• Cell lines expressing DLC-1 were found to be UNMETHYLATED.