[V]. Process of Transcription and Transcriptional Control of Gene Expression

RNA polymerases and Initiation of transcription Transcriptional elongation and termination Gene promoter, enhancer and silencers DNA binding by transcription factors Activation and repression of transcription Regulation at transcriptional elongation Regulation of transcription by RNA

polymerases I and III

Overview of Control of Gene Expression

• Regulation at transcriptional level: Regulation of initiation of transcription

Chromatin-mediated transcriptional control Activators and repressors interaction with transcription

complex

• Regulation at post-transcriptional level: Regulation of alternative splicing leading to production of

multiple isoforms of proteins Regulation of transport of mRNA into cytoplasm

• Regulation at the translational or post-translational level Modification of the translational apparatus or specific

protein factors Micro RNAs RNA intereference (RNAi or siRNA) Cytoplasmic polyadenylation mRNA degradation Localization of mRNA in the cytoplasm

+1

Transcription start site

TATA Box -25 - -35 bp

TranscriptionProximal promoter

Distal promoter

Regulatory region (regulatory cis element) Structural gene

Structure of Protein Coding Gene

• Binding of transcription factors (proteins) to regulatory regions of genes and resulted in changing of chromatin structure (epigenetics and chromatin structure)

• Specific proteins that bind to a gene’s regulatory sequences determines where transcription will start and either via activating or repressing its transcription

Two key features of transcription control:

Conventions for Describing RNA Transcription

Regulatory region

Direction of transcription (RNA synthesis)

Polymerization of Ribonucleotides by RNA Polymerase during Transcription

• In transcription, the sequence of the RNA strand is copied from one strand of the DNA (Template Strand)

• The ribonucleotide is added at the 3’ end of the growing RNA strand, i.e., RNA strand grows from 5’ to 3’ direction

• The DNA strand used as the template runs in the direction of 3’ to 5’

• By denoting the position where RNA polymerase starts to make RNA as +1. Down stream sequence is toward the 5’ direction of the template while upstream is toward the 3’end

RNA Polymerase in Prokatrotes

• In prokaryotes, there is only one type of RNA polymerase that is responsible for the synthesis of rRNA, tRNA and mRNA

The Structure of Bacterial RNA Polymerase

• Generally speaking, similar structures of RNA polymerases are found in bacteria, archaea and eukaryotic cells

• Bacterial RNA polymerase consists of two large submits (b’ and b), two small subunits (a), one sigma factor, and one omaga (w) submit

Stages of Transcription

• RNA polymerase binds to the promoter region

• Requires transcription factors to locate the promoter region

• Melts the double stranded DNA (12 to 14 base pairs) in order to expose the template strand

• Template strand enters into the active site of RNA polymerase that catalyzes the formation of phosphodieaster bond between two molecules of ribonucleotide triphosphate that are complementary to the promoter template strand at the start site of transcription

• Strand elongation

• Transcription termination

Stages in Transcription (I)

Promoter; Transcription factors; Transcription bubble

Stages in Transcription (II)

Rate of Elongation: 1000 NT/min at 37oC

Active RNA Polymerase in Bacterial Cells

• For active transcription in eubacteria, the RNA polymerase needs to bind to a protein, factor (70), to form a complete complex

• Sigma factor (70) binds to the promoter DNA at -10 (six bases) and -35 (seven bases) to bring the core enzyme of RNA polymerase to initiate transcription at +1 position

-35 -10

TTGACAT--------16 – 18 bp-------TATAAT

-35 element TATA Box

T82T84G78A65C54A43 T80A93T45A60T96

• Sigma factor (70) acts as an initiation factor for transcription since it falls off from the RNA polymerase once the first few bases are transcribed. This factor is not required for elongation of the transcription

Gene Organization, Transcription & Translation in Prokaryotes

• Genes encoding proteins devoted to a single metabolic goal are most arranged in a contiguous array

• Such arrangement of genes in a functional group is called “operon”

• Transcription of genes in an operon results in a long mRNA (polycistronic mRNA)

• Translation of the polycistronic mRNA gives rise to different peptides

Poly-cistronic mRNA

Translation initiates at five different sites

Gene Organization, Transcription and Translation in Eukaryotes

• No polycistronic mRNA was found in eukaryotes

• Genes devoted to a single metabolic pathway are physically separated

• Each gene contains exons and introns

• Precursor mRNA initially transcribed from the gene and the intron regions are spliced out to form a mature mRNA

• Introns are rarely found in bacteria, archaea and uncomon to unicellular eukryotes such as yeast

Assigned Reading: --Structural basis of eukaryotic gene transcription. FEBS Letters 579: 879-903, 2005.

Eukaryotic RNA Polymerases

In eukaryotes, three different RNA polymerases are found. These are:

• All eukaryotic RNA polymerases have ~12 subunits and are complexes of ~500 kD.

• Some subunits are common to all three RNA polymerases.• The largest subunit in RNA polymerase II has a CTD (carboxy-

terminal domain) consisting of multiple repeats of seven amino acids (Tyr-Ser-Pro-Thr-Ser-Pro-Ser)

Eukaryotic RNA Polymerases

RNA Polymerases of Prokaryotes &

Eukaryotes

• There are three different RNA polymerases responsible for transcription of different genes. These different RNA polymerases share some common features. Each polymerase contains:

Two large submits ( and -like)

Two -like submits, the submits in Pol I and Pol III are identical

One subunit Identical common submits Additional enzyme-specific

submits

Three RNA Polymerases in Eukaryotes

• Three different RNA polymerases can be separated by chromatography on DEAE cellulose column

• Polymerase II is very sensitive to -aminitin (I mg/ml), polymerase III is less sensitive to -aminitin (10 mg/ml) and polymerase I is not sensitive to -aminitin

• RNA pol I: pre-rRNA• RNA pol II: pre-mRNA,

snRNA, miRNA• RNA pol III: tRNA, 5S

rRNA, snRNA U6, 7S RNA

Separation of RNA polymerases on a DEAE Cellulose column

Transcription by RNA Polymerase I (1)

• Transcription of rRNA genes by RNA polymerase I is relatively simple. rRNA genes are arranged in tandem repeat flanked by non-transcribed spacers. The promoter of rRNA gene consists of two separate regions. The core promoter surrounds the start site extending from -45 to +20

• It has a G-C - rich and an A-T – rich regions in the core promoter region

• The efficiency of the core promoter is enhanced by the upstream promoter element (UPE or UCE). UPE is another G-C-rich sequence extending from -180 to -107 bp

• UBF: upstream transcription factor that binds to UPE, responsible for high efficiency of transcription

• RNA pol I requires two ancillary transcription factors to recognize the promoter sequence. These factors are TATA-binding protein (TBP) and TFIIP

Transcription of Repeated rRNA Genes by Pol I

To initiate the transcription, the TATA-binding protein (TBP) is required. Although the same TBP is required for initiating transcription by RNA polymerase II and RNA polymerase III, it does not bind to DNA directly in this case

A protein factor, upstream binding factor (UBP) recognizes and binds to the upstream promoter element. This complex will recruit other transcription factors (SL1), TBP, and RNA polymerase I

By doing so, the entire transcription initiation complex is formed and transcription initiated

Transcription by RNA Polymerase I (2)

Transcription by RNA Polymerase III

• RNA polymerase III has two types of promoters:

Internal promoters (types1 & 2; 5S and tRNA genes) containing consensus sequences located within the transcription unit and cause initiation to occur at a fixed distance upstream (Promoters for 5S rRNA and tRNA genes belong this type)

Upstream promoters contain three short consensus sequences (Oct, PSE and TATA) upstream of the start-point that are bound by transcription factors (promoter for snRNA belongs to this type)

Transcription-Control Elements of Genes

Transcribed by Pol III

Transcription by RNA Polymerase II

• Genes encoding proteins are transcribed by RNA polymerase II• RNA polymerase II requires general transcription factors called

TFIIX to initiate transcription.• RNA polymerase II promoters frequently have a short conserved

sequence Py2CAPy5 (the initiator, Inr) at the startpoint.• The TATA box is a common component of RNA polymerase II

promoters and consists of an A-T-rich octamer located ~25 bp upstream of the startpoint.

• Transcription of genes by RNA polymerase II is regulated by binding of multiple transcription factors to the transcription-control regions: Promoter region Other transcription control elements

• TATA box, initiators and CpG islands function as promoters in eukaryotic DNA• TATA box is found 25-35 bp upstream of the transcription start

site

Consensus Sequence of TATA Box

RNA Polymerases in All Promoters Are Positioned by

Factors Containing TBP• TBP (TATA binding protein) 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 and ~14 TAFs, with a total mass ~800 kD

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

• TBP forms a saddle around the DNA and bends it by ~80°.

Saddle-Like Structure of TBP Bound to TATA Box

TFIID is composed of the TATA-binding protein (TBP) and a group of evolutionarily conserved proteins known as TBP-associated factors or TAFs

TBP has a saddle-like structure which binds to TATA box and bend the DNA as shown in figure

TFIIA binds to TBP at the N-terminus of TBP, and TFIIB binds at the C-terminus

TAFII250, one of the TBP associated factors which contains histone acetyltransferase activity, capable of acetylating histones leading to modulation of chromatin structure

Initiators and CpG Islands• Some eukaryotic genes, instead of containing a TATA box, they contain an

initiator element (Inr) with a consensus sequence as: (5’Y-Y-A+1-N-T/A-Y-Y3’) where C is at the -1 position and A at the transcription start site (+1) and Y is pyrimidine (C or T)

• CpG islands: Some genes contain CG rich stretch of 20 to 50 nucleotides with 100 bp upstream of the start site. Since CG is statistically under represented in the vertebrate genome, the presence of a stretch of CG-rich region or the CpG island in the upstream of the start site is a distinctly nonrandom distribution Therefore, the presence of a CpG island in the genomic DNA suggests that it may contain a transcription-initiation region

• In this case, TBP does not bind to DNA but recruited initiator binding protein (IN) which will bind to the initiator element and recruit TFIIB and the RNA polymerase II. Hence TBP plays a central role for the formation of transcription initiation complex

• Assigned Reading: Weight matrix description of four eukaryotic RNA polymerase II

promoter…..

Transcription of Protein Coding Genes by RNA Polymnerase II

• The TFIID-DNA complex will recruit TFIIA and TFIIB (as in b)

• Following this step, RNA polymerase II and TFIIF will be recruited to form the pre-transcription initiation complex

• TFIIE and TFIIH will be subsequently recruited to the complex to form the transcription initiation complex

• TFIIH contains kinase activity which phosphorylates the C terminus Tyr-ser-pro-Thr-Ser-Pro-Ser domains of RPBI of RNA polymerase II. This will allow transcription to occur and RNA polymerase II and TFIIF will move along the DNA molecule to continue the elongation of the RNA molecule

• Therefore phosphorylation of RNA polymerase II is critical for transcription to produce RNA product

In Vitro Assembly of RNA Pol II Preinitiation Complex

• TFIIB helps position RNA polymerase II

• 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

Pre-Initiation Complex of Transcription

TPB is a Universal Factor for All RNA

Polymerases

RNA polymerases are positioned at all promoters by a factor that contains TPB

Modification of the RNA Polymerase II CTD Heptapeptide During Transcription

• Phosphorylation of CTD is required for release of poly II from the promoter and poly II proceedes transcription

• Phosphorylation of serine 5 in CTD by TFIIH is required for Capping of the mRNA

• Phosphorylation of serine 2 in CTD by P-TEFb can recruit SCAFs essential for splicing of mRNA and for polyadenylation of mRNA

Promoter Clearance and Elogation

• TFIIH has several activities: (i). ATPase, (ii). Helicases of both polarity, (iii). Kinase activity that can phosphorylate CTD tail of poly II (serine 3 of the CTD)

• In addition, TFIIH may play a role in elongation, The interaction of TFIIH with DNA downstream of the start site is required for poly II to escape from the promoter

• TFIIH is also involved in repair of damage to DNA

• For poly II to move from the start site to down stream, hydrolysis of ATP by TFIIE and melting of the supercoiled DNA by TFIIH (XPB subnit) are required

• For successful elongation of the initiated transcript, a kinase (P-TEFb) is required to phosphorylate Serine 2 at the CTD

• The phosphorylation pattern of CTD is dynamic during elongation process which is controlled by multiple protein kinases and phosphatases. Transcription factors associate with poly II when CTD is un-phosphorylated and dissociated with poly II when CTD is phosphorylated

Elongation of Transcription• Following the initiation of transcription,

the synthesis of RNA will continue for 20-30 bases and the RNA polymerase II will pause

• Release of the pause will take place when the serine 2 at the C-terminus (tyr-ser-pro-thr-ser-pro-ser) of RNA polymerase II is phosphrylated

• The phosphorylation of serine 2 is closely linked to the modification of the free 5’ end of the nascent RNA molecule by addition of a modified G nucleotide in the process known as capping. This process promotes the binding of pTEF-b (positive transcription elongation factor) kinase which can phosphorylate the serine 2 on the RNA polymerase II allowing elongation to proceed

Addition of Poly (A) Tail to the mRN A

The initial RNA transcript is cleaved downstream of the poly-adenylation signal (AAUAAA) and a poly(A) tail added to the free 3’ end. Hence , the 3’ end of the mature mRNA is in significantly upstream of the transcriptional termination site.

Stages in the Transcriptional Process of Protein Coding Genes

• Initiation of transcription• Stalling of transcription• Elongation• Termination and polyadenylation

Some protein coding genes contain a TATA box located approximately 30 bases upstream of the transcription start site. Other protein coding genes utilize an initiator (Inr) sequence with the consensus 5’-YCANTYY-3’, with A residue being the first base which is transcribed (Y=C or T; N=any nucleotide)

Regulatory Elements of a Gene• Regulatory elements of a gene in eukaryotes often are many bases to

kilobases from the start site of the transcription of the gene

• Core promoter: A DNA sequences that specifies where RNA polymerase binds and initiates transcription of the gene

• Transcription factors: Protein factors necessary for transcription

• Transcription factor binding sites: Sites in the regulatory region where transcription factors bind to regulate the transcription. These binding sites are also called as cis-acting elements which usually located many bases upstream or downstream of the site of initiation of transcription (or promoter)

• Transcription from a single promoter may be regulated by binding of multiple transcription factors to alternative cis-acting elements, permitting complex control of gene expression

The Promoter Structure of Typical RNA Polymerase II Transcribed Gene

More precisely the promoter region should be described as: Core or basal promoter (proximal promoter) : serves to recruit

basal transcriptional complex to initiate basal transcription Upstream promoter elements (distal promoter or regulatory

region): Elements that regulate the rate of transcription

Identification of Promoter-Proximal Elements that Regulate Eukaryotic Genes

• There are elements in the promoter-proximal regions that regulate the expression of genes. These elements are termed as “promoter-proximal elements” or “promoter-proximal transcription regulatory elements”

• How are “promoter-proximal elements” determined?

5’ deletion analysis to determine the region that may contain the transcription regulatory site(s).

Linker scanning mutations to pinpoint the sequence with regulatory function. In this analysis, a series of constructs with contiguous overlapping mutations are assayed for their effect on expression of a reporter gene or production of a specific mRNA

The promoter-proximal regulatory region of the thymidine kinase (tk) was determined by this analysis

• Reading List An efficient protocol for linker scanning mutagenesis

Determination of TranspcritionControl Sequence

• TTR: transthyretin, a protein that transports thyroid hormone in the blood and the cerebrospinal fluid that surrounds the brain and spinal cord

• TTR is expressed in hepatocytes and in the choroid plexus in the brain

• The control elements (cis-acting elements) that control the transcription of TTR gene are identified by experiments outlined in the left of this slide

• Reporter gene: a gene that is used to report the activity of a promoter. e.g., green fluorescent protein gene (GFP), luciferase (lux), -galactosidase (lac Z)

• Two sites upstream of the transcription start sites are important (~ -2.1 – 1.8 kb; ~ -200 bp)

• Reading List: Deletion analysis identifies a region in the

upstream of a gene that regulate the expression of the gene

Linker Scanning Mutation Analysis

• To pin-point the exact location of a regulatory element, linker scanning mutation analysis should be conducted

Reading List:An efficient protocol for linker scanning mutagenesis…..

Enhancers• In eukaryotic promotrs there is a CAAT

box at -75 and a GGGCGG box at -90. These boxes function to increase efficiency of the promoter and they can function in either orentation

• Enhancer: A cis-acting element located at a distance from the start site of transcription that can influence gene expression in either orientation

• Example: a sequence at -100 upstream of histone H2 gene that is essential for high level expression of histone H2 gene

• An enhancer can be at (i) many bases upstream of the core promoter, (ii) many bases behind the transcription unit, or (iii) inside of the transcription unit

• Enhancer works in either orientation

Interactions of Promoter Regulatory

Elements and Regulatory Factors

Promoter regulatory elements act by binding factors which either affect chromatin structure and/or influence transcription directly

As shown in (a), binding of glucocorticoid hormone receptor-hormone complex to Glucocorticoid Response Element (GRE) will result in displacement of a nucleosome and generate a DNase I-hypersensitive site allowing easy access to the gene for transcription

A second example: As shown in (b), binding of HSF (heat shock factor) to HSE (heat shock element) will directly activate transcription

How could these factors be discovered?

1.Chromatin precipitation

2.Yeast two hybrid system

Chromatin Immunoprecipitation• This technique can be

used to detect protein-DNA interactions in the native chromatin context in vivo. The associated DNA is purified for analysis by identifying its specific sequence by PCR or by labeling the DNA and applying to a tiling array to detect genome wide interactions

• This assay can be used to test the presence of a specific gene interacting with a transcription factor

• Reading List: Chip assay, an overview

Using Yeast Two-hybrid System to Detect cDNAs Encoding Interacting Proteins

• Yeast two-hybrid system exploits the flexibility in activator structures to identify genes whose products bind to a specific protein of interest

• If one is interested in identifying the cDNA of GH receptor, the strategy is:• Bait hybrid: DNA binding

domain of UAS + liner sequence + GH

• Fish hybrid: cDNA (fish domain) + linker + activation domain of HIS gene

• Transfer both bait hybrid and fish hybrid into yeast cells and observe the proper phenotype

Using Yeast Two-hybrid System to Detect cDNAs Encoding Interacting Proteins

• Transfer both bait and fish hybrids into yeast cells carrying trp-, leu- and his- markers and select for trp+, leu+ and his+ phenotypes

• This system is widely used to detect cDNA encoding protein domains that bind to a specific protein of interest

• Protein factors that interacting with enhancers could be actovators or repressors

• Activators can either bind directly to specific DNA site and making contact with the basal machinery at the promoter; or bind to mediators to modify chromatin structure to allow transcription to occur

• A tissue-specific enhancer can activate the promoter of its own or another reporter gene only in one particular tissue but not the others

• In yeast, upstream activating sequence (UAS) has been found to have the same activity as typical “enhancer”

Proteins Interacting with Enhancers

Diagram depicted on the left demonstrated the function of a cell type specific enhancer

Enhancers• 50-200 bp length DNA in the regulatory region of genes that can enhance the

transcription of genes

• Enhancer sequences contain multiple binding sites for transcription factors to form enhancesome

• Enhancers work by increasing the concentration of activators near the promoter

• Example of formation of enhancesome in the -interferon enhancer: HMG1, IRF-3, IRF7, ATF-2, cJun, p50 and p65

How is the Enhancer Activity Detected?

• Enhancer elements located in far distance upstream or downstream of the TATA box that can stimulate the transcription of a gene

• The function of enhancer element was determined by the experiment outlined in this figure: Plasmid 1 contains an enhancer element from SV40 DNA and a globin gene, and plasmid 2 contains a globin gene but without an enhancer element

• Enhancer could be cell-type specific

• Cellular enhancer are composed of multiple elements that contribute to the overall activity of the enhancer

Silencers

Silencer: A cis-acting element that inhibit the expression of a gene Silencer element is present some distance away from the gene it

acts A silencer element acts by recruiting factors (i) to direct the tight

packing of the adjacent DNA (i.e., tight packing of chromatin) or (ii) to directly inhibiting transcription by interacting with RNA polymerase and its associated factors

Examples of Silencers

The silencer element appears to represent a site for attachment of the DNA to the nuclear matrix. It may act by promoting further condensation of the 30 nm chromatin fiber to form a loop of DNA attached to the nuclear matrix to a most condensed structure

This is achieved by recruiting protein factors which direct tight packing of the adjacent DNA

Examples: Polycomb inducing the ubiquitination of histone H2A and the methylation of histone H3 on lysine 9 and 27 leading to tight packing of chromatin

Polycomb-response element / trithorex-response element: When polycomb protein binds to this site, it will lead to tight packing of chromatin. When trithorex protein binds to this site, it direct synthesis of non-coding RNA and leading to activation of protein coding gene

Sequences Regulating a Typical RNA Polymerase II Gene

• CP: Basal promoter• UP: Upstream promoter• LCR: Locus control region• E: Enhancer• I: Insulator

The Promoter of Human hsp 70 Gene

These motifs are also found in a variety of the promoters of other genes, but not HSE (heat-shock element) which is only found in hsp70 gene

Experiment Confirming the Role of HSE

HSE is required for the expression of hsp genes in response to heat induction

Experiment described in the left proves that HSE is required to direct the expression of any genes in response to heat induction

Heat shock factor 1 (HSF-1) is the major regulator of heat shock protein transcription in eukaryotes. In the absence of cellular stress, HSF-1 is inhibited by association with heat shock proteins and is therefore not active. Cellular stresses, such as increased temperature, can cause proteins in the cell to misfold.

How are regulatory factors (trans-acting factors) identified?

• Activator• Repressor

Methods Used to Determine the Presence of Specific Transcription Factors

• For transcription factors to elicit their activity in controlling transcription, these factors have to interact with regulatory elements first

• Two techniques commonly used to identify transcription factors:

Electrophoretic mobility shift assay (EMSA; gel-shift assay, band-shift assay or DNA mobilityshift assay)

This technique is based on the fact that if a piece of DNA containing an identified regulatory element is bound to a transcription factor, it will migrate slower than the necked DNA on the agarose gel during electrophoresis (see example in next slide)

Footprinting assay (DNase 1 protecting assay or DNase 1 foot-printing assay)

This technique is based on the fact that if an identified regulatory element is bound to a specific transcription factor, it will be resistant to digestion by DNase 1. The protect region can be revealed if the product is analyzed on a denaturing polyacrylamid gel

• The specific transcription factor can be isolated by various types of chromatography and the resulting fractions assayed by EMSA and Footing assay

Electrophoretic Mobility Shift Assay (EMSA)

• EMSA is also called as gel-shift assay or band-shift assay. This method is used for identifying protein(s) that binds to the regulatory element(s). It can also be used for quantitative determination of DNA-binding proteins

• The assay is based on the fact that if an end labeled DNA fragment that contain regulatory element(s) is bound to protein factors, its electrophoretic mobility is reduced and thus causes a shift in the location of the radio-labeled DNA band. Thus this method is commonly used to detect transcription factors that may regulate the transcription of a gene

• If the proteins are fractionated by column chromatography, each fraction can be assayed by the same method to determine the binding characteristics to the DNA fragment. This method is commonly used to identify the DNA binding component during its purification

DNA Mobility-Shift Assay

Autoradiogram

Electrophoretic Mobility Shift Assay (EMSA)

• The same assay principle can be used to develop a “sequence-specific DNA affinity chromatography” technique for isolating protein factors that bind to specific sequence DNA

• Reading List: Purification and characterization of transcription factor IIIC2

• Protein sample containing specific transcription factor is separated by chromatography and the resulting fractions are incubated with 32P-labeled DNA fragment containing identified regulatory element, and resolved on an agarose gel by electrophoresis

DNase I Footprinting Assay• Various transcription-control elements (cis-acting elements) in

eukaryotic genes are binding sites for regulatory proteins (trans-acting factors)

• DNase 1 footprinting assay: When a DNA fragment is bound by a specific protein, it will be resistant to digestion by DNase I. This method is used to identify specific protein factors that may bind to a specific region of the DNA fragment. The assay is done as the following: Label the DNA fragment, containing identified regulatory

elements, at one end with 32P-ATP by kinase reaction Incubate the labeled DNA fragment with or without proteins to be

tested Digest the DNA-protein complex with DNase I and recover the

DNA by phenol/chloroforme extraction and precipitation in ethyl alcohol

Separate the DNA fragments on a denaturing polyacrylamide gel and visualize the DNA bands by autoradiography

DNase I Footprinting Analysis

• This assay is under the condition that low concentration of DNase I is used in the analysis

• The sequence of the protein-binding region can be determined by comparing with DNA markers of known sizes in (M)

• Suggested Reading List III:• DNase footprinting assay

Region of DNA protected from DNAse I

How do transcription factors (trans acting factors) interact with regulatory elements (cis-acting elements) to regulate the expression of genes?

• The expression of genes present in particular cell types or tissues is regulated by DNA motifs (cis-acting elements) present in the regulatory region (core promoter and enhancers). These elements control the alteration of the chromatin structure where the genes reside or by increasing the transcription efficiency

• These cis-acting elements are believed to act by binding to regulatory proteins (trans-acting factors) which is only synthesized in a particular tissue or is present in an active form in the tissue

• The binding of trans-acting factors to cis-acting elements will result in the expression of genes

• While identification of the presence of the cis-acting factors is proven to be easier, identification, isolation and purification of the trans-acting factors is much harder

• The main reason is that these factors are present in very small quantities

Approach I for Isolation and Purification of Transcription Factors

• Sp1 (Specificity Protein 1) is a human transcription factor involved in gene expression in the early development of an organism

• Sp1 protein was purified by affinity chromatography as described in the left figure

• Determine the partial A.A. sequence of Sp1

• Develop oligo-nucleotide to serve as a probe to screen the cDNA library

• Pick and characterize the isolated cDNA clones

• Reading List: Affinity purification of sequence-specific DNA binding proteins

In Vitro Assay of Transcription Factors

• Sp1 is a transcription factor that binds to GC-rich sequence, thereby activating transcription from a near by promoter in SV40

• The activity Sp1 was confirmed by the following in vitro assay:

Production of recombinant Sp1 in E. coli cells

Carry out an in vitro transcription reaction by incubating the purified SP1, with a DNA template, RNA polymerase II, the associated general transcription factors and 32P-UTP The labeled RNA product was resolved on agarose gels and visualized by autoradiography

Measure Transcription Activity by In Vitro Transfection Assay

• Once the transcription factor is identified and purified, the partial amino acid sequence can be determined and the cDNA can be cloned

• The activity of the protein encoded by the cDNA clone can be further confirmed by the in vitro transfection assay as described in the left panel

Alternative Method to Clone Transcription Factor

“Cloning of NFkB” cDNA

Mapping of the DNA Binding Domain of a

Transcription Factor (I)

• The DNA binding domain of a transcription factor can be mapped by the approach described in this figure

• Cut the cDNA gene to be mapped into smaller pieces, clone the DNA fragments into lambda phage for expression

• Screen the binding• To confirm the DNA fragment isolated

in this figure is indeed the real DNA binding domain, the sequence in this piece of DNA can be mutated and re-confirm its binding to the transcription factor

• By this approach, large amounts of information have been accumulated on individual transcription factors

Mapping of the DNA Binding Domain and the Activation Domain of a Transcription Factor (II)

• Activators are modular proteins composed of distinct functional domains and promote transcription

• a, Reporter gene construct containing a reporter gene, TATA box and a USAgal that contains several GAL4 binding sites

• b, Introduce wild type and deletion mutant of GAL4 protein into yeast cells and assay for binding GAL4 protein to UASGAL and also assay for expression of lacZ gene

• Amino acids 1-50 in the N-terminus is responsible for binding to UASGAL

• Amino acids 126-189 in the C-terminus of the GAL4 is responsible for the activation of transcription

• Results of the experiment in the previous slide showed that GAL4 protein contains two domains: DNA binding domain and activation domain

• When the N-terminus DNA binding domain of the GAL4 was fused to various lengths of its own C-terminal region and the truncated proteins retained the ability to stimulate the expression of the reporter gene in an in vivo assay. These results suggest that the internal portion of the GAL4 protein is not required for functioning of GAL4 as a transcription factor

• Similar results were found in a transcription factor, GCN4, that regulates the transcription of genes for amino acid synthesis in yeast: A DNA binding domain with 60 A.A. in its C-terminus An activation domain with 20 A.A. near the middle of

its sequence

Modular Structure of Eukaryotic Transcription Activators

• GAL4: Regulatory region controlling the expression of genes metabolizing galactose; GCN4: controlling genes for synthesis of amino acids; GR: Glucocorticoid responsive genes

• These transcription factors may contain more than one activation domains but rarely contain more than one DNA binding domain

• GAL4 and GCN4 are yeast transcription factors• GR: the glucocorticoid receptor, promotes transcription of target genes

when hormone binds to the C -terminal activation domain• SP1: Binds to G-C rich promoter elements in mammalian genes

Repressors

• Repressors are the functional converse of activators

• Repressors repress the transcription of genes

• Constitutive expression: the transcription of a gene is on all the time

• Repressor-binding sites in DNA have been identified by systematic linker scanning mutation analysis

• Like activators, most eukaryotic repressors are modular proteins that have two functional domains; DNA binding domain and a repression domain

• The repression domain continues to function when it is fused to another type of DNA-binding domain

The Control Region of the Gene Encoding EGR-1

• EGR 1 (early growth response protein 1): C2H2 Zn++ finger transcription activator

• There are several elements in the control region of EGR-1 gene that regulate the expression of EGR-1 gene

• WT1: Wilm’s tumor gene, a repressor for EGR-1 gene

(Repressor binding site)

(Activator binding site)

(Activator binding site)

Control regions could containing multiple regulatory regions (multiple activators and repressors)

DNA Binding Domains of Activators or Repressors

• The DNA-binding domains of eukaryotic activators or repressors contain a variety of structural motifs

• DNA-binding proteins bind to DNA at the major groove , the sugar-phosphate backbone or the minor groove

• In bacterial system, each -helix of the dimeric form of the repressors bind to the major grooves of the DNA (sequence-reading helix or recognition helix)

• This helix motif is called helix-turn-helix motif• In eukaryotic system, there are many different types

transcription factors that contain different DNA binding motifs. These motifs are: homeodomain proteins, Zin-finger proteins, Leucine-zipper proteins, basic helix-loop-helix proteins. These DNA binding motifs have characteristic consensus amino acid sequences

• Eukaryotic genomes encode dozens of classes of DNA-binding domains and hundreds to thousands of transcription factors. Human genome encodes about 2000 transcription factors

Eukaryotic DNA Binding Domains

• In eukaryotes, the DNA binding domains of transcription factors (i.e., activators or repressors) are classified into: Homeodomain proteins: helix-turn-helix domain containing

proteins Zn++ finger proteins Leucine zipper proteins Basic-helix-loop-helix (bHLH) proteins

Homeotic Mutation in Drosophila melanogaster

• These proteins are homeodomain proteins, containing a conserved 60 amino acid residues forming two - helical structures at both termini

• These proteins are like the helix-turn-helix family of DNA-binding proteins of bacterial system

• These proteins were first found in Drosophila but also fund in vertebrates as well

Helix-Turn-Helix Domain (Homeo Domain Protein)

• The second helix is the recognition helix which binds to the major groove of the DNA.

Examples of Helix-turn-Helix Proteins (I)

• Fushi tarazu (ftz) protein is encoded by ftz gene. Mutation of ftz gene will produce flies with half numbers of body segments

• Ftz protein binds to DNA with sequence 5’TCAATTAAATGA3’

• Mutation of this sequence will result in lose of binding to this DNA

• Introduction of Ftz gene into Drosophila cells containing a marker gene with 5’TCAATTAAATGA3’ sequence, the expression of the marker gene is elevated

• Engrailled protein, another homeotic gene product, bind to the same sequence. Introduction of engrailled protein gene will block the expression of the same marker gene

Amino Acid Sequences of POU Proteins

• POU (pronounced pow; pit-oct-unc) domain transcription factors contain a conserved sequence of 150-160 amino acids, which has two boxes: POU-specific box and POU homeodomain

• In mammals, Pit-1, Oct1, Oct2 and Unc all contain POU domain

• Although the POU homeodomain alone is sufficient to bind to the specific DNA sequence, the addition of POU-specific domain will increase the affinity of the binding

• Like POU homeodomain, the POU-specific domain also form helix-turn-helix motif

• When Oct1 protein binds to “ATGCAAAT” of cellular genes, it activates only low level of expression. However when it binds to a different target sequence “TAATGART” where R is purine, it results in high level expression

• When Pit-1 binds to prolactin gene, it activates prolactin gene expression. But when Pit-1 binds to growth hormone gene , binding of nuclear receptor co-repressor Nco-R will result in repression of growth hormone gene. Hence the DNA-binding site can affect the configuration of the bound factor and therefore its ability to recruit other regulatory factor to activate or repress transcription

• Degenerate oligonucleotide primers have been developed from the highly conserved sequence of POU domain proteins have been used to isolate novel members of the POU family by RT-PCR and results are promising

C2H2 Zinc Finger Binding Protein Motif• C2H2 zinc finger is the most common DNA-binding

motif encoded in the human genome and the genomes of most other multicellular animals.

• It contains nine repeats of a 30 amino acid sequence of the form Tyr/Phe-X-Cys-X-Cys-X2-4-Cys-X3-Phe-X2-Leu-x2-His-X2-4-His-X5, where X is a variable amino acid• The repeating structure contains two pairs Cys and His residues which can bind to Zn++. Thus it is called zinc finger. TFIIIA contains zinc finger

• The 30 a.a repeating unit of zinc finger sits on the basis of a loop of 12 a.a containing conserved Leu and Phe as well as several basic residues projects from the surface of the protein

• The finger is anchored at its base by a conserved Cys and His residues which directly co-ordinated an atom of zinc

• The finger consists of two antiparallel -sheets and an -helix packed against one of the -sheet, with the -helix contacting the major grove of the DNA. Hence zinc finger can mediate the DNA binding of transcription factors

Nuclear Receptor and Four Cysteine Zinc Finger

Steroid hormone nuclear receptors such as glucocorticoid hormone receptor (GR), estrogen receptor (ER), progesterone receptor (PR) thyroid hormone receptor (TR) contain a consensus DNA binding sequence, Cys-X2-cys-X13-Cys-X2-Cys-X15-17-Cys-X9-Cys-X2-Cys-X4-Cys

The binding of this peptide to DNA requires the presence of Zn++ or related heavy metal such as Cd++. This element can be drawn as a conventional two zinc fingers

The two zinc fingers are separated by a linker region containing 15-17 variable amino acids

In Cys2His2 zinc finger, each finger forms a separate unit, in Cys4 zinc finger, two fingers interact with one another to form a single structural element. The Cys4 zinc finger can not be converted into Cys2His2 zinc finger

Cys2His2 Zinc Finger Cys4 Zinc FingerGR

Cys4 Zinc FingerER

The multiple cysteine zinc finger DNA binding domain has also been identified in many other DNA binding proteins such as GAL4, PRL, LAC9 etc.

Nuclear Receptor Response Elements Contain Inverted or Direct Repeats

• The site on the 5’ upstream region of a gene that nuclear receptor-ligand complexes bind is termed as response element

• GRE and ERE are bound by homodimers of their respective receptors. The DNA binding sites are in palindromic sequence

• VDRE (Vitamin D receptor); RARE (retinoid acid receptor)

• RXR: a common nuclear receptor monomer which binds to direct repeat DNA sequence

• VDRE binds to heterodimer of RXR-VDR; TRE binds to TR-VDR; RARE binds to RXR-RAR

Binding of Cysteine Zinc Finger to Direct Repeat DNA Sequence

Leucine Zipper DNA Binding Proteins

Another group DNA binding proteins contain a region of 35 amino acids in which every seventh amino acid is leucine, called leucine zipper domain

Transcription factors that contain leucine zipper are C/EBP, GCN4, and proto-oncogenes such as Myc, Fos and Jun

The leucine zipper region, the amino acids will form -helical structure with leucine in every 2 turns. Two molecules of this domain will form a zipper

Binding of Leucine Zipper Containing Protein to DNA

Unlike helix-turn-helix motif, the leucine zipper motif does not bind to DNA directly. Rather, by facilitating the dimerization of the protein, it provides the correct protein structure for DNA binding by the adjacent region of the protein which is rich in basic amino acids that can interact directly with DNA. This basic amino acid region is termed as “basic DNA-binding domain”

Mutation in the basic DNA binding domain will abolish the DNA binding activity

Leucine Zipper Proteins and Basic Helix-Loop Helix (bHLH) Proteins

• (a), Leucine zipper protein: the DNA-binding domains of transcription factors. Each -helix binds to an adjacent major groove of the DNA backbone

• These proteins bind to DNA as dimers, and leucine is required for dimerization

• Example: GCN4 transcription factor in yeast

• (a), The DNA-binding domain of the yeast GCN4 transcription factor contains a leucine-zipper binding domain that bind to the major groove of the DNA. Basic Zipper (bZip)

• (b), Basic Helix-Loop Helix (bHLH) proteins: The DNA-binding domain of another class of transcription factors containing a structural motif very similar to basic leucine zipper, can form heterodimer. MyoD is the example

Dimerization Between Factors Provide Additional Levels of Regulation

Since leucine zipper proteins and helix-loop-helix proteins can form dimerization and the dimerized molecule can bind to DNA via basic DNA binding domain, therefore formation of homodimer or heterodimer of these transcription factors can provide an additional level of gene regulation

Examples given above are homodimer formation between Jun and heterodimer formation between Jun and Fos which can all recognize “TGAGTCAG”

Other DNA Binding Domains

There are some transcription factors that their DNA binding domains do not fall into the four classes that were discussed. Examples are: ets DNA binding domain, winged helix-turn-helix, forkhead DNA-binding domain and Pax (that contains a homeodomain and a paired domain) etc.

Summary of Common DNA-Binding Motiefs and Dimerization Domains

General Mechanisms of Transcription Activation and Repression

• Two mechanisms that activators and repressors regulate the expression of the associated protein coding genes:

Chromatin-mediated transcriptional control: These regulatory proteins act in concert with other proteins to modulate chromatin structure, thereby influencing the ability of general transcription factors to bind to promoters

Activators and repressors interaction with transcription complex: Activators and repressors interact with a large multi-protein complex, mediator of transcription complex (or mediator), leading to binding to the RNA Pol II to regulate assembly of transcription pre-initiation complex

Activation of Transcription

• Activation domains can be identified by “domain swap experiment”

• Activators can interact with TFIID

• Activators can interact with TFIIB

• Activators can interact with the mediator and SAGA

• Activators can interact with co-activators

• Activators can interact with modulators of chromatin structure

• Activators have a multitude of targets

Reporter GeneAssaying construct

DNA binding site

DNA bindingdomain

Functional domainIn question

Basal promoter

Testing construct

Identification of Activation Domain by Domain-Swap Experiment (I)

Structurally Diverse Activation and Repression Domains Regulate Transcription (I)

• Experiments with fusion proteins composed of the GAL4 DNA-binding domain and random segments of E. coli proteins demonstrated that a diverse group of amino acid sequences can function as activation domains, ~1% of all E. coli sequences even though they have evolved to perform other functions GAL4, GCN4 and most other yeast transcription factors are rich in acidic amino

acids (Asp and Glu), these transcription factors can stimulate transcription of nearly all types of eukaryotic cells [Acid Activation Domains]

Among activation domains of Drosophila and mammalian transcription factors, some are glutamine-rich, some are proline-rich; and some are rich in serine and threonine. However, some activation domains are not rich in any particular amino acid

Structurally Diverse Activation and Repression Domains Regulate Transcription (II)

Acidic activation domains have unstructured, random-coil conformation. Upon binding with co-activator proteins, these domains become active in stimulating transcription

Binding of co-activator proteins to acidic activator domains make these domains to assume a more structured -helical confirmation in the complex

A well studied example of this is CREB (cAMP responsive element binding) protein, a mammalian transcription factor with acidic activation domain. Upon phosphorylation by cAMP, CREB protein binds to its co-activator protein, CBP (CREB binding protein), resulting in transcription of genes that their control regions contain a CREB binding site

Another example is shown in the right figurein which binding of VP16 to TAFn31 will result in a tighter binding because of the -helical structure

Some activation domains are larger and more highly structured than acidic activation domains. For example, the ”ligand-binding domains” of nuclear receptors function as activation domains when they bind to their ligands, leading to conformational change to allow interaction with the co-activator and activate the gene

Interactions of Transcription Factors Increase Gene-Control Options

• Basic-zipper proteins and bHLH proteins often exist in alternative heterodimeric combinations of monomers

• If each monomer has similar characteristics, the heterodimer formed will not affect it specificity

• If each monomer in the heterodimer possesses different DNA-binding specificity, the combination will increase the DNA sequences that the factor can bind

• This phenomenon is demonstrated in the next slide

Combination of DNA Binding

Factors

Cooperative Binding of Two Unrelated Transcreption Factors in a Composite Control Element

• Two structurally unrelated transcription factors work together to regulate a promoter by binding to closely spaced DNA binding sites

• Example: NFAP and AP1 both bind to the proximal promoter region of interleuken-2 gene with low affinity

• However binding of NFAP and AP1 simultaneously to their respective binding sites increase the stability of the binding complex.

• Such cooperative DNA binding of various transcription factors results in considerable combinatorial complexity of transcription control

• There are more than 2000 transcriptional factors encoded in human genome are for this purpose

Molecular Mechanisms of Transcription Activation and Repression

Chromatin mediated transcriptional control

Transcription control through mediator

Epigenetic control through DNA methylation

Transcription of Many Genes Requires Ordered Binding & Function of Activators and Co-activators

• Results of various studies showed that multiple activators bind to a mediator complex and thus regulate the transcription of a gene from a promoter

• Some of this type of complex could comprise >100 polypeptides

Model of several DNA-bound activators interacting with a single mediator complex

Transcription of Many Genes Requires Ordered Binding of Activators and Action of Co-Activators

• Ordered binding of activators and co-activators leading to transcription of the yeast HO gene (encoding an endonuclease)

• Initially HO gene is packaged in to condensed chromatin

• Activation of the gene begins with binding of SWI5 to the enhancer site 1.2 to 1.4 kb upstream• SWI5 activator interacts with chromatin-

remolding complex, SWI/SNF.

Ordered Binding of Activators and Co-Activators Leading to Transcription of the Yeast HO Gene

• SW/SNF acts to decondense the chromatin

GCN5-containing SAGA histone acetylase complex

Ordered Binding of Activators and Co-Activators Leading to Transcription of the Yeast HO Gene

• A GCN5-containing histone acetylase complex associates with bound SWI5 and acetylate histone tails in the HO gene as SWI/SNF continues to decondense adjunct chromatin

Ordered Binding of Activators and Co-Activators Leading to Transcription of the Yeast HO Gene

• SWI5 is released from the DNA, but the SWI/SNF and GCN5 complexes remain associate with the HO control region. This action allows SBF activator to bind to several sites in the proximal promoter region

• SBF: an DNA binding activator

Ordered Binding of Activators and Co-activators Leading to Transcription of the Yeast HO Gene

SBF binds to mediator complex

Ordered Binding of Activators and Co-activators Leading to Transcription of the Yeast HO Gene

Subsequent binding of Pol II and general transcription factors results in assembly of a transcription preinitiation complex

Transcription-Control Region of the Mouse Transthyretin (TTR) Gene

Binding sites for the five activators required for the transcription of TTR gene in hepatocytes in mouse are indicated. The complete set of activators is expressed at the required concentrations. A different set of activators are required for stimulating the expression of TTR in choroid plexus cells

Regulation of Transcription-Factor Activity

• Developmental stage and cell type specific expression of a gene in a multi-cellular organism is dependent upon the concentrations and activities of the transcription factors that interact with the regulatory cis-acting elements

• The types and the amounts of transcription factors produced are determined by multiple regulatory interactions between transcription-factor genes that occur during the development and differentiation of the particular cell type

• The amounts and the time of appearance of these transcription-factors are also regulated by signals (including hormones) from neighboring cells and humeral source

• One major group of extracellular signals comprise peptides and proteins that can bind to receptors on the cell membrane. Binding of ligands to receptors trigger intracellular signal transduction cascades

Regulation of Transcription Factor Activity

Covalent modification (phosphorylation, acetylation or ubiquitination)

By binding to ligand (nuclear receptor)

Examples of Hormones that Bind to Nuclear Receptors

• A class of extracellular signals that bind to receptors residing in the nucleus are cortisol, retinoic acid, thyroxin, testostrone and estrogen

• These nuclear receptors will function as transcription factors after binding to the corresponding ligands

• These nuclear receptors are called as nuclear receptor superfamily

All Nuclear Receptors Share a Common Domain Structure

Activation Domain

Hormone Binding to a Nuclear Receptor Regulates its Activity as a Transcription Factor

• Heterodimeric Nuclear Receptors: (e.g., RXR-VDR, RXR-TR and RXR-RAR) are located exclusively in the

nucleus. In the absence of their ligands, these receptors repress transcription when bound to their cognate sites in DNA by deacetylating nearby nucleosomes

In the ligand-bound conformation, RXR in the ligand-receptor complex directs hyperacetylation of the nearby nucleosomes to reverse the repression effect. In addition, the ligand-receptor complex also binds to mediator, stimulating preinitiation complex assembly

• Homodimeric Nuclear Receptors: Homodimeric nuclear receptors are found in the cytoplasm in the

absence of ligands Binding of ligands to these receptors lead to translocation of the

complex to the nucleus The translocated ligand-nuclear receptor complex will bind to the

hormone response element and initiate transcription of the regulated genes with other transcription factors

Model of Hormone-Dependent Gene Activation by Homodimeric Nuclear Receptor

Repression of Transcription

• Repressor can act by inhibiting the positive effect of activator

• Repressors can act directly by inhibiting the assembly or activity of the basal transcriptional complex

Repressors Act Indirectly by Inhibiting the

Positive Effect of Activator

(a). Repressor binds to the site where activator binds(b). Repressor binds to activator to block its action

Degradation of Activators by Repressors

1. Ubiquitination of p53 activator by MDM2 lead to degradation of p53 activator by a protease (P). Another example, binding of a repressor AEBP1 to activator will lead to degradation of activator

2. MDM2: an ubiquitin ligase for p53 protein; AEBP1: A transcription factor which regulate the differentiation of adipocytes

Functions of p53 protein (Tumor Suppressor)

Repressors Can Act Directly by Inhibiting the Assembly or Activity of the Basal Transcriptional Complex

• Repressors can exert their repression effects by (i) neutralizing the action of a positively acting factor, (ii) by preventing either its DNA binding or its activation of transcription, by inducing its degradation

• Repressors can also inhibit transcription directly

• Examples: eve (eve-skipped) protein and c-erbA gene are this type of repressor

• c-erbA encodes thryoid hormone nuclear receptor. Upon binding to its co-repressor, NCoR, it inhibits

transcription of thyroid hormone responsive genes. Upon binding of thyroid hormone to its receptor-corepressor complex, the NCoR will dissociate from thyroid hormone-receptor complex and initiate transcription

There are two isoforms of c-erbA mRNAs, c-erbA and c-erbA-2

c-erbA-1 can bind to thyroid hormone but c-erbA-2 can not

Since both isoforms contain DNA binding site, they can bind to thyroid hormone responsive element. However only c-erbA-1 contains activating

domain so it can activate the transcription of thyroid hormone responsive gene. Binding of cerbA-2 to the thyroid hormone responsive element will result in inactivating the transcription of the thyroid hormone responsive gene These two alternatively spliced forms of the transcription factor which are made in different amounts in different tissues, therefore mediating thyroid hormone dependent gene

Another Forms of Repressor Action Another form of repression by

repressor is binding of repressor to a specific site on the DNA, and the binding of R to the DNA will result in inhibiting activity of the basal transcription activity. Example: Binding of the thyroid hormone receptor to the silencer in the chicken lysozyme gene

Alternatively, R can bind directly to the basal transcription unit and thus inhibit the transcription.

Example to this is Dr1 or Mot1 which binds to TBP and prevents TFIIB from binding, thereby preventing assembly of basal trqanscriptional complex on TATA box-containing promoter (see next slide)

The basal transcriptional complex can be inhibited by the binding to TBP by Dr1, thus preventing binding of TFIIB to TBP. Binding of Mot1 will displace TBP from the DNA (TATA box)

Regulated Elongation and Termination of Transcription

• Mechanisms for terminating transcription for each RNA polymerase are different: Termination of transcription of RNA polymerase I requires binding of

polymerase-specific termination factor to a site downstream of the transcription unit

RNA polymerase III terminates after polymerasing a series of U residues.

The deoxy(A)n – ribo(U)n DNA-RNA hybrid is particularly unstable and thus easily melted

Once RNA polymerase II initiates transcription (beyond 50 bases), further elongation is highly processive and does not terminate until after a sequence that directs cleavage and polyadenylation is reached. RNA polymerase II can terminate transcription at multiple sites located over a distance of 0.5 to 2.0 kb beyond the polyadenylation signal. Protein complex that cleaves and polyadenylates the 3’end of the RNA at the specific sequence associates with the phosphorylated carboxyl-terminal domain (CTD) of the Pol II. This protein complex consists of CPSF, CStF, CF1 and CF2

Chromatin Remodeling

• Chromatin remodeling – The energy-dependent displacement or reorganization of nucleosomes that occurs in conjunction with activation of genes for transcription.

• This is the process to make the template available for transcription

• Energy (in the form of ATPs) required to disrupt the interaction of proteins with the DNA in the neucleasomes to free DNA for transcription

Chromatin Remodeling Is an Active Process

• There are numerous ATP-dependent chromatin remodeling complexes that use energy provided by hydrolysis of ATP.

• Chromatin Remolding Complex: RSC is a 17-submit complex with the capacity to remodel the structure of chromatin. It exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to perturb nucleosome structures. It is at least 10-fold more abundant than the SWI/SNF complex and is essential for mitotic growth

• Chromatin remodeling is an active process

• All remodeling complexes contain a related ATPase catalytic subunit, and are grouped into subfamilies containing more closely related ATPase subunits.

• Remodeling complexes can alter, slide, or displace nucleosomes

Remodeling Changes Nucleosome Organization

• Some remodeling complexes can exchange one histone for another in a nucleosome.

Remodeling Complexes Are Classified by Their ATPase Subunits

Remodeling complexes bind via activators

• How are remodeling complexes targeted to specific sites on chromatin?

• A remodeling complex does not itself have specificity for any particular target site, but must be recruited by a component of the transcription apparatus

• Remodeling complexes are recruited to promoters by sequence-specific activators

• The factor may be released once the remodeling complex has bound

• Example: Swi5 is a transcription factor regulating

the expression of HO gene in yeast Swi5 bind to HO promoter, then recruit

SWI/SNF Swi5 is released from the promoter and

leaving the SWI/SNF

• Transcription activation often involves nucleosome displacement at the promoter

• Promoters contain nucleosome-free regions flanked by nucleosomes containing the H2A variant H2AZ (Htz1 in yeast)

Nucleosomes are Displaced from Promoters during Activation

• PHO5 promoter contains nucleosomes positioned over TATA box and one of the binding sites for the Pho4 and Pho2 activators. When PHO5 is induced by the phosphate starvation, promoter nucleosomes are displaced

The Nucleosome is Required for NF1

Binding

• The MMTV promoter requires a change in rotational positioning of a nucleosome to allow an activator to bind to DNA on the nucleosome.

Histone Acetylation Is Associated with Transcription Activation

• Newly synthesized histones are acetylated at specific sites, then deacetylated after incorporation into nucleosomes. Histone acetylation is associated with activation of gene expression.

• Lysine (K) acetyltransferase (KAT) – An enzyme (HAT, typically present in large complexes) that acetylates lysine residues in histones (or other proteins).

• Transcription activators are associated with histone acetylase activities in large complexes.

• Trichostatin and butyric acid are two inhibitors for HAT. These inhibitors are instrumental in understanding the importance of acetylation in regulation of gene expression

• Example: In yeast, Gcn5 is a prototype catalytic HAT subunit of the 1.8 MDa Spt-Ada-Gcn5 acetyltransferase (SAGA) complex that is involved transcription. Taf1 submit of TFIID is also an acetyltransferase which has functional overlap with SAGA. It suggests that an acetyltransferase is essential for gene expression in yeast that can be provided by TFIID or SAGA

Coactivators May Have HAT Activities

• One of the first general activators to be characterized as HAT was p300/CREB-binding protein (CBP). It is a coactivaor that links an activator to the basal apparatus

• p300/CREB interacts with various activators, including hormone receptors, AP-1 (c-Ju8n and c-Fos), and MyoD

• It acetylate multiple histone targets, with preference for H4 tail

• p300/CREB also interacts with another coactivator , PCAF (lysine acetyle transferase 2),

which is related to Gcn5 and preferentially acetylates H3 in nucleosomes. P300/CREB and PCAF form a complex that functions in transcription activation

Complexes that Control Acetylation Levels

• Transcription activators are associated with histone acetylase activities in large complexes.

• Group A HATs, like ATP-dependent remolding enzymes, are typically found in large complexes as shown in the left

• The complex contains site-specific DNA binding protein, Target submit, acetylase acts on histone tails and effector submit act on chromatin

• The effect of acetylation may be both quantitative and qualitative

• Deacetylation is associated with repression of gene activity.

• Histone deacetylase (HDAC) – Enzyme that removes acetyl groups from histones; may be associated with repressors of transcription.

• A repressor complex contains three components: a DNA binding submit, a corepressor, and a histone deacetylase

• Rpd3 and its homologes are present in mutiple HDACn complex found in yeast to human

Deacetylation is Associated with Repression

• In mammalian cells, Sin3 is part of a repressive complex that includes histone binding proteins and the Rpd3m homologs to HDAC1 and HDAC2, and this complex can be recruited by a variety of repressors to specific genes

Methylation of Histones and DNA Is Connected

• Methylation of both DNA and specific sites on histones is a feature of inactive chromatin. DNA methylation is associated with transcriptional inactivity, histone methylation can be linked either to activae or inactive regions, depending on specific site of methylation

• There are several lysine methylation sites in the tail and core H3, and a single lysine in H4 and 3 arginine in H3 and one arginine in H4 that can be methylated

• The SET domain is part of the catalytic site of protein methyltransferases.

• Like acetylation, methylation is reversible by two families of demethylases; lysine-specific demethylase 1 and Jumonji family

• Different classes of enzymes demethylate arginine

• In silent or heterochromatic regions, the methylation of H3K9 is linked to DNA methylation. The enzyme responsible for this is called Suv39h1, and deacetylation of H3K9 must occur prior to the methylation. HP1 is recruited by H3K9me to the chromatin and target the activity of DNA methyltransferases on GpC island

Promoter Activation Involves Mutiple Changes in Chromatin

• Active chromatin is acetylated on the tails of H3 and H4

• Inactive chromatin is methylated on specific lysine such as K9 of H3

• Inactive chromatin is methylated on the C of CpG island

• In some cases, the enzyme involves in activation of gene (acetyltransferase) is mutually exclusive with inactivating events (methyltransferase). Example: silencing methylation of H3K9 and the activating acetylation of H3 at K9 and K14 are mutually anatagonistic

• Histones are phosphorylated (i) cyclically during the cell cycle; (ii) in association with chromatin remodeling during transcription and DNA repair

• Histone H1 is as good substrate for Cdc2 kinase suggesting that phosphorylation of H1 may be important for cell division

• Phosphorylation of ser 10 of H3 is linked to transcriptional activation, chromosome condensation and mitotic progression

• JIL-1, a kinase that phosphorylate histone H3S10, null mutant in Drosophila showed abnormal structure (unextended structure ) of polytene chromosomes

Histone Phosphorylation Affects Chromatin Structure

Summary (I)

• Transcription factors: basal factors, activators and co-activators

• Basal factors interact with RNA polymerase at the start-point within the promoter

• Activators bind to specific cis-acting elements (including enhancers) in the regulatory region

• Activators function by making protein-protein interaction with basal apparatus

• Activators have modular construction, DNA binding domain and activation domain

• The main function of the DNA binding domain is to position the activation domain close to the vicinity of the initiation complex

• Some of the activators are tissue specific or developmental stage specific while others are ubiquitous

• In the promoters of genes transcribed by poly II, there are several cis-acting elements each is recognized by trans-acting factors. These cis-acting elements can be located in the upstream of TATA box in orientation or down stream of the start site

Summary (II)

• These elements are recognized by activators or repressors that interact with the basal transcription complex to determine the efficiency of the promoter

• Some activators interact with the basal apparatus directly, while others interact via the mediation of co-activators. The target of the basal apparatus are TFIID, TFIIB or TFIIA. The interaction stimulates assembly of the basal apparatus

• DNA binding domains of transcription factors are catalogued into (i) homeodain, (ii) zinc finger domain, (iii) leucine zipper and (iv) helix-loop-helix

• Many transcription factors function as dimers, homodimers or heterodimers

• The control regions of some genes organized in nucleosomes are usually not expressed. Chromatin remodeling is required to expose the control region for regulating gene expression

• Chromatin remodeling involves hydrolysis of ATP, acetylation of lysine residues in histone, phosphorylation of serine residues and methylation/demethylation of lysine residues

Assigned Reading (V)

1. Structural basis of eukaryotic gene transcription. FEBS Letters 579: 879-903, 2005.

2. Weight matrix description of four eukaryotic RNA polymerase II promoter…..

3. An efficient protocol for linker scanning mutagenesis 4. Deletion analysis identifies a region in the upstream of a gene

that regulate the expression of the gene5. Purification and characterization of transcription factor IIIC26. Affinity purification of sequence-specific DNA binding proteins 7. CTD of RNA poly II8. DNase foot-printing assay