Chapter 11:

Transcription in Eukaryotes

… the modern researcher in transcriptional control has much to think about.

James T. Kadanoga, Cell (2004), 116:247

11.1 Introduction

Eukaryotic gene regulation involves:

• DNA-protein interactions

• Protein-protein interactions

• Chromatin structure

• Nuclear architecture

• Cellular compartmentalization

11.2 Overview of transcriptional regulation

Transcription and translation are uncoupled in eukaryotes

• Transcription takes place in the nucleus and translation takes place in the cytoplasm.

• The whole process may take hours, or in some cases, months for developmentally regulated genes.

• Gene expression can be controlled at many different levels.

Transcription is mediated by:

• Sequence-specific DNA-binding transcription factors.

• The general RNA polymerase II (RNA pol II) transcriptional machinery.

• Coactivators and corepressors.• Elongation factors.

Chromosomal territories and transcription factories

• Chromosome “painting” has shown that each chromosome occupies its own distinct territory in the nucleus.

• Transcription decondenses chromatin territories.

• The DNA loops that form in decondensed regions are proposed to be associated with transcription “factories.”

• Transcriptionally active genes also appear to be preferentially associated with nuclear pore complex.

Eukaryotes have different types of RNA polymerase

• Bacteria have one type of RNA polymerase that is responsible for transcription of all genes.

• Eukaryotes have multiple nuclear DNA-dependent RNA polymerases and organelle-specific polymerases.

• Focus here on regulation of transcription of protein-coding genes by RNA polymerase II.

11.3 Protein-coding gene regulatory elements

The big picture:

• Transcription factors interpret the information present in gene promoters and other regulatory elements and transmit the appropriate response to the RNA pol II transcriptional machinery.

• What turns on a particular gene in a particular cell is the unique combination of regulatory elements and the transcription factors that bind them.

• Regulatory regions of unicellular eukaryotes such as yeast are usually only composed of short sequences located adjacent to the core promoter.

• Regulatory regions of multicellular eukaryotes are scattered over an average distance of 10 kb of genomic DNA.

Variation in:

• Whether a particular element is present or absent.

• The number of distinct elements.

• Their orientation relative to the transcriptional start site.

• The distance between them.

• Gene regulatory elements are specific cis-acting DNA sequences that are recognized by trans-acting transcription factors.

• Two broad categories of cis-acting regulatory elements.

– Promoter elements.

– Long-range regulatory elements.

Structure and function of promoter elements

The gene promoter is the collection of cis-regulatory elements that are:

• Required for the initiation of transcription.

• Increase the frequency of initiation only when positioned near the transcriptional start site.

• The recognition site for RNA pol II general transcription factors.

The gene promoter region

• Core promoter elements.

• Proximal promoter elements.

Core promoter elements

• Approximately 60 bp DNA sequence overlapping the transcription start site.

• Serves as the recognition site for RNA pol II and the general transcription factors.

• All core promoter elements, except for BRE, are recognized by TFIID.

• A particular core promoter many contain some, all, or none of the common motifs.

The TATA box

• First core promoter element identified in a eukaryotic protein-coding gene.

• Key experiment by Pierre Chambon and colleagues demonstrated that a viral TATA box is both necessary and sufficient for specific initiation of transcription by RNA pol II in vitro.

• Sequence database analysis suggests the TATA box is present in only 32% of potential core promoters.

Promoter proximal elements

• Regulation of TFIID binding to the core promoter in yeast depends on an upstream activating sequence (UAS).

• Multicellular eukaryotic genes are likely to contain several promoter proximal elements.

e.g. CAAT box and the GC box

Promoter proximal elements

• Transcription factors that bind promoter proximal elements do not always directly activate or repress transcription.

• Transcription factors may serve as “tethering elements.”

Structure and function of long-range regulatory elements

• Additional regulatory elements in multicellular eukaryotes that can work over distances of 100 kb or more from the gene promoter.

• Enhancers and silencers

• Insulators

• Locus control regions (LCRs)

• Matrix attachment regions (MARs)

Enhancers and silencers

• Usually 700 to 1000 bp or more away from the start of transcription.

• Increase or repress gene promoter activity either in all tissues or in a regulated manner.

• Typically contain ~10 binding sites for several different transcription factors.

• How can you tell an enhancer from a promoter?

Insulators

• Chromatin boundary markers.

• Enhancer or silencer blocking activity.

• Insulator elements are recognized by specific DNA-binding proteins.

Locus control regions (LCRs)

• Organize and maintain a functional domain of active chromatin.

• Prototype LCR characterized in the mid-1980s as a cluster of DNase I-hypersensitive sites upstream of the -globin gene cluster.

-globin gene LCR is required for high-level transcription

• Physiological levels of expression of the embryonic, fetal, and adult -globin genes only occurs when they are downstream of the LCR.

• The DNase I hypersensitive sites contain clusters of transcription factor-binding sites and interact via extensive protein-DNA and protein-protein interactions.

Hispanic thalassemia and DNase I hypersensitive sites

• Analysis of patients with -thalassemia has led to significant advances in understanding of the LCR of the -globin gene locus.

• Partial or complete deletion of the LCR leads to reduced amounts of hemoglobin in the blood.

Hispanic thalassemia

• ~35 kb deletion of the LCR.

• The Hispanic locus is transcriptionally silent.

• The entire region of the -globin gene cluster is DNase I-resistant.

Analysis of DNase I sensitivity

• Transcriptionally active genes are more susceptible to deoxyribonuclease (DNase I) digestion.

Matrix attachment regions (MARs)

• Organize the genome into loop domains.

• Typically AT rich sequences located near enhancers in 5′ and 3′ flanking sequences.

• Confer tissue specificity and developmental control of gene expression.

• “Landing platform” for transcription factors.

• Attach to the nuclear matrix.

Position effect and long-range regulatory elements

• The function of many long-range regulatory elements was confirmed by their effect on gene expression in transgenic animals.

• Protect transgenes from the negative or positive influences exerted by chromatin at the site of integration.

Position effect

• Expression of a transgene is unpredictable in a transgenic organism.

• Varies with the random chromosomal site of integration.

• Can long-range regulatory elements protect transgenes from position effect?

Intron enhancers contribute to tissue-specific gene expression

• Does the enhancer located in the second intron of the apolipoprotein B gene play a role in gene regulation?

• What do the results suggest?

• Why do you think a reporter gene was used in the experiment?

MARs promote formation of independent loop domains

• Experiment to test the importance of MARs in transcriptional regulation of the whey acidic protein (WAP) gene.

• Analyzed by Southern blot (DNA) and Northern blot (RNA).

• What do the results suggest?

Is there a nuclear matrix?

• The nuclear matrix is operationally defined as “a branched meshwork of insoluble filamentous proteins within the nucleus that remains after digestion with high salt, nucleases, and detergent.”

• What forms the branching filaments remains unknown.

What does the nuclear matrix do?

• Proposed to serve as a structural organizer within the cell nucleus.

• Interaction of MARs with the nucleus is proposed to organize chromatin into loop domains and maintain chromosomal territories.

• Active genes are found associated with the nuclear matrix only in cell types in which they are expressed.

What are the components of the nuclear matrix?

• >200 types of proteins associated with the nuclear matrix.

• What forms the branching filaments remains unknown.

• General components include the heterogeneous nuclear ribonucleoprotein (hnRNP) complex proteins and the nuclear lamins.

What are the components of the nuclear matrix?

• The nuclear lamina is a protein meshwork underlying the nuclear membrane.

• Composed of the intermediate filament proteins lamins A, B, and C.

• Internal lamins form a “veil” that branches throughout the interior of the nucleus.

Hutchinson-Gilford progeria syndrome

• A premature aging syndrome.

• Splicing mutation in the lamin A gene.

• Patient cells have altered nuclear sizes and shapes, disrupted nuclear membranes, and extruded chromatin.

Is there a nuclear matrix?

• Established by nuclear functions?

• Present as a structural framework which then promotes functions?

11.4 The general transcriptional machinery

• General, but diverse, components of large multi-protein RNA polymerase machines required for promoter recognition and the catalysis of RNA synthesis.

Three major classes of proteins that regulate transcription

• The general (basal) transcription machinery

• Transcription factors

• Transcriptional coactivators and corepressors

Components of the general transcription machinery

• RNA polymerase II

• General transcription factors: TFIIB, TFIID, TFIIE, TFIIF, and TFIIH

• Mediator

Four major steps of transcription initiation

1.Preinitiation complex assembly

2.Initiation

3.Promoter clearance and elongation

4.Reinitiation

Structure of RNA polymerase II

• A 12 subunit polymerase capable of synthesizing RNA and proofreading nascent transcript.

Crystal structure for Saccharomyces cerevisiae RNA polymerase II

• 12 subunits total (Rpb1 to 12).

• 10 subunit catalytic core.

• Heterodimer of Rpb4 and Rpb7.

• Unstructured C-terminal domain (CTD) of Rpb1 is not seen by X-ray crystallography.

RNA polymerase II catalytic core

• The wall prevents straight passage of nucleic acids through the cleft.

• The RNA-DNA hybrid is nearly 90 to that of the entering DNA duplex.

• A pore beneath the active site widens towards the outside like a funnel and includes two Mg2+

binding sites.

• Positively charge “cleft” occupied by nucleic acids.

• One side of cleft is formed by a massive, mobile “clamp.”

• The active site is formed between the clamp, a “bridge helix” and a “wall”.

RNA polymerase II C-terminal domain (CTD)

• Tail-like feature of the largest subunit.

• Consists of up to 52 heptapeptide repeats.

• Undergoes dynamic phosphorylation of serine residues at positions 2 and 5 in the repeats.

General transcription factors and preinitiation complex formation

• A set of five general transcription factors, denoted TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.

• Responsible for promoter recognition and unwinding of promoter DNA.

• Nomenclature denotes “transcription factor for RNA polymerase II.”

• RNA polymerase II is absolutely dependent on these auxiliary transcription factors for the initiation of transcription.

• TFIIA and its subunit TFIIJ are not absolutely required for transcription initiation in vitro, so are not considered general transcription factors.

• Transcription initiation requires an unphosphorylated CTD.

• Assembling the general transcription apparatus involves a series of highly ordered steps.

• Binding of TFIID provides a platform to recruit other general transcription factors and RNA pol II to the promoter.

TFIID recruits the rest of the transcriptional machinery

• Binding of TFIID to the core promoter is a critical rate limiting step.

• TFIID is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFs)

• TBP contains an antiparallel -sheet that sits on the DNA like a saddle in the minor groove and bends the DNA.

TFIIB orients the complex on the promoter

• TFIIB binds to one end of TBP and to a GC-rich DNA sequence after the TATA motif.

• The TFIID-TBP-DNA complex “signposts” the direction for the start of transcription.

• The complex indicates which strand acts as the template.

TFIIE, TFIIF, and TFIIH binding completes the preinitiation complex

formation

• RNA polymerase II joins the assemblage in association with TFIIF and Mediator.

• TFIIE binds and recruits TFIIH.

• Promoter melting is mediated by the helicase activity of TFIIH.

• Unwinding is followed by “capture” of the nontemplate strand by TFIIF.

• The template strand descends into the active site of RNA polymerase II.

• Because of the conserved spacing from the TATA box to the transcription start site, the start site is positioned in the polymerase active site.

TFIIH has both cyclin-dependent kinase activity and helicase activity

• Transcription elongation requires a phosphorylated CTD.

• TFIIH is the kinase that phosphorylates the CTD of RNA pol II.

• Landmark experiments showed that purified TFIIH could convert the unphosphorylated polymerase to its phosphorylated form in vitro.

• The ATP-dependent helicase activity of TFIIH was demonstrated using an in vitro assay.

Mediator: a molecular bridge

• A 20-subunit complex which transduces regulatory information from the activator and repressor proteins to RNA pol II.

• Mediator serves as a molecular bridge between the transactivation domain of various transcription factors and RNA pol II.

The discovery of Mediator

• In vitro transcription assays could support basal transcription but were not responsive to transcriptional activators.

– Minimum set of general transcription factors– Purified core RNA pol II– G-less cassette assay

• Mediator was shown to be required for activator-responsive transcription.

• Mediator is expressed ubiquitously in eukaryotes.

• At least seven different mammalian Mediator complexes with 25-30 protein subunits.

• Comprised of head, middle, and tail modules according to electron microscopy.

• Conserved flexible hinge in the middle of the MED7/MED 21 heterodimer may promote changes in Mediator structure upon binding RNA pol II or transcription factors.

• How does Mediator reach regulatory elements that are so far away from the gene promoter?

Initiation of transcription

• Assembly of the preinitiation complex.

• A period of abortive initiation.

• Promoter clearance.

• Elongation.

Abortive initiation

• RNA polymerase II synthesizes a series of short transcripts

• As it moves, the polymerase holds the DNA strands apart forming a transcription bubble.

• A transcript of >10 nucleotides and bubble collapse lead to promoter clearance.

Promoter clearance

• Requires phosphorylation of the C-terminal domain (CTD) of RNA pol II.

• Phosphorylation helps RNA pol II to leave behind most of the general transcription factors.

• TFIID remains bound at the promoter and allows the rapid formation of a new preinitiation complex.

Once phosphorylated, RNA polymerase II can:

• Unwind DNA.

• Polymerize RNA.

• Proofread.

11.5 The role of specific transcription factors in gene

regulation

Transcription factors influence that rate of transcription of specific genes either positively or negatively by:

• Specific interactions with DNA regulatory elements.

• Interaction with other proteins.

• Gene regulation at the transcriptional level generally occurs via changes in the amounts or activities of transcription factors.

• The genes encoding transcription factors may be activated or repressed by other regulatory proteins.

• Transcription factors themselves may be activated or deactivated by proteolysis, covalent modifications, ligand binding, etc.

Transcription factors mediate gene-specific transcriptional activation

or repression

• Transcription factors that serve as repressors block the general transcription machinery.

• Transcription factors that serve as activators increase the rate of transcription by several mechanism.

Mechanisms of action of transcriptional activators

1.Stimulate the recruitment and binding of general transcription factors and RNA polymerase II.

2. Induce a conformational change or post-translational modification that stimulates the enzymatic activity of the general transcription machinery.

3. Interact with chromatin remodeling and modification complexes to increase DNA accessibility to the transcription machinery.

Transcription factors are modular proteins

• Composed of separable, functional domains.

• The three major domains are a DNA-binding domain, a transactivation domain, and a dimerization domain.

DNA-binding domain motifs

• Hundreds of protein-DNA complexes have been analyzed by X-ray crystallography.

• NMR spectroscopy has been used to study complexes in solution.

• High affinity binding is dependent on overall 3-D shape and formation of specific hydrogen bonds.

• Loss of just a few hydrogen bonds or hydrophobic contacts from a protein-DNA complex will usually result in a large loss of specificity.

• The most common recognition pattern is an interaction between an -helical domain of the protein and about 5 bp within the major groove of the DNA double helix.

Some of the most common DNA-binding domain motifs:

• Helix-turn-helix

• Zinc finger

• Basic leucine zipper

• Basic helix-loop-helix

Helix-turn-helix (HTH)

• The first DNA-binding domain to be well characterized.

• The classic HTH is composed of three core -helices.

• The third “recognition” helix inserts into the major groove of the DNA.

• Other variant forms may contain additional features, such as in the winged HTH motif

Homeoboxes and homeodomains

• In the late 1970s, developmental biologists were studying the genes regulating pattern formation in the developing Drosophila embyro.

• Discovery of homeobox genes in Drosophila.

• Later discovered in many other organisms from humans to the plant Arabidopsis.

• Homeobox genes encode transcription factors with a homeodomain that regulate many developmental programs.

• The homeodomain is a variant of the classic HTH.

• The best known homeobox gene subclass is the Hox family.

Homeotic mutations

• Mutations in some of these homeobox genes result in homeotic transformation.

• A homeotic mutation is a mutation that transforms one body part into another part:

– Antennapaedia homeotic mutant in which the antennae are transformed into legs.

– In humans, a mutation in the Hoxd13 gene results in duplication of a digit and development of six fingers.

The Hox genes of Drosophila

• Eight Hox genes regulate the identity of regions within the adult fruitfly and embryo.

Colinear expression of homeobox genes

• Homeobox genes are organized in clusters.

• Differential gene expression in which an expression gradient is achieved either spatially, temporally, or quantitatively depending on the location of each gene in the cluster.

Spatial colinearity

• The position of a gene in a cluster correlates with its expression domain.

• 5′ and 3′ Hox genes are typically expressed in the posterior and anterior portions of developing embryos, respectively.

Temporal colinearity

• Genes at one end of the cluster are turned on first and genes at the opposite end are turned on last.

Quantitative colinearity

• The first gene in a cluster displays a maximum level of expression and downstream genes exhibit progressively lower expression.

The Rhox genes of mouse

• A cluster of twelve homeobox genes on the X-chromosome.

• Important regulatory role in reproduction.

• The genes are expressed in the order they occur on the chromosome during sperm differentiation.

Polycomb group proteins silence homeobox genes

• Mediate gene silencing by altering the higher order structure of chromatin.

Zinc finger (Zif)

• One of the most prevalent DNA-binding motifs.

• First described in 1985 for Xenopus laevis TFIIIA.

• A “finger” is formed by interspersed cysteines and/or histidines that covalently bind a central zinc (Zn2+) ion.

• The finger inserts its -helical portion into the major groove of the DNA.

• The number of fingers is variable between different transcription factors.

• Classic finger: Cys2-His2 pattern.

• Nuclear receptors have two fingers of a Cys2-Cys2 pattern.

Zinc finger DNA-binding domain of the glucocorticoid receptor (GR)

• Three to four amino acids at the base of the first finger confer specificity of DNA binding.

• A dimerization domain near the base of the second finger is the region that interacts with another GR to form a homodimer.

• Each protein in the pair recognizes half of a two-part DNA regulatory element called a glucocorticoid response element (GRE).

• GR distinguishes both the base sequence and the spacing of the half sites.

• Other nuclear receptors share the same sequence, but have different spacing in between.

Greig cephalopolysyndactyly syndrome and Sonic hedgehog signaling

• Autosomal dominant disease.

• Physical abnormalities affecting the fingers, toes, head, and face.

– Polydactyly: extra fingers or toes.– Syndactyly: webbing and/or fusion of the

fingers and toes.

• Mutations in the GLI3 gene.

• Mutation in the zinc finger protein GLI3 disrupts the Sonic hedgehog signaling pathway during development.

• Sonic hedgehog is one of three vertebrate homologs to the Drosophila hedgehog gene

• Named for “Sonic the Hedgehog,” a character in a popular video game.

• Encodes a secreted protein.

Sonic hedgehog signaling

• The transmembrane receptor protein, Patched-1 (PTC-1) inhibits downstream signaling by interaction with Smoothened.

• When the Sonic hedgehog signal (SHH) binds PTC-1 it relieves repression of Smoothened.

• Leads to the activation and repression of target genes by GLI family transcription factors.

Defective histone acetyltransferases in Rubinstein-Taybi syndrome

• Rubinstein-Taybi syndrome is a rare autosomal dominant disease characterized by facial abnormalities, broad digits, stunted growth, and mental retardation.

• The disease is due to mutations in the gene coding for CBP, a coactivator with histone acetyltransferase activity.

Basic leucine zipper (bZIP)

• bZIP motif is a stretch of amino acids that folds into a long -helix with leucines in every seventh position, forming a hydrophobic “stripe.”

• bZIP motif is not the DNA binding domain.

• Plays an indirect structural role in DNA binding by facilitating dimerization.

• Two polypeptides with a bZIP motif form a “coiled-coil” Y-shaped structure.

– Homodimer: two of the same polypeptide.

– Heterodimer: two different polypeptides.

• One end of each -helix protrudes into the major groove of the DNA.

• The two basic binding regions contact the DNA.

• The transcription factor AP-1 is a heterodimer of Fos and Jun.

• The bZIP domains are essential for binding.

• Jun can form both homodimers and heterodimers.

• Fos can only form heterodimers.

Basic helix-loop-helix (bHLH)

• Forms two amphipathic helices, containing all the charged amino acids on one side of the helix.

• Helices are separated by a loop.

• bHLH motif is not the DNA binding domain.

• Plays an indirect structural role in DNA binding by facilitating dimerization.

• When the BHLH protein Max binds to Myc, the Myc-Max complex is a transcriptional activator.

• When Max binds to Mad, the Mad-Max complex is a transcriptional repressor.

Transactivation domain

• Activates transcription via protein-protein interactions.

• Structurally more elusive than DNA-binding motifs.

– “Acid blobs”– Glutamine-rich regions– Proline-rich regions– Hydrophobic -sheets.

Dimerization domain

• The majority of transcription factors bind DNA as homodimers or heterodimers.

• What are two well-characterized dimerization domains?

11.6 Transcriptional coactivators and corepressors

Increase or decrease transcriptional activity without binding DNA directly by:

• Serving as scaffolds for recruitment of proteins with enzymatic activity.

• Having enzymatic activity themselves for altering chromatin activity.

• More difficult to study compared with transcription factors.

• In general, assays for protein-protein interactions are more difficult to perform than techniques for studying DNA-protein interactions.

Two main classes of coactivators

• Chromatin modification complexes.

• Chromatin remodeling complexes.

Chromatin modification complexes

• Multiprotein complexes that modify histones post-translationally, in ways that allow greater access of other proteins to DNA.

Post-translational modification of histone N-terminal tails

• The N-terminal tails of histones H2A, H2B, H3, and H4 are subject to a wide range of post-translational modifications.

• Function as master on/off switches that determine whether a gene is active or inactive.

• Recognition landmarks by other proteins that bind chromatin.

Four major types of modification

• Acetylation of lysines• Methylation of lysines and arginines• Ubiquitinylation of lysines• Phosphorylation of serines and threonines

Two less common types

• ADP-ribosylation of glutamic acid• Sumoylation of lysines

• Levels of specific histone modifications or “marks” are maintained by balanced activities of modifying and demodifying enzymes.

Histone acetyltransferases

• Histone acetyltransferase (HAT) directs acetylation of histones at lysine residues.

• Histone deacetylase (HDAC) catalyzes removal of acetyl groups.

• The addition of the negatively charged acetyl group reduces the overall positive charge of the histones.

• Decreased affinity of the histone tails for the negatively charged DNA.

• Acetylation of lysines provides a specific binding surface that can either recruit repressors or activators of gene activity.

Histone methyltransferases

• Histone methyltransferase (HMT) directs methylation of histones on both lysine and arginine residues.

• Histone demethylase removes methyl groups.

e.g. lysine-specific demethylase 1 (LSD-1)

• The methyl groups increase the bulk of histone tails but do not alter the electric charge.

• Histone methylation is linked to both activation and repression of transcription.

Ubiquitin-conjugating enzymes

• A ubiquitin-conjugating enzyme adds one ubiquitin to a lysine residue.

• Isopeptidase removes ubiquitin.

• Monoubiquitinylation of H2B is associated with activation or silencing.

• Monoubiquitinylation of linker histone H1 leads to its release from DNA and gene activation.

Kinases

• A specific kinase adds a phosphate group to one or more serine or threonine amino acids, adding a negative charge.

• Phosphatase removes phosphate groups.

• Phosphorylation of histone H3 or the linker histone H1 is associated with the activation of specific genes.

ADP-ribosyltransferases

• ADP-ribosyltransferase adds an ADP-ribose from NAD+ to a glutamic acid residue.

• ADP-ribosylhydrolase removes ADP-ribose.

• MacroH2A a histone variant associated with X-chromosome inactivation may function in ADP-ribosylation of histones.

SUMO-conjugating enzymes

• Small ubiquitin-like modifier (SUMO) is a 97 amino acid protein that has 20% identity with ubiquitin.

• SUMO-conjugating enzymes add SUMO to lysines.

• SUMO-specific proteases remove SUMO.

• In most cases, sumoylation is associated with transcription repression.

Linker histone variants

• Mammals contain eight histone H1 subtypes

• H1a through H1e and H1 in somatic cells

• Two germ-cell specific subtypes, H1t and H1oo.

Is there a histone code?

• Histone modifications are used as recognition landmarks by other proteins.

• Chromodomain motif targets proteins to methylated lysines

• Bromodomain motif targets proteins to acetlyated lysines.

The histone code hypothesis

• Covalent post-translational modifications of histone tails are read by the cell and lead to a complex, combinatorial transcriptional output.

• The hypothesis continues to be a subject of much debate:

– Some researchers conclude that if there is a “code” it is a simple one and not combinatorial.

– Other researchers conclude that histone modifications are more like a “complex language.”

Depression of the MyoD gene by the linker histone H1b

• The Msx1 homeodomain protein forms a complex with H1b.

• The complex binds to an enhancer element in the MyoD gene and inhibits gene expression.

• This prevents differentiation of muscle progenitor cells.

Chromatin remodeling complexes

• Use the energy from ATP hydrolysis to change the contacts between histones and DNA.

• Allow transcription factors to bind to DNA regulatory elements.

Mediate at least four different changes in chromatin structure:

• Nucleosome sliding

• Remodeled nucleosomes

• Nucleosome displacement

• Nucleosome replacement

Three main families defined by a unique subunit composition and the presence of a distinct ATPase

• SWI/SNF complex family

• ISWI complex family

• SWR1 complex family

Mode of action of SWI/SNF: nucleosome sliding and disassembly

• The SWI/SNF complex from the budding yeast was the first chromatin remodeling complex to be characterized.

• 2 MDa complex composed of at least 11 different polypeptides.

• Many other chromatin remodeling factors in this family have been identified, from Drosophila to humans.

ISWI chromatin remodeling complexes slide histone octamers

along DNA

• Change the position of a nucleosome on the DNA.

• Relocate nucleosomes by sliding the histone octamers along the DNA without perturbation of their structure.

Histone replacement with a variant histone by the SWR1 chromatin

remodeling complex

• Histone replacement with a variant histone in the core octamer.

• Can replace histone H2A-H2B dimers with H2A.Z-H2B dimers.

11.7 Transcription complex assembly: the enhanceosome model versus the

“hit-and-run” model

• The dynamic process by which transcription factors and coactivators interact on DNA to activate transcription is the subject of much study.

Order of recruitment of various proteins that regulate transcription

• No general rule for the order of recruitment

• Gene-specific order of events.

Yeast HO promoter:

• The Swi5p transcription factor recruits SWI/SNF and a HAT complex, followed by a second transcription factor, SBF, before assembly of the preinitiation complex.

Human -antitrypsin gene promoter:

• Multiple HAT complexes and SWI/SNF are recruited after preinitiation complex assembly.

Two models for binding of transcription factors and assembly of transcription complexes

• Enhanceosome model

• Hit and run model

Enhanceosome model

• Interactions among transcription factors promote their cooperative stepwise assembly on DNA.

• Exceptionally stable complex.

• Example: interferon- promoter.

Hit and run model

• The “hit”: Transcriptional activation reflects the probability that all components required for activation will meet at a certain site.

• The “run”: Binding is transient.

• Transient and dynamic binding can be observed by fluorescence recovery after photobleaching (FRAP) experiments.

• Example: The glucocortiocoid receptor binds and unbinds to chromatin in cycles of only a few seconds.

Merging of models

• The principles of combinatorial interaction and complex stability apply to hit and run models even if the complex itself has a very limited lifetime.

• Example: Interaction between high mobility group box 1 protein (HMGB1) and the glucocorticoid receptor lengthens each other’s residence time on chromatin.

11.8 Transcription elongation through nucleosomes

• Pausing of RNA pol II in early elongation plays an important role in gene regulation.

• In Drosophila, RNA pol II synthesizes 25-50 nt of RNA prior to heat shock and then pauses.

• Heat-shock jump starts the polymerase and it immediately begins elongation.

Transcription elongation

RNA transcript synthesis by RNA pol II occurs by a four step cycle:

1.A nucleoside triphosphate (NTP) enters the entry (E) site beneath the active center.

2.The NTP rotates into the nucleotide addition (A) site and is checked for mismatches.

3. Pretranslocation: phosphodiester bond formation.

4. Translocation and post-translocation: the NTP just added to the RNA transcript moves into the next position, leaving the A site open for entry of another NTP.

Proofreading and backtracking

• RNA polymerase has one “tunable active site” that switches between RNA synthesis and cleavage.

• How does this compare with DNA polymerase proofreading?

Proofreading

• RNA polymerization and cleavage both require metal ion “A” (e.g. Mg2+ ) in the active site.

• The differential positioning of metal ion “B” switches activity from polymerization to cleavage.

Backtracking

• When transcribing, if RNA pol II encounters an arrest site, the polymerase pauses.

• The polymerase then backtracks, and with the help of TFIIS cleaves the unpaired 3′ end of the transcript.

• Transcription then continues on past the arrest site.

Role of TFIIS in RNA cleavage

• TFIIS is proposed to insert an acidic -hairpin loop into the active center of RNA pol II to position metal B and a nucleophilic water molecule for RNA cleavage.

Transcription elongation through the nucleosomal barrier

• Most of the factors discussed so far are required for the initiation of transcription but not for elongation.

• RNA polymerase encounters a nucleosome approximately every 200 bp.

• Other factors are needed for the polymerase to move through the nucleosomal array.

Two distinct mechanisms for the progression of RNA polymerases through chromatin

• Nucleosome mobilization or “octamer transfer.”

• Histone H2A-H2B dimer removal.

Nucleosome mobilization

• Mechanism for RNA polymerase III and bacteriophage SP6 RNA polymerase.

• Nucleosomes are translocated without release of the core octamer.

• May be facilitated by the elongation factor FACT.

Histone H2A-H2B dimer removal

• Mechanism for RNA polymerase II.

• Requires a number of auxiliary factors, including:

– FACT (facilitates chromatin transcription)– Elongator– TFIIS

FACT promotes nucleosome displacement

• Experiments have shown that FACT mediates displacement of an H2A-H2B dimer, leaving a “hexasome” on the DNA.

• FACT helps to redeposit the dimer after passage of RNA pol II.

Elongator facilitates transcript elongation

• Human Elongator is composed of six subunits, including a HAT with specificity for histone H3.

• Interacts directly with RNA pol II and facilitates transcription.

TFIIS relieves transcriptional arrest

• The elongation factor, TFIIS, facilitates passage of RNA pol II through regions of DNA that can cause transcription arrest.

– AT-rich sequences.

– The presence of DNA-binding proteins.

– Lesions in the transcribed strand.

Defects in Elongator and familial dysautonomia

• Familial dysautonomia is a disorder of the sensory and autonomic nervous system.

• Autosomal recessive.

• Common in Ashkenazi Jewish populations.

Symptoms of familial dysautonomia

• Absence of tears when crying.

• Decreased perception of heat, pain, taste:– e.g. an individual leaning on a pot of boiling

water may not feel it and could be seriously burned.

• Breath-holding episodes.

• Vomiting in response to stress.

A defect in the IKBKAP gene causes familial dysautonomia

• Mutations in IKBKAP, the gene encoding one subunit of Elongator.

• Splice site mutation results in tissue-specific exon-skipping.

• Brain cells are particularly sensitive.

• Link between gene mutation and symptoms is not yet clear.

11.9 Nuclear import and export of proteins

• Protein synthesis occurs in the cytoplasm.

• How do transcription factors get into the nucleus?

• Trafficking between the nucleus and cytoplasm occurs via the nuclear pore complexes (NPCs).

The nuclear pore complex

• Large multiprotein complexes embedded in the nuclear envelope.

• Eight-fold radial symmetry.

• Composed of about 30 different nucleoporins.

• Central 9-11 nm channel that can increase to an effective diameter of 45-50 nm.

• The NPC allows bidirectional passive diffusion of ions and small molecules.

• Nuclear proteins, RNAs, and RNPs larger than ~9 nm in diameter (~40-60 kD) selectively, and actively, enter and exit the nucleus by a signal-mediated and energy-dependent process.

• Proteins are targeted to the nucleus by a specific amino acid sequence called a nuclear localization sequence (NLS).

• Some shuttling proteins also have a nuclear export sequence (NES).

• Nuclear import and export are mediated by a family of soluble receptors, collectively called karyopherins.

• The presence of several different NLSs and NESs and multiple karyopherins suggest the existence of multiple pathways for nuclear localization.

Karyopherins mediate nuclear import and export

• Karyopherins are composed of helical molecular motifs called HEAT repeats.

• Form highly flexible superhelical or “snail-like” structures.

• Karyopherins that mediate nuclear import are called importins.

• Karyopherins that mediate export are called exportins.

• Importin-1 is one of the predominant karyopherins that drives import.

• Most cargoes require the adaptor protein importin

• Seven different importin- adapters have been characterized in mammals.

Nuclear import pathway

• Signal sequences targeting proteins to the endoplasmic reticulum or mitochondrion are removed during transit.

• In contrast, nuclear proteins retain their NLSs.

• There is no real consensus sequence, but many are lysine and arginine-rich.

• Some are bipartite.

The process of nuclear import involves three main steps:

1.Cargo recognition and docking.

2.Translocation through the nuclear pore complex.

3.Cargo release and receptor recycling.

Cargo recognition and docking

• The import receptor for the classic, lysine/arginine-rich NLS is a complex of importin- and importin-1.

• Importin binds directly to the NLS of the cargo.

• Importin 1 binds to both importin and to NPC proteins.

• This step does not require energy.

Translocation through the nuclear pore complex

• The exact mechanism for cargo translocation is poorly understand.

• Weak hydrophobic interactions between importins and the FG repeat domains of nucleoporins seem to be essential.

• This step does not require energy and transport occurs via diffusion.

Two current models for translocation through the nuclear pore complex

• Affinity gate model

• Selective phase model

• Nuclear import (and export) occurs against a concentration gradient .

• The energy source and directional cue are provided by the small GTPase Ran.

• RanGTP is at a high concentration within the nucleus and a low concentration within the cytoplasm.

• Ran belongs to a superfamily of GTP-binding proteins that act as molecular switches cycling between GDP- and GTP-bound states.

• The conversion from the GDP- to GTP-bound state involves nucleotide exchange.

• The conversion from the GTP- to GDP-bound state occurs by removal of the terminal phosphate from the bound GTP.

• The RanGTP gradient is maintained by an asymmetric distribution of auxiliary factors.

• The Ran guanosine-nucleotide exchange factor RanGEF is a resident nuclear protein that binds chromatin.

• The Ran-specific GTPase-activating protein (RanGAP) is excluded from the nucleus and is at its highest concentration at the outer face of the NPC.

Cargo release and receptor recycling

• Once the cargo-import receptor complex reaches the nuclear side of the NPC, RanGTP binds to the importin and dislodges it from the cargo.

• The RanGTP-importin complex is exported to the cytoplasm.

• After GTP hydrolysis, the export complex dissociates.

• The importins are recycled for another round of import.

• RanGDP is rapidly imported into the nucleus by transport factor NTF2.

• RanGDP is converted to RanGTP by nucleotide exchange with the aid of RanGEF.

Nuclear export pathway

• The best characterized nuclear export sequences (NESs) are leucine-rich.

• First described in the HIV-1 Rev protein.

• The classic Rev-type NES functions by interaction with the export factor CRM1.

• Exportins bind to their cargo in the nucleus in the presence of RanGTP.

• In the cytoplasm, the “spring-loaded” complex disassembles spontaneously upon GTP hydrolysis.

• The export receptor is recycled.

11.10 Regulated nuclear import and signal transduction

pathways

• Localization of a protein at “steady” state depends on the balance between import, retention, and export, and which signal is dominant.

• A transcription factor may be sequestered in the cytoplasm until an extracellular signal induces its nuclear import.

Regulated nuclear import of NF-B

• NF- B is a dimeric transcription factor that is a central mediator of the human stess response.

• Plays a key role in regulating cell division, apoptosis, and immune and inflammatory responses.

The events leading to signal-mediated nuclear import of NF-B involve three main stages:

1.Cytoplasmic retention of NF-B by I-B.

2.A signal transduction pathway that induces phosphorylation and degradation of I-B.

3. I-B degradation results in exposure of the NLS on NF-B, allowing nuclear import of NF-B.

Cytoplasmic retention by I-B

• In a resting B lymphocyte, NF-B subunits (e.g. p50 and p65) form homodimers or heterodimers in the cytoplasm.

• The dimers are retained in the cytoplasm by an anchor protein called I-B.

• I-B contains a stretch of 5-7 ankyrin repeat domains that mask the NLS of NF-B.

Signal transduction pathways induce phosphorylation and degradation

of I-B

• Upon receipt of an extracellular signal (e.g. tumor necrosis factor ), a signal transduction pathway is triggered.

• Leads to activation of the serine-specific I-B kinase (IKK) complex.

• I-B is phosphorylated at two conserved serines.

I-B degradation results in exposure of the NLS on NF-B

• Phosphorylation of I-B triggers release from NF-B and proteasome-mediated degradation.

• The NLS of NF-B is exposed.

• In the nucleus NF-B activates target genes by binding to specific DNA regulatory elements.

Regulated nuclear import of the glucocorticoid receptor

• The glucocorticoid receptor mediates a highly abbreviated signal transduction pathway.

• The receptor for the extracellular signal is cytoplasmic and carries the signal directly to the nucleus.

• Leads to many diverse cellular responses ranging from increases in blood sugar to anti-inflammatory actions.

In the absence of hormone

• GR remains cytoplasmic, bound in a complex with Hsp90 and p59.

In the presence of hormone

• Ligand-induced conformational change releases GR from the Hsp90-p59 complex.

• Recent evidence suggests that Hsp90 may play a role in efficient nuclear entry.

• Two GRs form a homodimer, which undergoes nuclear import.

• GR activates target genes by binding to specific DNA regulatory elements.