Transcript Transcription in Eukaryotes

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:
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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 DNAdependent RNA polymerases and organellespecific 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 transacting 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 cisregulatory 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 mid1980s as a cluster of DNase Ihypersensitive 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 proteinprotein 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 tissuespecific 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 activatorresponsive 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 genespecific 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 posttranslational 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 DNAbinding 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 Xchromosome.
• 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 twopart 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 Nterminal tails
• The N-terminal tails of histones H2A, H2B, H3,
and H4 are subject to a wide range of posttranslational 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 Xchromosome inactivation may function in ADPribosylation 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 2550 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 energydependent 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.