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Part IV => DNA and RNA
§4.6 GENE REGULATION
§4.6a Chromatin Remodeling
§4.6b Transcriptional Control
Overview of Gene regulation
- While the expression of the so-called “housekeeping” genes (eg basal
transcription factors) occurs in a constitutive manner (they are expressed
at a constant level—so as to maintain basic cellular functions—as opposed
to being turned on or off as the circumstances demand), the expression of
many critical genes is tightly regulated lest the chaos take over
- Simply put, gene regulation is the process by which the cell controls:
(a) When to turn on or off a gene
(b) The rate at which to turn on or off a gene
(c) The duration for which the gene is to be turned on
- Such gene regulation is hierarchical in that it occurs at multiple levels—ie
it can be tweaked at various stages such as chromatin level or
transcriptional level
- In particular, chromatin remodeling and transcriptional control represent
two key stages at which the expression of a gene can be tightly regulated
Hierarchy of Gene Regulation
Chromatin
Remodeling
Chromatin
DNA
Transcriptional Control
RNA 5’-Cap
Intron
Poly(A)
RNA Splicing
mRNA 5’-Cap
Poly(A)
mRNA
Degradation
Translation
Protein
Degradation
Post-translational
Modification (PTM)
Protein
Exon
5’-Cap
Poly(A)
Section 4.6a:
Chromatin Remodeling
Synopsis 4.6b
- In eukaryotes, DNA does not exist in isolation but rather it is wound/wrapped
(or negatively supercoiled) around bead-like protein complexes called
“histones” to form what has come to be known as a “nucleosome”
- Nucleosomes represent the first building blocks for packaging/folding DNA
into a higher-order architecture called the “chromatin”
- Chromatin does not only serve to package DNA into a highly condensed form
but it also serves to protect the DNA as well as act as a “gatekeeper” to
regulate the access of other proteins such as RNA polymerases and
transcription factors, thereby tightly controlling the expression of genes
- In order to allow gene expression in a regulated manner, chromatin must
undergo transient “opening” and “closing” according to cellular demands—
such ability of chromatin to undergo dynamic and flexible structural changes
is referred to as “chromatin remodeling”
- Chromatin remodeling is under the control of a wide array of so-called
“chromatin-remodeling” protein complexes that in part execute their function
via covalent modification of histones and/or DNA
DNA Packaging: The Conundrum
40,000km
1nm = 10 Å
1m = 109 nm
1km = 5/8 mile
How long is human DNA?! Is there really a need to package it into a condensed form?
Within average human cell:
Human genome size
Helical rise (assuming B-DNA)
Contour (fully extended) length
Diameter of human cell nucleus
= 3.2 x 109 bp (from 46 DNA molecules/cell = 46 chromosomes/cell)
= 3.4Å/bp => 0.34nm/bp
= (0.34nm/bp)*(3.2 x 109 bp) => 1.1 x 109 nm => 1.1m (1.1 x 106 µm)
= 10µm => 1.1m DNA thus has to be tightly packaged!
Within average human body:
Number of cells in human body = 10 x 1012 (~10 trillion) cell
(cf: US$20x1012 national debt!)
Total length of human DNA
= (10 x 1012 cell)*(1.1m/cell) => 11 x 1012 m => 11,000 x 106 km
Celestial distances:
Earth circumference
Earth  Moon
Earth  Sun
= 0.04 x 106 km => Human DNA wraps around the earth ~300,000 times!
= 0.40 x 106 km => Human DNA stretches back-and-forth ~30,000 times!
= 150 x 106 km => Human DNA stretches back-and-forth ~100 times!
DNA Packaging: The Big Picture
Heterochromatin—highly-condensed and transcriptionally-inactive form of chromatin
Euchromatin—loosely-packed and transcriptionally-active form of chromatin
DNA Packaging: The Histones
Histone
Length / aa
Mass / kD
Arg / %
Lys / %
Basic / %
H1
215
23
1
29
30
H2A
129
24
9
11
20
H2B
125
14
6
16
22
H3
135
15
13
10
23
H4
102
11
14
11
25
- Histones are small proteins that engage in non-specific interactions with DNA (cf transcription
factors), thereby allowing it to be packaged into a condensed form called “chromatin”—with
five major classes of histones being H1, H2A, H2B, H3, and H4
- How do histones interact with DNA?—Histones are highly basic due to their evolutionary
enrichment with basic amino acid residues such as Lys and Arg
- The basic/alkaline character of histones is a perfect compliment to the negatively charged
DNA—a marriage surely made in heavens!
- Histones are among the evolutionarily most conserved proteins across species—eg histone H4
from cow and pea (species that apparently diverged over a billion years ago!) share a
remarkable 98% identity at amino acid level
- While H4 is the most conserved of all histones, H1 displays highest variability between species
DNA Packaging: The Nucleosome
DNA (2nd turn)
90°
2x H3
2 x H2A
2 x H4
Nucleosome Cartoon
(H2A)2(H2B)2(H3)2(H4)2
2 x H2B
DNA (1st turn)
- Nucleosome represents the basic building block or unit of DNA
packaging with a contour (extended) length of 80Å
- A single nucleosome is comprised of a histone octamer—made up
of two copies of each of H2A, H2B, H3, and H4—around which
double-helical DNA wraps around twice or makes two left-handed
superhelical (coiled double-helix) turns involving ~200bp
- Such negative supercoiling represents a compression of the contour
length of B-DNA by nearly an order of magnitude—200bp equates
to 680Å (200bp∗3.4Å/bp) => 680Å/80Å=9
- H1 histone is not an integral component of nucleosome—H1 docks
at the base of the nucleosome close to the DNA entry and exit so as
to serve as a “glue” or “linker” to bring individual nucleosomes
together into a condensed form (eg the 30-nm fiber—vide infra!)
Nucleosome Structure
Left-handed
Turn (-ve)
Right-handed
Turn (+ve)
DNA Packaging: From DNA to Chromosome
- The continuous left-handed winding
of DNA onto multiple histone octamers
yields a loosely defined “10-nm fiber” with
a “beads-on-a-string” appearance—H1 histone
not needed!
- The 10-nm fiber undergoes further
condensation in the presence of H1 histone to
form what is called the “30-nm fiber”
- Within the 30-nm fiber, the 10-nm fiber winds
up into a solenoid-like coiled conformation
(though it could also fold into the 30-nm fiber in
a zigzag fashion)
- Addition of non-histone “scaffold” proteins to
the 30-nm fiber allows further compaction of
DNA into a higher-order structural organization
called “chromatin”
- Chromatin (harboring 1.1m/46 ≈ 24,000µm of
double-helical DNA) finally packs into an Xshaped chromosome (transiently visible only
during mitosis) roughly measuring 5µm-by-1µm
10 nm
Chromatin Modification: Overview
Temporal—DNA only temporarily or transiently exposed at a time
Spatial—only specific sequences/regions within DNA exposed
- In order to allow cellular processes such as RNA transcription, DNA replication, and DNA repair in a
regulated manner, chromatin must undergo transient “opening” and “closing”—such ability of
chromatin to undergo dynamic and flexible structural changes is called “chromatin remodeling”
- Chromatin remodeling is under the control of a wide array of so-called “chromatin-remodeling”
protein complexes (eg SWI/SNF and RSC )—interactions between histones and DNA within
nucleosomes are disrupted to render the DNA more accessible in a highly temporal and spatial manner
- Simply put, chromatin remodeling complexes facilitate the transient release of DNA from histone
octamers so that it can be accessed by other proteins and enzymes such as RNA polymerase to initiate
gene expression—how?!
Chromatin Modification: Epigenetic Control
- DNA and histones within the chromatin are subject to a wide variety of chemical
modifications that allow the chromatin to undergo remodeling and thereby directly
impinge upon the ability of genes to be switched on or off
- The study of such chromatin modifications—executed by specific protein domains that
form an integral part of chromatin remodeling protein complexes—that affect the
ability of genes to be expressed without altering the DNA sequence itself has come to
be known as “epigenetics”—epi meaning “beyond” or “above”
- Two major epigenetic mechanisms involved in the control of gene expression are:
(1) Histone modification
(2) DNA methylation
Chromatin Modification: Histone Modification
- Histone tails are highly decorated with residues
such as Lys, Arg, Ser, Thr and Tyr
- Many of these residues in histones are
subject to post-translational modification
(PTM) by enzymes that couple the extra/
intra-cellular needs of the cell with the state
of its chromatin—such that gene expression
can be turned on or off in an “epigenetic” manner
- Major histone PTMs include (in the order of prevalence):
- Acetylation (Ac) of Lys residues
- Methylation (Me) of Lys/Arg residues
- Phosphorylation (P) of Ser/Thr/Tyr residues
- Such PTMs not only alter the net charge on histones but also provide docking sites for the
recruitment of chromatin remodeling complexes
- Histone modification thus plays a central role in the alteration of histone-DNA interactions—
reducing the positive charge on histones generally correlates with loosening of chromatin
structure and thus facilitating gene expression and vice versa
Chromatin Modification: Histone Acetylation
HATs
HDACs
Lysine (K)
Acetllysine (AcK)
- Acetylation on Lys is catalyzed by histone acetyltransferases (HATs)—using acetyl-CoA as an acetyl donor
- Lys residues can be deacetylated by histone deacetylases (HDACs)
- Histone acetylation reduces net positive charge on histones—thereby mitigating histone-DNA
interactions, loosening up chromatin structure (favors euchromatin), and making it more accessible to
RNA polymerase and transcription factors
- Acetyl moiety on Lys residues in histone also serves as a docking site for bromodomain—a highly
ubiquitous protein domain found in transcriptional co-activators such as PCAF (a component of
chromatin remodeling protein complexes)
- Although histone acetylation generally correlates with transcriptional activation, it can also result in
transcriptional repression depending on the context of target lysine
Chromatin Modification: Histone Methylation
Lysine (K)
HMTs
HMTs
HMTs
HDMs
HDMs
HDMs
Monomethyllysine (MeK)
Dimethyllysine (Me2K)
Trimethyllysine (Me3K)
- Mono-, di-, or tri-methylation on Lys/Arg is catalyzed by histone methyltransferases (HMTs)—using
S-adenosylmethionine (SAM) as a methyl donor
- Lys/Arg residues can be demethylated by histone demethylases (HDMs)
- Unlike acetylation, histone methylation does not reduce net positive charge on histones—thus it
may loosen up chromatin (favor euchromatin) and make it transcriptionally-accessible, or
alternatively, it may induce condensation of chromatin (favor heterochromatin) and render it
transcriptionally-inaccessible
- Methyl moieties on Lys/Arg residues in histone also provide docking sites for chromodomain—a
highly ubiquitous protein domain found in transcriptional co-activators and co-repressors such as
HP1 (a component of chromatin remodeling protein complexes)
- Histone methylation can correlate both with transcriptional activation and transcriptional
repression depending on the location of Lys/Arg target residue and the state of modification of
other residues within its vicinity
Chromatin Modification: Histone Phosphorylation
Kinases
Phosphatases
Serine (S)
Phosphoserine (pS)
- Phosphorylation on Ser/Thr/Tyr residues is catalyzed by a plethora of protein kinases—using ATP as a
phosphoryl donor
- Ser/Thr/Tyr residues can be dephosphorylated by protein phosphatases
- Histone phosphorylation imparts negative charge on histones—this can have profound effects on
histone conformation leading to both chromatin condensation and relaxation
- Phosphorylated tyrosine (pY) residues in histone may also serve as a docking site for SH2 domain—a
modular component of many cellular proteins as well as transcriptional regulators
- Histone phosphorylation can correlate both with transcriptional activation and transcriptional
repression depending on the cellular context
Chromatin Modification: DNA Methylation
Cytosine (C)
5-methylcytosine (M5C)
- Of the four DNA bases, two are subject to methylation—cytosine and adenine (adenine methylation
only occurs in prokaryotes such as during DNA replication)
- In eukaryotes, only cytosine base in DNA is subject to methylation—
particularly within CG-rich sequences called the “CpG” islands
- Cytosine methylation is catalyzed by DNA methyltransferase (DNMT)—
using S-adenosylmethionine (SAM) as a methyl donor and releasing
S-adenosylhomocysteine (SAH) as a by-product
- Cytosine can also be demethylated by DNA demethylases
SAM
- Unlike the more subtle nature of histone modification, DNA methylation switches off eukaryotic gene
expression by virtue of its ability to induce chromatin condensation
Chromatin Modification: Transcriptional Initiation Complex
Transcription Initiation site
Binding of TAF1 via its bromodomain to
acetylated histones can result in the recruitment
of basal transcriptional factors such as TBP
required for the formation of transcriptional
initiation complex at gene promoters
Exercise 4.6a
- Explain why histones from different species are so similar
- What is the role of histones in compacting DNA?
- Compare the binding of histones and transcription factors to DNA
- Describe the levels of DNA packaging in eukaryotic cells
- Why is chromatin remodeling necessary for efficient gene expression?
- List three major ways that histones can be covalently modified
- Describe how histone modifications can affect the structure of nucleosomes
and the function of transcription factors
- Summarize the role of DNA methylation in epigenetic control
Section 4.6b:
Transcriptional Control
Synopsis 4.6b
- Since the transcriptional machinery is largely dependent upon
transcription factors, modulating the activity of transcription factors
presents a key step in the decision to turn on or off a target gene at
transcriptional level
- In particular, the so-called gene-specific transcription factors (as
opposed to basal transcription factors) are subject to a wide array of
post-translational modifications (PTMs)
- Such PTMs not only upregulate but can also downregulate the activity
of specific transcription factors depending on the state of the cell
- PTMs thus tightly control protein-DNA interactions pertinent to gene
transcription
- In particular, the “modular” design of transcription factors befits their
role as regulators of gene transcription in that they not only become
subject to PTM but may also recruit “co-activators” or “co-repressors”
depending on the cellular needs
Modular Proteins
- With the exception of small proteins designed for simple tasks, a vast array of more
complex and regulatory proteins are not monolithic but rather modular—ie they can be
divided into constituent parts or regions specialized for specific roles
- Such specialized parts/regions of modular proteins are referred to as “modules”, or more
commonly as “domains”
- Modular proteins are a hallmark of the eukaryotic cell—by virtue of such domains, a single
protein may not only accomplish multiple tasks but its function may also be tightly
regulated—eg ligand binding or PTM of one domain within a transcription factor may
control the ability of its DNA-binding (DB) domain to affect the expression of a target gene
Transcription Factors: Modular Design
Transcription
Factor
DNA
Promoter
mRNA
Gene
- The DNA-binding (DB) proteins or Transcription factors harbor a set of well-defined structural
motifs (as a constituent component of the so-called DB domains) such as:
(1) Helix-Turn-Helix
(2) Helix-Loop-Helix
(3) Zinc Finger
(4) Leucine Zipper
- Such structural motifs usually bind to the major groove of DNA by virtue of their ability to
recognize sequence-specific motifs (or cis-acting elements) located within target gene promoters
- Because of the (pseudo)palindromic nature of cis-acting elements within gene promoters, DB
domains usually bind to DNA as dimers—one monomer recognizes the element on one strand and
the other on the opposite within the major groove of the double helix!
- Such dimeric interaction also accounts for the high specificity of protein-DNA interactions in that
the binding of two monomers simultaneously (and usually symmetrically) doubles the free energy
- Upon binding to DNA promoters via their DB domains, transcription factors activate or repress
expression of target genes, usually by recruiting and interacting with other cellular proteins
termed co-activators and co-repressors
Transcription Factors: Ligand-Activated
- SHRs usually exist as monomers in
complex with heat shock proteins
(HSPs) in the cytoplasm
Steroid
Hormone
- After diffusion through the cell
membrane, the binding of the
hormone to the LB domain
results in its dimerization
- Dimeric SHR translocates to the
nucleus and binds to the target
gene promoters via its DB domain
LB
LB
LB LB
+
TA
- Recruitment of cellular factors required
to assemble the transcriptional
machinery at the gene promoters is
aided by the LB and TA domains
- This turns on gene expression of
specific proteins—which in turn set
about causing changes to the cell in
response to the hormone
DB DB
DB
DB
HSPs
SHR
TA
TA
TA
Nucleus
LB LB
mRNA
DB DB
DNA
TA
TA
Transcriptional
machinery
Transcription Factors: PTM-Activated
- Ligand binding to RTK induces receptor
dimerization and/or autophosphorylation
Ligand
- Activated RTK serves as a binding site for
the recruitment of adaptors such as GRB2 (via its P
SH2 domain) to the inner membrane surface
P
(IMS) in a phosphorylation(Tyr)-dependent manner
Extracellular
Ras
RTK
GDP
GTP
P
P
- Since GRB2 adaptor exists in complex with SOS exchange
factor, the recruitment of guanine nucleotide exchange
factor SOS to the IMS catalyzes GDP-GTP exchange in
Ras GTPase, thereby resulting in its activation
- Next, activated Ras binds and activates Raf kinase
SH3
SH2
Raf
SOS
GRB2 SH3
MEK
MAPK
Jun
P
P
Cytoplasm
Nucleus
MAPK
- Raf kinase then activates the kinase MEK via Ser/Thr
phosphorylation
- This is followed by the activation of the MAP kinases (MAPKs)
such as ERK2 by MEK, also via Ser/Thr phosphorylation
Ras
P
mRNA
P
Jun
- Activated MAPK translocates to the nucleus and phosphorylates specific transcription factors (eg
Jun/Fos/Myc)
- Phosphorylated Jun binds to its promoter within the target genes and turns on gene expression of
specific proteins—which in turn set about causing changes to the cell in response to the ligand
Transcription Factors: (1) Helix-Turn-Helix
turn
Helix-Turn-Helix (HTH)
CAP RESPONSE ELEMENT
5’-AATGTGATCTAGATCACATT-3’
3’-TTACACTAGATCTAGTGTAA-5’
- HTH motif is usually comprised of two
consecutive α-helices interrupted by a turn
- Within the inter-helical turn, the polypeptide
chain adopts an extended and ordered structure
so as to reverse its direction and allow the two
helices to pack together
- Examples include bacterial CAP (catabolite
Two HTH motifs (blue and red helices)
activator protein) and viral λ (Lambda repressor) binding to two consecutive major grooves
Transcription Factors: (2) Helix-Loop-Helix
HIF1 RESPONSE ELEMENT
5’-ACGTG-3’
3’-TGCAC-5’
Helix-Loop-Helix (HLH)
Two HLH motifs (green and cyan
helices) binding to a major groove
- HLH motif is usually comprised of two consecutive α-helices interrupted by a loop
- Within the inter-helical loop, the polypeptide chain harbors a highly flexible or disordered
structure so as to allow the two helices to fold back onto each other (without the
polypeptide chain necessarily having to reverse its direction!)
- Examples include HIF1 (hypoxia-inducible factor 1) and Myc (myelocytomatosis oncogene
homolog)
Transcription Factors: (3) Zinc Finger
ERα RESPONSE ELEMENT
5’-AGGTCANNNTGACCT-3’
3’-TCCAGTNNNACTGGA-5’
Zinc Finger (ZF)
Two ZF motifs (green and blue) binding to
two consecutive major grooves
- ZF motif is usually comprised of a Zn2+ divalent ion sandwiched between a two-stranded
antiparallel β-sheet (β-hairpin) and an α-helix (ββα topology)
- Zn2+ ion is tetrahedrally coordinated by four ligands in the form of either four cysteine
residues (C4-type) or two cysteine and two histidine residues (C2H2-type)
- Examples include nuclear/steroid receptors such as ERα (estrogen receptor α) and EGR1
(early growth response 1)
Transcription Factors: (4) Leucine Zipper
- LZ motif is usually characterized by a leucine residue at every
seventh position within 4-5 successive heptads of amino
acids with each of two constituent polypeptide chains
- Inter-chain van der Waals contacts and ionic interactions
drive dimerization of the two amphipathic LZ polypeptide
chains so as to enable them to adopt α-helices packed
against each other
LZ
LZ
- LZ motifs harbor a highly basic region (BR) at the N-terminus
that enables them to adopt continuous α-helices and wrap
around each other into a coiled coil
- LZ motifs together with their BR segments are collectively
referred to as “basic zipper (bZIP)” domains
BR
AP1
RESPONSE
ELEMENT
5’-TGACGTCA-3’
3’-ACTGCAGT-5’
BR
- While bZIP domains contact the major groove
within the DNA via their BR segments, LZ motifs
are critical for their structural integrity
- Examples include Jun and Fos—collectively
referred to as activator protein 1 (AP1)
Leucine Zipper (LZ)
Two bZIP domains (yellow and green)
binding to a major groove
Transcription Factors: Electrostatic Polarization
ZFI
ZFI
ZFI
Positive charge
Negative charge
ZFII
90°
Neutral charge
ZRE
duplex
ZFII
90°
ZFII
ZFIII
ZRE
duplex
ZFIII
ZFIII
Front View
Side View
ZRE
duplex
Back View
Electrostatic surface potential map of a DB domain in complex with DNA
- DB domains are basic proteins in that they carry a net positive charge with pK values
typically greater than 9—and so they should be if they are to be “attracted” to a negativelycharged DNA!
- Not only are they basic but DB domains generally tend to be electrostatically polarized—
they harbor a net positive charge on one face of the molecule that docks into the major
groove (the minor groove is too narrow and thus spatially-restrictive) of DNA
- Ionic (not electrostatic!) interactions thus play a key role in driving protein-DNA
interactions though van der Waals contacts and hydrogen bonding by and large account for
the specificity of such unions of the opposites
Exercise 4.6b
-
Describe the types of interactions between nucleic acids and proteins
-
Compare the major structural motifs within transcription factors involved
in the recognition of DNA
-
Why do transcription factors usually bind to DNA as dimers? Why do they
only bind to the major groove?