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REGULATION OF GENE EPRESSION IN EUKARYOTES
The organizational structure of an eukaryotic cell determines the
mode of gene regulation:
Chromatin packaging into nucleosomes and other organized
structures → possible control at the chromatin structure level
Compartmentalization of the cell→ need of internal signaling
system to communicate between different compartments
Multicellular organism→ need of intercellular communication
system
Differentiation of a totipotent cell into different cell types during
body formation → spatial and temporal regulation
The organizational structure of an eukaryotic cell determines the mode of gene
regulation :
Due to organizational characteristics of eukaryotic cell
and organism, and the spatial and temporal separation
of transcription and translation, the regulation of gene
expression in eukaryotes can be exerted at more levels
than in prokaryotes,.
CONTROL AT THE LEVEL OF CHROMATIN AND GENOME STRUCTURE
Chromatin structure and organization fundamentally
affect gene expression by changing the chromatin
structure, especially its compaction state.
The degree of chromatin compaction essentially relies
on histone modifications and DNA methylation.
Active chromatin regions usually contain high rate of
acetylated histones and unmethylated DNA whereas
inactive regions are associated with nonacetylated
histones and methylated DNA.
Histone modifications and DNA methylation constitute the base of a
special mechanism of gene expression control called epigenetic
inheritance.
Epigenetic inheritance refers to inherited gene expression pattern
independent of modifications in DNA sequence. It concerns
alternative heritable expression of genes that occur throughout the
whole life of an organism and usually expand to its offspring.
Epigenetic inheritance is essential to the normal development of
eukaryotes. Some phenomena considered as epigenetic regulation
involve X chromosome inactivation and genomic imprinting.
Epigenetic inheritance is crucial for normal embryonic development,
plays important roles in cancerogenesis and other biological
processes.
What is Epigenetics?
• Study of heritable changes in gene function
that do not involve changes to the nucleotide
sequence of DNA
• When a cell undergoes mitosis or meiosis, the
epigenetic information is stably transmitted to
the subsequent generation
• Epigenetic controls add an ‘extra layer’ of
transcriptional control
Epigenetics was coined by C. H. Waddington in 1942.
Epigenesis is an old word which has more recently been used to
describe the differentiation of cells from their initial totipotent state
in embryonic development.
When Waddington coined the term the physical nature of genes and
their role in heredity was not known; he used it as a conceptual
model of how genes might interact with their surroundings to
produce a phenotype.
Robin Holliday defined epigenetics as "the study of the mechanisms
of temporal and spatial control of gene activity during the
development of complex organisms.“
Thus epigenetic can be used to describe anything other than DNA
sequence that influences the development of an organism.
The molecular basis of epigenetics is complex. It involves modifications of
the activation of certain genes, but not the basic structure of DNA.
Additionally, the chromatin proteins associated with DNA may be
activated or silenced.
This accounts for why the differentiated cells in a multi-cellular organism
express only the genes that are necessary for their own activity.
Epigenetic changes are preserved when cells divide. Most epigenetic
changes only occur within the course of one individual organism's
lifetime, but, if a mutation in the DNA has been caused in sperm or egg
cell that results in fertilization, then some epigenetic changes are
inherited from one generation to the next.
This raises the question of whether or not epigenetic changes in an
organism can alter the basic structure of its DNA, a form of Lamarckism.
Specific epigenetic processes include paramutation, bookmarking,
imprinting, gene silencing, X chromosome inactivation, position
effect, reprogramming, transvection, maternal effects, the progress
of carcinogenesis, many effects of teratogens, regulation of histone
modifications and heterochromatin, and technical limitations
affecting parthenogenesis and cloning.
Epigenetic research uses a wide range of molecular biologic
techniques to further our understanding of epigenetic phenomena,
including chromatin immunoprecipitation, fluorescent in situ
hybridization, methylation-sensitive restriction enzymes, DNA
adenine methyltransferase identification (DamID) and bisulfite
sequencing.
Furthermore, the use of bioinformatic methods is playing an
increasing role (computational epigenetics).
Three major epigenetic processes we will
discuss
• DNA Methylation
• Histone modifications
• RNA-mediated phenomena
DNA methylation is a biochemical process that is important for normal
development in higher organisms.
It involves the addition of a methyl group to the 5 position of the cytosine
pyrimidine ring or the number 6 nitrogen of the adenine purine ring (cytosine
and adenine are two of the four bases of DNA). This modification can be
inherited through cell division.
DNA methylation is a crucial part of normal organismal development and
cellular differentiation in higher organisms. DNA methylation stably alters the
gene expression pattern in cells such that cells can "remember where they
have been" or decrease gene expression; for example, cells programmed to be
pancreatic islets during embryonic development remain pancreatic islets
throughout the life of the organism without continuing signals telling them
that they need to remain islets.
DNA methylation is typically removed during zygote formation and reestablished through successive cell divisions during development. However,
the latest research shows that hydroxylation of methyl group occurs rather
than complete removal of methyl groups in zygote.
DNA Methylation
Most well-studied epigenetic tag/mark; best understood
epigenetic cause of disease
Conserved across various kingdoms of life
SAM – S-adenosylmethionine
SAH – S-adenosylhomocystine
So, G, A, T, C…. and the fifth base, mC in
mammalian genome
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Distribution of DNA methylation
• In mammals, in the context of CpG dinucleotides (plants have
other types too)
• Methylated CpGs are associated with silenced DNA, eg.
Transposons, inactive X chromosome, imprinted genes
• “CpG islands”, associated with promoters of 40% of
mammalian genes, are generally free of methylation
eg. housekeeping genes, tissue-specific genes
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DNA methyltransferases (DNMTs)
2 major classes of enzymes in mammalian systems
De novo
methylases
Maintenance
methylase
Mouse knockouts of these genes tell us they
are necessary for the survival and proper
development of the organism.
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In mammalian cells, DNA methylation occurs mainly at the C5 position of
CpG dinucleotides and is carried out by two general classes of enzymatic
activities – maintenance methylation and de novo methylation.
Maintenance methylation activity is necessary to preserve DNA
methylation after every cellular DNA replication cycle.
Without the DNA methyltransferase (DNMT), the replication machinery
itself would produce daughter strands that are unmethylated and, over
time, would lead to passive demethylation.
DNMT1 is the proposed maintenance methyltransferase that is
responsible for copying DNA methylation patterns to the daughter
strands during DNA replication.
It is thought that DNMT3a and DNMT3b are the de novo
methyltransferases that set up DNA methylation patterns early in
development.
DNMT3L is a protein that is homologous to the other DNMT3s but
has no catalytic activity. Instead, DNMT3L assists the de novo
methyltransferases by increasing their ability to bind to DNA and
stimulating their activity.
Finally, DNMT2 (TRDMT1) has been identified as a DNA
methyltransferase homolog, containing all 10 sequence motifs
common to all DNA methyltransferases; however, DNMT2 (TRDMT1)
does not methylate DNA but instead methylates cytosine-38 in the
anticodon loop of aspartic acid transfer RNA.
How does DNA methylation affect gene
transcription?
Unmethylated (or
hypomethylated) promoter
allows gene transcription
Methylated CpGs block
binding of TFs; hence,
transcription blocked
Me-CpG binding proteins
also preclude TF binding to
the promoter region
Other ways too…
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Role of DNA methylation
• Tight control for maintaining gene silencing (vertebrate genes
are less “leaky” compared to bacterial)
• Transcriptional silencing of transposons (‘genome defense’
model)
• Genomic imprinting – one of the alleles of a gene is silenced,
depending on the parent of origin
• X inactivation – all but one of the X chromosomes in
female is inactivated – methylation of the inactive X copy
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Three major epigenetic processes
• DNA Methylation
• Histone modifications
• RNA-mediated
phenomena
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For gene expression, eukaryotic DNA must be decompacted to
become accessible to transcription initiators. The decompaction
process is ensured by nucleosome modifiers.
Nucleosome modifiers are classified into two groups :
1. Enzymes that modify the amino-terminal tails of histones such as
histone
deacetylases,
histone
acetylases
and
histone
methyltransferases
2. Remodeler complexes that “loosen” the interaction between DNA
and histones
Modifications of the chromatin can activate gene expression by two
ways :
1. “Loosening” the chromatin structure, thus liberating binding sites
for regulatory proteins
2. Enhancing the binding of some particular regulatory proteins to
the modified chromatin
Structural organization of the genome
Unless the genome is
accessible
by
the
transcription machinery of
the cell, the genome cannot
be functional!
Hence, the utilization of the
biological information in the
genome is dependent on
the chromatin organization.
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Structure of a nucleosome
~146 bp
DNA
Histone octamer
core
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Post-translational histone modifications
The amino-terminal tails of nucleosomal histones protrude from the
DNA and are subject to covalent modifications.
These modifications include lysine acetylation, lysine and arginine
methylation, serine and threonine phosphorylation, ADP-ribosylation,
and ubiquitination.
Histone lysine methylation can have different effects depending on
the residue that is modified: methylation of histone H3 at Lys4 (H3K4)
is associated with gene activation, whereas methylation of H3K9,
H3K27, and H4K20 generally correlates with transcriptional
repression.
The roles of H3K36 and H3K79 methylation remain elusive; indeed,
these modifications are associated with both transcriptional activation
and repression.
Different modifications of histone amino-terminal tails constitute the
so-called 'histone code‘ .
Indeed, specific combinations of histone modifications can alter
chromatin structure to allow transcription or to repress it, either
reversibly or stably.
Chromatin modifications confer a unique identity on the nucleosomes
involved. The composite pattern of modifications regulates the
binding and activities of other chromatin-associated components.
Indeed, modifications of histones at a specific nucleosome very likely
influence subsequent modifications, regulated by both cis and trans
mechanisms.
Characterizing such modifications could provide insight into the roles
of chromatin-binding proteins
Post-translational histone modifications
A = acetylation
M = methylation
P = phosphorylation
U = ubiquitination
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Consequences of tail modifications
• Higher order chromatin structure is affected
eg. Addition of acetyl groups (-ve) neutralizes the positive
charge on lysine
=> affinity of the histone to bind tightly to DNA is reduced
=> chromatin becomes less compact
=> transcription of the associated gene is favored
Vice versa for deacetylation (the gene is repressed)
• Other proteins are attracted to these sites of
odifications….which, in turn, affect gene expression
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Enzymes catalyze these covalent tail
modifications
• Histone Acetyl Transferases (HATs)
function as large, multiprotein complexes, eg. SAGA,
ADA complexes (yeast), TFTC complexes (humans);
associated with transciptional activation.
• Histone Deacetylases (HDACs)
part of multiprotein complexes, eg.Sin3, NuRD;
associated with transcriptional repression.
• Histone Methyl Transferases (HMTs)
• Histone Demethylases
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Comparing chromatin types
Transcriptionally active
chromatin/euchromatin
Transcriptionally inactive
chromatin/
heterochromatin
Chromatin conformation
Open, extended
conformation
Highly condensed
conformation
DNA CpG methylation
Relatively unmethylated,
especially at promoter
regions
Methylated, including at
promoter regions
Histone acetylation
Acetylated histones
Deacetylated histones
Histone methylation
H3-K4me3, R17me2
H3-K9me
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Crosstalk between DNA methylation and
chromatin modification
DNA
methylation
Self-reinforcing
repressive cycle
Histone
deacetylation
Histone H3-K9
methylation
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Three major epigenetic processes
• DNA Methylation
• Histone modifications
• RNA-mediated
phenomena
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RNA interference (RNAi) is a system within living cells that
takes part in controlling which genes are active and how active
they are.
Two types of small RNA molecules – microRNA (miRNA) and
small interfering RNA (siRNA) – are central to RNA
interference.
RNAs are the direct products of genes, and these small RNAs
can bind to other specific RNAs (mRNA) and either increase or
decrease their activity, for example by preventing a messenger
RNA from producing a protein.
RNA interference has an important role in defending cells
against parasitic genes – viruses and transposons – but also in
directing development as well as gene expression in general.
RNA interference (RNAi) causes gene
silencing
RNAi initiates heterochromatin formation in fission
yeast and DNA methylation in plants.
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Epigenetics in human disease
Association with various cancers – stomach, kidney,
colon, pancreas, liver, uterus, lung and cervix
ICF syndrome
Fragile X syndrome
Angelman’s syndrome
Rett Syndrome
HUMAN “EPIGENOME”
PROJECT
Coffin-Lowry Syndrome
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BIOLOGICAL MEANINGS OF EPIGENETIC INHERITANCE
Epigenetic inheritance play crucial roles in normal growth and
development of multicellular eukaryotic organisms :
In embryonic development, epigenetic abnormalities can lead to genetic
disorders such as Prader Willi and Angelman syndromes.
Babies with Prader-Willi and Angelman syndromes are born with both
alleles expressed, an abnormal active paternal allele (Prader-Willi) or an
abnormal active maternal allele (Angelman) of the same gene.
In Assisted Reproductive Technologies, epigenetic inheritance is thought
to be associated with abnormal embryonic development due to loss of
maternal/paternal selective allele expression and high rate of embryonic
losses.
It is thought that imprinting is a tentative of the mother to protect
herself from her fetus. Silencing of maternal alleles limit the fetus
growth.
DNA hypermethylation cause tumor suppressot gene silencing
whereas DNA hypomethylation favorize oncogene expression.
These are the cause of many cancer types, e.g the aberrant
methylation pattern of Igf2 and H19 genes give rise to
simultaneous expression of maternal and paternal alleles and
are the cause of many human cancers.
Demethylating agents and agents promoting histone
acetylation constitute possible therapeutic approaches for
certain cancers.
Epigenetic control is thought to be used by cells to silencing
some regions in the genome containing repetitive “useless”
DNA, e.g inserted “foreign” (viral) sequences (transposon).
Most of these transposons are methylated