Structure of genome and its interaction with
environmental factors
Epigenetics
Department of Pathophysiology, MED MUNI Mgr. Martina Raudenská, Ph.D. / Ing. Hana Holcová Polanská, Ph.D.
Genome structure
Human genome
• Humans have two genomes, nuclear
and mitochondrial.
• Normal diploid cells contain two copies
of the nuclear genome and a much
larger but variable number of copies of
the mitochondrial genome.
• 23 chromosome pairs in cell nuclei. In
a pair of chromosomes, one
chromosome is always inherited from
the mother and one from the father.
• Small DNA molecules found within
individual mitochondria. Each
mitochondrion contains 2-10
mitochondrial DNA copies.
Structure of
protein-coding
genes
• each gene has its own unique
position or locus on
chromosome.
• The different versions of the
gene are known as alleles.
Structure of
protein-coding
genes
• Regulatory sequence controls
when and where expression
occurs for the protein coding
region (red).
• Promoter and enhancer
regions (yellow) regulate the
transcription of the gene into
a pre-mRNA which is modified
to remove introns (light grey)
and add a 5' cap and poly-A
tail (dark grey).
• The mRNA 5' and 3'
untranslated regions (UTR;
blue) regulate translation into
the final protein product.
Alternative splicing
• Alternative splicing is a process that
enables a messenger RNA (mRNA)
to direct synthesis of different
protein variants (isoforms) that may
have different cellular functions or
properties.
• It occurs by rearranging the pattern
of intron and exon elements that are
joined by splicing to alter the mRNA
coding sequence.
Mutations
• A genetic mutation is a permanent
alteration in the DNA sequence (DNA
sequence differs from what is found in most
people).
• Mutations range in size; they can affect
anywhere from a single DNA building block
(base pair) to a large segment of a
chromosome that includes multiple genes
(point mutations, chromosomal mutations,
copy number variation).
Mutations
• Hereditary mutations are inherited from a
parent and are present in virtually every cell
in the body. These mutations are also called
germline mutations because they are present
in the parent’s egg or sperm cells.
• Acquired (or somatic) mutations occur at
some time during a person’s life and are
present only in certain cells, not in every cell
in the body. These changes can be caused by
environmental factors such as ultraviolet
radiation, ionizing radiation, chemicals, or can
occur if an error is made as DNA copies itself
during cell division. Acquired mutations in
somatic cells cannot be passed to the next
generation.
Mutations
• Mutations can result from many events,
including unequal crossing-over during
meiosis. In addition, some areas of the
genome simply seem to be more prone to
mutation than others. These "hot spots" are
often a result of the DNA sequence itself
being more accessible to mutagens. Hot spots
include areas of the genome with highly
repetitive sequences, such as trinucleotide
repeats, in which a sequence of three
nucleotides is repeated many times. During
DNA replication, these repeat regions are
often altered because the polymerase can
"slip" as it disassociates and reassociates with
the DNA strand.
Consequences of DNA
mutations depend on their
position in the gene region
• m1: mutations in the promoter
region may affect gene
transcription. May lead to nonfunctional
(null) alleles.
• m2: mutations in exons, if they
result in the substitution of an
amino acid in the active site or
other critical region of the protein,
also lead to alleles with changed or
null functionality.
Consequences of DNA
mutations depend on their
position in the gene region
• m3: exon mutations that result in
changes outside the active sites or
synonymous mutations (do not alter
the amino acid encoded by the
affected codon due to the
degeneracy of the genetic code but
change the DNA and RNA sequence)
may have little or no effect on gene
function. These mutations are called
silent (if the amino acid is
unchanged) or neutral (if the
change has no effect).
Consequences of DNA
mutations depend on their
position in the gene region
• m4: mutations at critical positions near
intron / exon junctions may affect
mRNA splicing and lead to the deletion
or retention of entire exons, and result
in null alleles.
• m5: mutations that occur in non-coding
introns, may have little or no effect on
gene function.
• m6: mutations that occur in 5' or 3'
UTR may have little or no effect on
gene function but may affect the level
of translation.
Epigenetics
If all cells are created from the same genetic material,
why are there so many different cell types?
Epigenetics
• Epigenetics is defined as the study of changes in
gene expression that are not derived from a
change in the underlying DNA sequence i.e. a
change in the phenotype without changing the
genotype.
• Epigenomics – the study of the complete set of
epigenetic alterations.
• Epigenetic code – epigenetic features that
maintain different phenotypes in different cells.
Epigenetic
mechanisms
A. DNA modifications
B. Chromatin modifications
C. Non-coding RNAs
D. RNA modifications
A. DNA
modifications
• DNA can be modified at cytosine and adenine
residues by the addition of chemical groups.
• Cytosines can be modified by methylation,
hydroxymethylation, formylation and
carboxylation, while adenines can be modified
by methylation.
• In humans, cytosine methylation is the most
frequent, occurring often at CpG sites (cytosine
followed by a guanine base in the DNA
sequence). DNA methylation can also be found at
cytosines followed by i.e., adenine, cytosine, or
thymine, such non-CpG methylation is an
abundant modification in neural tissues and
increases during development.
DNA methylation
• DNA methylation is a process by
which methyl groups are added
to the DNA molecule.
Methylation can change the
activity of a DNA segment
without changing the sequence.
• In humans, cytosine methylation
is the most frequent.
• Cytosine methylation is
catalysed by enzymes known as
DNA methyltransferases (DNMT)
on position 5 (C5) of cytosine
residues to form 5-methyl
cytosine (5-mC).
DNA methylation
• Maintenance DNMT1 activity is necessary to preserve DNA methylation after every cellular DNA
replication cycle.
• Each tissue and cell type has unique DNA methylation profiles.
• DNA methylation patterns change during ontogenesis and are largely erased and then re-established
between generations in mammals. Almost all parental methylation patterns are erased during
gametogenesis and in early embryogenesis.
Dynamic of DNA methylation
during mouse embryonic
development. E3.5-E6, etc., refer
to days after fertilization. PGC:
primordial germ cells.
Consequences of
DNA methylation
• Transcription of most genes encoding
proteins is initiated at promoters rich in
CpG sequences. These sections of CpG-rich
DNA are known as CpG islands.
• When the CpG island in the promoter
region of the gene is methylated, the
expression of the gene is suppressed. CpGdense
promoters of actively transcribed
genes are never methylated.
• The methylation of DNA may physically
impede the binding of transcriptional
factors.
Consequences of
DNA methylation
• Methylated DNA may be bound by
proteins known as methyl-CpG-binding
domain proteins (MBDs). MBD proteins
then recruit chromatin remodeling
proteins that can modify histones.
• Repression of transposable elements.
• High CpG methylation increases the
frequency of spontaneous mutations.
(Methylated C residues can
spontaneously deaminate to
T residues).
B. Chromatin
modifications
• The primary protein components of chromatin are
histones, which bind to DNA and function as
"anchors" around which the DNA strands are wound.
• Regions of chromatin containing genes which are
actively transcribed are less tightly compacted and
closely associated with RNA polymerases in a
structure known as euchromatin.
• Regions containing transcriptionally inactive genes
(heterochromatin) are generally more condensed.
Chromatin
• Chromatin structure is not rigid and
can be modified by protein complexes
that specify the location, composition,
and modification state of nucleosomes,
ultimately regulating access to the
underlying DNA sequence.
• Canonical histones (H3, H4, H2A, H2B,
and linker histone H1) package the
newly replicated genome.
• They can be replaced with histone variants
that alter nucleosome structure, stability,
dynamics, and DNA accessibility.
Histon modifications
• Posttranslational modification of
histone proteins alters chromatin
function and DNA accessibility.
• N-termini of histones (called histone
tails) are particularly highly modified.
• Histon modifications include
acetylation, methylation,
ubiquitylation, phosphorylation,
sumoylation, ribosylation and
citrullination. Acetylation is the most
highly studied.
Histon modifications
• Histone acetylation is performed by
histone acetyltransferases (HATs)
which add an acetyl group to lysine
residues in the histone tail which
masks the positive charge of the
residue, loosening the electrostatic
interaction between DNA and histones,
causing decondensation of chromatin
thereby allowing gene transcription.
• Histone deacetylases (HDACs) remove
the acetylation mark of histone lysine
residues.
• The HDAC-containing enzyme complex binds to
methylated DNA via MeCP1 and MeCP2
binding proteins. Thus, DNA methylation and
histone modifications are interconected
processes.
Histon modifications
• Methylation of lysine 9 of histone H3
has been associated with constitutively
transcriptionally silent chromatin
(constitutive heterochromatin).
• Phosphorylation (the addition of a
phosphate group from ATP) is possible
on serine residues in histone tails.
C. RNA
modifications
• Epigenetic modifications occur not only in DNA but also in
RNA (the epitranscriptome).
• mRNA, tRNA, rRNA and non-coding RNAs contain
thousands of post-transcriptional chemical modifications.
• N⁶-methyl-adenosine (m6A) modification is the most
abundant.
• m6A modification is recognized by families of RNA binding
proteins that affect many aspects of mRNA function.
• RNA modifications represent another layer of epigenetic
regulation of gene expression, analogous to DNA
methylation and histone modification.
D. Non-coding
RNAs
• RNA interference is a biological process in which
RNA molecules inhibit gene expression or
translation, by neutralizing targeted mRNA
molecules.
miRNA
• MicroRNAs (miRNAs) are short (∼22
nucleotides in length), single-stranded,
noncoding RNAs which regulate mRNAs
via degradation or inhibition of their
translation into proteins. Each miRNA
may target about 100 to 200 mRNAs.
Many miRNAs are epigenetically
regulated. About 50 % of miRNA genes
are associated with CpG islands, that
may be repressed by epigenetic
methylation.
lncRNA
• Long noncoding RNAs (lncRNAs) are
a large family (∼50,000 in the human
genome) of RNAs that are >200 base
pairs in length. lncRNAs are abundant
in neural tissues. lncRNAs reduce
transcription, regulate the processing
of mRNAs to modulate the abundance
of subtly different transcripts from the
same gene (alternative splicing), and
influence the activity of miRNAs.
X chromosome
inactivation
• In female somatic cells, one of the two
X chromosomes is inactivated to
equalize the dose of sex-linked gene
products between female and male
cells.
• The most iconic of all long non-coding
RNAs, the X-inactive specific transcript
(Xist), mediates X chromosome
silencing.
• Xist RNA was found to physically coat
the inactive-X chromosome.
• Father and mother have different epigenetic
patterns for specific genomic loci in their
germ cells.
• Some loci are expressed only paternally, this
is referred to as maternal imprinting. Other
loci are expressed exclusively maternally,
indicating paternal imprinting. The second
allele of the gene is inactive (imprinted).
• This phenomenon is estimated to affect
several hundred human genes under
physiological conditions.
• In the case of imprinted alleles, the identity
of reciprocal crosses found by Gregor Mendel
does not apply, because paternal and
maternal information are not equivalent.
Imprinting
• The epigenetic imprints are established
during male and female gametogenesis,
passed to the zygote through fertilization,
maintained throughout development and
adult life, and erased in primordial germ
cells before the new imprints are set.
• The best-known case of imprinting in human
disorders is that of Angelman syndrome and
Prader-Willi syndrome.
• Both can be produced by the same genetic
mutation (chromosome 15q partial
deletion). The particular syndrome that
will develop depends on whether the
mutation is inherited from the child's
mother or father.
• Loss of the maternal contribution is linked
to Angelman syndrome and the loss of the
paternal contribution is linked to PWS.
Imprinting
Epigenetics
and disease
• Epigenome is intricately linked to the environment and
disease state. Many environmental exposures can induce
changes in the epigenome which alters patterns of gene
expression without directly altering the underlying DNA
sequence to induce disease states.
• The combination of epigenetic marks has been shown to
predispose an individual to certain disease states such as
diabetes, cancer, cardiovascular disease and obesity.
• For example, the environment you were exposed to in the
uterus (poor maternal diet, maternal smoking, high stress
levels, pollution etc.) directly affects the epigenome.
• Epigenetic modification act as mediators or risk factors
showing the interconnected relationship between the
environment and disease.
Cancer
• A variety of epigenetic mechanisms can be
perturbed in different types of cancer.
• Epigenetic alterations of DNA repair genes
or cell cycle control genes are very frequent
in sporadic (non-germ line) cancers.
• Epigenetic alterations are important in
malignant transformation and their
manipulation holds great promise for
cancer prevention, detection and therapy.
Example: Histone modification profiles Normal vs Cancer
Thank you for your attention