basic epigenetics

1. Name- Mugdha Rangnath
Class- M.Sc. Biotechnology- semester
2 (1st year)
Seminar Topic- EPIGENETICS

2. INTRODUCTION
 The term Epigenetics refers to the heritable changes in gene expression, that does not
involve changes in DNA sequence; i.e. heritable changes in trait or phenotype
without a change in genotype.
 The term epigenetics was coined by Conrad H. Waddington in 1942.
 It literally means above or on top of genetics. It refers to external modifications of DNA
that turn genes on or off.
 Genome is an organism’s complete set of DNA, including all of it’s gene.
 An epigenome consists of a record of the
chemical changes to the DNA and histone
proteins of an organism; these changes can be
passed down to an organism's offspring.

3.  Epigenetic change is a regular and natural occurrence but can also be influenced by
several factors including age, the environment/lifestyle, and disease state. Epigenetic
modifications can manifest as commonly as the manner in which cells terminally
differentiate to end up as skin cells, liver cells, brain cells, etc.
 When epigenomic compounds attach to DNA and modify its function, they are said to
have "marked" the genome. These marks do not change the sequence of the DNA.
Rather, the change the way cells use the DNA's instructions. The marks are sometimes
passed on from cell to cell as cells divide. They also can be passed down from one
generation to the next.
 DNA holds the instructions for building the proteins that carry out a variety of functions
in a cell. The epigenome is made up of chemical compounds and proteins that can attach
to DNA and direct such actions as turning genes on or off, controlling the production of
protiens in particular cells.

4. MECHANISM OF
EPIGENETICS
Epigenetic regulation:-
DNA methylation
(modifications at the DNA
level).
Histone modifications and
chromatin remodeling.
Non-coding RNA
mediated pathway (nc-
RNA associated gene).

5. DNA methylation
 DNA methylation is one of the most commonly occuring epigenetic events taking place in
mammalian genome.
 It is biochemical process involving the addition of methyl group to C-5 position of cytosine residue.
Most cytosine methylation occurs in the sequence context 5’CG3’.
 Methyl group is mostly attached to base cytsosine C that is followed by base guanine G. These are
called CpG (cytosine phosphate guanine) sites or motifs.
 C5-methylcytosine(5-mc). {In mammals, DNA methylation occurs mainly on the fifth carbon of
the cytosine base, forming what is known as 5-methylcytosine or 5-methylcytidine (5-mC)}.
Silencing: Methylation of CpG sites within the promoters of
genes can lead to their silencing, a feature found in a number
of human cancers (eg. silencing of tumor suppressor genes).
Activation: In contrast, the hypomethylation of CpG
sites has been associated with the over-expression of
oncogenes within cancer cells

6. Methyl groups are transferred from S-adenosyl methionine (SAM) in reaction catalysed by DNA methyl
tranferases (DNMT) or methylases.
SAM is then converted to SAH (S-adenosyl homocytosine).

7. Enzymes involved in DNA
methylation
 DNA METHYLTRANSFERASES (DNMTs—catalzye this reaction at different times
during the cell cycle).
1. DNMT1- Maintainance methylase
2. DNMT 2
3. DNMT3a and DNMT3b -‘de novo ’ methylases
4. DNMT3L
 Enzymes
1) DNMT1:---
 maintains the pattern of DNA methylation after DNA replication.
 requires hemimethylated DNA substrate and will faithfully reproduce the pattern of
DNA methylation on the newly synthesized strand.
 DNA methylation- ‘an automatic semi conservative mechanism.’
2) DNMT3a and DNMT3b:---
 Will add methyl groups to CG dinucleotides which are previously unmethylated on both
the strands.
 Re-establish the methylation pattern.

9. Role of DNA methylation
 Plays a role in long term silencing of gene.
 Plays a role in transposon silencing of repetitive elements (eg;- deactivation of parasitic
transposans).
 Plays a role in X-chromosome inactivation.
 In the establishment and maintenance of imprinted genes (genomic imprinting)
 Suppresses the expression of viral genes and other deletorious elements that have been
incorporated into the genome of the host over time.
 Inactivation of tumor suppressor genes (p16, BRCA1).
 METHYLATION IMBALANCE may contribute to
TUMOR PROGRESSION.
Genomic instability.
Abnormal chromosomal
structures.
Activating oncogenes.
Inactivation of DNA repair genes
(MLH1, MGMT).

10. histone modification
 Histones are responsible for basic level of chromosome packing. Histone
modifications are post-translational modifications that regulate gene
expression.
 Post-translational modifications to histones – referred to as marks – regulate
gene expression by organizing the genome into active regions of euchromatin,
where DNA is accessible for transcription, or inactive heterochromatin regions,
where DNA is more compact and less accessible for transcription.
 Histones pack and order DNA into structures known as nucleosomes so that it
fits within a cell’s nucleus. Each nucleosome contains two subunits, both made
of histones H2A, H2B, H3 and H4 – known as core histones – with the linker
histone H1 acting as a stabilizer.
 Histone H3 is the most modified histone.
 Post transitional modification (PTM) of histons is crucial step in epigenetic
regulation of gene.

11. Histone Core
• A histone octamer of 4 histone proteins- H2A, H2B, H3 and
H4.
• Each histone protein has a structured domain,
‘Histone Fold’ and unstructured ‘N- terminal tail’.

12. Steps involved:-
Acetylation, Methylation, Phosphorylation,
Ubiquitination/Ubiquitylation.

13. Histone acetylation
 It is the introduction of an Acetyl functional group to the Lysine amino acid of
the histone tail.
 Acetylation on lysine residues leads to relaxation of the chromatin structure and
allows the binding of transcription factors and significantly increases gene
expression.
 These reactions are catalyzed by enzymes with "histone acetyltransferase"
(HAT) or "histone deacetylase" (HDAC) activity.
 Acetylation is one of the most widely studied histone modifications, as it
was one of the first described and linked to transcriptional regulation.

14. Histones are acetylated by HAT (histone acetylases) which are parts of
many chromatin remodeling and transcription complexes.
Histones are deacetylated by HDAC (histone deacetylase)
proteins.

15. Histone methylation
 It is the introduction of an
Methyl functional group to
Lysine or Arginine of the
histone tail.
 Tri-methylation on K4 of
Histone H3 (H3K4me3) is
generally associated with
transcriptional activation,
whereas tri-methylation on
K9 and K27 of histone H3
(H3K9me3 & H3K27me3)
are generally associated
with transcriptional
repression
 Methylation promotes
transcriptional activation.

16. Histone phosphorylation
 Phosphorylation of core histones is a critical intermediate step in chromosome
condensation during cell division, transcriptional regulation, and DNA
damage repair.
 Histone H2B phosphorylation;- recent findings suggest that this modification
facilitates apoptosis-related chromatin condensation, DNA fragmentation and
cell death.
 Known markers for mitosis are phosphorylation of histone H3 at S10, involved
in chromatin compaction and phospho-T120 in histone H2A, linked to
regulation of chromatin structure and function during mitosis

17. Histone ubiquitylation
 Histone H2A and H2B are two of the most abundant ubiquitylated proteins
found in the nucleus (addition of ubiquitin protien).
 Histone (H3 and H4) ubiquitylation plays a central role in the DNA damage
response (DNA repair- CUL40).

18.  Chromatin remodeling is the dynamic modification of chromatin architecture to allow access of
condensed genomic DNA to the regulatory transcription machinery proteins, and thereby control
gene expression.
 To overcome DNA sequence accessibility problems, cells have developed mechanisms to open
higher order structures of chromatin and to disrupt nucleosomes allowing the binding of sequence
specific regulators.
Cromatin remodeling
dna+histon
e

19. Non-Coding RNAs
 A non-coding RNA (ncRNA) is a
functional RNAmolecule that is
transcribed from DNA but not translated
into proteins.
 ncRNA represent small RNA molecules
encoded in the genomes of plants and
animals. These highly conserved 22
nucleotides long RNA sequences regulate
the expression of genes by binding to the
3'-untranslated regions (3'-UTR) of
specific mRNAs.
 Epigenetic related ncRNAs
include miRNA, siRNA, piRNA and lnc
RNA. Both major groups are shown to
play a role
in heterochromatin formation, histone
modification, DNA
methylation targeting, and gene
silencing.

20. Role of ncRNAs:--
Analysis of ncRNA
expression may provide valuable information, as
dysregulation of its function can lead to human
diseases such as cancer, cardiovascular and
metabolic diseases, liver conditions and immune
dysfunction.
A growing body of evidence
shows that ncRNAs are one of the key players in
cell differentiation and growth, mobility and
apoptosis (programmed cell death).

21. Basic concepts of RNAi
 Silencing of homologous gene
expression triggered by double-stranded
RNA is called RNA-mediated
interferance (RNAi).
 It was first discovered in 1998 by
Andrew Fire and Craig Mello in the
nematode worm Caenorhabditis elegans
and later found in a wide variety of
organisms, including mammals.
 RNA interference is an RNA-dependent
gene silencing process.
For example;- preventing the messenger
RNA (mRNA) from producing a protein

22. MECHANISM OF RNAi
conversion of dsRNA input into 21-23bp small
fragments by the enzyme Dicer;
loading of small RNAs into large multiprotein
complex RISC
sequence specific silencing of the cognate gene
by RISC that is guided by the small RNA
fragment

23. dsRNAs are cleaved into 21-23 nt segments (“small
interfering RNAs”, or siRNAs) by an enzyme called
Dicer. (It functions to generate siRNA molecules. Role in loading one of
the two siRNA strands onto RISC complex).

24.  The silencing mechanism of
RNAinterference following steps;-
 Initiation step: - first the dsRNA get
processed into 21-23 nucleotides small
interfering RNAs (siRNAs), which have
also been called “guide RNAs, by an
RNase III like enzyme called Dicer.
 Effecter step: - Then, the siRNAs
assemble into endoribonuclease-
containing complexes known as RNA-
induced silencing complexes.
 (RISCs), unwinding in the process.An
ATP-dependent unwinding of the siRNA
duplex is required for activation of the
RISC.
 The active RISC then targets the
homologous transcript by base pairing
interactions and cleaves the mRNA ~12
nucleotides from the 3' terminus of the
siRNA and destroys the cognate RNA.

26. references
 Biotechnology by B.D. Singh.
 Rna interferance by Gregory Hannon.
 Links;- https://www.whatisepigenetics.com/non-coding-rna/
 https://en.wikipedia.org/wiki/Epigenetics
 https://www.ncbi.nlm.nih.gov/pubmed/20143452
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174260
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