Gene silencing is a molecular process that down regulates gene expression at the transcriptional or translational level. It allows researchers to study essential genes and provides insight into disease development since many diseases are associated with reduced gene expression. Gene silencing occurs through various mechanisms including histone modification, DNA methylation, and small RNA pathways. It has applications in research areas like producing virus resistant plants and modifying food quality and plant traits.

1. GENE SILENCING
and its
SIGNIFICANCE

2. • defined as a molecular process involved in the down
regulation of specific genes.
• Interruption or suppression of the expression of a gene at transcriptional or translational
levels.
• “switching off”
• Gene silencing is same as gene knock down but is totally different from gene knock out.
• When genes are knock down ,there expression is reduced by at least 70% , where in
contrast when genes are knocked out, they are completely eliminate from organism’s
genome.
• allow researchers to study essential genes that are required for the animal models to
survive and cannot be removed.
• they provide a more complete view on the development of diseases since diseases are
generally associated with genes that have a reduced expression.
• probably evolved as a genetic defense system against viruses and invading nucleic acids
(Brigneti et al., 1998; Voinnet et al., 2000; Waterhouse et al., 2001; Wassenegger, 2002)

4. GENE “SILENCING” BY
MODIFICATION OF
HISTONES AND DNA

5. Silencing is a position effect---a gene is
silenced because of where it is located, not
in response to a specific environmental signal.
Also, silencing can “spread” over large
stretches of DNA ,switching off multiple
genes, even ones quite distant from the
initiating event.
The most common form of silencing is
associated with a dense form of chromatin
called heterochromatin.
Both activation and repression of
transcription often involve modification of
nucleosomes to alter the accessibility of a
gene to the transcriptional machinery and
other regulatory proteins.
Transcription can also be silence by
methylation of DNA by enzymes called DNA
methylases.

7. Rap1 recruits SIR complex to the telomere.
SIR2, a component of that complex, deacetylates nearby nucleosomes.
The unacetylated tails themselves then bind Sir3 and SIR4, recruiting
more SIR complex, allowing the SIR2 within it to act on
nucleosomes further away, and so on.
This explains the spreading of the silencing effect produced by deacetylation.
The telomeres, the silent mating-type locus, and the rDNA genes are all “silent” regions in
Saccharomyces cerevisiae.
Silencing in Yeast Is Mediated by Deacetylation and
Methylation of Histones.

8. Consider the telomere as an example.
•The final 1-5 kb of each chromosome is found in a folded , dense
structure.
•Genes taken from other chromosomal locations and moved to this
region are
often silenced, particularly if they are only weakly expressed in
their usual location.
•Mutations have been done , in which a gene placed at the telomere
is expressed at higher levels.
•These studies implicate three genes encoding regulators of
silencing:
Silent information regulator(SIR)2,3 and 4.
 The three proteins encoded by these genes form a complex that
associates with silent chromatin, and Sir2 is a histone deacetylase.
•The silencing complex is recruited to the telomere by a DNA-
binding protein(RAP1) that recognizes the telomere’s repeated
sequences.
•Histone methyl transferases attach methyl groups to histone tails.
•Just as acetylated residues within histones are recognized by
proteins bearing bromodomains, methylated residues bind proteins
with chromodomains.

9. Position effect
• Position effect is the effect
on the expression of a
gene when its location in a
chromosome is changed,
often by translocation.
• described in Drosophila
with respect to eye colour
and is known as
position effect variegation
(PEV).

11. Repression by polycomb also uses histone methylation
Polycomb repressive complex 1
Polycomb Response Elements (PREs)
DNA binding subunit (PHO or PHOL - Repressive Complex )
Polycomb protein (PHO) = Protein pleiohomeotic

12. Switching a gene off through DNA methylation
and histone midification

13. DNA Methylation Is Associated
with Silenced Genes in Mammalian Cells
Some mammalian genes are kept silent by methylation of nearby DNA sequences.
Methylation of DNA can mark sites where heterochromatin subsequently forms.
DNA methylation lies at the heart of a phenomenon called imprinting.
Two regulatory sequences are critical for the differential expression of the human
H19 and Igf2 genes:
An enhancer and an insulator.

14. Imprinting
Two examples of genes controlled by imprinting- the mammalian Igf2 and H19 genes.
The H19 genes is expressed from only the matermal chromosome, Igf2 from the paternal
chromosome.
The methylation state of the insulator element determines whether or not the insulator binding
protein( CTCF) can bind and block activation of the H19 gene from the downstream enhancer.

15. Some States of Gene Expression Are
Inherited through Cell Division even when
the Initiating Signal Is No Longer Present
Patterns of DNA methylation can be maintained
through cell division

16. TRANSCRITIONAL
SILENCING

17. Paramutation :
• In epigenetics, a paramutation is an
interaction between two alleles at a single
locus, whereby one allele induces a heritable
change in the other allele.
• The change may be in the pattern of DNA
methylation or histone modifications.
• For example – Anthocyanin pigment in corn
plant
• B allele – Anthocyanin pigment coded
• Paramutagenic allele at this locus(B’) cause
reduced pigment production
• B allele is silenced by the B’ allele in the first
generation
• In next generation, the newly silence B allele is
paramutagenic and silence.

18. Transposon
silencing:
• is a product of histone
modification that prevent
the transcription of that
area of DNA.
• The “jumping” of
transposon generates the
genomic instability and
cause the extremely
deleterious mutations.

19. Transgene silencing:
• insertion of transgene in to a
transcriptionally inactive part
of genome.
When an insertion of any
transgene it does not show
activity as per desire and this is
because of it’s instability.
• The lose of transgene stability is
because of gene silencing.
• E.g. slow fruit softening tomato,
by reducing expression of
polygalactouronase enzyme.

20. RNA Directed DNA
Methylation:
• an epigenetic process
first elucidated in plants
where by small double-
stranded RNAs (dsRNA's)
are processed to guide
methylation to
complementary DNA
loci.
• In Arabidopsis thaliana

21. POST -TRANSCRITIONAL
SILENCING

23. RNA i
(RNA interference):
• it is a post
transcriptional process
triggered by the
introduction of double
stranded RNA (ds RNA)
which leads to the gene
silencing in a sequence
specific manner.
• It is also known as post
transcriptional gene
silencing / co
suppression and
quelling.

26. Non sense
Mediated Decay:
• is a cellular mechanism
of mRNA surveillance
that functions to detect
nonsense mutations and
prevent the expression
of truncated or
erroneous proteins.
• NMD is triggered by
exon junction complexes
(EJCs) (components of
the assembled RNP) that
are deposited during pre-
mRNA processing.

27. Anti sense RNA
technology:
• It blocks the activity of the
mRNA in a stoichiometric
manner.
• Antisense RNA has the opposite
sense to m RNA.
• The presence of complimentary
sense and antisense RNA in the
in the same cell can lead to the
formation of a stable duplex
which interferes with gene
expression at the level of RNA
processing or possible
translation.
• This technology widely used in
plants for gene inhibition.

28. Strategies for G S in research

30. GENE SILENCING IN PLANTS
Currently, there are several routes of GS identified in plants, such as:
• transcriptional gene silencing (Vaucheret & Fagard, 2001),
• post-transcriptional gene silencing or RNA interference (PTGS or
RNAi) (Vaucheret et al., 2001),
• microRNA silencing (Bartel, 2004),
• and virus induced gene silencing (Burch-Smith et al., 2004).
• All these pathways play an important role at the cellular level,
affecting differentiation, gene regulation (Bartel, 2004), and
protection against viruses and transposons (Waterhouse et al.,
2001).

33. Applications for GS in plants
• the production of virus resistant plants through genetic
transformation.
• used in food quality modification such as the reduction of
caffeine levels in coffee beans and to increase the
nutritional value of corn protein and tomato.
• Research on forest tree yield and quality has included the
study of GS related to lignin synthesis.
• research on fruit crops has targeted applications of GS on
viral and bacterial resistance, and physiological aspects
such as self fertility.

34. Advantages of gene silencing:
• Downregulation of gene expression simplifies "knockout" analysis.
• Easier than use of antisense oligonucleotides. Si RNA more effective and sensitive at lower concentration.
• Cost effective
• Can be labelled Ease of transfection by use of vector
• blocking expression of unwanted genes and undesirable substances.
• Inducing viral resistance
• Powerful tool for analysing unknown genes in sequenced genomes.
• Useful approach in future gene therapy.
• Oligonucleotides can be manufactured quickly, some within one week; the sequence of the mRNA is all that is
needed.
• Cancer treatments
• Modulation of HIV-I replication by RNA i.
• Small RNA and it’s application in andrology and urology.

35. Disadvantages of gene silencing:
• ”High pressure injection” and electroporation can cause significant
damage to the integrity of the normal tissues and organs and thus
preclude the utilisation in a clinical set-up.
• Liposomes/cationic encapsulated Si RNA may also be toxic to the
host and may cause severe host immune responses.
• Other emerging strategies includes chemical modification of Si RNA
molecules and encapsulated with different molecules are still in
their infancy and need to be thoroughly investigated before used in
therapeutic applications.