Gene Silencing

W Filipowicz, Friedrich Miescher Institute for Biomedical Reserach, Basel, Switzerland

J Paszkowski, University of Geneva, Geneva, Switzerland
© 2013 Elsevier Inc. All rights reserved.

Glossary Histone modifications Covalent modifications of amino

Chromatin A structure consisting of DNA and associated acids in histone proteins by acetylation, methylation,
proteins that builds eukaryotic chromosomes. The basic phosphorylation, ubiquitination, and sumoylation.
unit of chromatin is a nucleosome in which 147 base pairs Particular histone modifications mark transcriptionally
of DNA is wrapped around an octamer of histone proteins active euchromatin and others mark transcriptionally
consisting of four core histones (H2A, H2B, H3, and H4). silent heterochromatin. Therefore, it has been postulated
Chromatin structure can alternate between a condensed that patterns of histone modifications form a histone code
form of transcriptionally silent heterochromatin and that can be linked to the structural features and
decondensed euchromatin, which packages actively transcriptional activity of chromatin.
transcribed regions. RNA interference (RNAi) The dsRNA-induced, sequence
DNA methylation Covalent modification of DNA by homology-dependent gene silencing mechanism. The
addition of methyl groups to DNA bases, which in dsRNA is processed to small noncoding RNAs (siRNAs),
eukaryotes are cytosines. In mammalian DNA, methylated which, in association with specialized proteins, guide the
cytosines are found predominantly in symmetric CG endonucleolytic cleavage of target RNAs, leading to either
sequences, which permit propagation of DNA transcriptional or posttranscriptional gene silencing. In
methylation patterns through DNA replication. In plants, ciliates like Tetrahymena or Paramecium, a mechanism
methylated cytosines are found in other sequence contexts which involves small noncoding RNAs (related to siRNAs)
(non-CG methylation) in addition to CGs. Recently, induces heterochromatin formation, which is followed by
non-CG methylation has been detected in mammalian elimination of DNA sequences which often interrupt
embryonic stem cells prior to their differentiation. protein-coding regions. RNAi offers an efficient way of
Double-stranded RNA (dsRNA) RNA forms usually specific inactivation of genes of interest, providing a
resulting from hybridization of two complementary powerful tool for investigating gene function.
chains. dsRNA can be formed as a result of transcription of Small noncoding RNAs Regulatory RNAs of 20–30
DNA in both sense and antisense directions or nucleotides in length which are involved in both
DNA-containing inverted repeats, which upon transcriptional and posttranscriptional silencing of genes.
transcription fold into dsRNA hairpins. dsRNA is often The regulators include small interfering RNAs (siRNAs),
formed during viral infection and RNA interference microRNAs (miRNAs), or Piwi-associated RNAs
(RNAi), through cleaving the viral dsRNA and producing (piRNAs). They are all generated from different forms of
siRNAs, represents an important antiviral defence dsRNA accumulating in cells. Particularly important are
mechanism. miRNAs which regulate, posttranscriptionally, expression
Epigenetics Study of heritable phenotypes or traits that of ∼50% of human genes. miRNAs are generated from
are not encoded in the DNA sequence. dsRNA hairpins encoded in a genome.

Gene silencing is defined as an epigenetic modification of gene regulators such as microRNAs (miRNAs) and small interfering
expression leading to inactivation of previously active genes. RNAs (siRNAs). Aberrant silencing of genes may lead to disease
Epigenetic modification does not alter the DNA sequence and, in mammals and generate developmental variants in plants.
although it is heritable, variable frequencies of reversions to For example, methylation of tumor suppressor genes contri­
expression are observed. Gene silencing is used in the course of butes to the onset and progression of cancer, while methylation
normal development and differentiation to repress genes of genes controlling flower development results in heritable
whose products are not required in specific cell types or tissues. changes of flower morphology.
This may apply to individual genes or larger chromosome Gene silencing can act at the transcriptional or posttran­
regions. In some special situations, such as chromosome scriptional level; the two phenomena being referred to as
dosage compensation in mammals, one of the two female X transcriptional gene silencing (TGS) and posttranscriptional
chromosomes is almost completely repressed. Mechanisms gene silencing (PTGS). Genes affected by TGS are not tran­
responsible for repression of genes involve changes in levels scribed at all, or transcripts are produced at very low levels.
of DNA methylation, alterations in covalent modifications of TGS has been observed in fungi, plants, and animals. It is
histone proteins, chromatin compaction, or destabilization of probably triggered by redundancies of genetic information,
mRNA. Particular patterns of modifications of chromatin pro­ since its occurrence correlates well with the presence of
teins and DNA template make genes inaccessible to the repeated genes or gene fragments. As shown in plants,
transcription machinery. mRNA destabilization and repression increased levels of ploidy may likewise act as a trigger of TGS.
of mRNA translation are often mediated by small RNA In organisms that are able to methylate their DNA, levels of

Brenner’s Encyclopedia of Genetics, 2nd edition, Volume 3 doi:10.1016/B978-0-12-374984-0.00603-3 221

222 Gene Silencing

DNA methylation are significantly increased in genes silenced endogenous mRNAs and targeting them for degradation. In
by TGS. In the fungus Neurospora crassa, DNA methylation of both plants and C. elegans, the PTGS/RNAi effect spreads across
redundant sequences is followed by a modification of their cellular and tissue boundaries and small RNA fragments are the
nucleotide sequences in a process referred to as repeat-induced best candidates for the diffusible silencing signals. RNAi also
point mutation (RIP). In the fungus Ascobolus nidulans, methy­ functions in vertebrates although there is no evidence that it
lation and inactivation of redundant genes occurs in a specific spreads across cellular boundaries also in these organisms.
phase of the life cycle, in a process called methylation induced Genetic screens have identified several genes essential for estab­
premeiotically (MIP). TGS has been well studied genetically in lishing and/or maintaining PTGS. Some of them are also
yeast, Drosophila, and plants. These studies revealed a number required for RNAi, indicating that the two phenomena are
of genes that are required for establishment and maintenance mechanistically related. Like TGS, PTGS may also represent a
of silencing. Their protein products are either chromatin com­ mechanism to defend the organism and its genome against
ponents or posttranslational modifiers of DNA and chromatin invasive nucleic acids such as transposons, retroelements, and
proteins. In organisms that are able to methylate DNA, TGS viruses. Certain forms of PTGS, in particular RNAi, offer a
regulators also include DNA methyltransferases and proteins targeted and efficient way of inactivating genes of interest,
recognizing methylated DNA. DNA methyltransferases respon­ providing a powerful tool for investigating gene function.
sible for maintenance of DNA methylation are conserved Other forms of PTGS are mediated by miRNAs, ∼20­
between plants and mammals. Acquisition of new methylation nucleotide-long small RNA regulators encoded by independent
patterns requires de novo methyltransferases, which in plants are genes or excised from introns of protein-coding transcripts.
guided by a dedicated class of siRNAs in a process called
Several hundred different miRNAs are expressed in plants and
RNA-directed DNA methylation (RdDM). The biological role
metazoan animals. By hybridizing to mRNAs, miRNAs inhibit
of TGS is still under debate. One postulated function is to
protein synthesis by either repressing mRNA translation or
extinguish transcription of transposable elements in order to
inducing mRNA degradation. Over 50% of human genes are
prevent their movement and propagation in chromosomal
predicted to undergo regulation by miRNAs. Recently, a new
DNA. In plants, TGS is also able to affect single-copy genes,
class of small RNAs, the Piwi-associated RNAs (piRNAs), has
giving rise to semi-stable epigenetic variants. Creation of rever­
been discovered. PiRNAs function in germ cells of metazoan
sible epialleles adds to the phenotypic variability important
animals and their main role is to repress expression of transpo­
in evolving plant populations. In the fission yeast
sons, thereby protecting the genome of the gametes from
Schizosaccharomyces pombe, siRNAs participate at formation
insertional mutagenesis by mobile genetic elements.
and maintenance of heterochromatin at centromeric region of
chromosomes.
In PTGS, also referred to as cosuppression in plants, quel­
See also: Epigenetics; Macronuclear Development, Ciliates;
ling in N. crassa, or RNA interference (RNAi) in animals, the
Transposable Elements; X-Chromosome Inactivation.
affected gene is transcriptionally active but its transcripts
undergo rapid degradation, resulting in the absence of transla­
table mRNA. PTGS is frequently observed in transgenic
organisms, in particular when multiple copies of the transgene Further Reading
are present. Transcripts of both the transgene and the host
genes having ~80% or more sequence identity with the trans- Bourc’his D and Voinnet O (2010) A small-RNA perspective on gametogenesis,
gene are subject to the degradation. In plants, infection with fertilization, and early zygotic development. Science 330(6004): 617–622.
Ding SW and Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130(3):
RNA viruses engineered to express sequences homologous to
413–426.
host genes will likewise result in specific degradation of Feng S, Jacobsen SE, and Reik W (2010) Epigenetic reprogramming in plant and animal
host and viral RNAs. Available evidence indicates that development. Science 330(6004): 622–627.
double-stranded RNAs (dsRNAs) and siRNAs, which represent Ghildiyal M and Zamore PD (2009) Small silencing RNAs: An expanding universe.
Nature Reviews Genetics 10(2): 94–108.
20- to 25-nucleotide-long products of dsRNA fragmentation,
Grewal SI (2010) RNAi-dependent formation of heterochromatin and its diverse
are responsible for specific RNA degradation. dsRNAs may be functions. Current Opinion in Genetics & Development 20(2): 134–141.
formed as a result of the artifactual bidirectional transcription Matzke M, Kanno T, Daxinger L, Huettel B, and Matzke AJ (2009) RNA-mediated
from the transgene loci, or may be produced from endogenous chromatin-based silencing in plants. Current Opinion in Cell Biology 21: 367–376.
genes modified by ectopic interactions with homologous trans- Matzke M, Kanno T, Huettel B, Daxinger L, and Matzke AJ (2007) Targets of
RNA-directed DNA methylation. Current Opinion in Plant Biology 10: 512–519.
genes. Injection of dsRNA into the nematode Caenorhabditis Siomi MC, Miyoshi T, and Siomi H (2010) piRNA-mediated silencing in Drosophila
elegans or into the eggs of Drosophila melanogaster leads to germlines. Seminars in Cell & Developmental Biology 21(7): 754–759.
potent and sequence-specific PTGS/RNAi. Injected dsRNA is White SA and Allshire RC (2008) RNAi-mediated chromatin silencing in fission yeast.
fragmented into siRNAs which act as guides hybridizing to Current Topics in Microbiology and Immunology 320: 157–183.