Silencer (genetics)

In genetics a silencer is a DNA sequence capable of binding transcription regulation factors termed repressors. DNA (deoxyribonucleic acid) contains genes which are the hereditary material located in the nuclei of eukaryotic and prokaryotic cells. DNA sequences are made up of four chemical bases: adenine, guanine, cytosine, and thymine. These nucleotides form base pairs, adenine with thymine and guanine with cytosine, held together by hydrogen bonds along the double helix structure of DNA. The two DNA strands of the double helix are antiparellel to each other and each strand consists of a 5' (five prime) end with a terminal phosphate group and a 3' (three prime) end with a terminal hydroxy group.

See also: Molecular Biology

Chemical structure of DNA with hydrogen bonds between nucleotides represented as dotted lines.

DNA consists of many other regulatory regions including the promoter (genetics), enhancer (genetics), and insulator (genetics). Promoters are the regions of DNA that initiates the transcription of a gene, enhancers are the antagonists of silencers, and insulators are regions that block the interaction between enhancers and silencers. DNA also contains exons which are regions that remain in the mature messenger RNA, or mRNA, product and introns which are removed by RNA splicing during the process of transcription.

RNA polymerase, a DNA-dependent enzyme, produces RNA (ribonucleic acid) by transcribing the DNA sequences, nucleotides, in the 3' to 5' direction while the complementary RNA is synthesized in the 5' to 3' direction. RNA is similar to DNA except that RNA contains uracil, instead of thymine, which forms a base pair with adenine. The RNA produced, referred to as messenger RNA or mRNA, will be used to convey the genetic information from DNA to ribosomes to specify amino acids for the translation of proteins that will activate or inactivate gene expression in cells.

Once a repressor protein binds to the silencer region of DNA, RNA polymerase is prevented from binding to the promoter region of DNA and transcription cannot be initiated, thus decreasing or fully suppressing RNA synthesis. If transcription is prevented, proteins cannot be synthesized because mRNA will be absent to initiate the translation of proteins.

Mechanisms of Silencers

Locations of Silencers

A silencer is a sequence-specific element that induces a negative effect on the transcription of its particular gene. As a DNA base pair sequence, there are many positions that a silencer element can be located in DNA. The most common position is found upstream of the target gene where it can help repress the transcription of the gene. This distance can vary greatly between approximately -20 bp to -2000 bp upstream of a gene. Certain silencers can be found downstream of a promoter, located within the intron or exon of the gene itself. Silencers have also been found within regions of the mRNA, such as the 3' untranslated region.

A simple image of how an enhancer and a silencer affect the function of a promoter region

Type of Silencers

Currently, there are two main types of silencers in DNA. There is the classical silencer element and the non-classical negative regulatory element (NRE). In classical silencers, the gene is actively repressed by the silencer element, mostly by interfering with general transcription factor (GTF) assembly. NREs passively repress the certain gene, usually by inhibiting other elements upstream of the gene. Of the NREs, there are certain silencers that are orientation-dependent, meaning that the binding factor binds in a particular direction relative to other sequences. Promoter-dependent silencers are understood to be silencer elements because they are position- and orientation-dependent but must also use a promoter-specific factor. There has been a recent discovery of Polycomb-group Response Elements (PREs) that can repress and inhibit repression depending on the protein bound to it and the presence of non-coding transcription.

Mechanisms of Silencers

For classical silencers, the signaling pathway is relatively simple. Since repression is active, silencer elements target the assembly of GTFs, necessary for transcription of the gene. These silencer elements are mostly located upstream of the gene and can vary between short and long distances. For long-range silencers, it has been observed that the DNA will form a loop in order to bring the silencer closer to the promoter and loop out the interfering DNA. Silencers also target helicase sites in the DNA that are rich in adenine and thymine (AT). These sites are prone to unwinding of the DNA, giving room to initiate transcription. This helicase activity is inhibited, thus inhibiting transcription. This is commonly seen in the human thyrotropin-β gene promoter. For NREs, they can induce a bend in the promoter region to block interactions, as seen when an NRE binds to Yin-Yang 1 (YY1).They can flank regulatory signals or promoter regions as well. When the silencer region is located within an intron, there can be two types of repression. First, there can be a physical blockage of a splice site. Second, there can be a bend in the DNA that will inhibit RNA processing. When located in the exon or the untranslated region, the silencer will mainly be classical or position-dependent. These silencers, though, can carry out their activity prior to transcription. Most silencers are constitutively expressed in organisms, only allowing activation of a gene by either inhibiting the silencer or activating an enhancer region. The best example of this is the Neuronal-Restrictive Silencer Factor (NRSF) that is produced by the REST gene. The REST gene produces NRSF in order to repress the transcription of neuronal genes. This is essential for localization of neuronal tissue. When a silencer represses REST, NRSF is also inhibiting, allowing for the transcription of neuronal genes.

Similarities with Enhancers

See also: Enhancers

Another regulatory element located upstream of the DNA is an enhancer. Enhancers function as a "turn on" switch in gene expression and will activate the promoter region of a particular gene, while silencers act as the "turn off" switch. Though these two regulatory elements work against each other, both sequence types affect the promoter region in very similar ways. Because silencers have not been thoroughly identified and analyzed, extensive research in enhancers have aided biologists in understanding the mechanics of the silencer. Enhancers can be found in many of the certain areas that silencers are found, such as upstream of the promoter by many kilobase pairs or even downstream within the intron the gene. DNA looping is also a model function used by enhancers in order to shorten the proximity of the promoter to the enhancer. Enhancers also function with transcription factors in order to initiate expression, much like silencers with repressors.

Functionality and Evolution of Silencers

Prokaryotes

1: RNA Polymerase, 2: Repressor (LacI), 3: Promoter, 4: Operator, 5: Lactose, 6: lacZ, 7: lacY, 8: lacA. Top: lac operon is initially repressed because lactose is not present to inhibit the repressor. Bottom: Repressor LacI is inhibited because it binds to lactose and transcription of the lac operon is initiated for the breakdown of lactose.

There are several differences in the regulation of metabolic control in eukaryotes and in prokaryotes. Prokaryotes can vary the numbers of specific enzymes made in their cells in order to regulate gene expression which is a slow metabolic control. They can also regulate enzymatic pathways through mechanisms such as feedback inhibition and allosteric regulation which is under a rapid metabolic control. Feedback inhibition is a system of self regulation in which the result of a system or process will influence the process itself to reduce and suppress its activity while allosteric regulation is a regulation of an enzyme by an effector molecule at the protein's allosteric site. The genes of prokaryotes are grouped together based on similar functions into units called operons. Operons consist of a promoter and an operator which is the binding site for the repressor and regulates gene expression. When the repressor protein is bound to the operator, RNA polymerase cannot bind to the promoter to initiate the transcription of the operon.

The operator of the lac operon in the prokaryote E. coli which consists of genes that produce enzymes to break down lactose is an example of a prokaryotic silencer. The three functional genes in this operon are lacZ which produces β-galactosidase, lacY which produces permease, and lacA which produces β-galactosidase transacetylase. The repressor gene, lacI, will produce the repressor LacI which is under allosteric inhibition. These genes are activated by the presence of lactose in the cell which acts as an effector molecule that binds to repressor LacI. When the repressor is bound to lactose, it will not bind to the operator which allows RNA polymerase to bind to the operator to initiate transcription of the operon. When the repressor's allosteric site is not bound to lactose, its active site will bind to the operator to prevent RNA polymerase from transcribing the genes of the lac operon.

Eukaryotes

Eukaryotes have a much larger genome and thus have different methods of gene regulation than do prokaryotes. All cells in a eukaryotic organism have the same DNA--a human cell is capable of having up to 35,000 genes. This phenomenon is known as genetic totipotency in which a single cell is able to be differentiated through the genes it expresses. However, in order for a cell to express the genes for proper functioning, the genes must be closely regulated to express the correct properties. Genes in eukaryotes are controlled on the transcriptional, post-transcriptional, translational, and post-translational levels. On the transcriptional level, gene expression is regulated by altering transcription rates. Genes that encode proteins include exons which will encode the polypeptides, the introns that are removed from mRNA before the translation of proteins, a trasnscriptional start site in which RNA polymerase binds, and a promoter.

DNA is transcribed into mRNA, introns are spliced during post-transcriptional regulation, and the remaining exons in the mRNA are translated into proteins.

See also: Regulation of gene expression

Silencers in eukaryotes control gene expression on a transcriptional level in which the mRNA is not transcribed. These DNA sequences may act as either silencers or enhancers based on the transcription factor that binds to the sequence. Silencers bind to specific transcription factors called repressors that prevent RNA polymerase from binding to the promoter of a gene that can be located up to thousands of base pairs upstream or downstream from the silencer to transcribe the mRNA product. Eukaryotic genes contain a an upstream promoter and a core promoter also referred to as a basal promoter. A common basal promoter is the TATAAAAAA sequence known as the TATA box. The TATA box is a complex with several different proteins including transcription factor II D (TFIID) which includes the TATA-binding protein (TBP) which binds to the TATA box along with 13 other proteins that bind to TBP. The TATA box also includes the transcription factor II B (TFIIB) which binds to both the DNA and RNA polymerase. While there is little variation of the basal promoter from gene to gene, upstream promoters which are located up to 2000 base pairs upstream have binding factors that differ from gene to gene.

TATA box, a common basal promoter in eukaryotes. The TATA box is grouped with the TFIIB and the transcription initiator site and the downstream promoter element are located several base pairs away

Repressors bind to silencers that can be thousands of base pairs away from the genes it controls. Repressors may have regions that bind to the DNA sequence as well as regions that bind to the transcription factors assembled at the promoter of the gene which would create a chromosome looping mechanism. Looping may bring the silencers in close proximity to the promoters to ensure that groups of genes needed for optimal gene expression will work together.

Mutated silencers, hereditary diseases, and their effects

Genetic mutations occur when nucleotide sequences in an organism are altered. These mutations lead to not only observable phenotypic influences in an individual but also alterations that are undetectable phenotypically. The sources for these mutations can be errors during replication, spontaneous mutations, or caused chemical and physical mutagens (UV and ionizing radiation, heat). Silencers, being encoded in the genome, are susceptible to such alterations, which, in many cases, lead to severe phenotypical and functional abnormalities. In general terms, mutations in silencer elements or regions could lead to either the inhibition of the silencer’s action or to the persisting repression of a necessary gene. Thus, leading to the expression or suppression of an undesired phenotype, which then translates into impacts on the normal functionality of certain systems in the organism. Among the many silencer elements and proteins, REST/NSRF is an important silencer factor that has a variety of impacts not only in neural but also in other areas of development. In fact, in many cases, REST/NSRF acts in conjunction with RE-1/NRSE to repress and influence non-neuronal cells. Its effects ranges from frogs, Xenopus laevis to be more specific, to humans having innumerous repercussions not only in phenotype but also in development. In Xenopus laevis, REST/NRSF malfunction or damage has been associated to abnormal ectodermal patterning during development and significant consequences in neural tube, cranial ganglia, and eye development. Finally, in humans, a deficiency in the REST/NSRF silencer element has been correlated to Huntington’s disease, due to the decrease in the transcription of BDNF. Furthermore, ongoing studies indicate that NRSE is involved in the regulation of the ANP gene, which when overexpressed can lead to ventricular hypertrophy. Hence, modification in silencer elements and sequences can result in either devastating changes or unnoticeable ones.

See also: RE1-silencing transcription factor

REST/NRSF in Xenopus laevis

The effects and influences of RE1/NRSE and REST/NRSF are significant in non-neuronal cells that require the repression or silencing of neuronal genes. These silencer elements also regulate the expression of genes that do not induce neuron-specific proteins, and studies have shown the extensive impact these factors have in cellular processes. In Xenopus laevis, RE1/NRSE and REST/NRSF dysfunction or mutation demonstrated significant impact on neural tube, cranial ganglia, and eye development. All of these alterations can be traced to an improper patterning of the ectoderm during Xenopus development. Thus, a mutation or alteration in either the silencing region Re1/NRSE or silencer REST/NRSF factor can disrupt the proper differentiation and specification of the neuroepithelial domain, and also hinder the formation of skin or ectoderm. Furthermore, the lack of these factors result in a decreased production of bone morphogenetic protein (BMP), which translates into a deficient development of the neural crest. Hence, the effects of NRSE and NRSF are of fundamental importance for neurogenesis of the developing embryo, and also in the early stages of ectodermal patterning. Ultimately, inadequate functioning of these factors can result in aberrant neural tube, cranial ganglia, and eye development in Xenopus.

REST/NRSF and Ventricular Hypertrophy in Mammals

See also: Atrial natriuretic peptide

REST/NRSF in conjunction with RE1/NRSE also acts outside the nervous system by acting as regulators and repressors. Researches have linked RE1/NRSE activity with the regulation of expression of the atrial natriuretic peptide (ANP) gene.A NRSE regulatory region is present in the 3’untranslated region of the ANP gene, and acts as a mediator for the appropriate expression of the gene. The protein encoded by the ANP gene is important during embryonic development for the maturation and development of cardiac myocytes. However, during early childhood and throughout adulthood, ANP expression is suppressed or kept to a minimum in the ventricle. Thus, an abnormal induction of the ANP gene can lead to ventricular hypertrophy, and severe cardiac consequences. In order to maintain the gene repressed, NRSF (neuron-restrictive silencer factor) or REST binds to the NRSE region in the 3’untranslated region of the ANP gene. Furthemore, the NRSF-NRSE complex recruits a transcriptional corepressor known as mSin3. Thus, leading to the activity of histone deacetylase in region, and the repression of gene expression. Therefore, studies have revealed the correlation between REST/NRSF and RE1/NRSE in regulating the ANP gene expression in ventricular myocytes. A mutation in either the NRSF or NRSE can lead to an undesirable development of ventricular myocytes, due to lack of repression, which can then cause ventricular hypertrophy. Left ventricular hypertrophy, for example, increases an individual’s chance of sudden death due to a ventricular arrhythmia resulting from the increased ventricular mass. In addition to the influence on the ANP gene, the NRSE sequence regulates other cardiac embryonic genes, such as brain natriuretic peptide BNP, skeletal α-actin, and Na, K – ATPase α3 subunit. Hence, the regulatory activity of both NRSE and NRSF in mammals prevents not only neural dysfunctions, but also physiological and phenotypical abnormalities in other non-neuronal regions of the body.

REST/NSRF and Huntington’s Disease

See also: Huntingtin

Huntington’s disease (HD) is an inherited neurodegenerative disorder, which has the emergence of its symptoms during an individual’s mid-adulthood. The most noticeable symptoms of this progressive disease are cognitive and motor impairments as well as behavioral alterations. These impairments can develop into dementia, chorea, and eventually death. In the molecular level, HD results from a mutation in the huntingtin protein (Htt). More specifically, there is an abnormal repetition of a CAG sequence towards the 5’-end of the gene, which then leads to the development of a toxic polyglutamine (polyQ) stretch in the protein. The mutated Htt protein affects an individual’s proper neural functions by inhibiting the action of REST/NRSF.

REST/NRSF is an important silencer element that binds to regulatory regions to control the expression of certain proteins involved in neural functions. The mechanistic actions of huntingtin are still not fully understood, but a correlation between Htt and REST/NRSF exists in HD development. By attaching to the REST/NRSF, the mutated huntingtin protein inhibits the action of the silencer element, and retains it in the cytosol. Thus, REST/NRSF cannot enter the nucleus and bind to the 21 base-pair RE-1/NRSE regulatory element. An adequate repression of specific target genes are of fundamental importance, since many are involved in the proper development of neuronal receptors, neurotransmitters, synaptic vesicle proteins, and channel proteins. A deficiency in the proper development of these proteins can cause the neural dysfunctions seen in Huntington’s disease. In addition to the lack of repression due to the inactive REST/NRSF, mutated huntingtin protein can also decrease the transcription of the brain-derived neurotropic factor (BDNF) gene. BDNF influences the survival and development of neurons in the central nervous system as well as the peripheral nervous system. This abnormal repression occurs when the RE1/NRSE region within the BDNF promoter region is activated by the binding of REST/NRSF, which leads to the lack of transcription of the BDNF gene. Hence, the anomalous repression of the BDNF protein suggests a significant impact in Huntington’s disease.

Mutations in Polycomb-group Response Elements (PREs)

The Polycomb-group (PcG) regulatory complexes are widely known for their influence in epigenetic regulation of stem cells, and to be more specific in hematopoietic stem cells. The Polycomb Repressive Complex 1 (PRC 1) is directly involved in the process of hematopoiesis, and functions together, for example, with the PcG gene “Bmi1”. Studies in mice indicate that organisms with mutated “Bmi1” demonstrate deficient mitochondrial functioning, and also hindered the ability of hematopoietic cells to self-renew. Likelise, mutations in PRC2 genes were related to hematological conditions such as acute lymphoblastic leukemia (ALL), which is a form of leukemia. Hence, Polycomb-group genes and proteins are involved in the proper maintenance of hematopoiesis in the body.

External links

• Silencer+Elements at the U.S. National Library of Medicine Medical Subject Headings (MeSH)