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An insulator is a type of
cis-regulatory element ''Cis''-regulatory elements (CREs) or ''Cis''-regulatory modules (CRMs) are regions of non-coding DNA which regulate the transcription of neighboring genes. CREs are vital components of genetic regulatory networks, which in turn control morphoge ...
known as a long-range
regulatory element A regulatory sequence is a segment of a nucleic acid molecule which is capable of increasing or decreasing the expression of specific genes within an organism. Regulation of gene expression is an essential feature of all living organisms and v ...
. Found in
multicellular A multicellular organism is an organism that consists of more than one cell, in contrast to unicellular organism. All species of animals, land plants and most fungi are multicellular, as are many algae, whereas a few organisms are partially uni- ...
eukaryote Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
s and working over distances from the promoter element of the target gene, an insulator is typically 300 bp to 2000 bp in length. Insulators contain clustered
binding sites In biochemistry and molecular biology, a binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. The binding partner of the macromolecule is often referred to as a ligand. Ligands may inclu ...
for sequence specific
DNA-binding proteins DNA-binding proteins are proteins that have DNA-binding domains and thus have a specific or general affinity for DNA#Base pairing, single- or double-stranded DNA. Sequence-specific DNA-binding proteins generally interact with the major groove ...
and mediate intra- and inter-
chromosomal A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
interactions. Insulators function either as an enhancer-blocker or a barrier, or both. The mechanisms by which an insulator performs these two functions include loop formation and
nucleosome A nucleosome is the basic structural unit of DNA packaging in eukaryotes. The structure of a nucleosome consists of a segment of DNA wound around eight histone proteins and resembles thread wrapped around a spool. The nucleosome is the fundamen ...
modifications. There are many examples of insulators, including the
CTCF Transcriptional repressor CTCF also known as 11-zinc finger protein or CCCTC-binding factor is a transcription factor that in humans is encoded by the ''CTCF'' gene. CTCF is involved in many cellular processes, including transcriptional regulatio ...
insulator, the ''gypsy'' insulator, and the β-globin locus. The CTCF insulator is especially important in
vertebrate Vertebrates () comprise all animal taxa within the subphylum Vertebrata () ( chordates with backbones), including all mammals, birds, reptiles, amphibians, and fish. Vertebrates represent the overwhelming majority of the phylum Chordata, ...
s, while the ''gypsy'' insulator is implicated in ''
Drosophila ''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
.'' The β-globin locus was first studied in chicken and then in humans for its insulator activity, both of which utilize CTCF. The genetic implications of insulators lie in their involvement in a mechanism of imprinting and their ability to regulate
transcription Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, the fir ...
. Mutations to insulators are linked to
cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
as a result of
cell cycle The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and subs ...
disregulation,
tumourigenesis Carcinogenesis, also called oncogenesis or tumorigenesis, is the formation of a cancer, whereby normal cells are transformed into cancer cells. The process is characterized by changes at the cellular, genetic, and epigenetic levels and abno ...
, and silencing of growth suppressors.


Function

Insulators have two main functions: # Enhancer-blocking insulators prevent distal
enhancers In genetics, an enhancer is a short (50–1500 bp) region of DNA that can be bound by proteins ( activators) to increase the likelihood that transcription of a particular gene will occur. These proteins are usually referred to as transcriptio ...
from acting on the promoter of neighbouring genes # Barrier insulators prevent silencing of
euchromatin Euchromatin (also called "open chromatin") is a lightly packed form of chromatin ( DNA, RNA, and protein) that is enriched in genes, and is often (but not always) under active transcription. Euchromatin stands in contrast to heterochromatin, whic ...
by the spread of neighbouring
heterochromatin Heterochromatin is a tightly packed form of DNA or '' condensed DNA'', which comes in multiple varieties. These varieties lie on a continue between the two extremes of constitutive heterochromatin and facultative heterochromatin. Both play a role ...
While enhancer-blocking is classified as an inter-chromosomal interaction, acting as a barrier is classified as an intra-chromosomal interaction. The need for insulators arises where two adjacent
gene In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
s on a
chromosome A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
have very different
transcription Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, the fir ...
patterns; it is critical that the inducing or repressing mechanisms of one do not interfere with the neighbouring gene. Insulators have also been found to cluster at the boundaries of
topologically associating domain A topologically associating domain (TAD) is a self-interacting genomic region, meaning that DNA sequences within a TAD physically interact with each other more frequently than with sequences outside the TAD. The median size of a TAD in mouse cells ...
s (TADs) and may have a role in partitioning the genome into "chromosome neighborhoods" - genomic regions within which regulation occurs. Some insulators can act as both enhancer blocker and barriers, and some just have one of the two functions. Some examples of different insulators are: * ''
Drosophila melanogaster ''Drosophila melanogaster'' is a species of fly (the taxonomic order Diptera) in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the "vinegar fly" or "pomace fly". Starting with Ch ...
'' insulators ''gypsy'' and ''scs scs'' are both enhancer-blocking insulators * ''
Gallus gallus The red junglefowl (''Gallus gallus'') is a tropical bird in the family Phasianidae. It ranges across much of Southeast Asia and parts of South Asia. It was formerly known as the Bankiva or Bankiva Fowl. It is the species that gave rise to the ...
'' have insulators, ''Lys 5' A'' that have both enhancer-blocking and barrier activity, as well as ''HS4'' that have only enhancer-blocking activity * ''
Saccharomyces cerevisiae ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have been o ...
'' insulators STAR and UASrpg are both barrier insulators * ''
Homo sapiens Humans (''Homo sapiens'') are the most abundant and widespread species of primate, characterized by bipedalism and exceptional cognitive skills due to a large and complex brain. This has enabled the development of advanced tools, culture, ...
'' ''HS5'' insulator acts as an enhancer-blocker


Mechanism of action


Enhancer-blocking insulators

Similar mechanism of action for enhancer-blocking insulators; chromatin loop domains are formed in the nucleus that separates the enhancer and the promoter of a target gene. Loop domains are formed through the interaction between enhancer-blocking elements interacting with each other or securing
chromatin Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...
fibre to structural elements within the
nucleus Nucleus ( : nuclei) is a Latin word for the seed inside a fruit. It most often refers to: *Atomic nucleus, the very dense central region of an atom *Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA Nucle ...
. The action of these insulators is dependent on being positioned between the promoter of the target gene and the upstream or down stream enhancer. The specific way in which insulators block enhancers is dependent on the enhancers mode of action. Enhancers can directly interact with their target promoters through looping (direct-contact model), in which case an insulator prevents this interaction through the formation of a loop domain that separates the enhancer and promoter sites and prevents the promoter-enhancer loop from forming. An enhancer can also act on a promoter through a signal (tracking model of enhancer action). This signal may be blocked by an insulator through the targeting of a nucleoprotein complex at the base of the loop formation.


Barrier insulators

Barrier activity has been linked to the disruption of specific processes in the heterochromatin formation pathway. These types of insulators modify the nucleosomal substrate in the reaction cycle that is central to heterochromatin formation. Modifications are achieved through various mechanisms including
nucleosome A nucleosome is the basic structural unit of DNA packaging in eukaryotes. The structure of a nucleosome consists of a segment of DNA wound around eight histone proteins and resembles thread wrapped around a spool. The nucleosome is the fundamen ...
removal, in which nucleosome-excluding elements disrupt heterochromatin from spreading and silencing (chromatin-mediated silencing). Modification can also be done through recruitment of
histone acetyltransferase Histone acetyltransferases (HATs) are enzymes that acetylate conserved lysine amino acids on histone proteins by transferring an acetyl group from acetyl-CoA to form ε-''N''-acetyllysine. DNA is wrapped around histones, and, by transferring an ...
(s) and ATP-dependent nucleosome remodelling complexes.


CTCF insulator

The
CTCF Transcriptional repressor CTCF also known as 11-zinc finger protein or CCCTC-binding factor is a transcription factor that in humans is encoded by the ''CTCF'' gene. CTCF is involved in many cellular processes, including transcriptional regulatio ...
insulator appears to have enhancer blocking activity via its 3D structure and have no direct connection with barrier activity. Vertebrates in particular appear to rely heavily on the CTCF insulator, however there are many different insulator sequences identified.
Insulated neighborhood In mammalian biology, insulated neighborhoods are chromosomal loop structures formed by the physical interaction of two DNA loci bound by the transcription factor CTCF and co-occupied by cohesin. Insulated neighborhoods are thought to be structu ...
s formed by physical interaction between two CTCF-bound DNA loci contain the interactions between enhancers and their target genes.


Regulation

One mechanism of regulating CTCF is via
methylation In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These t ...
of its
DNA sequence DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. Th ...
. CTCF protein is known to favourably bind to unmethylated sites, so it follows that methylation of
CpG islands The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG isl ...
is a point of epigenetic regulation. An example of this is seen in the ''Igf2-H19'' imprinted locus where methylation of the paternal imprinted control region (ICR) prevents CTCF from binding. A second mechanism of regulation is through regulating proteins that are required for fully functioning CTCF insulators. These proteins include, but are not limited to
cohesin Cohesin is a protein complex that mediates sister chromatid cohesion, homologous recombination, and DNA looping. Cohesin is formed of SMC3, SMC1, SCC1 and SCC3 ( SA1 or SA2 in humans). Cohesin holds sister chromatids together after DNA rep ...
,
RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template. Using the enzyme helicase, RNAP locally opens the ...
, and CP190.


''gypsy'' insulator

The insulator element that is found in the ''gypsy'' retrotransposon of ''
Drosophila ''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
'' is one of several sequences that have been studied in detail. The ''gypsy'' insulator can be found in the 5'
untranslated region In molecular genetics, an untranslated region (or UTR) refers to either of two sections, one on each side of a coding sequence on a strand of mRNA. If it is found on the 5' side, it is called the 5' UTR (or leader sequence), or if it is foun ...
(UTR) of the
retrotransposon Retrotransposons (also called Class I transposable elements or transposons via RNA intermediates) are a type of genetic component that copy and paste themselves into different genomic locations (transposon) by converting RNA back into DNA through ...
element. ''Gypsy'' affects the expression of adjacent genes pending insertion into a new
genomic Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, three-dim ...
location, causing mutant
phenotype In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
s that are both tissue specific and present at certain developmental stages. The insulator likely has an inhibitory effect on enhancers that control the spatial and temporal expression of the affected gene.


β-globin locus

The first examples of insulators in vertebrates was seen in the chicken β-globin locus, ''cHS4''. ''cHS4'' marks the border between the active euchromatin in the β-globin locus and the upstream heterochromatin region that is highly condensed and inactive. The ''cHS4'' insulator acts as both a barrier to chromatin-mediated silencing via heterochromatin spreading, and blocks interactions between enhancers and promoters. A distinguishing characteristic of ''cHS4'' is that it has a repetitive heterochromatic region on its 5' end. The human β-globin locus homologue of ''cHS4'' is ''HS5''. Different from the chicken β-globin locus, the human β-globin locus has an open chromatin structure and is not flanked by a 5' heterochromatic region. HS5 is thought to be a genetic insulator ''in vivo'' as it has both enhancer-blocking activity and transgene barrier activities. CTCF was first characterized for its role in regulating β-globin gene expression. At this locus, CTCF functions as an insulator-binding protein forming a chromosomal boundary. CTCF is present in both the chicken β-globin locus and human β-globin locus. Within cHS4 of the chicken β-globin locus, CTCF binds to a region (FII) that is responsible for enhancer blocking activity.


Genetic implications


Imprinting

The ability of enhancers to activate imprinted genes is dependent on the presence of an insulator on the unmethylated allele between the two genes. An example of this is the ''Igf2-H19'' imprinted locus. In this locus the CTCF protein regulates imprinted expression by binding to the unmethylated maternal imprinted control region (ICR) but not on the paternal ICR. When bound to the unmethylated maternal sequence, CTCF effectively blocks downstream enhancer elements from interacting with the ''Igf2'' gene promoter, leaving only the ''H19'' gene to be expressed.


Transcription

When insulator
sequences In mathematics, a sequence is an enumerated collection of objects in which repetitions are allowed and order matters. Like a set, it contains members (also called ''elements'', or ''terms''). The number of elements (possibly infinite) is called t ...
are located in close proximity to the promoter of a gene, it has been suggested that they might serve to stabilize enhancer-promoter interactions. When they are located farther away from the promoter, insulator elements would compete with the enhancer and interfere with activation of
transcription Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, the fir ...
. Loop formation is common in eukaryotes to bring distal elements (enhancers, promoters, locus control regions) into closer proximity for interaction during transcription. The mechanism of enhancer-blocking insulators then, if in the correct position, could play a role in regulating transcription activation.


Mutations and cancer

CTCF insulators affect the expression of genes implicated in
cell cycle The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and subs ...
regulation processes that are important for cell growth,
cell differentiation Cellular differentiation is the process in which a stem cell alters from one type to a differentiated one. Usually, the cell changes to a more specialized type. Differentiation happens multiple times during the development of a multicellula ...
, and programmed cell death (
apoptosis Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...
). Two of these cell cycle regulation genes that are known to interact with CTCF are ''hTERT'' and ''C-MYC.'' In these cases, a
loss of function In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mitos ...
mutation to the CTCF insulator gene changes the expression patterns and may affect the interplay between cell growth, differentiation and apoptosis and lead to
tumourigenesis Carcinogenesis, also called oncogenesis or tumorigenesis, is the formation of a cancer, whereby normal cells are transformed into cancer cells. The process is characterized by changes at the cellular, genetic, and epigenetic levels and abno ...
or other problems. CTCF is also required for the expression of tumour repressor
retinoblastoma Retinoblastoma (Rb) is a rare form of cancer that rapidly develops from the immature cells of a retina, the light-detecting tissue of the eye. It is the most common primary malignant intraocular cancer in children, and it is almost exclusively fo ...
(Rb) gene and mutations and deletions of this gene are associated with inherited
malignancies Malignancy () is the tendency of a medical condition to become progressively worse. Malignancy is most familiar as a characterization of cancer. A ''malignant'' tumor contrasts with a non-cancerous ''benign'' tumor in that a malignancy is not s ...
. When the CTCF binding site is removed expression of Rb is decreased and tumours are able to thrive. Other genes that encode cell cycle regulators include
BRCA1 Breast cancer type 1 susceptibility protein is a protein that in humans is encoded by the ''BRCA1'' () gene. Orthologs are common in other vertebrate species, whereas invertebrate genomes may encode a more distantly related gene. ''BRCA1'' is a h ...
, and
p53 p53, also known as Tumor protein P53, cellular tumor antigen p53 (UniProt name), or transformation-related protein 53 (TRP53) is a regulatory protein that is often mutated in human cancers. The p53 proteins (originally thought to be, and often s ...
, which are growth suppressors that are silenced in many cancer types, and whose expression is controlled by CTCF. Loss of function of CTCF in these genes leads to the silencing of the growth suppressor and contributes to the formation of cancer.


References


External links

* {{Transcription Gene expression