Insulator (genetics)

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An insulator is a type of cis-regulatory element known as a long-range regulatory element. Found in multicellular eukaryotes and working over distances from the promoter element of the target gene, an insulator is typically 300 bp to 2000 bp in length. [1] Insulators contain clustered binding sites for sequence specific DNA-binding proteins [1] and mediate intra- and inter-chromosomal interactions. [2]

Contents

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 modifications. [3] [4] There are many examples of insulators, including the CTCF insulator, the gypsy insulator, and the β-globin locus. The CTCF insulator is especially important in vertebrates, while the gypsy insulator is implicated in Drosophila. The β-globin locus was first studied in chicken and then in humans for its insulator activity, both of which utilize CTCF. [5]

The genetic implications of insulators lie in their involvement in a mechanism of imprinting and their ability to regulate transcription. Mutations to insulators are linked to cancer as a result of cell cycle disregulation, tumourigenesis, and silencing of growth suppressors.

Function

Insulators have two main functions: [3] [4]

  1. Enhancer-blocking insulators prevent distal enhancers from acting on the promoter of neighbouring genes
  2. Barrier insulators prevent silencing of euchromatin by inhibiting the spread of neighbouring heterochromatin

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 genes on a chromosome have very different transcription patterns; it is critical that the inducing or repressing mechanisms of one do not interfere with the neighbouring gene. [6] Insulators have also been found to cluster at the boundaries of topologically associating domains (TADs) and may have a role in partitioning the genome into "chromosome neighborhoods" - genomic regions within which regulation occurs. [7] [8]

Some insulators can act as both enhancer blocker and barriers, and some just have one of the two functions. [3] Some examples of different insulators are: [3]

Mechanism of action

Enhancer-blocking insulators

Gene enhancer.svg

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 fibre to structural elements within the nucleus. [4] 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 [9] (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. [4] 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. [4]

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. [4] Modifications are achieved through various mechanisms including nucleosome 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(s) and ATP-dependent nucleosome remodelling complexes. [4]

CTCF insulator

The CTCF insulator appears to have enhancer blocking activity via its 3D structure [10] and have no direct connection with barrier activity. [11] Vertebrates in particular appear to rely heavily on the CTCF insulator, however there are many different insulator sequences identified. [2] Insulated neighborhoods formed by physical interaction between two CTCF-bound DNA loci contain the interactions between enhancers and their target genes. [12]

Regulation

One mechanism of regulating CTCF is via methylation of its DNA sequence. CTCF protein is known to favourably bind to unmethylated sites, so it follows that methylation of CpG islands is a point of epigenetic regulation. [2] 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. [13] 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, RNA polymerase, and CP190. [2] [14]

gypsy insulator

The insulator element that is found in the gypsy retrotransposon of Drosophila is one of several sequences that have been studied in detail. The gypsy insulator can be found in the 5' untranslated region (UTR) of the retrotransposon element. Gypsy affects the expression of adjacent genes pending insertion into a new genomic location, causing mutant phenotypes 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. [15]

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. [5]

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. [5]

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. [13] 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. [5]

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. [13]

Transcription

When insulator sequences 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. [3] Loop formation is common in eukaryotes to bring distal elements (enhancers, promoters, locus control regions) into closer proximity for interaction during transcription. [4] The mechanism of enhancer-blocking insulators then, if in the correct position, could play a role in regulating transcription activation. [3]

Mutations and cancer

CTCF insulators affect the expression of genes implicated in cell cycle regulation processes that are important for cell growth, cell differentiation, and programmed cell death (apoptosis). 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 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 or other problems. [2]

CTCF is also required for the expression of tumour repressor retinoblastoma (Rb) gene and mutations and deletions of this gene are associated with inherited malignancies. When the CTCF binding site is removed expression of Rb is decreased and tumours are able to thrive. [2]

Other genes that encode cell cycle regulators include BRCA1, and p53, 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. [2]

The aberrant activation of insulators can modulate the expression of cancer-related genes, including matrix metalloproteinases involved in cancer cell invasion. [16]

Related Research Articles

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 reinforcing the DNA during cell division, preventing DNA damage, and regulating gene expression and DNA replication. During mitosis and meiosis, chromatin facilitates proper segregation of the chromosomes in anaphase; the characteristic shapes of chromosomes visible during this stage are the result of DNA being coiled into highly condensed chromatin.

Heterochromatin is a tightly packed form of DNA or condensed DNA, which comes in multiple varieties. These varieties lie on a continuum between the two extremes of constitutive heterochromatin and facultative heterochromatin. Both play a role in the expression of genes. Because it is tightly packed, it was thought to be inaccessible to polymerases and therefore not transcribed; however, according to Volpe et al. (2002), and many other papers since, much of this DNA is in fact transcribed, but it is continuously turned over via RNA-induced transcriptional silencing (RITS). Recent studies with electron microscopy and OsO4 staining reveal that the dense packing is not due to the chromatin.

In molecular biology and genetics, transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA (transcription), thereby orchestrating gene activity. A single gene can be regulated in a range of ways, from altering the number of copies of RNA that are transcribed, to the temporal control of when the gene is transcribed. This control allows the cell or organism to respond to a variety of intra- and extracellular signals and thus mount a response. Some examples of this include producing the mRNA that encode enzymes to adapt to a change in a food source, producing the gene products involved in cell cycle specific activities, and producing the gene products responsible for cellular differentiation in multicellular eukaryotes, as studied in evolutionary developmental biology.

HHV Latency Associated Transcript is a length of RNA which accumulates in cells hosting long-term, or latent, Human Herpes Virus (HHV) infections. The LAT RNA is produced by genetic transcription from a certain region of the viral DNA. LAT regulates the viral genome and interferes with the normal activities of the infected host cell.

The histone code is a hypothesis that the transcription of genetic information encoded in DNA is in part regulated by chemical modifications to histone proteins, primarily on their unstructured ends. Together with similar modifications such as DNA methylation it is part of the epigenetic code. Histones associate with DNA to form nucleosomes, which themselves bundle to form chromatin fibers, which in turn make up the more familiar chromosome. Histones are globular proteins with a flexible N-terminus that protrudes from the nucleosome. Many of the histone tail modifications correlate very well to chromatin structure and both histone modification state and chromatin structure correlate well to gene expression levels. The critical concept of the histone code hypothesis is that the histone modifications serve to recruit other proteins by specific recognition of the modified histone via protein domains specialized for such purposes, rather than through simply stabilizing or destabilizing the interaction between histone and the underlying DNA. These recruited proteins then act to alter chromatin structure actively or to promote transcription. For details of gene expression regulation by histone modifications see table below.

A locus control region (LCR) is a long-range cis-regulatory element that enhances expression of linked genes at distal chromatin sites. It functions in a copy number-dependent manner and is tissue-specific, as seen in the selective expression of β-globin genes in erythroid cells. Expression levels of genes can be modified by the LCR and gene-proximal elements, such as promoters, enhancers, and silencers. The LCR functions by recruiting chromatin-modifying, coactivator, and transcription complexes. Its sequence is conserved in many vertebrates, and conservation of specific sites may suggest importance in function. It has been compared to a super-enhancer as both perform long-range cis regulation via recruitment of the transcription complex.

The family of heterochromatin protein 1 (HP1) consists of highly conserved proteins, which have important functions in the cell nucleus. These functions include gene repression by heterochromatin formation, transcriptional activation, regulation of binding of cohesion complexes to centromeres, sequestration of genes to the nuclear periphery, transcriptional arrest, maintenance of heterochromatin integrity, gene repression at the single nucleosome level, gene repression by heterochromatization of euchromatin, and DNA repair. HP1 proteins are fundamental units of heterochromatin packaging that are enriched at the centromeres and telomeres of nearly all eukaryotic chromosomes with the notable exception of budding yeast, in which a yeast-specific silencing complex of SIR proteins serve a similar function. Members of the HP1 family are characterized by an N-terminal chromodomain and a C-terminal chromoshadow domain, separated by a hinge region. HP1 is also found at some euchromatic sites, where its binding can correlate with either gene repression or gene activation. HP1 was originally discovered by Tharappel C James and Sarah Elgin in 1986 as a factor in the phenomenon known as position effect variegation in Drosophila melanogaster.

<span class="mw-page-title-main">CTCF</span> Transcription factor

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 regulation, insulator activity, V(D)J recombination and regulation of chromatin architecture.

<span class="mw-page-title-main">KLF1</span> Protein-coding gene in the species Homo sapiens

Krueppel-like factor 1 is a protein that in humans is encoded by the KLF1 gene. The gene for KLF1 is on the human chromosome 19 and on mouse chromosome 8. Krueppel-like factor 1 is a transcription factor that is necessary for the proper maturation of erythroid cells.

<span class="mw-page-title-main">Chromosome conformation capture</span>

Chromosome conformation capture techniques are a set of molecular biology methods used to analyze the spatial organization of chromatin in a cell. These methods quantify the number of interactions between genomic loci that are nearby in 3-D space, but may be separated by many nucleotides in the linear genome. Such interactions may result from biological functions, such as promoter-enhancer interactions, or from random polymer looping, where undirected physical motion of chromatin causes loci to collide. Interaction frequencies may be analyzed directly, or they may be converted to distances and used to reconstruct 3-D structures.

<span class="mw-page-title-main">EZH2</span> Protein-coding gene in the species Homo sapiens

Enhancer of zeste homolog 2 (EZH2) is a histone-lysine N-methyltransferase enzyme encoded by EZH2 gene, that participates in histone methylation and, ultimately, transcriptional repression. EZH2 catalyzes the addition of methyl groups to histone H3 at lysine 27, by using the cofactor S-adenosyl-L-methionine. Methylation activity of EZH2 facilitates heterochromatin formation thereby silences gene function. Remodeling of chromosomal heterochromatin by EZH2 is also required during cell mitosis.

<span class="mw-page-title-main">USF1</span> Protein-coding gene in the species Homo sapiens

Upstream stimulatory factor 1 is a protein that in humans is encoded by the USF1 gene.

<span class="mw-page-title-main">HMGB2</span> Protein-coding gene in the species Homo sapiens

High-mobility group protein B2 also known as high-mobility group protein 2 (HMG-2) is a protein that in humans is encoded by the HMGB2 gene.

SilentInformationRegulator (SIR) proteins are involved in regulating gene expression. SIR proteins organize heterochromatin near telomeres, ribosomal DNA (rDNA), and at silent loci including hidden mating type loci in yeast. The SIR family of genes encodes catalytic and non-catalytic proteins that are involved in de-acetylation of histone tails and the subsequent condensation of chromatin around a SIR protein scaffold. Some SIR family members are conserved from yeast to humans.

Epigenetics of human development is the study of how epigenetics effects human development.

<span class="mw-page-title-main">Topologically associating domain</span> Self-interacting genomic region

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 is 880 kb, and they have similar sizes in non-mammalian species. Boundaries at both side of these domains are conserved between different mammalian cell types and even across species and are highly enriched with CCCTC-binding factor (CTCF) and cohesin. In addition, some types of genes appear near TAD boundaries more often than would be expected by chance.

<span class="mw-page-title-main">Nuclear organization</span> Spatial distribution of chromatin within a cell nucleus

Nuclear organization refers to the spatial distribution of chromatin within a cell nucleus. There are many different levels and scales of nuclear organisation. Chromatin is a higher order structure of DNA.

<span class="mw-page-title-main">Insulated neighborhood</span>

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 structural and functional units of gene control because their integrity is important for normal gene regulation. Current evidence suggests that these structures form the mechanistic underpinnings of higher-order chromosome structures, including topologically associating domains (TADs). Insulated neighborhoods are functionally important in understanding gene regulation in normal cells and dysregulated gene expression in disease.

DXZ4 is a variable number tandemly repeated DNA sequence. In humans it is composed of 3kb monomers containing a highly conserved CTCF binding site. CTCF is a transcription factor protein and the main insulator responsible for partitioning of chromatin domains in the vertebrate genome.

Human epigenome is the complete set of structural modifications of chromatin and chemical modifications of histones and nucleotides. These modifications affect according to cellular type and development status. Various studies show that epigenome depends on exogenous factors.

References

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  2. 1 2 3 4 5 6 7 Yang, Jingping; Corces, Victor G. (2011). "Chromatin Insulators: A Role in Nuclear Organization and Gene Expression". Advances in Cancer Research. 110: 43–76. doi:10.1016/B978-0-12-386469-7.00003-7. ISBN   9780123864697. ISSN   0065-230X. PMC   3175007 . PMID   21704228.
  3. 1 2 3 4 5 6 West, Adam G.; Gaszner, Miklos; Felsenfeld, Gary (2002-02-01). "Insulators: many functions, many mechanisms". Genes & Development. 16 (3): 271–288. doi: 10.1101/gad.954702 . ISSN   0890-9369. PMID   11825869.
  4. 1 2 3 4 5 6 7 8 Gaszner, Miklos; Felsenfeld, Gary (September 2006). "Insulators: exploiting transcriptional and epigenetic mechanisms". Nature Reviews Genetics. 7 (9): 703–713. doi:10.1038/nrg1925. ISSN   1471-0064. PMID   16909129. S2CID   31291034.
  5. 1 2 3 4 Wai, Albert W.K.; Gillemans, Nynke; Raguz-Bolognesi, Selina; Pruzina, Sara; Zafarana, Gaetano; Meijer, Dies; Philipsen, Sjaak; Grosveld, Frank (2003-09-01). "HS5 of the human β-globin locus control region: a developmental stage-specific border in erythroid cells". The EMBO Journal. 22 (17): 4489–4500. doi:10.1093/emboj/cdg437. ISSN   0261-4189. PMC   202379 . PMID   12941700.
  6. ↑ Burgess-Beusse B, Farrell C, Gaszner M, Litt M, Mutskov V, Recillas-Targa F, Simpson M, West A, Felsenfeld G (December 2002). "The insulation of genes from external enhancers and silencing chromatin". Proc. Natl. Acad. Sci. U.S.A. 99 (Suppl 4): 16433–7. Bibcode:2002PNAS...9916433B. doi: 10.1073/pnas.162342499 . PMC   139905 . PMID   12154228.
  7. ↑ Perkel J (1 June 2015). "Mapping chromosome neighborhoods". BioTechniques. 58 (6): 280–284. doi: 10.2144/000114296 . PMID   26054763.
  8. ↑ Ong CT; Corces VG (April 2014). "CTCF: an architectural protein bridging genome topology and function". Nat Rev Genet. 15 (4): 234–46. doi:10.1038/nrg3663. PMC   4610363 . PMID   24614316.
  9. ↑ Deng, W; Lee, J; Wang, H; Miller, J; Reik, A; Gregory, P. D.; Dean, A; Blobel, G. A. (2012). "Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor". Cell. 149 (6): 1233–44. doi:10.1016/j.cell.2012.03.051. PMC   3372860 . PMID   22682246.
  10. ↑ Phillips JE, Corces VG (June 2009). "CTCF: master weaver of the genome". Cell. 137 (7): 1194–211. doi:10.1016/j.cell.2009.06.001. PMC   3040116 . PMID   19563753.
  11. ↑ Phillips, Jennifer E.; Corces, Victor G. (2009-06-26). "CTCF: Master Weaver of the Genome". Cell. 137 (7): 1194–1211. doi:10.1016/j.cell.2009.06.001. ISSN   0092-8674. PMC   3040116 . PMID   19563753.
  12. ↑ Dowen, JM; Fan, ZP; Hnisz, D; Ren, G; Abraham, BJ; Zhang, LN; Weintraub, AS; Schuijers, J; Lee, TI; Zhao, K; Young, RA (9 October 2014). "Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes". Cell. 159 (2): 374–87. doi:10.1016/j.cell.2014.09.030. PMC   4197132 . PMID   25303531.
  13. 1 2 3 Allison, Lizabeth A. (2012). Fundamental Molecular Biology. New Jersey: John Wiley & Sons, Inc. p. 367. ISBN   9781118059814.
  14. ↑ Kim, Somi; Yu, Nam-Kyung; Kaang, Bong-Kiun (June 2015). "CTCF as a multifunctional protein in genome regulation and gene expression". Experimental & Molecular Medicine. 47 (6): e166. doi:10.1038/emm.2015.33. ISSN   2092-6413. PMC   4491725 . PMID   26045254.
  15. ↑ Gdula, David A.; Gerasimova, Tatiana I.; Corces, Victor G. (1996). "Genetic and Molecular Analysis of the gypsy Chromatin Insulator of Drosophila". Proceedings of the National Academy of Sciences of the United States of America. 93 (18): 9378–9383. Bibcode:1996PNAS...93.9378G. doi: 10.1073/pnas.93.18.9378 . JSTOR   39717. PMC   38435 . PMID   8790337.
  16. ↑ Llinàs-Arias P, Ensenyat-Mendez M, Íñiguez-Muñoz S, Orozco J, Valdez B (November 2023). "Chromatin insulation orchestrates matrix metalloproteinase gene cluster expression reprogramming in aggressive breast cancer tumors". Molecular Cancer. 22 (4): 190. doi: 10.1186/s12943-023-01906-8 . PMC   10683115 . PMID   38017545.
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