The barrier function of an insulator couples high histone acetylation levels with specific protection of promoter DNA from methylation

Mutskov, Vesco; Farrell, Catherine; Wade, Paul A.; Wolffe, Alan P.; Felsenfeld, Gary

doi:10.1101/gad.988502

Cited by 175 publications

(172 citation statements)

References 43 publications

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“…We infer that the insulator-induced changes in CpG methylation at this site do not determine expression of the transgene, but the insulator-induced changes in expression determine CpG methylation of the promoter site. This inference is compatible with a model proposed by Felsenfeld and his associates (12,13) based on their analysis of chromatin modifications associated with a transgene flanked with two copies of cHS4. Our experiments show that two flanking copies of cHS4 are more potent than a single flanking copy in protecting the transgene from repression and its promoter from CpG methylation.…”

Section: Dna Methylation Of the Transgene On Xa But Not XI Is Affectesupporting

confidence: 71%

“…Flanking copies of cHS4 without the CTCF binding site can still protect randomly integrated transgenes from position effects. In a position effect assay the cHS4 insulator itself and transgenes flanked with it are associated with acetylated histones, histone H3 methylated at lysine 4, and reduced CpG methylation relative to the methylation of uninsulated transgenes (11)(12)(13).…”

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confidence: 99%

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A DNA insulator prevents repression of a targeted X-linked transgene but not its random or imprinted X inactivation

Ciavatta¹,

Kalantry

Magnuson

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Some genes on the inactive X chromosome escape silencing. One possible escape mechanism is that heterochromatization during X inactivation can be blocked by boundary elements. DNA insulators are candidates for blocking because they shield genes from influences of their chromosomal environment. To test whether DNA insulators can act as boundaries on the X chromosome, we inserted into the mouse X-linked Hprt locus a GFP transgene flanked with zero, one, or two copies of a prototypic vertebrate insulator from the chicken ␤-globin locus, chicken hypersensitive site 4, which contains CCCTC binding factor binding sites. On the active X chromosome the insulators blocked repression of the transgene, which commences during early development and persists in adults, in a copy number-dependent manner. CpG methylation of the transgene correlated inversely with expression, but the insulators on the active X chromosome were not methylated. On the inactive X chromosome, insulators did not block random or imprinted X inactivation of the transgene, and both the insulator and transgene were almost completely methylated. Thus, the chicken hypersensitive site 4 DNA insulator is sufficient to protect an X-linked gene from repression during development but not from X inactivation. CCCTC binding factorE ukaryotic genomes contain interspersed domains of transcriptionally active euchromatin and inactive heterochromatin. Heterochromatin can encroach into transcriptionally active euchromatin and silence adjoining genes, as illustrated by position effect variegation in Drosophila (1, 2). Maintaining these chromosomal domains and preventing the spread of heterochromatin implies the existence of boundary elements that act as barriers (3). One class of boundary element candidates consists of DNA insulators because they can block the positive effects of an adjacent enhancer and protect a gene from negative position effects (4, 5).One of the best-characterized vertebrate DNA insulators is from the chicken ␤-globin locus. In chicken erythrocytes, DNaseI hypersensitive site 4 upstream of the ␤-globin locus marks the transition between a euchromatic region that contains the ␤-globin genes and upstream heterochromatin (6, 7). Chicken hypersensitive site 4 (cHS4), a 1.2-kb fragment that encompasses hypersensitive site 4, has both insulator properties: it can block an enhancer in an enhancer blocking assay, and it has barrier activity in a position effect assay (8). A binding site for the transcription factor CTCF (CCCTC binding factor) within cHS4 is necessary and sufficient for its enhancer blocking activity, but is not necessary for its barrier function (9, 10). Flanking copies of cHS4 without the CTCF binding site can still protect randomly integrated transgenes from position effects. In a position effect assay the cHS4 insulator itself and transgenes flanked with it are associated with acetylated histones, histone H3 methylated at lysine 4, and reduced CpG methylation relative to the methylation of uninsulated transgenes (11-13).A specialized for...

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Section: Dna Methylation Of the Transgene On Xa But Not XI Is Affectesupporting

confidence: 71%

mentioning

confidence: 99%

A DNA insulator prevents repression of a targeted X-linked transgene but not its random or imprinted X inactivation

Ciavatta¹,

Kalantry

Magnuson

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…2,10,[15][16][17][18][19] Previous reports have shown that HS4 insulator protects transgenes from position effect variegation when introduced into MoMLV-based retroviral vectors. 20,21 Since the HS4 fragment is active in restricted cell types, 21 a quest for alternative insulators is necessary to propagate the use of the insulator in various cell types and tissues.…”

Section: Introductionmentioning

confidence: 99%

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

et al. 2004

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Suppressed expression of transgenes in vivo is the major obstacle in the gene therapy. For the long-term expression, we utilized a chromatin insulator from sea urchin arylsulfatase (Ars) gene locus (Ars insulator, ArsI), which has been shown to epigenetically regulate gene expression across species. ArsI was able to prevent silencing of the transgene in a myeloid cell line, HL-60, and a murine embryonic stem cell line, CCE, in an orientation-dependent manner, but not in Huh-7, K562 and MCF-7 cells, indicating that the effect of ArsI on gene silencing was cell type dependent. Although anti-silencing effect of ArsI was almost equivalent to that of chicken b-globin insulator, incorporation of ArsI into lentiviral vector had little effect on the virus titer compared with chicken b-globin insulator. Clonal analysis of transduced HL-60 cells revealed that ArsI protects the lentiviral vector from position effects regardless of its orientation. Furthermore, chromatin immunoprecipitation assays revealed that a high acetylation level was observed in the promoter of the insulated vector, whereas that of ArsI was independent of its anti-silencing capacity. In addition to it having little deteriorative effect on the virus titer, the identified anti-silencing effect of ArsI suggested its possibility for application in gene therapy.

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“…It has been shown in turn in the fungus Neurospora that the ability to methylate histone Lys 9 is essential for DNA methylation 28 , suggesting that local methylation at Lys 9 may provide a signal for methylation of the underlying DNA. Furthermore, in a different reaction pathway, maintenance of histone acetylation at promoters can lead to inhibition of DNA methylation 29 .…”

Section: Epigenetic Inheritancementioning

confidence: 99%

Controlling the double helix

Felsenfeld

Groudine

2003

Nature

Self Cite

975

782

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development in the sea urchin have delineated a regulatory network that has significant predictive power 16 (Fig. 2). Finally, systems approaches to metabolism in an archaeal halobacterium (an organism thriving in up to five-molar salt solutions, such as the Dead Sea) have revealed new insights into the inter-relationships among several modules controlling energy production in the cell 18 .The study of cellular and organismal biology using the systems approach is at its very beginning. It will require integrated teams of scientists from across disciplines -biologists, chemists, computer scientists, engineers, mathematicians and physicists. New methods for acquiring and analysing high-throughput biological data are needed. A powerful computational infrastructure must be leveraged to generate more effective approaches to the capture, storage, analysis, integration, graphical display and mathematic formulation of biological complexity. New technologies must be integrated with each other. Finally, hypothesis-driven and discovery science must be integrated. In short, both new science and technology must emerge for the systems biology approach to realize its promise. A cultural shift in the biological sciences is needed, and the education and training of the next generation of biologists will require significant reform.Gordon Moore, the founder of Intel, predicted that the number of transistors that could be placed on a computer chip would double every 18 months. It has for more than 30 years. This exponential growth has been a driver for the explosive growth of information technology. Likewise, the amount of DNA sequence information available to the scientific community is following a similar, perhaps even steeper, exponential increase. The critical issue is how sequence information can be converted into knowledge of the organism and how biology will change as a result. We believe that a systems approach to biology is the key. It is clear, however, that this approach poses significant challenges, both scientific and cultural 19 . The discovery of DNA structure started us on this journey, the end of which will be the grand unification of the biological sciences in the emerging, information-based view of biology.s s

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The barrier function of an insulator couples high histone acetylation levels with specific protection of promoter DNA from methylation

Cited by 175 publications

References 43 publications

A DNA insulator prevents repression of a targeted X-linked transgene but not its random or imprinted X inactivation

A DNA insulator prevents repression of a targeted X-linked transgene but not its random or imprinted X inactivation

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

Controlling the double helix

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