Recruitment of Histone Modifications by USF Proteins at a Vertebrate Barrier Element

West, Adam G.; Huang, Suming; Gaszner, Miklos; Litt, Michael D.; Felsenfeld, Gary

doi:10.1016/j.molcel.2004.10.005

Cited by 236 publications

(313 citation statements)

References 35 publications

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“…Our results suggest that TPE is counteracted by an active histone methylation of the transgene and not histone acetylation. This contrasts with previous reports that demonstrated the importance of histone acetylation in insulation-mediated protection of random integrants from CPE (13,24). We propose that insulators may adapt themselves to block different chromatin propagation signals in distinct insertional epigenome environments.…”

Section: Enrichment Of Histone Methylation Open Marks Over the Telomericcontrasting

confidence: 99%

“…Recent evidence supports an active role of insulators in the optimal topology conformation of chromosomal domains in the nucleus (10)(11)(12). In particular, the chicken cHS4 ␤-globin insulator can protect a transgene against CPE via the recruitment of histone acetyltransferases and methyltransferases by USF1 (5,13). This process further supports the idea that the cHS4 insulator is capable of blocking heterochromatin propagation.…”

supporting

confidence: 54%

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Protection against telomeric position effects by the chicken cHS4 β-globin insulator

Rincón‐Arano

Furlan-Magaril

Recillas‐Targa

2007

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

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Epigenetic silencing of genes relocated near telomeres, termed telomeric position effect, has been extensively studied in yeast and more recently in vertebrates. However, protection of a transgene against telomeric position effects by chromatin insulators has not yet been addressed. In this work we investigated the capacity of the chicken ␤-globin insulator cHS4 to shield a transgene against silencing by telomeric heterochromatin. Using telomeric repeats, we targeted transgene integration into telomeres of the chicken cell line HD3. When the chicken cHS4 insulator is incorporated to the transgene, we observe a sustained gene expression of singlecopy integrants that can be maintained for >100 days of continuous culture. However, uninsulated single-copy clones showed an accelerated gene expression extinction profile. Unexpectedly, telomeric silencing was not reversed with trichostatin A or nicotidamine. In contrast, significant reactivation was obtained with 5-aza-2 -deoxycytidine, consistent with the subtelomeric DNA methylation status. Strikingly, insulated transgenes integrated into telomeric regions were enriched in histone methylation, such as H3K4me2 and H3K79me2, but not in histone acetylation. Furthermore, the cHS4 insulator counteracts telomeric position effects in an upstream stimulatory factor-independent manner. Our results suggest that this insulator has the capacity to adapt to different chromatin propagation signals in distinct insertional epigenome environments.chromatin insulator ͉ DNA methylation ͉ heterochromatin ͉ histone modifications ͉ epigenetic silencing

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Section: Enrichment Of Histone Methylation Open Marks Over the Telomericcontrasting

confidence: 99%

supporting

confidence: 54%

Protection against telomeric position effects by the chicken cHS4 β-globin insulator

Rincón‐Arano

Furlan-Magaril

Recillas‐Targa

2007

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

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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: 90%

“…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).…”

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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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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“…This novel concept of a delocalized synthetic enhancer has no precedent insofar as we are aware. Recent studies have also described the existence of domain boundaries, which is established by an array of cis elements (including insulators, boundary elements) that serve to delimit an active transcriptional state in a given region of the genome (25)(26)(27)(28). Thus a possible scenario is that there is a single Gata2 ureteric epithelial enhancer, which requires the full complement of domain boundaries present only in the linked BAC to activate GATA-2 expression in the ureteric epithelium.…”

Section: Discussionmentioning

confidence: 99%

Defining the Functional Boundaries of the Gata2 Locus by Rescue with a Linked Bacterial Artificial Chromosome Transgene

Brandt

Khandekar

Suzuki

et al. 2008

Journal of Biological Chemistry

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Transcription factor GATA-2 is vital for both hematopoietic progenitor cell function and urogenital patterning. Transgenic mapping studies have shown that the hematopoietic and urogenital enhancers are located hundreds of kbp 5 and 3 to the Gata2 structural gene, and both are vital for embryonic development. Because the size of mammalian genes, including all of their associated regulatory elements, can exceed a megabase, transgenic complementation in mice has, in specific instances, proven to be a formidable hurdle. After incorporating the Gata2 structural gene as well as the distant hematopoietic and urogenital enhancers into a single, contiguous piece of DNA by fusing two bacterial artificial chromosomes (BACs) into one, we formally tested the hypothesis that the functional boundaries of this locus are contained within this contiguous genomic span. We show that two independent lines of transgenic mice bearing a multicopy 413-kbp-linked Gata2 BAC transgene (bearing sequences from ؊187 to ؉226 kbp of the locus) are able to fully rescue Gata2 null mutant embryonic lethality and that the rescued animals behave and reproduce normally. Surprisingly, the linked BAC confers expression in the ureteric epithelium, whereas sequences within any of the overlapping parental BACs and a yeast artificial chromosome that were originally tested do not, and thus these experiments also define a novel synthetic enhancer activity that has not been previously described. These genetic complementation studies define the required outer limits of the Gata2 locus and formally demonstrate that enhancers lying beyond those boundaries are not necessary for Gata2-regulated viability or fecundity.Transcription factor GATA-2 is exemplary of a vital developmental factor whose regulation reveals many of the intricacies of the controlling apparatus that is required for proper temporal and tissue-specific expression of a gene in many different tissues and organs that are critical for mammalian development. The evolutionarily conserved C 4 zinc finger GATA transcription factors play demonstrably crucial roles in embryogenesis. GATA-2, originally cloned from a chicken cDNA library (1), was shown to be essential for the proliferation and/or differentiation of early hematopoietic progenitors, as Gata2 null mutant mice die around mid-gestation from a block in primitive hematopoiesis (2). Further examination of Gata2 gain-of-function mice and in vitro differentiation of Gata2 null mutant ES cells underscored the fundamental conclusions from the initial loss of function experiments, and showed that GATA-2 plays a pivotal role in the proliferation of very early hematopoietic progenitors (3-5).We showed several years ago that Gata2 null mutant embryonic lethality could be rescued by complementation with a 247-kbp transgenic yeast artificial chromosome (YAC) 3 bearing sequences from Ϫ174 to ϩ73 kbp (revised endpoints relative to the Gata2 translational initiation site); the YAC contains all of the regulatory information required to rescue Gata2 function in p...

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Recruitment of Histone Modifications by USF Proteins at a Vertebrate Barrier Element

Cited by 236 publications

References 35 publications

Protection against telomeric position effects by the chicken cHS4 β-globin insulator

Protection against telomeric position effects by the chicken cHS4 β-globin insulator

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

Defining the Functional Boundaries of the Gata2 Locus by Rescue with a Linked Bacterial Artificial Chromosome Transgene

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