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Protection Against Telomeric Position Effects by the Chicken Chs4 ß-Globin Insulator

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Protection Against Telomeric Position Effects by the Chicken Chs4 ß-Globin Insulator Protection against telomeric position effects by the chicken cHS4 ␤-globin insulator He´ ctor Rinco´ n-Arano, Mayra Furlan-Magaril, and Fe´ lix Recillas-Targa* Instituto de Fisiologı´aCelular, Departamento de Gene´tica Molecular, Universidad Nacional Auto´noma de Me´xico, Apartado Postal 70-242, 04510 Me´xico, D.F., Me´xico Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved July 18, 2007 (received for review May 28, 2007) Epigenetic silencing of genes relocated near telomeres, termed chromosomal domains in the nucleus (10–12). In particular, the telomeric position effect, has been extensively studied in yeast and chicken cHS4 ␤-globin insulator can protect a transgene against more recently in vertebrates. However, protection of a transgene CPE via the recruitment of histone acetyltransferases and meth- against telomeric position effects by chromatin insulators has not yltransferases by USF1 (5, 13). This process further supports the yet been addressed. In this work we investigated the capacity of idea that the cHS4 insulator is capable of blocking heterochro- the chicken ␤-globin insulator cHS4 to shield a transgene against matin propagation. silencing by telomeric heterochromatin. Using telomeric repeats, Here we ask whether the cHS4 insulator is able to protect a we targeted transgene integration into telomeres of the chicken transgene against a dominant source of constitutive heterochro- cell line HD3. When the chicken cHS4 insulator is incorporated to matin, like telomeres. To this end we targeted the integration of the transgene, we observe a sustained gene expression of single- a transgene into telomeric regions by incorporating telomeric copy integrants that can be maintained for >100 days of contin- (TTAGGG)n repeats on one side of our constructs. Our results uous culture. However, uninsulated single-copy clones showed an show that the cHS4 insulator is able to protect a transgene accelerated gene expression extinction profile. Unexpectedly, te- against TPE. Reactivation experiments demonstrated that DNA lomeric silencing was not reversed with trichostatin A or nicoti- methylation is one of the predominant causes of uninsulated damine. In contrast, significant reactivation was obtained with transgene silencing. Surprisingly, the insulated transgene is 5-aza-2؅-deoxycytidine, consistent with the subtelomeric DNA enriched in histone methylation, which contrasts with an absence methylation status. Strikingly, insulated transgenes integrated of histone acetylation marks. Furthermore, we demonstrate that into telomeric regions were enriched in histone methylation, such this insulator protects from TPE independent of upstream as H3K4me2 and H3K79me2, but not in histone acetylation. Fur- stimulatory factor (USF). This study provides evidence that a thermore, the cHS4 insulator counteracts telomeric position effects chromatin insulator protects a transgene against TPE and sug- in an upstream stimulatory factor-independent manner. Our re- gests that insulators are able to adapt themselves to protect a sults suggest that this insulator has the capacity to adapt to transgene against distinct epigenomic environments. different chromatin propagation signals in distinct insertional epigenome environments. Results Experimental System to Study CPE. To study CPE and TPE we used chromatin insulator ͉ DNA methylation ͉ heterochromatin ͉ the enhanced EGFP as a reporter gene under the control of the histone modifications ͉ epigenetic silencing chicken adult ␣D gene promoter, which is susceptible to strong CPE (Fig. 1A and data not shown). We first validated our assay by flanking the transgene on both sides with two copies of the he eukaryotic genome is partitioned in two classes of chro- ϫ ␤ Tmatin: euchromatin and heterochromatin. Centromeric and core (2 250 bp) chicken -globin cHS4 insulator element (7). telomeric sequences represent the main source of constitutive This reporter was randomly integrated into the avian trans- heterochromatin. Telomeres, which are composed of TTAGGG formed erythroblast HD3 cell line. Southern blot analysis con- repeats and subtelomeric regions, are gene-poor genomic areas firmed the integrity and copy number of the transgene in 10 that adopt particular heterochromatin conformation (1, 2). In independently isolated lines (data not shown). We performed addition to their contribution to genome stability and their fluorescence cytometry to measure GFP expression in individ- protective role, telomeres can also influence the expression of ual, stable HD3 clones (Fig. 1B). Using this reporter gene, genes integrated nearby through a phenomenon known as promoter, and cell line, we confirmed the previous observation that the core cHS4 insulator is capable of contributing to telomeric position effect (TPE) (3, 4). Ͼ There are two sorts of position effects, position effect varie- sustained expression over 100 days of continuous cell culture gation and chromosomal position effect (CPE) (4). Position in the absence of drug selection (7). As expected, uninsulated GFP effect variegation is defined as the variegated pattern of expres- transgenes showed a gradual silencing of expression began after Ϸ40–50 days of continuous cell culture (data not shown). sion from cell to cell when a gene is translocated into the To validate the progressive epigenetic silencing of the uninsu- proximity of dominant heterochromatin (3, 4), whereas CPE are lated transgene, we performed reactivation experiments using alterations of transgene expression associated with a distinct insertion into the epigenome milieu (5). In both cases, transgene expression is affected by changes in chromatin conformation, Author contributions: H.R.-A. and F.R.-T. designed research; H.R.-A. and M.F.-M. performed such as those associated with histone deacetylation, H3K9me3, research; H.R.-A., M.F.-M., and F.R.-T. analyzed data; and H.R.-A. and F.R.-T. wrote the and DNA methylation, that can extend over considerable paper. genomic distances (3, 4). Many studies have focused on trying to The authors declare no conflict of interest. understand such phenomena, but little has been done to study This article is a PNAS Direct Submission. the capacity of vertebrate chromatin insulators to protect against Abbreviations: CPE, chromosomal position effect; TPE, telomeric position effect; TSA, TPE (5–10). trichostatin A; 5-azadC, 5-aza-2Ј-deoxycytidine; USF, upstream stimulatory factor. Chromatin domain boundaries or insulators are epigenomic *To whom correspondence should be addressed. E-mail: [email protected]. components that contribute to the formation and maintenance This article contains supporting information online at www.pnas.org/cgi/content/full/ of genomic domains (9, 10). Recent evidence supports an active 0704999104/DC1. role of insulators in the optimal topology conformation of © 2007 by The National Academy of Sciences of the USA 14044–14049 ͉ PNAS ͉ August 28, 2007 ͉ vol. 104 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0704999104 Downloaded by guest on September 25, 2021 Fig. 2. Telomeric insertion of insulated and uninsulated transgenes. (A) Transgene vector with the telomeric (TTGAAA)n repeats. (B) Colocalization of GENETICS transgene with telomeric repeats. In situ hybridization was performed with the clones 613 and 615 and the random integrated clone 1001. Transgene signal was amplified and detected with a FITC-labeled antibody against Fig. 1. The cHS4 insulator protects against CPE. (A) Scheme of the vectors anti-digoxigenin (green). Telomeric repeats were hybridized with biotin- used for stable transfections. (B) Stable integrant of HD3 cells transfected with ylated oligonucleotides and identified with streptavidin coupled to Alexa an uninsulated or insulated GFP transgene was maintained in continuous cell Fluor 568 (red). Cells were counterstained with DAPI. Arrows indicate the culture for 100 days (d100). FACS analysis was performed for each clone every location of the transgene. (C) Sequence specificity of the purification of 2 weeks. Representative FACS profiles are shown for several single or multi- telomeres. DraIII-digested HD3 genomic DNA was annealed to telomeric- copy integrants. (C) Reactivation assays were performed with silenced unin- specific biotinylated oligonucleotides, and telomere–oligonucleotides com- sulated clones by incubating with TSA or 5-azadC for 24 and 48 h, respectively. plexes were captured with streptavidin-coated magnetic beads. The bound DNA was resolved on an agarose gel and probed with a 32P-labeled GFP probe or with an 32P-labeled (TTAGGG) oligonucleotide. histone deacetylase [trichostatin A (TSA)] and DNA methyl- 7 ation [5-aza-2Ј-deoxycytidine (5-azadC)] inhibitors. Our results showed that both DNA methylation and histone deacetylation (17). One possibility is that the transgene is integrated near an are responsible for maintaining silencing of uninsulated trans- interstitial (TTAGGG) repeat region (15) (SI Fig. 7). We believe genes (Fig. 1C), and the core cHS4 insulator is able to counteract that the avian genome represents an attractive model for TPE CPE (6, 7). because of its high density of telomeric (TTAGGG)n repeats in macrochromosomes and microchromosomes (15). Transgene Targeting to Telomeric Regions in Chicken HD3 Cells. To analyze the effects of an insulated transgene in the context of The Core cHS4 Insulator Protects Against TPE. We next analyzed the telomeric heterochromatin, we targeted our constructs to telo- effects of insulators flanking both sides of the transgene
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