Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press The barrier function of an insulator couples high histone acetylation levels with specific protection of promoter DNA from methylation Vesco J. Mutskov,1 Catherine M. Farrell,1 Paul A. Wade,2 Alan P. Wolffe,3 and Gary Felsenfeld1,4 1Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA; 2Emory University School of Medicine, Department of Pathology and Laboratory Medicine, Atlanta, Georgia 30322, USA Stably integrated transgenes flanked by the chicken ␤-globin HS4 insulator are protected against chromosomal position effects and gradual extinction of expression during long-term propagation in culture. To investigate the mechanism of action of this insulator, we used bisulfite genomic sequencing to examine the methylation of individual CpG sites within insulated transgenes, and compared this with patterns of histone acetylation. Surprisingly, although the histones of the entire insulated transgene are highly acetylated, only a specific region in the promoter, containing binding sites for erythroid-specific transcription factors, is highly protected from DNA methylation. This critical region is methylated in noninsulated and inactive lines. MBD3 and Mi-2, subunits of the Mi-2/NuRD repressor complex, are bound in vivo to these silenced noninsulated transgenes. In contrast, insulated cell lines do not show any enrichment of Mi-2/NuRD proteins very late in culture. In addition to the high levels of histone acetylation observed across the entire insulated transgene, significant peaks of H3 acetylation are present over the HS4 insulator elements. Targeted histone acetylation by the chicken ␤-globin insulator occurs independently of gene transcription and does not require the presence of a functional enhancer. We suggest that this acetylation is in turn responsible for the maintenance of a region of unmethylated DNA over the promoter. Whereas DNA methylation often leads to histone deacetylation, here acetylation appears to prevent methylation. [Key Words: Chromatin; histone acetylation; DNA methylation; insulator; methyl-binding domain (MBD) proteins; silencing] Received March 1, 2002; revised version accepted May 1, 2002. The chromosomes of higher eukaryotes are subdivided boundary elements have been described in Drosophila, into discrete functional domains in which gene expres- yeast, mammalian cells, chicken, and Xenopus (Geyer sion is either repressed or facilitated. Active or poten- 1997; Bi and Broach 2001; West et al. 2002). They possess tially active genes are packaged into a chromatin struc- at least one of two properties potentially related to ture, defined as euchromatin, that exhibits increased boundary formation. First, insulators can block commu- nuclease sensitivity and reduced amounts of linker his- nication between an enhancer and a promoter if they are tones such as H1 and H5, and is subject to several types located between them, thereby preventing inappropriate of chemical modifications. The inactive chromatin, of- gene activation. A second activity of some chromatin ten described as condensed chromatin, is relatively in- insulators is their ability to protect a stably integrated accessible to DNA-modifying reagents, and it contains gene from local position effects, as well as from gradual hypoacetylated histones and methylated DNA. Euchro- extinction of activity during long-term propagation of matic and condensed chromatin regions are interspersed the transformed cells. along the chromosome, and boundary elements can The first insulator described in vertebrates—the mark the border between adjacent domains. These DNA chicken ␤-globin 5ЈHS4 element (Chung et al. 1993)— sequences can functionally isolate neighboring genes, has been successfully used to protect transgenes against thereby blocking their interactions. Several insulators or position effects and prevent their silencing in different cell lines and transgenic animals (Bell et al. 2001). When two copies of the 1.2-kb ␤-globin boundary element sur- 3A.P.W. was at Sangamo BioSciences, Inc., Richmond, CA, USA until his death in May 2001. round an erythroid-specific reporter gene in immature 4Corresponding author. erythroid 6C2 chicken cells, expression is uniform E-MAIL [email protected]; FAX (301) 496-0201. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ among different lines and is maintained after 80 d of gad.988502. incubation in the absence of selection (Pikaart et al. 1540 GENES & DEVELOPMENT 16:1540–1554 © 2002 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/02 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Barrier function of the chicken ␤-globin insulator 1998). In contrast, noninsulated cell lines display varying role in the ability of the chicken ␤-globin insulator to levels of expression after selection, and silencing of the protect against silencing. transgene frequently occurs in less than 80 d. The silent transgene loses several epigenetic hallmarks of active chromatin, including DNA hypomethylation and his- Results tone hyperacetylation. This 5ЈHS4 function has also The chicken ␤-globin insulator prevents DNA been confirmed in transgenic mice, rabbits, and cell lines methylation of specific CpG dinucleotides (for review, see Bell et al. 2001). However, the biological in the transgene promoter mechanisms of action of the chicken ␤-globin insulator in protection against position-dependent transgene si- To determine how the chicken ␤-globin insulator keeps lencing have not yet been established. At least two pos- the chromatin of insulated genes in an open and tran- sible models have been suggested (Pikaart et al. 1998). scriptionally active state, we applied the model system One model proposes that the insulator can exclude or developed in our laboratory for studying the function of block access of repressor complexes to the promoter of the 5ЈHS4 insulator (Pikaart et al. 1998). The gene en- the flanked transgene. These repressor complexes can coding the Tac subunit of the interleukin 2 receptor include histone deacetylases (HDACs), DNA methyl- (IL2R) under the control of the chicken ␤A-globin pro- transferases, methyl-binding domain (MBD) proteins, moter and the ␤/␧ enhancer (Fig. 1A) was stably inte- transcription repressors, and proteins such as HP1 that grated into the chicken early erythroid 6C2 cell line. are associated with condensed chromatin. A second pos- After withdrawal of hygromycin, which was used for se- sibility is that insulators can recruit chromatin remod- lection of the transformed cells, there was a gradual ex- eling complexes and histone acetyltransferases (HATs), tinction of activity over a period of 20–80 d of growth although it has been shown that the 5ЈHS4 insulator (data not shown; Pikaart et al. 1998). However, when the does not function independently as a transcription acti- transgene was flanked by two copies of the 1.2-kb vation complex. It is possible that the above models chicken insulator (Fig. 1B), position effects were sup- function simultaneously. pressed and extinction of expression was prevented (data In this paper, we explore the question of how insula- not shown; Pikaart et al. 1998). tors keep the chromatin of insulated genes in an open Whereas it has been shown previously that the silent and transcriptionally active state, and how they prevent transgene loses several epigenetic hallmarks of active gene silencing. To address these questions, we examined chromatin, including DNA hypomethylation and his- the patterns of DNA methylation and histone acetyla- tone hyperacetylation (Pikaart et al. 1998), there was no tion across insulated and noninsulated transgenes, and clear correlation between the presence of the insulators, tested for the presence of repressor complexes. We found transcriptional activity, and DNA methylation of three that the chicken ␤-globin insulator prevented DNA detectable HpaII restriction sites over the promoter. methylation of specific CpG dinucleotides; this effect Some insulated cell lines after long-term growth in cul- was largely confined to a promoter region that is critical ture, even with high expression levels of the IL2R cell for transgene expression. In noninsulated and inactive surface marker, showed resistance to HpaII digestion due lines, this region was methylated. We found that MBD3 to DNA methylation at these three sites (data not and Mi-2, which are subunits of the Mi-2/NuRD repres- shown; Pikaart et al. 1998). To resolve this apparent sor complex, were bound to silenced noninsulated trans- paradox and determine precisely how the DNA methyl- genes in vivo. This was accompanied by histone un- ation pattern contributes to the function of the chicken deracetylation over the entire transgene, except at the ␤-globin insulator, we used the bisulfite genomic se- ␤/␧ enhancer. In the insulated lines, neither MBD3 nor quencing method, which, unlike the HpaII digestion Mi-2 proteins were present, and specific hyperacetyla- method, reveals the methylation state of every CpG. We tion patterns of histones H3 and H4 were observed across amplified a region covering the main CpG cluster of the the insulated domains. These and other results reported ␤A-globin promoter, including the transcription start site here indicate that histone acetylation plays a dominant and part of the IL2R cDNA (Fig. 2A). For each cell line,