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

Abstract

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...

X chromosome inactivation: new players in the initiation of gene silencing

“…Rather, this promotes illegitimate escape of neighboring genes. On the other hand, flanking GFP transgenes inserted in the Hprt locus with CTCF-binding sites did not prevent its silencing upon Xist expression 40 . Taken together, these data suggest that CTCF might not be sufficient to explain escape.…”

Dosage compensation and gene expression on the mammalian X chromosome: one plus one does not always equal two

“…Nevertheless, regulatory elements that rely on distance to distinguish genes cannot account for the close juxtaposition of some escape and inactivated genes (Tsuchiya et al 2004; Carrel and Willard 2005), but could explain large Mb sized domains with large transition regions elsewhere on the X. ii ) Sequences such as insulators, boundary elements or barriers ( red octagon ) flank coordinately regulated genes and protect them from silencing. This model, or at least this model with respect to the CTCF protein (Filippova et al 2005), also cannot fully explain escape gene expression (Ciavatta et al 2006). iii ) Incorporation of both models; way stations propagate XCI which is prevented from reaching escape genes by boundary elements.…”

“…At a minimum, the story is clearly not so simple. CTCF binding sites alone are not sufficient for escape expression, as a reporter gene, flanked by insulators that included CTCF binding sites, was still silenced by XCI (Ciavatta et al 2006). Nonetheless, this result may not be completely surprising, since CTCF has many functions, binds many locations, and the distribution of sites on the X is inconsistent with a role solely in escape gene regulation (Kim et al 2007).…”

Escape from X chromosome inactivation is an intrinsic property of the Jarid1c locus

“…CTCF is proposed to isolate escape genes from the surrounding inactive heterochromatin (6). However, CTCF binding alone is not sufficient for escape gene expression because a reporter gene flanked by CTCF binding sites was silenced by XCI (7).…”