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Insulators can block an enhancer of one gene from activating a promoter on another nearby gene. Almost all described vertebrate insulators require binding of the regulatory protein CTCF for their activity. We show that CTCF copurifies with the nucleolar protein nucleophosmin and both are present at insulator sites in vivo. Furthermore, exogenous insulator sequences are tethered to the nucleolus in a CTCF-dependent manner. These interactions, quite different from those of the gypsy insulator element in Drosophila, may generate similar loop structures, suggesting a common theme and model for enhancer-blocking insulator action.

DNA-dependent ATPases participate in a broad range of biological processes that include transcription, DNA repair, and chromatin dynamics. Mutations in the HARP ATPase are responsible for Schimke immuno-osseous dysplasia (SIOD), but the function of the protein is unknown. Here we report that HARP is an ATP-dependent annealing helicase. HARP rewinds single-stranded DNA bubbles that are stably bound by replication protein A. Other related ATPases, including the DNA translocase Rad54, do not exhibit annealing helicase activity. Analysis of mutant HARP proteins suggests that SIOD is caused by a deficiency in annealing helicase activity. Moreover, the pleiotropy of HARP mutations is consistent with the function of HARP as an annealing helicase that acts throughout the genome to oppose the action of DNA-unwinding activities in the nucleus.HARP (HepA-related protein; also known as SMARCAL1 and DNA-dependent ATPase A) is a member of the SNF2 family of ATP-driven molecular motor proteins (1-4). The biological importance of HARP was revealed by the discovery that mutations in HARP are responsible for a pleiotropic disorder known as Schimke immuno-osseous dysplasia (SIOD) (5). However, the molecular function of the HARP ATPase activity is unknown.We investigated how human HARP functions as an ATP-dependent molecular motor by synthesizing and purifying human HARP protein ( fig. S1). By using the gel mobility shift assay, we determined that HARP binds with higher affinity to fork DNA than to single-stranded DNA or to double-stranded DNA (Fig. 1A). In addition, the ATPase activity of HARP is stimulated to a much greater extent by fork DNA than by single-or double-stranded DNA (Fig. 1B) (6). These results are consistent with the finding that the HARP ATPase is activated by M13 single-stranded DNA (4), which probably contains hairpin structures, as well as the observation that the HARP ATPase domain is stimulated by DNA structures that contain both single-and double-stranded DNA (7,8).The stimulation of the HARP ATPase activity upon binding to fork DNA suggested that HARP may be an ATP-driven helicase that unwinds DNA. Helicases generate single-stranded DNA regions that can be bound by single-stranded DNA-binding proteins, such as replication protein A (RPA) in eukaryotes (see, for example, 9). However, we tested the ability of HARP to function as a helicase with several different assays and substrates, but did not observe any detectable helicase activity (for example, see fig. S2). Thus, HARP does not appear to be a helicase.We therefore considered the possibility that HARP is an ATP-driven annealing helicase that anneals complementary RPA-bound single-stranded DNA. To test this hypothesis, we devised an assay for annealing helicase activity ( fig. S3). We generated a stable, partially unwound DNA substrate by adding RPA to plasmid DNA in the presence of topoisomerase I (10,11).

HepA-related protein (HARP) (also known as SMAR-CAL1) is an ATP-driven annealing helicase that catalyzes the formation of dsDNA from complementary Replication protein A (RPA)-bound ssDNA. Here we find that HARP contains a conserved N-terminal motif that is necessary and sufficient for binding to RPA. This RPAbinding motif is not required for annealing helicase activity, but is essential for the recruitment of HARP to sites of laser-induced DNA damage. These findings suggest that the interaction of HARP with RPA increases the concentration of annealing helicase activity in the vicinity of ssDNA regions to facilitate processes such as DNA repair.Supplemental material is available at http://www.genesdev.org.Received June 12, 2009; revised version accepted August 7, 2009. Proteins in the SNF2 family of ATPases participate in a variety of nuclear processes, such as chromatin assembly and remodeling, transcription, DNA repair, and recombination (Gorbalenya and Koonin 1993;Eisen et al. 1995;Flaus et al. 2006). The HepA-related protein (HARP, also known as SMARCAL1) is a distant member of the SNF2 family (Coleman et al. 2000;Flaus et al. 2006). HARP is present in many eukaryotes but appears to be absent in fungi. In humans, mutations in HARP contribute to the pleiotropic disorder known as Schimke immunoosseous dysplasia (SIOD) (Boerkoel et al. 2002).HARP is an ATP-dependent annealing helicase (Yusufzai and Kadonaga 2008). Specifically, HARP is able to rewind complementary ssDNA that is bound by the ssDNA-binding protein Replication protein A (RPA). HARP binds preferentially to fork DNA relative to ssDNA or to dsDNA (Yusufzai and Kadonaga 2008). The ATPase activity of HARP is also stimulated preferentially by fork DNA relative to ssDNA or dsDNA (Yusufzai and Kadonaga 2008). This property is consistent with the observation that the ATPase activity of HARP is enhanced by DNA species with ssDNA-dsDNA junctions (Hockensmith et al. 1986;Muthuswami et al. 2000). These findings suggest a model wherein the binding of HARP to a DNA fork activates its ATP-driven motor with which it catalyzes the rewinding of DNA.The DNA rewinding activity of HARP could potentially participate in many different processes such as transcription, DNA replication, and DNA repair, in which ssDNA regions are generated by the action of helicases or polymerases (for example, see Liu and Wang 1987;Kowalski et al. 1988;Havas et al. 2000;Pyle 2008). These ssDNA regions can be stabilized by ssDNAbinding proteins (SSBs) such as RPA, the major SSB in eukaryotes (for example, see Wold 1997;Iftode et al. 1999;Zou et al. 2006). RPA is a heterotrimer (RPA1 [70 kDa], RPA2 [32 kDa], and RPA3 [14 kDa]) that binds stably to ssDNA and prevents complementary DNA from reannealing. RPA is required for many cellular processes, including replication, recombination, and repair, during which it stabilizes ssDNA intermediates. Because HARP catalyzes the regeneration of dsDNA from complementary RPA-bound ssDNA, it is possible that there is a specific link between HARP and RPA...

The insulator element at the 5 end of the chicken ␤-globin locus acts as a barrier, protecting transgenes against silencing effects of adjacent heterochromatin. We showed earlier that the transcription factor USF1 binds within the insulator and that this site is important for generating in adjacent nucleosomes histone modifications associated with active chromatin and, by inference, with barrier function. To understand the mechanism of USF1 action, we have characterized USF1-containing complexes. USF1 interacts directly with the histone H4R3-specific methyltransferase PRMT1. USF1, PRMT1, and the histone acetyltransferases (HATs) PCAF and SRC-1 form a complex with both H4R3 histone methyltransferase and HAT activities. Small interfering RNA downregulation of USF1 results in localized loss of H4R3 methylation, and other histone modifications associated with euchromatin, at the insulator. A dominant negative peptide that interferes with USF1 binding to DNA causes silencing of an insulated reporter construct, indicating abolition of barrier function. These results show that USF1 plays a direct role in maintaining the barrier, supporting a model in which the insulator works as a barrier by maintaining a local environment of active chromatin.Within the nucleus, heterochromatic and euchromatic domains may lie next to one another. In the absence of constraints, a variety of mechanisms may allow the extension of repressive heterochromatic structures into adjacent euchromatin (14,20,28). Barrier insulators are capable of preventing this heterochromatic encroachment. They are distinct in properties and composition from enhancer-blocking insulators, which prevent inappropriate interactions of neighboring gene systems (6, 41).Although there may be more than one way to block the propagation of condensed chromatin structures into an active chromatin domain, experiments both in yeast (30) and in vertebrates (42) have suggested that elements which recruit high levels of histone modifications associated with transcriptional activation may establish such a barrier. The 5ЈHS4 insulator at the 5Ј end of the chicken ␤-globin locus lies immediately downstream of an ϳ16-kb condensed chromatin domain and is thus in a position to protect the globin locus against the extension downstream of this heterochromatic region (24,25,34). The nucleosomes adjacent to the insulator site are highly enriched in active histone modifications, including acetylation of histones H3 and H4 and methylation of lys4 on histone H3 and of arg3 on H4 (15,24,25). Consistent with barrier function, we have shown that a 250-bp core sequence, derived from the ␤-globin insulator, can protect a stably integrated transgene from silencing by endogenous heterochromatic sequences at the site of integration (32,36). This barrier assay enabled us to show that a single factor binding site within the core was required to maintain high levels of histone acetylation as well as H3K4 methylation over the protected sequences and that this site was essential for insulation. We identi...

The neurodevelopmental disorder known as Rett syndrome has recently been linked to the methyl-CpG-binding transcriptional repressor, MeCP2. In this report we examine the consequences of these mutations on the function of MeCP2. The ability to bind specifically to methylated DNA and the transcription repression capabilities are tested, as well as the stability of proteins in vivo. We find that all missense mutations (R106W, R133C, F155S, T158M) within the methyl-binding domain impair selectivity for methylated DNA, and that all nonsense mutations (L138X, R168X, E235X, R255X, R270X, V288X, R294X) that truncate all or some of the transcriptional repression domain (TRD) affect the ability to repress transcription and have decreased levels of stability in vivo. Two missense mutations, one in the TRD (R306C) and one in the C-terminus (E397K), had no noticeable effects on MeCP2 function. Together, these results provide evidence of how Rett syndrome mutations can affect distinct functions of MeCP2 and give insight into these mutations that may contribute to the disease.

The protein CTCF plays an essential role in the action of a widely distributed class of vertebrate enhancer-blocking insulators, of which the first example was found in a DNA sequence element, HS4, at the 5 end of the chicken ␤-globin locus. HS4 contains a binding site for CTCF that is necessary and sufficient for insulator action. Purification of CTCF has revealed that it interacts with proteins involved in subnuclear architecture, notably nucleophosmin, a 38-kDa nucleolar phosphoprotein that is concentrated in nuclear matrix preparations. In this report we show that both CTCF and the HS4 insulator element are incorporated in the matrix; HS4 incorporation depends on the presence of an intact CTCF-binding site. However the DNA sequence in the neighborhood of HS4 is not like that of canonical matrix attachment regions, and its incorporation into the matrix fraction is not sensitive to ribonuclease, suggesting that the insulator is a distinct matrix-associated element.I nsulators are DNA sequence elements that can act either to block the extension of a condensed chromatin domain into a transcriptionally active region (barrier activity), or to prevent the interaction of a distal enhancer with a promoter when placed between the two (1, 2). Elements with the latter property, called enhancer blocking insulators, have been found in Drosophila and in vertebrates. In flies the most studied insulator element is gypsy, which when placed between two enhancers in a series of enhancers found in the yellow locus, blocks the action of all enhancers distal to the insertion but has no effect on those more proximal to the promoter (3). It has been shown that the insulator action of gypsy is mediated by a DNA-binding protein, Suppressor of Hairy wing [Su(Hw)], and a cofactor, Mod(mdg4) (4). Gypsy elements appear to localize to the nuclear envelope, where they cluster and organize the neighboring chromatin into loop domains (5). It is thought that the loop domain structure gives rise to the insulating activity either by preventing regulatory elements on different loops from interacting or by interfering with a ''tracking'' signal that would ordinarily proceed from enhancer to promoter (6-8). Loop domains can be established by attachment to other fixed sites in the nucleus. For example, a barrier function that prevents heterochromatinization of an active gene can be generated by tethering DNA elements to nuclear pore proteins (9). Loop domains can also arise simply from interactions that cause the insulator-bound proteins to stick to each other.A different enhancer blocking insulator activity has been described in vertebrates. First found at the 5Ј end of the chicken ␤-globin locus, it is part of a compound element (HS4) at that site that has both barrier and enhancer-blocking action (10). These two activities are separable; the enhancer-blocking insulation arises from a single DNA site that binds the protein CTCF (11). Insulator elements that bind CTCF have also been found at many other loci including the human and mouse ␤-globin cl...

We have investigated the properties of mutant forms of the methyl-CpG binding transcriptional repressor MeCP2 associated with Rett syndrome, a childhood neurodevelopmental disorder. We find that four Rett syndrome mutations at known sites within the methyl-CpG binding domain (MBD) impair binding to methylated DNA, but have little effect on nonspecific interactions with unmethylated DNA. Three of these mutations (R106W, R133C, and F155S) have their binding affinities for methylated DNA reduced more than 100-fold; this is consistent with the hypothesis that impaired selectivity for methylated DNA of mutant MeCP2 contributes to Rett syndrome. However, a fourth mutant, T158M, has its binding affinity for methylated DNA reduced only 2-fold, indicative either of additional distinct regulatory functions associated with the MBD or of an exquisite sensitivity of developing neurons to the selective association of MeCP2 with methylated DNA.