Although it is now well-established that boundary elements/insulators function to subdivide eukaryotic chromosomes into autonomous regulatory domains, the underlying mechanisms remain elusive. One idea is that boundaries act as barriers, preventing the processive spreading of "active" or "silenced" chromatin between domains. Another is that the partitioning into autonomous functional units is a consequence of an underlying structural subdivision of the chromosome into higher order "looped" domains. In this view, boundaries are thought to delimit structural domains by interacting with each other or with some other nuclear structure. The studies reported here provide support for the looped domain model. We show that the Drosophila scs and scs boundary proteins, Zw5 and BEAF, respectively, interact with each other in vitro and in vivo. Moreover, consistent with idea that this protein:protein interaction might facilitate pairing of boundary elements, we find that that scs and scs are in close proximity to each other in Drosophila nuclei. The chromosomes of higher eukaryotes are subdivided into functionally autonomous domains that have distinct properties depending on whether they are transcriptionally active or silent. A good example of an "active" chromatin domain is the 35-kb ␤-globin locus in chicken erythrocytes. In addition to being considerably more sensitive to DNase I digestion than the flanking silenced domains (Bellard et al. 1980;Stalder et al. 1980), chromatin from the ␤-globin locus has a reduced ability to form pseudo-higher order structures and a two-to threefold lower level of the linker histone H5 (Verreault and Thomas 1993). There are also striking differences in the patterns of histone acetylation and methylation. Histones in the ␤-globin domain are hyperacetylated and have a relatively high level of methylation at Lys 4 of histone H3 (Hebbes et al. 1992; Litt et al. 2001a,b). In contrast, histones in the flanking silenced domains are hypoacetylated and are enriched in histone H3 methylated at Lys 9. The features that distinguish active and inactive domains in chicken erythrocytes are evident in other eukaryotes. For example, the silenced mating type loci of yeast are located in chromatin domains that are resistant to nuclease and restriction enzyme digestion, are highly compacted, have hypoacetylated histones, are enriched in histone H3 that is methylated on Lys 9, and have a special set of nonhistone chromosomal proteins (Grewal 2000;Huang 2002).The subdivision of eukaryotic chromosome into domains that have a distinct chromatin organization, biochemical composition, and genetic activity requires a mechanism to separate one domain from another. Special elements called boundaries or insulators are thought to serve this purpose (Bell et al. 2001;Gerasimova and Corces 2001). Elements that function as boundaries of chromatin domains were first identified in Drosophila (Gyurkovics et al. 1990;Holdridge and Dorsett 1991;Kellum andSchedl 1991, 1992;Geyer and Corces 1992) and have subsequently been foun...

We have identified a novel gene named grappa (gpp) that is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development.

Adherens junctions are multiprotein complexes mediating cell-cell adhesion and communication. They are organized around a transmembrane cadherin, which binds a set of cytoplasmic proteins required for adhesion and to link the complex to the actin cytoskeleton. Three components of Drosophila adherens junctions, analogous to those in vertebrates, have been identified: Armadillo (homolog of ␤-catenin), Drosophila E-cadherin (DE-cadherin), and ␣-catenin. We carried out the first analysis of the interactions between these proteins using in vitro binding assays, the yeast two-hybrid system, and in vivo assays. We identified a 76-amino acid region of Armadillo that is necessary and sufficient for binding ␣ Adherens junctions consist of transmembrane cadherins and a set of cytoplasmic proteins associated with cadherin cytoplasmic domains (reviewed in Refs. 1 and 3). The extracellular domains of cadherins interact homotypically with cadherins of neighboring cells. The cytoplasmic proteins ␣-catenin, ␤-catenin, and plakoglobin (or ␥-catenin) are required for cadherin adhesive function and anchor the actin cytoskeleton. The Src tyrosine kinase substrate p120cas is also present in adherens junctions (4, 5); its function remains unknown. Changes in tyrosine phosphorylation of ␤-catenin (reviewed in Ref.2) and p120cas (6) correlate with transformation and associated changes in cell adhesion.To understand the cell biological function of adherens junctions, we must determine how interactions among different adherens junction proteins mediate assembly. ␤-Catenin and plakoglobin bind directly to the E-cadherin cytoplasmic domain in a mutually exclusive fashion (7,8). ␤-Catenin and plakoglobin are 70% identical in amino acid sequence; their central regions, containing ϳ13 copies of the 42-amino acid Arm 1 repeat (9), are particularly well conserved (ϳ80% amino acid identity). These highly conserved Arm repeats mediate interaction with cadherin (10 -12), suggesting that ␤-catenin and plakoglobin compete for the same binding site. The N-terminal regions of both ␤-catenin and plakoglobin bind to ␣-catenin; ␣-catenin does not bind cadherin directly (11,(13)(14)(15). ␣-Catenin, in turn, links adherens junctions to actin, directly (16) or via ␣-actinin (17). p120 cas also binds directly to E-cadherin (18), but likely to a site distinct from that bound by ␤-catenin/ plakoglobin (4, 5). p120cas does not interact with ␣-catenin (18), however, and thus does not appear to mediate interaction with actin. The core cadherin-catenin complex forms higher order assemblies such as the zonula adherens. Both E-and N-cadherins dimerize (19,20), and association with the cytoskeleton may help form larger assemblies.Adherens junctions were first described in vertebrates, but precisely analogous structures exist in Drosophila. The Drosophila homolog of ␤-catenin is Armadillo, first discovered because of its role in transducing the Wingless cell-cell signal (reviewed in Ref. 3). Arm is structurally similar to ␤-catenin and plakoglobin (it is 73% identical ...

Boundary elements or insulators subdivide eukaryotic chromosomes into a series of structurally and functionally autonomous domains. They ensure that the action of enhancers and silencers is restricted to the domain in which these regulatory elements reside. Three models, the roadblock, sink/decoy, and topological loop, have been proposed to explain the insulating activity of boundary elements. Strong predictions about how boundaries will function in different experimental contexts can be drawn from these models. In the studies reported here, we have designed assays that test these predictions. The results of our assays are inconsistent with the expectations of the roadblock and sink models. Instead, they support the topological loop model.

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