Academic journal article Genetics

Investigation of the Properties of Non-Gypsy Suppressor of Hairy-Wing-Binding Sites

Academic journal article Genetics

Investigation of the Properties of Non-Gypsy Suppressor of Hairy-Wing-Binding Sites

Article excerpt

ABSTRACT

Insulators define interactions between transcriptional control elements in eukaryotic genomes. The gypsy insulator found in the gypsy retrovirus binds the zinc-finger Suppressor of Hairy-wing [Su(Hw)] protein that associates with hundreds of non-gypsy regions throughout the Drosophila genome. Models of insulator function predict that the gypsy insulator forms chromatin loop domains through interactions with endogenous Su(Hw) insulators (SIs) to limit the action of transcriptional control elements. Here we study SI 62D and show that interactions occur between two SI 62D elements, but not between SI 62D and the gypsy insulator, limiting the scope of genomic gypsy insulator interactions. Enhancer blocking by SI 62D requires fewer Su(Hw)-binding sites than needed for gypsy insulator function, with these target regions having distinct zinc-finger requirements for in vivo Su(Hw) association. These observations led to an investigation of the role of the Su(Hw) zinc-finger domain in insulator function. Using a combination of in vitro and in vivo studies, we find that this domain makes sequence-dependent and -independent contributions to in vivo chromosome association, but is not essential for enhancer or silencer blocking. These studies extend our understanding of the properties of Su(Hw) and the endogenous genomic regions to which this protein localizes.

EUKARYOTIC genomes contain multiple classes of DNA elements that regulate transcription. One class includes insulators that restrict and define interactions between enhancers, silencers and promoters. Insulators have been identified on the basis of one of two properties (Kuhn and Geyer 2003; Capelson and Corces 2004; Recillas-Targa et al. 2004; Brasset and Vaury 2005; Valenzuela and Kamakaka 2006). First, insulators block enhancers when placed between an enhancer and a promoter (Geyer and Corces 1992; Kuhn and Geyer 2003). Enhancer blocking does not inactivate transcriptional regulatory elements, but prevents communication between enhancers and the target promoter (Cai and Levine 1995; Scott and Geyer 1995). Second, insulators act as barriers that protect the expression of transgenes from chromatin-silencing effects, including restricting the action of silencers and inhibiting the spread of heterochromatin (Roseman et al. 1995; Festenstein et al. 1996; Mallin et al. 1998; Pikaart et al. 1998; Yannaki et al. 2002; Jakobsson et al. 2004). Insulators are fundamental components of eukaryoticgenomes that are involved in multipleprocesses, ranging from centromere organization in yeast to imprinting inmammals (Nomaet al. 2001;Engel et al. 2006; Yoon et al. 2007).

The gypsy insulator is a well-characterized element in the Drosophila genome. This insulator resides in the 59 untranslated region of the gypsy retrovirus and is responsible for mutations caused by insertion of this retrovirus into the regulatory regions of several genes (Modolell et al. 1983; Geyer et al. 1988; Peifer and Bender 1988; Spana et al. 1988). The gypsy insulator consists of 12 direct repeats of a YRYTGCATAYYY motif, where Y represents a pyrimidine and R represents a purine, separated by an AT-rich spacer. Direct tests have shown that the gypsy insulator blocks a wide variety of enhancers (Dorsett et al. 1989; Geyer and Corces 1992; Scott andGeyer 1995;Hagstrom et al. 1996; Cai and Levine 1997), protects against the repressive effects of a Polycomb response element (PRE; Sigrist and Pirrotta 1997; Mallin et al. 1998), and partially prevents heterochromatic silencing of transgenes inserted into centric regions (Roseman et al. 1993, 1995). These observations demonstrate that the gypsy insulator has both properties of insulators and shows a versatile capacity for defining regulatory interchanges.

Several proteins that are required for gypsy insulator function have been identified. An essential component is the Suppressor of Hairy-wing [Su(Hw)] protein that binds this insulator through a centrally located 12-zinc-finger domain (Parkhurst et al. …

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