Academic journal article Genetics

The Enhancer-Blocking Activity of the Fab-7 Boundary from the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites

Academic journal article Genetics

The Enhancer-Blocking Activity of the Fab-7 Boundary from the Drosophila Bithorax Complex Requires GAGA-Factor-Binding Sites

Article excerpt

ABSTRACT

In the work reported here we have analyzed the role of the GAGA factor [encoded by the Trithorax-like (Trl) gene] in the enhancer-blocking activity of Frontabdominal-7 (Fab-7), a domain boundary element from the Drosophila melanogaster bithorax complex (BX-C). One of the three nuclease hypersensitive sites in the Fab-7 boundary, HS1, contains multiple consensus-binding sequences for the GAGA factor, a protein known to be involved in the formation and/or maintenance of nucleosome-free regions of chromatin. GAGA protein has been shown to localize to the Fab-7 boundary in vivo, and we show that it recognizes sequences from HS1 in vitro. Using two different transgene assays we demonstrate that GAGA-factor-binding sites are necessary but not sufficient for full Fab-7 enhancer-blocking activity. We show that distinct GAGA sites are required for different enhancer-blocking activities at different stages of development. We also show that the enhancer-blocking activity of the endogenous Fab-7 boundary is sensitive to mutations in the gene encoding the GAGA factor Trithorax-like.

EUKARYOTIC chromosomes are subdivided into functionally and structurally autonomous chromatin domains. This was first recognized more than a half century ago in cytological studies of lampbrush chromosomes in amphibian oocytes and polytene chromosomes in insects (ALFERT 1956; Gall 1956; Gall and CALLAN 1962; RITOSSA 1962). More direct evidence for the subdivision of eukaryotic chromosomes into discrete domains has recently come from a combination of genetic, molecular, and biochemical experiments. These studies demonstrate that domains correspond to units of independent genetic activity, and that each domain has its own distinct nucleoprotein composition and chromatin structure depending upon the developmental stage, tissue, or cell type and whether the domain is active or inactive (KELLUM and ELGIN 1998; GERASIMOVA and CORCES 1999; UDVARDY 1999; GREWAL 2000; WEST et al. 2002). Critical to ensuring the autonomy of each chromatin domain are special cis-acting elements called boundaries or insulators. While domain boundaries were first discovered in Drosophila (WELSHONS and KEPPY 1975; UDVARDY et al. 1985; GYURKOVICS et al. 1990; HOLBRIDGE and DORSETT 1991; KELLUM and SCHEDL 1991, 1992; GEYER and CORCES 1992), they have since been identified in organisms ranging from yeast to humans (GERASIMOVA and CORCES 2001; WEST et al. 2002). These elements define the limits of chromosomal domains and function to establish independent units of gene activity, insulating genes or regulatory elements within a domain from regulatory elements located in adjacent domains.

One of the better-characterized boundaries in Drosophila is the Fab-7 element from the bithorax complex (BX-C). BX-C contains three homeotic genes, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B), which specify segment identity in the posterior parasegments (PS) 5-14 of the fly. The parasegment-specific expression of these three homeotic genes is controlled by an ~300-kb cis-regulatory region that is subdivided into nine para-segment-specific cis-regulatory domains: abx/bx, bxd/pbx, and iab2-9 (LEWIS 1978; SANCHEZ-HERRERO et al. 1985; KARCH et al. 1985; CASANOVA and WHITE 1987; DUNCAN 1987; CELNIKER et al. 1990; SANCHEZ-HERRERO 1991). Each regulatory domain directs the appropriate parasegment specific pattern of expression of one of the BX-C homeotic genes and the domains are sequentially activated, going from anterior to posterior parasegments. For example, the iab6 cis-regulatory domain is activated in PS11 where it promotes an Abd-B expression pattern that confers PS11 identity. In contrast, the cis-regulatory domain immediately distal to iab -6 in BX-C, iab -7, is silenced in PS11. In the next posterior parasegment, PS12, iab-7 is activated and in this parasegment it, not iab-6, controls Abd-B expression. Mutations that disrupt the activity of any of the nine PS-specific regulatory regions result in a phenotypic transformation of the corresponding parasegment into the parasegment immediately anterior (KARCH et al. …

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