Studies of the Role of the Drosophila scs and scs′ Insulators in Defining Boundaries of a Chromosome Puff

Abstract

Insulators are DNA elements that establish independent transcriptional domains within eukaryotic genomes. The Drosophila scs and scs insulators localize near the borders of a structural domain in the polytene chromosomes, known as a puff, produced by transcription of the 87A heat shock protein (hsp) genes. It has been suggested that scs and scs are boundary elements that delimit this decondensed chromatin domain, reflecting the mechanism by which these sequences act to constrain regulatory interactions. This model was tested using transposons that carried a yellow gene to assess enhancer blocking and an hsp70-lacZ gene to examine the structure of a heat shock puff in the presence and absence of insulators. We found that although scs and scs blocked enhancer function, these sequences did not prevent the spread of decondensation resulting from hsp70-lacZ transcription. Further analysis of the endogenous 87A locus demonstrated that scs and scs reside within, not at, the borders of the puff. Taken together, our studies suggest that scs and scs are not boundary elements that block the propagation of an altered chromatin state associated with puff formation. We propose that these insulators may have a direct role in limiting regulatory interactions in the gene-dense 87A region.Eukaryotic genomes are assembled into chromatin to establish the platform for accurate transcriptional regulation. Chromatin is not uniformly organized along the length of the chromosome but is folded into regions of distinct higher-order structure. For example, the Drosophila polytene chromosomes display a reproducible dark and light banding pattern, reflective of an underlying structural organization that aligns the 500 to 1,000 chromatids (49, 62). Specialized DNA elements, known as boundary elements, are proposed to generate topologically independent structural domains by assembling protein complexes that interact with each other or nuclear substructures to form loop domains (16,43,46). Structural domains have been linked to independent gene expression, postulating that higher-order chromatin structures constrain regulatory interactions such that they occur only within, but not between, domains (reviewed in references 18 and 36). While recent studies suggest that 20 to 30 percent of genes in the Drosophila genome are organized into coexpressed clusters, a correlation between coregulated domains and the banding pattern of polytene chromosomes was not found (7, 13, 60). These observations suggest that a more detailed analysis of independent structural domains is required to fully understand the connection between structural and functional chromosomal domains.One well-characterized structural domain in the polytene chromosomes resides in the 87A region of the third chromosome (Fig. 1). This 15-kb region contains a pair of divergently transcribed heat shock protein 70 (hsp70) genes (Fig. 1). Under non-heat shock conditions, histones H3 and H4 within the 87A region are acetylated, suggesting that the locus is primed for transcriptional activation...

The Drosophila Boundary Element-Associated Factors BEAF-32A and BEAF-32B Affect Chromatin Structure

“…''Sibling'' lines in which the insulator was removed by the Cre recombinase were also used as controls for the presence and absence of an insulator at the same chromosomal locus. Using these fly lines, it was previously found that insulators do not block the propagation of heat-shock puffs in polytene chromosomes (Kuhn et al 2004). To use this assay, an appropriate GAL4 driver line was required.…”

“…The following yellow enhancer-blocking lines, described in Kuhn et al (2004), were used: 2scs9 inserted at 19D; scs inserted at 60A; gypsy inserted at 25C. The ey-GAL4/TM6b line was kindly provided by T. E. Haerry (Florida Atlantic University).…”

Insulators are fundamental components of the eukaryotic genomes

“…As an example, the first insulators identified, the Drosophila specialized chromatin structures, scs and scs 0 Schedl, 1991, 1992), are boundaries surrounding the 87A7 locus where two hsp70 genes reside. As proposed above for the gypsy elements, interaction between scs and scs 0 could explain their insulator function; however, this interaction fails to explain why interaction between scs and scs 0 is not a general property of these elements but depends on sequences located outside the specific domain bearing the insulator function (Kuhn et al, 2004). Additionally, Majumder and Cai have tested the effect of pairing on enhancer-blocking activity of 11 homologous Boundaries interact with nuclear pore proteins by the nuclear pore complex (from Ishii et al, 2002).…”

Chromatin domain boundaries: insulators and beyond

“…More recently, another possible role of the two insulator elements was demonstrated. Kuhn et al suggest that the two elements do not limit the extent of heat shock-induced chromatin decondensation at region 87 of Drosophila polytene chromosomes and that scs and scs' may play a role in maintaining the transcriptional fidelity in this gene-dense region [21].…”

“…They are located ~15 kb apart and flanking one of the hsp70 loci [20,21]. The proteins involved in their functions have also been identified [20,21]. The two elements contain binding sites for proteins Zw-5 and boundary element-associated factor (BEAF), respectively.…”