The enhancer-blocking suppressor of Hairy-wing zinc finger protein of Drosophila melanogaster alters DNA structure.

Abstract

(6,10,11,13,14). The SUHW-binding sites are the only part of gypsy required for enhancer blocking. For example, heat shock transcription is repressed when SUHW-binding sites are inserted in the hsp70 promoter between the two heat shock elements or between the heat shock elements and the TATA box (13). Similarly, insertion of SUHW-binding sites at various positions in the yellow gene prevents different enhancers from activating gene expression (10).The mechanism of enhancer blocking by SUHW is unknown but is likely to provide insight into how enhancers activate transcription over long distances. SUHW blocks an enhancer only when positioned between the enhancer and promoter, yet enhancer blocking is also distance independent, even for distances approaching 100 kb (6). Thus, SUHW bound just upstream of an embryonic enhancer in the cut locus does not interfere with that enhancer, yet it effectively blocks all upstream enhancers, including an enhancer nearly 50 kb further upstream (6,14). Furthermore, the embryonic enhancer is effectively blocked when SUHW is bound 40 kb downstream. Therefore, it is extremely unlikely that SUHW blocks by interacting with the transcription-activating proteins that bind to enhancers. SUHW enhancer blocking is also immediate and reversible (6), indicating that blocking does not result from formation of quasi-stable heterochromatin-like structures.An additional clue to the mechanism of enhancer blocking is the inability of SUHW to effectively block activation by the GAL4 protein in the yeast Saccharomyces cerevisiae when bound between GAL4 and the promoter (19). SUHW produced in yeast is indistinguishable from Drosophila SUHW and is present in the yeast nucleus at high levels. The GAL4 protein activates transcription in D. melanogaster (8) and is blocked by bacterial LexA protein in yeast cells (4), indicating that the failure of SUHW to block GAILA in yeast cells is not because GAIA activates through an unusual mechanism or because GAL4 is refractory to blocking. A key difference between enhancer-promoter interactions between S. cerevisiae and higher eukaryotes is that activator proteins do not function when more than a few hundred base pairs away from the promoter in yeast cells. Therefore, it is plausible that SUHW blocks enhancers by interfering with proteins or chromatin structures found only in higher eukaryotes, which allows enhancers to function several kilobases away from the promoter. For example, it is possible that SUHW disturbs a processive mechanism whereby enhancers find promoters by tracking along the chromatin fiber.The problem in long-distance activation is to bring the enhancer physically close to the promoter to allow interactions between the proteins bound to them. We considered the 5645 Vol.

Two modes of transvection: Enhancer action intransand bypass of a chromatin insulator incis

“…Finally, the insulator may be compromised by constraints imposed by the looped structure. This interpretation is in line with proposals that binding of su(Hw) protein to gypsy is sensitive to DNA topology (63) and that insulator function entails an increase in DNA flexibility (64). Although our models focus on the promoter, enhancer, and insulator elements and on loop formation, they are also compatible with pairing exerting its effects on other genetic elements or via other topologies, such as the structural alterations of chromatin and DNA that accompany the transcriptionally active state (65).…”

The DNA-Binding and Enhancer-Blocking Domains of the Drosophila suppressor of Hairy-wing Protein

“…All mutant SUHW proteins were expressed in yeast cells by galactose induction, and extracts were prepared as previously described (37,58). The amounts of the mutant proteins in the yeast extracts were determined by Western blots (immunoblots) with anti-GST antiserum (provided by M. Nussenzweig and Z. Misulovin, Rockefeller University).…”

“…Gel mobility shift assays were performed with crude yeast extracts (37,58), Schneider 2 cell nuclear extracts, and pupal nuclear extracts (11) with 32 P-labeled BaBx fragment of bx 34e gypsy containing eight consensus SUHW-binding repeats ( Fig. 1), as described previously under conditions in which the total amount of SUHW-BaBx complex formed is linearly dependent on the amount of SUHWbinding activity (11,37,58). Because the BaBx fragment contains multiple binding sites, multiple complexes can be formed.…”

“…There is significant evidence that DNA bends or loops out to allow contacts between regulatory proteins (7,14,22,49), and therefore it is conceivable that a protein that alters DNA bending could block enhancer-promoter interactions in a position-dependent manner. SUHW distorts DNA upon binding, and although the zinc fingers alone are sufficient to bind DNA, the distortion requires most of the flanking C-terminal domain (58). It is difficult to imagine, however, how a localized DNA distortion alone can block an interaction between an enhancer and promoter separated by approximately 90 kbp (12,32).…”

“…1) and XhoI and SalI overhangs were subcloned into a modified pBend2 plasmid (58). Plasmid fragments (166 bp) were used for the binding experiments.…”