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

Abstract

Mutations in the suppressor of Hairy-wing [su(Hw)] gene of Drosophila melanogaster can cause female sterility and suppress mutations that are insertions of the gypsy retrotransposon. Gypsy binds the protein (SUHW) encoded by su(Hw), and SUHW prevents enhancers promoter-distal to gypsy from activating gene transcription. SUHW contains 12 zinc fingers flanked by acidic N-and C-terminal domains. We examined the roles of each of the 12 zinc fingers in binding gypsy DNA and classified them into four groups: essential (fingers 6 through 10); beneficial but nonessential (fingers 1, 2, 3, and 11); unimportant (fingers 5 and 12); and inhibitory (finger 4). Because finger 10 is not required for female fertility but is essential for binding gypsy, these results imply that the SUHW-binding sites required for oogenesis differ in sequence from the gypsy-binding sites. We also examined the functions of the N-and C-terminal domains of SUHW by determining the ability of various deletion mutants to support female fertility and to alter expression of gypsy insertion alleles of the yellow, cut, forked, and Ultrabithorax genes. No individual segment of the N-and C-terminal domains of SUHW is absolutely required to alter expression of gypsy insertion alleles. However, the most important domain lies between residues 737 and 880 in the C-terminal domain. This region also contains the residues required for female fertility, and the fertility domain may be congruent with the enhancer-blocking domain. These results imply that SUHW blocks different enhancers and supports oogenesis by the same or closely related molecular mechanisms. Several spontaneous mutations in Drosophila melanogasterare insertions of the gypsy retrotransposon into or next to a gene (42). Expression of the phenotypes of gypsy insertion alleles can be suppressed to wild-type or near wild-type levels by suppressor of Hairy-wing [su(Hw)] mutations (42, 54). Other than suppression of gypsy insertions, female sterility is the only known phenotype associated with su(Hw) mutations (38).su(Hw) encodes a protein (SUHW) with 12 zinc finger motifs (44) that binds to a repeated consensus sequence downstream of the 5Ј long terminal repeat (LTR) of gypsy (11,61). Gypsy and the SUHW protein bound to gypsy can alter gene expression by multiple mechanisms. When gypsy is in a transcribed region of a gene and oriented in the same (parallel) direction, primary gene transcripts can be truncated and processed at the polyadenylation sites in the gypsy LTRs (13). Binding of SUHW to gypsy increases polyadenylation of gene transcripts in the 5Ј LTR (11, 13). Truncation of gene transcripts in gypsy occurs in parallel gypsy insertions in the achaete-scute complex (3) and the forked gene (29). The gypsy LTR poly(A) sites may also explain why parallel gypsy insertions in an intron of the Ultrabithorax gene display stronger phenotypes than antiparallel insertions (47).SUHW also alters gene expression by preventing enhancers and silencers from activating or repressing transcription (1,2,12,19,20,28,3...

An endogenous Su(Hw) insulator separates theyellowgene from theAchaete-scutegene complex inDrosophila

“…2B) probably corresponds to a complex of the 125 bp DNA fragment with one Su(Hw) protein. The inability of the Su(Hw) protein to simultaneously bind two closely spaced sites was previously described by Kim et al (Kim et al, 1996) who suggested that the Su(Hw) protein interferes with binding to neighboring sites. To show that both putative binding sites in the 125 bp fragment can interact with Su(Hw), we subcloned smaller fragments that contain only the first or the second Su(Hw) binding site ( Fig.…”

“…One is Suppressor of Hairy wing [Su(Hw)], a twelve zinc finger protein encoded by the suppressor of Hairy wing [su(Hw)] gene, which binds to the repeated sequence motifs in the gypsy insulator (Dorsett, 1990;Spana and Corces, 1990). Of the protein domains comprising Su(Hw), 9 out of 12 zinc fingers and a domain of approximately 150 amino acids including the C-terminal leucine zipper, but not the N-and C-terminal acidic regions, are required for enhancer blocking (Harrison et al, 1993;Kim et al, 1996).…”

Chip interacts with diverse homeodomain proteins and potentiates Bicoid activity in vivo

“…Su(Hw):706-944 was made by cloning the PvuII to HindIII 713-bp fragment of the su(Hw) cDNA into the EcoRV and HindIII sites of pING14.1. Mutant su(Hw) zinc finger domains described previously (21) were amplified by PCR with a 5Ј-primer with an NcoI site and an initiation codon, and a 3Ј-primer with an EcoRV site, and the resulting DNA fragments were cloned upstream of the HindIII (blunted) to SspI su(Hw) cDNA fragment with the termination codon into the NcoI and BamHI (blunted) sites of a pGEM3Z (Promega) vector modified to lack the polylinker HindIII site.…”

Insulators: many functions, many mechanisms

“…Of the protein domains comprising su(Hw), 9 out of 12 Zinc fingers and the C-terminal leucine zipper, but not the N-and Cterminal acidic regions, are required for enhancer blocking (Harrison et al 1993;Kim et al 1996). Molecular analysis of gypsy-induced mutations suggested that the retrotransposon had position-dependent enhancer-blocking activity.…”