A complex array of DNA-binding proteins required for pairing-sensitive silencing by a polycomb group response element from the Drosophila engrailed gene.
Regulatory DNA from the Drosophila gene engrailed causes silencing of a linked reporter gene (mini-white) in transgenic Drosophila. This silencing is strengthened in flies homozygous for the transgene and has been called "pairing-sensitive silencing." The pairing-sensitive silencing activities of a large fragment (2.6 kb) and a small subfragment (181 bp) were explored. Since pairing-sensitive silencing is often associated with Polycomb group response elements (PREs), we tested the activities of each of these engrailed fragments in a construct designed to detect PRE activity in embryos. Both fragments were found to behave as PREs in a bxd-Ubx-lacZ reporter construct, while the larger fragment showed additional silencing capabilities. Using the mini-white reporter gene, a 139-bp minimal pairing-sensitive element (PSE) was defined. DNA mobility-shift assays using Drosophila nuclear extracts suggested that there are eight protein-binding sites within this 139-bp element. Mutational analysis showed that at least five of these sites are important for pairing-sensitive silencing. One of the required sites is for the Polycomb group protein Pleiohomeotic and another is GAGAG, a sequence bound by the proteins GAGA factor and Pipsqueak. The identity of the other proteins is unknown. These data suggest a surprising degree of complexity in the DNA-binding proteins required for PSE function.
Americo
Jeffrey
J
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Whiteley
Mary
M
Brown
J Lesley
JL
Fujioka
Miki
M
Jaynes
James B
JB
Kassis
Judith A
JA
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila / Q000196:embryology / Q000235:genetics; D029721:Drosophila Proteins; D005786:Gene Expression Regulation; D020868:Gene Silencing / Q000502:physiology*; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010641:Phenotype; D020218:Response Elements; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D014157:Transcription Factors