The activity of the scs and scs' insulator elements is not dependent on chromosomal context.
Sequence elements that protect a reporter gene from chromosomal position effects or that block enhancer-activated transcription are called insulators. Using a plasmid-based microinjection assay with Xenopus laevis oocytes, we show that the heterologous Drosophila melanogaster scs and scs' insulator elements do not require chromosomal context to block enhancer-activated transcription. A single insulator element partially blocks enhancer-activated transcription, indicating that each element operates independently rather than as part of a pair. Deletion analysis of the 1.8-kb scs element identified a 220-bp fragment from one of the DNase I-hypersensitive regions that has full blocking activity in the oocyte assay. This fragment corresponds to the critical region of the scs mapped in previous studies with Drosophila. A time course of transcription shows that the scs blocks enhancer-activated transcription as early as transcription can be detected, about 30 min after injection. Complete assembly of the DNA template into nucleosomes requires 4 h. The scs and scs' sequences do not block site-specific recombination by FLP recombinase, implying that insulators do not operate by a general mechanism that physically sequesters the DNA. These data are most consistent with a model for insulator action in which direct interaction between the insulator and either the enhancer or promoter confers directionality to enhancer-activated transcription.
Dunaway
M
M
Department of Molecular and Cell Biology, University of California-Berkeley 94720-3202, USA. marietta@mendel.berkeley.edu
Hwang
J Y
JY
Xiong
M
M
Yuen
H L
HL
D000818:Animals; D002875:Chromosomes / Q000235:genetics*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D003850:Deoxyribonuclease I; D004331:Drosophila melanogaster / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D009707:Nucleosomes / Q000378:metabolism; D009865:Oocytes; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D015183:Restriction Mapping; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics*; D014982:Xenopus laevis
D009707:Nucleosomes; D012335:RNA, Ribosomal; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D003850:Deoxyribonuclease I