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Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three essential cis-acting sequences
- Publication Year :
- 2000
- Publisher :
- ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2000.
-
Abstract
- Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or their ability to protect adjacent transgenes from position effects. We have previously reported the identification of a 265 bp sns DNA fragment at the 3' end of the sea urchin H2A early histone gene that blocked expression of a reporter gene in transgenic embryos when placed between the enhancer and the promoter. Here we show that sns interferes with enhancer-promoter interaction in a directional manner. When sns is placed between the H2A modulator and the inducible tet operator, the modulator is barred from interaction with the basal promoter. However, the tet activator (tTA) can still activate the promoter, even in the presence of sns, demonstrating that sns does not interfere with activity of a downstream enhancer. In addition, the H2A modulator can still drive expression of a divergently oriented transcription unit, suggesting that sns does not inhibit binding of transcription factor(s) to the enhancer. To identify cis-acting sequence elements within sns which are responsible for insulator activity, we have performed in vitro DNase I footprinting and EMSA analysis, and in vivo functional assays by microinjection into sea urchin embryos. We have identified three binding sites for protein complexes: a palindrome, a direct repeat, and a C+T sequence that corresponds to seven GAGA motifs on the transcribed strand. Insulator function requires all three cis-acting elements. Based on these results, we conclude that sns displays properties similar to the best characterized insulators and suggest that directional blocking of enhancer-activated transcription by sns depends on the assembly of distinct DNA-protein complexes. (C) 2000 Academic Press.
- Subjects :
- animal structures
enhancer blocking
Molecular Sequence Data
DNA Footprinting
Settore BIO/11 - Biologia Molecolare
Biology
Regulatory Sequences, Nucleic Acid
insulator
Binding, Competitive
Histones
Structural Biology
Transcription (biology)
Gene cluster
microinjection
Animals
Deoxyribonuclease I
H2A enhancer
Gene Silencing
Transgenes
Enhancer
Downstream Enhancer
Promoter Regions, Genetic
Molecular Biology
Transcription factor
Repetitive Sequences, Nucleic Acid
Sequence Deletion
Reporter gene
Base Sequence
Activator (genetics)
Promoter
DNA
histone genes
Molecular biology
Cell biology
DNA-Binding Proteins
Enhancer Elements, Genetic
Multigene Family
Sea Urchins
Protein Binding
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....fd354458f1617124a67d8ccab4d98f24