title: Down-regulation of early sea urchin histone H2A gene relies on cis regulative sequences located in the 5' and 3' regions and including the enhancer blocker sns.
abstract: The tandem repeated sea urchin alpha-histone genes are developmentally regulated by gene-specific promoter elements. Coordinate transcription of the five genes begins after meiotic maturation of the oocyte, continues through cleavage, and reaches its maximum at morula stage, after which these genes are shut off and maintained in a silenced state for the life cycle of the animal. Although cis regulative sequences affecting the timing and the level of expression of these genes have been characterized, much less is known about the mechanism of their repression. Here we report the results of a functional analysis that allowed the identification of the sequence elements needed for the silencing of the alpha-H2A gene at gastrula stage. We found that important negative regulative sequences are located in the 462 bp sns 5 fragment located in the 3' region. Remarkably, sns 5 contains the sns enhancer blocking element and the most 3' H2A codons. In addition, we made the striking observation that inhibition of the anti-enhancer activity of sns, by titration of the binding proteins in microinjected embryos, also affected the capability of sns 5 to down-regulate transgene expression at gastrula stage. A further sequence element essential for repression of the H2A gene was identified upstream of the enhancer, in the 5' region, and contains four GAGA repeats. Altogether these findings suggest that down-regulation of the alpha-H2A gene occurs by the functional interaction of the 5' and 3' cis sequence elements. These results demonstrate the involvement of a genomic insulator in the silencing of gene expression.
authors.affiliation: Dipartimento di Biologia Cellulare e dello Sviluppo (Alberto Monroy), Università di Palermo, Parco d'Orleans II, 90128 Palermo, Italy.
authors.forename: Domenica
authors.initials: D
authors.lastname: Di Caro
authors.forename: Raffaella
authors.initials: R
authors.lastname: Melfi
authors.forename: Claudia
authors.initials: C
authors.lastname: Alessandro
authors.forename: Gaetana
authors.initials: G
authors.lastname: Serio
authors.forename: Valentina
authors.initials: V
authors.lastname: Di Caro
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cavalieri
authors.forename: Franco
authors.initials: F
authors.lastname: Palla
authors.forename: Giovanni
authors.initials: G
authors.lastname: Spinelli
mesh_terms: D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D015536:Down-Regulation; D004625:Embryo, Nonmammalian / Q000502:physiology; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005775:Gastrula; D020868:Gene Silencing; D006657:Histones / Q000235:genetics*; D008845:Microinjections; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012617:Sea Urchins / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D019076:Transgenes
chemical_list: D006657:Histones; D015500:Chloramphenicol O-Acetyltransferase