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The modulator of the sea urchin alpha-H2A histone gene promoter is the only enhancer identified in the alpha-histone gene cluster. Binding of a single factor, denoted MBF-1, has previously detected in nuclear extracts from morula and gastrula embryos. Here, we describe the cloning of MBF-1 by screening a cDNA expression library with a tandem array of modulator binding sites. MBF-1 presents no similarity with other DNA binding proteins and contains nine Krüppel like Zn fingers. In vitro translated proteins and a factor from nuclear extracts interact with the modulator with identical specificity. In addition, MBF-1 expressed in human cells transactivates a reporter gene driven by an array of modulator sites. The DNA binding domain consists of the Zn fingers plus an adjacent basic region, while sequences in the N-terminal region mediates the transactivation function. MBF-1 is expressed in the unfertilized egg and in early and late developmental stages thus confirming that it is not a stage specific enhancer binding factor and that silencing of the alpha-H2A gene after hatching is not due to the lack of the transactivator.

Insulators are a new class of genetic elements that attenuate enhancer function directionally. Previously, we characterized in sea urchin a 265-bp-long insulator, termed sns. To test insulator activity following stable integration in human cells, we placed sns between the CMV enhancer and a tk promoter up-stream of a GFP transgene of plasmid or retroviral vectors. In contrast to controls, cells transfected or transduced with insulated constructs displayed a barely detectable fluorescence. Southern blot and PCR ruled out vector rearrangement following integration into host DNA; RNase protection confirmed the enhancer blocking activity. Finally, we demonstrate that two cis-acting sequences, previously characterized in sea urchin, are also specific binding sites for human proteins. We conclude that sns interferes with enhancer promoter interaction also in a human chromatin context. The relatively small size, evolutionary conservation and apparent lack of enhancer specificity might result useful in gene transfer experiments in human cells. © 2001 Academic Press.

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.