Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three essential cis-acting sequences.

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.

Regulation of the sea urchin early H2A histone gene expression depends on the modulator element and on sequences located near the 3' end.

Transcription of the sea urchin early histone genes occurs transiently during early cleavage, reaching the maximum at the morula stage and declining to an undetectable level at the gastrula stage. To identify the regulatory elements responsible for the timing and the levels of transcription of the H2A gene, we used promoter binding studies in nuclear extracts and microinjection of a CAT transgene driven by the early H2A promoter. We found that morula and gastrula nuclear proteins produced indistinguishable DNase I footprint patterns on the H2A promoter. Two sites of interactions, centred on the modulator/enhancer and on the CCAAT box respectively, were detected. Deletion of the modulator or coinjection of an excess of modulator sequences severely affected the expression of two transgenes driven by the enhancer-less and modulator-containing H2A promoter. Finally, a DNA fragment containing 3' coding and post-H2A spacer sequences, where upon silencing three micrococcal nuclease hypersensitive sites were previously mapped, specifically repressed at the gastrula stage the expression of the transgene driven by the H2A promoter. These results indicate that the modulator is essential for the expression of early H2A gene and that sequences for downregulation are localized near the 3' end of the H2A gene.

Enhancer blocking activity located near the 3' end of the sea urchin early H2A histone gene.

The sea urchin early histone repeating unit contains one copy of each of the five histone genes whose coordinate expression during development is regulated by gene-specific elements. To learn how within the histone repeating unit a gene-specific activator can be prevented to communicate with the heterologous promoters, we searched for domain boundaries by using the enhancer blocking assay. We focused on the region near the 3' end of the H2A gene where stage-specific nuclease cleavage sites appear upon silencing of the early histone genes. We demonstrated that a DNA fragment of 265 bp in length, defined as sns (for silencing nucleoprotein structure), blocked the enhancer activity of the H2A modulator in microinjected sea urchin embryos only when placed between the enhancer elements and the promoter. We also found that sns silenced the modulator elements even when placed at 2.7 kb from the promoter. By contrast, the enhancer activity of the modulator sequences, located downstream to the coding region, was not affected when sns was positioned in close proximity to the promoter. Finally, the H2A sns fragment placed between the simian virus 40 regulative region and the tk promoter repressed chloramphenicol acetyltransferase expression in transfected human cell lines. We conclude that 3' end of the H2A gene contains sequence elements that behave as functional barriers of enhancer function in the enhancer blocking assay. Furthermore, our results also indicate that the enhancer blocking function of sns lacks enhancer and species specificity and that it can act in transient assays.

Modulator factor-binding sequence of the sea urchin early histone H2A promoter acts as an enhancer element.

The sea urchin early H2A histone gene, like the other four members of the repeating units, is transiently expressed during very early development. To investigate the mechanisms underlying the faithful expression of the early H2A gene, we focused our attention on the modulator element. We showed by DNase I cleavage protection patterns that the modulator includes the upstream sequence element 1 (USE1) and mapped at nucleotides -137 to -108 in the early H2A gene promoter. Functional tests conducted by microinjection into sea urchin embryos then showed that the modulator element binds the transcriptional factor called modulator-binding factor 1 (MBF-1). We found in fact that coinjection of an excess of the MBF-1-binding site, either as the modulator or as the USE1, efficiently impaired the activity of the H2A promoter. An unexpected finding was the expression of the reporter gene from the early H2A promoter at the gastrula stage of embryonic development, when the early histone genes are transcriptionally silent. In addition, we also found that the modulator element was active at the gastrula stage. The potential enhancer activity of the modulator was tested by microinjecting several constructs containing single or multiple copies of the modulator element placed 5' or 3' to a thymidine kinase gene (tk) promoter in both sea urchin embryos and Xenopus laevis oocytes and determining the expression of a reporter chloramphenicol acetyltransferase gene under the control of the linked tk promoter. We found that an oligonucleotide bearing the MBF-1-binding site activates the expression of the reporter gene independently of the position and orientation. We conclude that the modulator binds the MBF-1 activator and that it is a transcriptional enhancer of the early H2A histone gene.

Sea urchin early histone H2A modulator binding factor 1 is a positive transcription factor also for the early histone H3 gene.

To shed some light on the mechanisms involved in the coordinate regulation of the early histone gene set during sea urchin development, we tested the hypothesis that the upstream sequence element USE1, previously identified in the early H2A modulator, could also participate in the transcription of the early histone H3 gene. We found by DNAse I protection analysis and by competition in electrophoretic mobility-shift experiments that two sequence elements of the H3 promoter closely resembled the USE1-H2A sequence in their binding activity for nuclear factors from 64-cell stage embryos. These modulator binding factor 1 (MBF-1)-related factors seem to recognize the ACAGA motif that is conserved between the USE1-like sequences of both H2A and H3 promoters. In fact, excess oligonucleotide containing a mutated USE1-H2A element in which the ACAGA sequence was mutated to AGTCA failed to compete with the USE1 sites of both H2A and H3 genes for interaction with MBF-1. Finally, in vivo transcriptional analysis in both Xenopus and sea urchin showed that an excess of USE1-H2A element efficiently competed for the activity of the H3 promoter. From these results we conclude that MBF-1 is a transcription factor conserved between sea urchin and frog and that MBF-1 or related transcription factors are involved in the coordinate expression of both H2A and H3 early histone genes.

Cis-acting elements of the sea urchin histone H2A modulator bind transcriptional factors.

Functional tests, performed by microinjection into Xenopus laevis oocytes, show that a DNA fragment containing the modulator of the early histone H2A gene of Paracentrotus lividus enhances transcription of a reporter gene when located, in the physiological orientation, upstream of the tk basal promoter. Gel retardation and DNase I footprinting assays further reveal that the H2A modulator contains at least two binding sites [upstream sequence elements 1 and 2 (USE 1 and USE 2)] for nuclear factors extracted from sea urchin embryos, which actively transcribe the early histone gene set. Interestingly, USE 1 is highly homologous to a cis-acting element previously identified in the H2A modulator of Psammechinus miliaris [Grosschedl, R., Mächler, M., Rohrer, U. & Birnstiel, M. L. (1983) Nucleic Acids Res. 11, 8123-8136]. Finally, a cloned oligonucleotide containing the USE 1 sequence competes efficiently in Xenopus oocytes with the H2A modulator to prevent enhancement of transcription of the reporter gene. From these results, we conclude that USE 1 and perhaps USE 2 in the H2A modulator are upstream transcriptional elements that are recognized by trans-acting factors common to Xenopus and sea urchin.

Different micrococcal nuclease cleavage patterns characterize transcriptionally active and inactive sea-urchin histone genes.

The micrococcal nuclease cleavage sites have been mapped in the H2A coding and flanking regions of the sea-urchin histone DNA chromatin. A hypersensitive area, centered around - 100 base pairs from the H2A starting site, is found only in embryos actively transcribing the alpha-subtype histone genes. In mesenchyme blastula embryos, upon inactivation of the H2A gene, this region becomes protected while two other areas, near the transcription starting site and in the proximity of the 3' palindromic sequence, become preferential targets for the enzyme. Analysis of the pattern of micrococcal nuclease cleavage on the same region of the histone gene cluster in sperm and late blastula chromatin and on the corresponding segment of protein-free DNA indicates that distinct nucleosomal arrangements characterize the histone genes in the two cell populations.

Chromatin organization in Paracentrotus lividus eggs.

After purification by buoyant density centrifugation in ethidium bromide - CsCl gradient and electrophoretic fractionation, the DNA fragments isolated from P. lividus egg nuclei incubated with micrococcal nuclease exhibit a typical oligomeric pattern. Analysis of chromatin samples digested to an increasing extent by micrococcal nuclease reveals that the structural organization of egg chromatin is heterogeneous, both in terms of repeat size and degree of sensitivity to nuclease attack. The nucleosomal repeats of P. lividus sperms and embryos up to the mesenchyme blastula stage have also been determined, for comparison.

Chromatin structure of histone genes in sea urchin sperms and embryos.

The nucleosomal organization of active and repressed alpha subtype histone genes has been investigated by micrococcal nuclease digestion of P. lividus sperm, 32-64 cell embryo and mesenchyme blastula nuclei, followed by hybridization with 32P-labeled specific DNA probes. In sperms, fully repressed histone genes are regularly folded in nucleosomes, and exhibit a greater resistance to micrococcal nuclease cleavage than bulk chromatin. In contrast, both coding and spacer alpha subtype histone DNA sequences acquire an altered conformation in nuclei from early cleavage stage embryos, i.e., when these genes are maximally expressed. Switching off of the alpha subtype histone genes, in mesenchyme blastulae, restores the typical nucleosomal organization on this chromatin region. As probed by hybridization to D.melanogaster actin cDNA, actin genes retain a regular nucleosomal structure in all the investigated stages.