The 5′ flanking region of the human alpha 1‐antitrypsin (alpha 1‐AT) gene contains cis‐acting signals for liver‐specific expression and, when fused to a reporter gene, is able to drive the expression of this gene specifically in liver cells. Here we report the results of a functional dissection of the alpha 1‐AT regulatory region. The expression of the bacterial chloramphenicol‐transacetylase (CAT) gene, fused to a set of alpha 1‐AT 5′ flanking regions shortened by progressive deletions or mutated by base pair substitutions, has been compared by transfection in HepG2 (hepatocyte) and HeLa (non‐hepatocyte) human cell lines. A minimal tissue‐specific element has been identified between the nucleotides −137 and −37 (from the transcriptional start site). This DNA segment activates the heterologous SV40 promoter in hepatoma cell lines but not in HeLa cells. This element contains at least two regions referred to as the A (‐125/‐100) and B (‐84/‐70) domains, both essential for transcription. There are at least two other regulatory domains located upstream of the ‘minimal element’; the most active of these is located between positions −261 and −210 from the cap site. These upstream elements activate the heterologous SV40 early promoter both in hepatoma cell lines and in HeLa cells. Upon fractionation of rat liver nuclear extracts two proteins have been identified, alpha 1TF‐A and alpha 1TF‐B, which bind specifically to the A and B domains respectively. Transcriptionally inactive A and B domain mutants are not able to bind these proteins.
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.
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 USEl-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 condude 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.The early (or a) and late histone gene families of sea urchin have been widely used as model systems to study the regulation of temporally and tissue-specific gene expression. Different approaches have been applied to identify cis-acting regulative sequences and their binding factors. Microinjection into Xenopus oocytes was first employed to dissect the regulatory region of the Psammechinis miliaris early histone H2A gene. By this analysis the positive role of the "modulator" in the expression of the H2A gene was established (1,2). This region, which seems to have a bipartite structure (3), in Paracentrotus lividus has been shown to bind at least two nuclear factors (4). More recently, microinjection of promoter constructs into sea urchin eggs or zygotes, and promoter binding studies with nuclear extracts, allowed the identification of specific transcriptional elements in several early, late, and tissue-specific histone genes. Thus, in Strongylocentrotus purpuratus five distinct binding sites are involved in the regulation of the early (or a) histone H3 gene (5), and an enhancer element determines the temporal activation of late 13-Hi (6, 7) and Li late H2B (8, 9) genes. Other studies, conducted in Ps. miliaris, confirmed that transcription of the early H2A gene depends on its 5' sequence (10). Furthermore, a repressor, the CCAAT displacement factor, of the sperm-specific H2B-1 gene and a tissue-specific activator oflate H2A-2 and H2B-2 genes have been identified (11, 12). As a complementary approach, Weinberg and colleaguesThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "...
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.
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 milians [Grosschedl, R., Machler, 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.The sea urchin genome contains several histone gene families that encode the protein subtypes of sperm (S-type) and of cleavage, early, and late stage embryo (1). The synthesis of specific histone protein variants during development is the result of regulatory mechanisms that operate at both the transcriptional and posttranscriptional level (2). The early histone genes in all sea urchin species are tandemly repeated 300-600 times and are organized in quintets (3,4). Transcription of this gene set occurs upon meiotic maturation and soon after fertilization (5-7). Newly synthesized early histone mRNAs accumulate at the 32-to 128-cell stage. Their transcription is shut off in mesenchyme blastula embryos (8-10). The transition from a transcriptionally active to an inactive state of the early histone genes is accompanied by structural alterations of their chromatin arrangement (11-13).Molecular genetic analysis of many polymerase II promoters has revealed that optimal and accurate initiation of transcription requires a cooperative interaction of transacting factors with multiple cis-acting transcriptional elements (14)(15)(16)(17).Recent studies on the factors involved in the transcriptional control of the sea urchin histone H1 and H2B genes have identified basal and ontogenic transcriptional elements involved in the transition from early to late gene expression during development (18,19). Furthermore, they have shown that an embryo-specific repressor element, the CCAAT displacement factor, prevents the interaction of a positive trans-acting factor with the CCAAT box ofthe sperm-specific H2B-1 gene (20) and in so doing may block transcription in the embryo.We have focused our attention on the early H2A gene ofthe sea urchin Paracentrotus lividus for several reasons. The 5' flanking region of this gene contains, upstream...
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