Identification of the enhancer binding protein MBF1 of the sea urchin modulator α-H2A histone gene (original) (raw)
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Proceedings of the National Academy of Sciences, 1994
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 ofthe 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 micronijection into sea urchin embryos then showed that the modulator element binds the transcriptional factor called modulator-binding fac-
Proceedings of the National Academy of Sciences, 1993
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.
Biological Chemistry, 1999
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 hyperse...
Journal of Molecular Biology, 2007
The tandemly repeated sea urchin α-histone genes are developmentally regulated. These genes are transcribed up to the early blastula stage and permanently silenced as the embryos approach gastrulation. As previously described, expression of the α-H2A gene depends on the binding of the MBF-1 activator to the 5′ enhancer, while down-regulation relies on the functional interaction between the 3′ sns 5 insulator and the GA repeats located upstream of the enhancer. As persistent MBF-1 binding and enhancer activity are detected in gastrula embryos, we have studied the molecular mechanisms that prevent the bound MBF-1 from trans-activating the H2A promoter at this stage of development. Here we used chromatin immunoprecipitation to demonstrate that MBF-1 occupies its site regardless of the transcriptional state of the H2A gene. In addition, we have mapped two nucleosomes specifically positioned on the enhancer and promoter regions of the repressed H2A gene. Interestingly, insertion of a 26 bp oligonucleotide between the enhancer and the TATA box, led to upregulation of the H2A gene at gastrula stage, possibly by changing the position of the TATA nucleosome. Finally, we found association of histone de-acetylase and de-acetylation and methylation of K9 of histone H3 on the promoter and insulator of the repressed H2A chromatin. These data argue for a role of a defined positioned nucleosome in the promoter and histone tail post-translational modifications, in the 3′ insulator and 5′ regulatory regions, in the repression of the α-H2A gene despite the presence of the MBF-1 activator bound to the enhancer.
Molecular and cellular biology, 1990
In the sea urchin embryo, late histone genes are transcribed at low levels during cleavage and blastula formation and at substantially higher levels in later stages of embryogenesis. To investigate the molecular basis of the stage-specific expression of a late H2B histone gene, we injected mutant genes lacking portions of 5'-and 3'-flanking regions into Lytechinus pictus embryos and monitored their expression by RNase protection. A 200-bp region located 489 bp downstream of the mRNA 3'terminus was necessary for the ...
Cis-acting elements of the sea urchin histone H2A modulator bind transcriptional factors
Proceedings of the National Academy of Sciences, 1989
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.
Transcriptional elements of early subtype sea urchin histone genes
Cell Biology International Reports, 1990
Transcriptional regulators are thought to play a key role in cell fate determination and territorial specification in sea urchin. Our goals are to clone transcription factors for studying embryonic development. One approach has been to use promoter binding and gene transfer technology to investigate the mechanisms of transcriptional activation and repression of the early H2A histone gene. By this analysis we identified a transcriptional activator, the MBF-I, that binds to the modulator element of the H2A gene and enhances the activity of the H2A promoter. However, the enhancer activity of the modulator and its interaction with MBF-I also occurs at the gastrula stage when the early histone genes are shut off. Therefore, the silencing of the early H2A histone gene at late stages of development requires the inactivation of the modulator function. To search for antimodulator sequence elements, we took advantage of our previous work showing the presence of phased nucleosomes specifically positioned on the 3'-spacer and in the modulator of the repressed H2A gene. Evidence is described indicating that a 3'-spacer DNA fragment cloned between the modulator and the basal promoter behaves as an antimodulator element. However, this element does not confer temporal capability to the modulator in its function, suggesting that other elements have to be involved in the regulation of the early H2A expression. The second approach relied on the cloning of genes controlling development using probes of regulators known to be involved in regional specification, such as the homeobox genes, with the aim to understand their possible role through the study of their temporal and territorial expression and through the analysis of the mechanism of regulation of their expression. We present evidence that PlHboxl2, a homeodomain encoding gene, is transiently expressed during the earlylmid cleavage stages. The abundance of the transcripts reach their maximum in embryos at the 641128-cell stage concomitantly with the segregation of the specified embryonic territories. Expression of PIHboxl2 is drastically reduced in the absence of cell contacts and/or Ca ions, suggesting that this gene is transcriptionally activated by signal transduction mechanisms. Whole mount in situ hybridization showed that PlHbox 12 transcripts are asymmetrically distributed along the A-V axis, being spatially localized in the blastomeres of the ectodermal lineage.We suggest that PlHboxl2 might be involved in the initial events that lead to the specification of the ectodermal territories.
Nucleic Acids Research, 2009
Core promoters and chromatin insulators are key regulatory elements that may direct a transcriptional enhancer to prefer a specific promoter in complex genetic loci. Enhancer and insulator flank the sea urchin (Paracentrotus lividus) a-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. This article deals with the specificity of interaction between the H2A enhancer-bound MBF-1 activator and histone gene promoters, and with the mechanism that leads the H1 transcripts to peak at about one-third of the value for nucleosomal H3 and H2A mRNAs. To this end, in vivo competition assays of enhancer and insulator functions were performed. Our evidence suggests that the MBF-1 transcription factor participates also in the expression of the H3 gene and that the sns5 insulator buffers the downstream H1 promoter from the H2A enhancer. Altogether, these results provide a clear demonstration of the enhancer-blocking function of a chromatin insulator in a natural gene context. In addition, they suggest that both the H2A enhancer and the sns5 insulator may account for the diverse accumulation of the linker H1 versus the core nucleosomal histones during early development of the sea urchin embryo.
Enhancer blocking activity located near the 3' end of the sea urchin early H2A histone gene
Proceedings of the National Academy of Sciences, 1997
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.
Development, Growth & Differentiation, 2016
Histone variant H2A.Z promotes chromatin accessibility at transcriptional regulatory elements and is developmentally regulated in metazoans. We characterize the transcriptional and post-transcriptional regulation of H2A.Z in the purple sea urchin Strongylocentrotus purpuratus. H2A.Z depletion by antisense translation-blocking morpholino oligonucleotides during early development causes developmental collapse, in agreement with its previously demonstrated general role in transcriptional multipotency. During H2A.Z peak expression in 24-h embryos, endogenous H2A.Z 3 0 UTR sequences stabilize GFP mRNAs relative to those with SV40 3 0 UTR sequences, although the 3 0 UTR of H2A.Z does not determine the spatial distribution of H2A.Z transcripts during embryonic and postembryonic development. We elaborated an H2A.Z::GFP BAC reporter that reproduces embryonic H2A.Z expression. Genome-wide chromatin accessibility analysis using ATAC-seq revealed a cisregulatory module (CRM) that, when deleted, causes a significant decline of the H2A.Z reporter expression. In addition, the mutation of a Sox transcription factor binding site motif and, more strongly, of a Myb motif cause significant decline of reporter gene expression. Our results suggest that an undetermined Myb-family transcription factor controls the transcriptional regulation of H2A.Z.