Differential stimulation of sea urchin early and late H2B histone gene expression by a gastrula nuclear extract after injection into Xenopus laevis oocytes.

Abstract

Sea urchin early histone genes are active in preblastula embryos; late histone genes are maximafly expressed during subsequent stages of embryogenesis. We used the Xenopus laevis oocyte to assay for trans-acting factors involved in this differential regulation. Sea urchin nuclear proteins were prepared by extracting gastrula-stage chromatin successively with 0.45, 1, and 2 M NaCl. We injected three fractions into oocytes along with plasmids bearing sea urchin early and late H2b histone genes. While neither the 0 to 0.45 M nor the 1 to 2 M salt fraction affected H2b gene expression, the 0.45 to 1 M salt fraction stimulated early and late H2b mRNA levels significantly. Late H2b gene expression was stimulated preferentially when the early and late genes were coinjected into the same oocytes. This extract did not stimulate the accumulation of transcripts of injected herpesvirus thymidine kinase genes or of the sea urchin Spec 1 gene, suggesting that the stimulatory activity is not a general transcription factor. We localized the DNA sequence required for the stimulatory effect to a region of the late H2b gene located between -43 and +62 relative to the transcription start site. A component of the 0.45 to 1 M salt wash fraction specifically bound to the 105-base-pair late gene DNA sequence and to the corresponding early gene fragment. The abundance of this binding activity decreased on a per genome basis during early development of the sea urchin.The sea urchin synthesizes several distinct sets of histone proteins during early embryogenesis (7, 32). These include early isotypes, synthesized from fertilization to the blastula stage, and late isotypes, synthesized from the blastula stage onward. Each set of proteins is encoded by a distinct class of genes. Early histone genes are repeated several hundredfold in the genome and are organized in tandem quintets, each comprising genes for the five major histone types. In contrast, late histone genes are present in only 6 to 12 copies per genome and are arranged in irregular clusters (6,21,22,27). The developmental switches in the synthesis of early and late histone isotypes are caused largely by changes in rates of transcription of the two gene sets (23,28,35).The Xenopus laevis oocyte has proven highly useful in the analysis of transcriptional processes (9, 10). A variety of genes, including histone genes, are accurately transcribed after microinjection into oocyte nuclei, and the injection of genes with mutated promoter regions has enabled the identification of elements required for transcription in the oocyte (16). The Xenopus oocyte system has also been used to identify a trans-acting factor involved in the processing of sea urchin histone mRNAs (34) and, more recently, to identify a factor capable of stimulating sea urchin early H2b gene transcription (30).We used the Xenopus oocyte to examine the mechanisms underlying the differential expression of sea urchin early and late histone genes. We found that an extract of sea urchin gastrula-stage chromatin stimulated the ...

A Repression-derepression Mechanism Regulating the Transcription of Human Immunodeficiency Virus Type 1 In Primary T Cells

“…Micro-injection of gene constructs with intact or mutant promoters or hybrid promoters containing individual control elements has been widely used in determining cis-active elements of a variety of genes, regardless of their species of origin (30,31). Coinjection of putative regulatory proteins, or cDNA coding for such factors, also allows functional testing of transactive components of gene regulation (31)(32)(33)(34)(35)(36).…”

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

“…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.…”

“…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.…”

Transcriptional characteristics of in vitro assembled chromatin assayed by microinjection into Xenopus laevis oocytes

“…It has been demonstrated that protein-free plasmid DNA injected into oocyte nuclei becomes organized in a nucleosomal chromatin structure, but only a very small fraction of these 'minichromosomes' is transcriptionally active whereas the majority of the molecules persists as inactive chromatin [4][5][6]. Recently, attention has been focussed on the interdependence of chromatin assembly with formation of stable transcription complexes using this type of in vivo gene expression assay [7,8]. Here, we used an S-150 extract prepared from X. laevis oocytes [9] for controlled in vitro chromatin assembly.…”