Unichrom, a novel nuclear matrix protein, binds to the Ars insulator and canonical MARs.

Eukaryotic genomic DNA is organized into loop structures by attachments to the nuclear matrix. These attachments to the nuclear matrix have been supposed to form the boundaries of chromosomal DNA. Insulators or boundary elements are defined by two characteristics: they interrupt promoter-enhancer communications when inserted between them, and they suppress the silencing of transgenes stably integrated into inactive chromosomal domains. We recently identified an insulator element in the upstream region of the sea urchin arylsulfatase (HpArs) gene that shows both enhancer blocking and suppression of position effects. Here, we report that Unichrom, originally identified by its G-stretch DNA binding capability, is a nuclear matrix protein that binds to the Ars insulator and canonical nuclear matrix attachment regions (MARs). We also show that Unichrom recognizes the minor groove of the AT-rich region within the Ars insulator, which may have a base-unpairing property, as well as the G-stretch DNA. Furthermore, Unichrom selectively interacts with poly(dG).poly(dC), poly(dA).poly(dT) and poly(dAT).poly(dAT), but not with poly(dGC).poly(dGC). Unichrom also shows high affinity for single-stranded G- and C-stretches. We discuss the DNA binding motif of Unichrom and the function of Unichrom in the nuclear matrix.

The Otx binding site is required for the activation of HpOtxL mRNA expression in the sea urchin, Hemicentrotus pulcherrimus.

Two distinct types of orthodenticle-related (HpOtxE and HpOtxL) mRNA are transcribed from a single HpOtx gene by altering the transcription start site and by alternative splicing, and their expressions are differentially regulated during early development of the sea urchin Hemicentrotus pulcherrimus. To understand the mechanism of this regulation, we screened for the enhancer element involved in the stage-specific expression of HpOtxL mRNA. Different portions of the HpOtx gene, including the 5'-flanking region and the first intron, were ligated to the minimal HpOtxL promoter driving a luciferase gene, and their constructs were introduced into fertilized eggs using a particle gun. The enhancer element responsible for proper expression consistent with that of the endogenous HpOtxL was found in the first intron of the HpOtx gene. External and internal deletion analyses showed that the 334 bp region (from +8838 bp to +9171 bp) was required for enhancer activity. In addition, deletion of an Otx binding site within the 334 bp region markedly reduced reporter expression. These results suggest that the Otx binding site within the HpOtxL enhancer is required for the activation of HpOtxL mRNA expression. The promoter preference of the HpOtxL enhancer is also discussed.

A new G-stretch-DNA-binding protein, Unichrom, displays cell-cycle-dependent expression in sea urchin embryos.

We report the identification and characterization of Unichrom, a gene encoding a new G-stretch-DNA-binding protein in the sea urchin embryo. The derived amino acid sequence of Unichrom contains plant homeodomain (PHD) finger and high mobility group (HMG) motifs as well as motifs required for cell-cycle-dependent degradation. The expression of a Unichrom-green fluorescent protein (GFP) fusion protein in sea urchin embryonic cells indicates that Unichrom protein accumulates in nuclei during interphase and disperses into the cytoplasm at mitosis. Overexpression of dominant negative Unichrom, which contains the DNA binding domain lacking the motif for cell-cycle-dependent degradation, causes impairment of chromosome segregation. These results suggest that Unichrom binds to genome DNA at G-stretch and that degradation of Unichrom is required for segregation of chromosomes.

Utilization of a particle gun DNA introduction system for the analysis of cis-regulatory elements controlling the spatial expression pattern of the arylsulfatase gene (HpArs) in sea urchin embryos.

We applied a particle gun method to introduce DNA into fertilized sea urchin eggs for the analysis of cis-regulatory elements responsible for spatial gene expression during development. We introduced HpArs (sea urchin arylsulfatase gene) -GFP and HpArs-LacZfusion constructs into the fertilized eggs and obtained high expression levels of the fusion genes. Using this assay system, we demonstrated that a fragment of HpArs (-3,484 to +4,636) is sufficient for aboral ectoderm-specific expression, and that the region in the first intron from +406 to +1,993 contains the control elements responsible for the repression of the HpArs promoter activity in secondary mesenchyme cells.

T-brain homologue (HpTb) is involved in the archenteron induction signals of micromere descendant cells in the sea urchin embryo.

Signals from micromere descendants play a crucial role in sea urchin development. In this study, we demonstrate that these micromere descendants express HpTb, a T-brain homolog of Hemicentrotus pulcherrimus. HpTb is expressed transiently from the hatched blastula stage through the mesenchyme blastula stage to the gastrula stage. By a combination of embryo microsurgery and antisense morpholino experiments, we show that HpTb is involved in the production of archenteron induction signals. However, HpTb is not involved in the production of signals responsible for the specification of secondary mesenchyme cells, the initial specification of primary mesenchyme cells, or the specification of endoderm. HpTb expression is controlled by nuclear localization of beta-catenin, suggesting that HpTb is in a downstream component of the Wnt signaling cascade. We also propose the possibility that HpTb is involved in the cascade responsible for the production of signals required for the spicule formation as well as signals from the vegetal hemisphere required for the differentiation of aboral ectoderm.