Nucleosome exclusion from the interspecies-conserved central AT-rich region of the Ars insulator

The Ars insulator is a boundary element identified in the upstream region of the arylsulfatase (HpArs) gene in the sea urchin, Hemicentrotus pulcherrimus, and possesses the ability to both block enhancer-promoter communications and protect transgenes from silent chromatin. To understand the molecular mechanism of the Ars insulator, we investigated the correlation between chromatin structure, DNA structure and insulator activity. Nuclease digestion of nuclei isolated from sea urchin embryos revealed the presence of a nuclease-hypersensitive site within the Ars insulator. Analysis of micrococcal nuclease-sensitive sites in the Ars insulator, reconstituted with nucleosomes, showed the exclusion of nucleosomes from the central AT-rich region. Furthermore, the central AT-rich region in naked DNA was sensitive to nucleotide base modification by diethylpyrocarbonate (DEPC). These observations suggest that non-B-DNA structures in the central AT-rich region may inhibit nucleosomal formation, which leads to nuclease hypersensitivity. Furthermore, comparison of nucleotide sequences between the HpArs gene and its ortholog in Strongylocentrotus purpuratus revealed that the central AT-rich region of the Ars insulator is conserved, and this conserved region showed significant enhancer blocking activity. These results suggest that the central AT-rich nucleosome-free region plays an important role in the function of the Ars insulator.     

Ars insulator identified in sea urchin possesses an activity to ensure the transgene expression in mouse cells

Sea urchin arylsulfatase (Ars) gene locus has features of an insulator, i.e., blocking of enhancer and promoter interaction, and protection of a transgene against positional effects [Akasaka et al. (1999) Cell. Mol. Biol. 45, 555-565]. To examine the effect of Ars insulator on long-term expression of a transgene, the insulator was inserted into LTR of retrovirus vector harboring hrGFP gene as a reporter, and then introduced into mouse myoblast cells. The isolated clones transduced with the reporter gene with or without Ars insulator were cultured for more than 20 wk in the absence of a selection reagent, and the expression of hrGFP was periodically determined. Expression of hrGFP in four clones transduced with the reporter gene without Ars insulator was completely silenced after 20 wk of culture. On the other hand, hrGFP was expressed in all clones with Ars insulator inserted in one of the two different orientations. Histone H3 deacetylation and DNA methylation of the 5'LTR promoter region, signs for heterochromatin and silencing, were suppressed in the clones that were expressing hrGFP. Ars insulator is effective in maintaining a transgene in mouse cells in an orientation-dependent manner, and will be a useful tool to ensure stable expression of a transgene.     

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.     

An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells

Specialized DNA sequences known as insulators protect genes from both the positive and negative influences of nearby chromatin. Many insulators have been identified in various species; however, few function in multiple species. We have shown that an insulator from the Ars (arylsulfatase) gene of the sea urchin Hemicentrotus pulcherrimus functions in plant cells. Normally, expression of an introduced chimeric GUS gene is inactivated in approximately 30% of transformed tobacco BY2 clones. Transgenes containing the Ars insulator, however, were expressed in all transformed tobacco BY2 cells. The insulator did not affect the copy number, the chromosomal position of transgene integration or maximum expression levels. These results suggest that the insulator functions to suppress the variation normally associated with transgene expression in tobacco BY2 cells.     

The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer-promoter communication

Chromatin insulators, or boundary elements, appear to control eukaryotic gene expression by regulating interactions between enhancers and promoters. Boundaries have been identified in the 3' cis-regulatory region of Abd-B, which is subdivided into a series of separate iab domains. Boundary elements such as Mcp, Fab-7, and Fab-8 and adjacent silencers flank the iab domains and restrict the activity of the iab enhancers. We have identified an insulator in the 755-bp Mcp fragment that is linked to the previously characterized Polycomb response element (PRE) and silences the adjacent genes. This insulator blocks the enhancers of the yellow and white genes and protects them from PRE-mediated repression. The interaction between the Mcp elements, each containing the insulator and PRE, allows the eye enhancer to activate the white promoter over the repressed yellow domain. The same level of white activation was observed when the Mcp element combined with the insulator alone was interposed between the eye enhancer and the promoter, suggesting that the insulator is responsible for the interaction between the Mcp elements.     

cis-Elements and Protein Factors Related to Regulation of Transcription of Arylsulfatase Gene during Sea Urchin Development

Eight restriction fragments (I–VIII) were prepared to cover a whole span of the enhancer region in the upstream of the Ars gene of the sea urchin, Hemicentrotus pulcherrimus , and their abilities to influence on the Ars gene expression were estimated by CAT assay. Only three fragments (III, IV and V) encompassing a 0.6 kb region between −2.8 kb and −2.2 kb stimulated CAT expression. By mobility shift assays, it was found that the Ars enhancer region is composed of multiple cis -acting elements that interact with nuclear proteins in a sequence-specific manner. Among them, two sequences, a G-string and a GATCTCCCC, were determined by DNA footprinting as sites of protein-DNA interaction. The DNA-binding factor prevalence changed ontogenically in three different patterns. Possible activation of DNA-binding proteins through their modification is discussed.     

Multiple Promoter Targeting Sequences exist in Abdominal-B to regulate long-range gene activation

In complex genomes, insulators set up chromatin domain boundaries and protect promoters from inappropriate activation by enhancers from neighboring genes. The Drosophila Abdominal-B locus uses insulator elements to organize its large regulatory region into several body segment-specific chromatin domains. This organization leads to a problem in enhancer-promoter communication, that is, how do distal enhancers activate the Abd-B promoter when there are several insulators in between? This issue is partially resolved by the Promoter Targeting Sequence, which can overcome the enhancer blocking effect of an insulator. In this study, we describe a new Promoter Targeting Sequence, PTS-6, from the Abd-B 3' regulatory region. PTS-6, comprised of approximately 200 bp, was found to bypass both homologous Abdominal-B insulators, such as Fab-7 and Fab-8, and a heterologous insulator, suHw. Most importantly, it also overcomes a combination of two insulators such as Fab-7/Fab-8. Thus, PTS-6 could, in principle, target remote enhancers that are separated from the Abd-B promoter by multiple insulators. In addition, PTS-6 selectively targets the distal enhancer to only one transgenic promoter, and it strongly facilitates Abd-B enhancers. These results suggest that promoter targeting is necessary for long-range enhancer-promoter communication in Abd-B, and PTS elements could be a common occurrence in large, complex genetic loci.