Enhancer blocking activity located near the 3′ end of the sea urchin early H2A histone gene

Abstract

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.

Down-regulation of Early Sea Urchin Histone H2A Gene Relies on cis Regulative Sequences Located in the 5′ and 3′ Regions and Including the Enhancer Blocker sns

“…As previously described, the sea urchin sns, when included in artificial constructs and tested in enhancer blocking assays, 37 displays directional enhancer blocking activity in either orientation, both at early and late developmental stages. 38 In addition, if sns intervenes between two enhancers, only the enhancer located distally from the promoter with respect to the site of insertion is attenuated. Furthermore, sns does not prevent the blocked enhancer from trans-activating a promoter in the other direction, suggesting that sns represses enhancer-promoter interaction neither by enhancer inactivation nor by inducing local assembly of a repressive chromatin structure.…”

“…17 sns 2 extends further upstream and further downstream than the sns enhancer blocking element. 38 It contains the last 90 bp of the protein-coding Figure 2. Determination of positional silencing capability of sns 2 in transgenic embryos.…”

“…Ligated Box CCT oligonucleotides were microinjected into sea urchin embryos together with the insulated 6XM30-sns-CAT DNA (6XM30 is the enhancer cassette, used for the enhancer blocking assays, 38 which contains six copies of the MBF-1 binding site) and the developmentally regulated H2A-CAT-sns5 (see drawing in Figure 5) at the molar ratio of 50 to 1. Because the enhancer blocking function of sns is not dependent on the developmental stage, 38 transgene expression was determined by RNase protection only at gastrula stage for the insulated and non-insulated constructs.…”

“…Because the enhancer blocking function of sns is not dependent on the developmental stage, 38 transgene expression was determined by RNase protection only at gastrula stage for the insulated and non-insulated constructs. The results of Figure 5 clearly indicate that co-injection of excess of the Box CCT binding site impaired the sns capability to block enhancer-promoter interaction (lanes 1 and 2).…”

Functional characterization of the sea urchin sns chromatin insulator in erythroid cells

“…When included in artificial constructs, sns displays directional enhancer-blocking activity in either orientation, both at early and late developmental stages [21,22]. In addition, if sns intervenes between two enhancers, only the enhancer located distally from the promoter with respect to the site of insertion is attenuated [22].…”