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

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

“…In addition, sns might protect the transgene from position effects of the surrounding chromatin at the integration site, suggesting a possible barrier function for the sns element in erythroid cells. These findings are in agreement with our recent observations that the sea urchin sns 5 fragment, which includes sns, involved in the silencing of the H2A histone gene in sea urchin [23], is capable of reducing the influence of the chromatin environment on an integrated retroviral transgene (unpublished results). The observation that only a slight rise in the number of neomycin-resistant clones is generated by construct D is in line with the evidence that the chicken h-globin 5VHS4 (cHS4) insulator also does not improve gene expression and position effect protection when it is in the presence of a strong enhancer [28,29].…”

“…We have previously described three cis-acting sequence elements, Box A, Box B, and Box C + T, that are each required for the enhancer-blocking function of sns in sea urchin embryos and are binding sites for specific protein complexes [22,23]. Recent evidence indicates that all three sequence elements are needed for the enhancer-blocking function of sns in transfected human H1299 cell line (unpublished observation).…”

“…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]. Recent evidence indicates that sns is part of a DNA fragment, defined as sns 5, needed for silencing the a-H2A histone gene at gastrula stage [23]. This result suggests that the sea urchin insulator, in the natural environment of the histone gene sequences, is involved in the mechanism of repression.…”

Constitutive Promoter Occupancy by the MBF-1 Activator and Chromatin Modification of the Developmental Regulated Sea Urchin α-H2A Histone Gene

“…At least four negative cis regulatory sequences are found in the 462 bp sns 5 fragment, which is comprised between the last H2A codons and 3′ spacer sequences. 33 Three micrococcal nuclease sites specifically appear at this position at gastrula stage. 34 Remarkably, sns 5 contains an enhancer blocking element, termed sns, that as the best characterized insulators displays the capability to block enhancer-activated transcription in a polar and directional manner, in both sea urchin and human cells.…”

“…All cis-acting sequences needed for insulator function as well as the 5′ GAGA repeats are required for the down-regulation of the H2A gene at gastrula stage. 33 Several lines of evidence suggest that downregulation of the H2A gene occurs by an active repression mechanism, namely, in the presence of the necessary MBF-1 activator. Firstly, the MBF-1 gene is constitutively transcribed 29 and persistent MBF-1 binding activity can be detected at early and late blastula/gastrula stages.…”