Enhancer-blocking insulators are DNA elements that disrupt the communication between a regulatory sequence, such as an enhancer or a silencer, and a promoter. Insulators participate in both transcriptional regulation and global nuclear organization, two features of chromatin that are thought to be maintained from one generation to the next through epigenetic mechanisms. Furthermore, there are many regulatory mechanisms in place that enhance or hinder insulator activity. These modes of regulation could be used to establish cell-type specific insulator activity that is epigenetically inherited along a cell and/or organismal lineage. This review will discuss the evidence for epigenetic inheritance and regulation of insulator function.

An essential node in the gene regulatory network (GRN) for endomesoderm specification in the sea urchin embryo lies within the regulatory system of the otx gene. According to the predictions of the GRN, based on perturbation analysis and expression data, the beta1/2 transcription unit of this gene is engaged during specification in interactions with two other regulatory genes, krox and gatae. It is predicted to be driven into activity by the krox gene, to form a positively reinforcing functional loop with the gatae gene, and to respond to its own output as well. Here we isolate the relevant otx cis-regulatory element, and examine the specific input predictions of the GRN at the level of its genomic DNA sequence. This beta1/2-otx regulatory module performs the necessary functions, as shown by use of expression constructs. It requires gatae, otx, and krox inputs, as predicted, and it operates as an "AND" logic processor in that removal of any one of these inputs essentially destroys activity. The necessary target sites were identified in the module sequence, and mutation of these sites was demonstrated to produce the same respective effects on construct expression as does blocking its regulatory inputs by treatment with morpholino antisense oligonucleotides. For spatial expression in the endoderm, one particular pair of Gata sites is essential and these function synergistically with an adjacent Otx site. We thus demonstrate directly the structure/function relationships of the genomic regulatory code, at this key node of the endomesoderm GRN.

Insulator elements can be classified as enhancer-blocking or barrier insulators depending on whether they interfere with enhancer-promoter interactions or act as barriers against the spreading of heterochromatin. The former class may exert its function at least in part by attaching the chromatin fiber to a nuclear substrate such as the nuclear matrix, resulting in the formation of chromatin loops. The latter class functions by recruiting histone modifying enzymes, although some barrier insulators have also been shown to create chromatin loops. These loops may correspond to functional nuclear domains containing clusters of co-expressed genes. Thus, insulators may determine specific patterns of nuclear organization that are important in establishing specific programs of gene expression during cell differentiation and development.

A specific prediction of our detailed cis-regulatory analysis of the Strongylocentrotus purpuratus (Sp) endo16 gene was that the later expression of this gene would be driven by a midgut-specific transcriptional regulator. We have now identified this factor and determined some of its functions. The cDNA sequence reveals it to be a POU domain factor related closely to the mammalian factors Brain-1, -2, and -4. The factor was termed SpBrn1/2/4 (henceforth Brn1/2/4). Quantitative measurements of transcript prevalence show that the gene is first activated in the 20-h blastula, but there remain only about 100 molecules of brn1/2/4 mRNA per embryo (only a few per endoderm cell) until an abrupt 10-fold increase occurs as gastrulation begins. Measured in the same embryos, the late rise in prevalence of endo16 transcripts follows that of brn1/2/4 transcripts. As predicted by the endo16 model, brn1/2/4 expression is confined perfectly to the midgut, coincident with the domain of endo16 expression. The kinetics of accumulation of these transcripts indicates that the switch into the late phase of endo16 expression occurs when the brn1/2/4 transcript level nears its plateau (2000 molecules mRNA per embryo), after which each endo16 gene produces about 1 mRNA every 2 min (about 380 molecules mRNA per min in the whole embryo). Arrest of Brn1/2/4 translation by MASO treatment blocks the late phase of endo16 expression and specifically abolishes expression of cis-regulatory Module B of endo16, while not affecting Module A, also as predicted. The brn1/2/4 gene lies downstream of the regulatory genes executing post-gastrular specification of the midgut, as shown by further gene expression perturbation experiments which provide an initial glimpse of the underlying network architecture.