Insulators: exploiting transcriptional and epigenetic mechanisms

Insulators are DNA sequence elements that prevent inappropriate interactions between adjacent chromatin domains. One type of insulator establishes domains that separate enhancers and promoters to block their interaction, whereas a second type creates a barrier against the spread of heterochromatin. Recent studies have provided important advances in our understanding of the modes of action of both types of insulator. These new insights also suggest that the mechanisms of action of both enhancer blockers and barriers might not be unique to these types of element, but instead are adaptations of other gene-regulatory mechanisms.     

CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species

Insulators can block an enhancer of one gene from activating a promoter on another nearby gene. Almost all described vertebrate insulators require binding of the regulatory protein CTCF for their activity. We show that CTCF copurifies with the nucleolar protein nucleophosmin and both are present at insulator sites in vivo. Furthermore, exogenous insulator sequences are tethered to the nucleolus in a CTCF-dependent manner. These interactions, quite different from those of the gypsy insulator element in Drosophila, may generate similar loop structures, suggesting a common theme and model for enhancer-blocking insulator action.     

CTCF-dependent enhancer blockers at the upstream region of the chicken alpha-globin gene domain

The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5′ flanking region of the chicken α-globin domain, 11–16 kb upstream of the embryonic α-type π gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two α-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific α-globin gene domain located in a permanently open chromatin area.     

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.     

绝缘子调控基因的表达

绝缘子在调控真核基因时空特异表达的过程中起着至关重要的作用.它的主要功能是增强子阻断和异染色质屏障.已经有竞争、阻断和成环等模型描述其增强子阻断功能;而它的异染色质屏障功能主要是通过影响染色质组蛋白的翻译后修饰来实现.已经确定的绝缘子包括果蝇基因组中的染色质特化结构(specialized chromatin structures, scs)和scs、gypsy、鸡珠蛋白β基因座上游的DNaseⅠ高敏感位点cHS4以及小鼠或人Igf2/H19基因座上的印记控制区(imprinting control region, ICR)和DNA甲基化区域(DNA methylated regions, DMR)元件等.许多转录因子参与绝缘子的基因调控作用,例如脊椎动物中的CCCTC结合因子(CCCTC binding factor,CTCF).利用基因组学和生物信息学等方法,还可以在基因组中发现新的绝缘子元件.     

Ibf1 and Ibf2 are novel CP190‐interacting proteins required for insulator function

Insulators are DNA‐protein complexes that play a central role in chromatin organization and regulation of gene expression. In Drosophila different proteins, dCTCF, Su(Hw), and BEAF bind to specific subsets of insulators most of them having in common CP190. It has been shown that there are a number of CP190‐binding sites that are not shared with any other known insulator protein, suggesting that other proteins could cooperate with CP190 to regulate insulator activity. Here we report on the identification of two previously uncharacterized proteins as CP190‐interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190‐binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNA‐binding proteins that form hetero‐oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancer‐blocking assays and Ibf2 null mutation cause a homeotic phenotype. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity.     

Mechanisms of distant enhancer action on DNA and in chromatin

Enhancers and insulators are regulatory DNA sequences that can work over a large distance. Efficient action over a distance clearly requires special mechanisms for facilitating communication between a regulatory region and its target. Studies from our laboratory identified DNA supercoiling as primary factor that mediates efficient enhancer-promoter communication over a distance in prokaryotes through a “DNA slithering” mechanism. These studies allowed rational design and construction of an insulator that can block enhancer action over a distance both in vitro and in vivo. Our most recent studies suggest that eukaryotic chromatin structure can support action over a distance using similar principles, but in a mechanistically distinct way. Published in Russian in Molekulyarnaya Biologiya, 2009, Vol. 43, No. 2, pp. 204–214. The article was translated by the author.     

Effects of functional interactions between nonhomologous insulators wari and Su(Hw)

Insulators are regulatory DNA elements restricting gene activation by enhancers. Interactions between insulators can lead to both insulation and activation of promoters by enhancers. In this work, we analyzed the effects of interaction of two Drosophila insulators, Wari and Su(Hw). The functional interaction between these insulators was found to enhance the activity of the Su(Hw) insulator only, but not of the Wari insulator. This suggests that the formation of a chromatin loop between interacting insulators is not a key factor for enhancement of insulation, which is in disagreement with the main idea of structural models. In addition, the effect of interaction between Wari and Su(Hw) depends on a distance between them and on the position in the system relative to other regulatory elements.