The role of insulators in transgene transvection inDrosophila

Transvection is the phenomenon where a transcriptional enhancer activates a promoter located on the homologous chromosome. It has been amply documented inDrosophilawhere homologues are closely paired in most, if not all, somatic nuclei, but it has been known to rarely occur in mammals as well. We have taken advantage of site-directed transgenesis to insert reporter constructs into the same genetic locus inDrosophilaand have evaluated their ability to engage in transvection by testing many heterozygous combinations. We find that transvection requires the presence of an insulator element on both homologues. Homotypictrans-interactions between four different insulators can support transvection: thegypsy insulator(GI),Wari, Fab-8and1A2;GIandFab-8are more effective thanWarior1A2. We show that in the presence of insulators, transvection displays the characteristics that have been previously described: it requires homologue pairing, but can happen at any of several loci in the genome; a solitary enhancer confronted with an enhancerless reporter is sufficient to drive transcription; it is weaker than the action of the same enhancer-promoter pair incisand it is further suppressed bycis-promoter competition. Though necessary, the presence of homotypic insulators is not sufficient for transvection; their position, number and orientation matters. A single GI adjacent to both enhancer and promoter is the optimal configuration. The addition of a heterologous insulator in one homolog can positively or negatively influence transvection strength. The local landscape of enhancers and promoters is also important, indicative of complex insulator-enhancer-promoter interactions.

The HumanGATA1Gene Retains a 5′ Insulator That Maintains Chromosomal Architecture andGATA1Expression Levels in Splenic Erythroblasts

GATA1 is a key transcription factor for erythropoiesis.GATA1gene expression is strictly regulated at the transcriptional level. While the regulatory mechanisms governing mouseGata1(mGata1) gene expression have been studied extensively, how expression of the humanGATA1(hGATA1) gene is regulated remains to be elucidated. To address this issue, we generatedhGATA1bacterial artificial chromosome (BAC) transgenic mouse lines harboring a 183-kbhGATA1locus covering thehGATA1exons and distal flanking sequences. TransgenichGATA1expression coincides with endogenousmGata1expression and fully rescues hematopoietic deficiency inmGata1knockdown mice. The transgene exhibited copy number-dependent and integration position-independent expression ofhGATA1, indicating the presence of chromatin insulator activity within the transgene. We found a novel insulator element at 29 kb 5′ to thehGATA1gene and refer to this element as the 5′ CCCTC-binding factor (CTCF) site. Substitution mutation of the 5′ CTCF site in thehGATA1BAC disrupted the chromatin architecture and led to a reduction ofhGATA1expression in splenic erythroblasts under conditions of stress erythropoiesis. Our results demonstrate that expression of thehGATA1gene is regulated through the chromatin architecture organized by 5′ CTCF site-mediated intrachromosomal interactions in thehGATA1locus.

Analysis on the Dynamic Responses of the Broken Conductors in Transmission Lines

Impact loads from the broken conductors are common for transmission lines, which can bring threaten to the safe operation of the transmission lines. Dynamic analysis of the conductors in transmission lines under broken load was carried out. A finite element model of seven span conductors in transmission line was established in general software ANSYS. The insulator and the phase spacer were considered in the FEA model. The broken load case can be realized by the birth-death element method in ANSYS. Stiffness of the broken conductor or insulator element was changed to be a near zero value in a very short time. Effect of the damping property of the conductors was considered by the Rayleigh damping method. Dynamic responses of displacements at the broken points and the reaction forces of the insulators were obtained. Dynamic responses for the broken conductors with different damping ratios and bundle numbers were compared.

Host Cell Detection of Noncoding Stuffer DNA Contained in Helper-Dependent Adenovirus Vectors Leads to Epigenetic Repression of Transgene Expression

ABSTRACT Helper-dependent adenovirus (hdAd) vectors have shown great promise as therapeutic gene delivery vehicles in gene therapy applications. However, the level and duration of gene expression from hdAd can differ considerably depending on the nature of the noncoding stuffer DNA contained within the vector. For example, an hdAd containing 22 kb of prokaryotic DNA (hdAd-prok) expresses its transgene 60-fold less efficiently than a similar vector containing eukaryotic DNA (hdAd-euk). Here we have determined the mechanistic basis of this phenomenon. Although neither vector was subjected to CpG methylation and both genomes associated with cellular histones to similar degrees, hdAd-prok chromatin was actively deacetylated. Insertion of an insulator element between the transgene and the bacterial DNA derepressed hdAd-prok, suggesting that foreign DNA nucleates repressive chromatin structures that spread to the transgene. We found that Sp100B/Sp100HMG and Daxx play a role in repressing transgene expression from hdAd and act independently of PML bodies. Thus, we have identified nuclear factors involved in recognizing foreign DNA and have determined the mechanism by which associated genes are repressed.