Variegated expression of a globin transgene correlates with chromatin accessibility but not methylation status.

There are now many mammalian examples in which single cell assays of transgene activity have revealed variegated patterns of expression. We have previously reported that transgenes in which globin regulatory elements drive the lacZ reporter gene exhibit variegated expression patterns in mouse erythrocytes, with transgene activity detectable in only a sub-population of circulating erythroid cells. In order to elucidate the molecular mechanism responsible for variegated expression in this system, we have compared the chromatin structure and methylation status of the transgene locus in expressing and non-expressing populations of erythrocytes. We find that there is a difference in the chromatin conformation of the transgene locus between the two states. Relative to active transgenes, transgene loci which have been silenced exhibit a reduced sensitivity to general digestion by DNase I, as well as a failure to establish a transgene-specific DNase I hypersensitive site, suggesting that silenced transgenes are situated within less accessible chromatin structures. Surprisingly, the restrictive chromatin structure observed at silenced transgene loci did not correlate with increased methylation, with transgenes from both active and inactive loci appearing largely unmethylated following analysis with methylation-sensitive restriction enzymes and by sequencing PCR products derived from bisulphite-converted genomic DNA.     

Cis- and trans-acting influences on telomeric position effect in Drosophila melanogaster detected with a subterminal transgene

One model of telomeric position effect (TPE) in Drosophila melanogaster proposes that reporter genes in the vicinity of telomeres are repressed by subterminal telomere-associated sequences (TAS) and that variegation of these genes is the result of competition between the repressive effects of TAS and the stimulating effects of promoters in the terminal HeT-A transposon array. The data presented here support this model, but also suggest that TPE is more complex. Activity of a telomeric white reporter gene increases in response to deletion of some or all of the TAS on the homolog. Only transgenes next to fairly long HeT-A arrays respond to this trans-interaction. HeT-A arrays of 6-18 kb respond by increasing the number of dark spots on the eye, while longer arrays increase the background eye color or increase the number of spots sufficiently to cause them to merge. Thus, expression of a subtelomeric reporter gene is influenced by the telomere structure in cis and trans. We propose that the forces involved in telomere length regulation in Drosophila are the underlying forces that manifest themselves as TPE. In the wild-type telomere TAS may play an important role in controlling telomere elongation by repressing HeT-A promoter activity. Modulation of this repression by the homolog may thus regulate telomere elongation.     

Two distinct domains in Drosophila melanogaster telomeres

Telomeres are generally considered heterochromatic. On the basis of DNA composition, the telomeric region of Drosophila melanogaster contains two distinct subdomains: a subtelomeric region of repetitive DNA, termed TAS, and a terminal array of retrotransposons, which perform the elongation function instead of telomerase. We have identified several P-element insertions into this retrotransposon array and compared expression levels of transgenes with similar integrations into TAS and euchromatic regions. In contrast to insertions in TAS, which are silenced, reporter genes in the terminal HeT-A, TAHRE, or TART retroelements did not exhibit repressed expression in comparison with the same transgene construct in euchromatin. These data, in combination with cytological studies, provide evidence that the subtelomeric TAS region exhibits features resembling heterochromatin, while the terminal retrotransposon array exhibits euchromatic characteristics.     

Telomere length homeostasis and telomere position effect on a linear human artificial chromosome are dictated by the genetic background

Telomere position effect (TPE) is the influence of telomeres on subtelomeric epigenetic marks and gene expression. Previous studies suggested that TPE depends on genetic background. As these analyses were performed on different chromosomes, cell types and species, it remains unclear whether TPE represents a chromosome—rather than genetic background-specific regulation. We describe the development of a Linear Human Artificial Chromosome (L-HAC) as a new tool for telomere studies. The L-HAC was generated through the Cre-loxP-mediated addition of telomere ends to an existing circular HAC (C-HAC). As it can be transferred to genetically distinct cell lines and animal models the L-HAC enables the study of TPE in an unprecedented manner. The HAC was relocated to four telomerase-positive cell lines via microcell-mediated chromosome transfer and subsequently to mice via blastocyst injection of L-HAC⁺-ES-cells. We could show consistent genetic background-dependent adaptation of telomere length and telomere-associated de novo subtelomeric DNA methylation in mouse ES-R1 cells as well as in mice. Expression of the subtelomeric neomycin gene was inversely correlated with telomere length and subtelomeric methylation. We thus provide a new tool for functional telomere studies and provide strong evidence that telomere length, subtelomeric chromatin marks and expression of subtelomeric genes are genetic background dependent.     

P-Element repression in Drosophila melanogaster by variegating clusters of P-lacZ-white transgenes.

In Drosophila, clusters of P transgenes (P-lac-w) display a variegating phenotype for the w marker. In addition, X-ray-induced rearrangements of chromosomes bearing such clusters may lead to enhancement of the variegated phenotype. Since P-lacZ transgenes in subtelomeric heterochromatin have some P-element repression abilities, we tested whether P-lac-w clusters also have the capacity to repress P-element activity in the germline. One cluster (T-1), located on a rearranged chromosome (T2;3) and derived from a line bearing a variegating tandem array of seven P-lac-w elements, partially represses the dysgenic sterility (GD sterility) induced by P elements. This cluster also strongly represses in trans the expression of P-lacZ elements in the germline. This latter suppression shows a maternal effect. Finally, the combination of variegating P-lac-w clusters and a single P-lacZ reporter inserted in subtelomeric heterochromatic sequences at the X chromosome telomere (cytological site 1A) leads to strong repression of dysgenic sterility. These results show that repression of P-induced dysgenic sterility can be elicited in the absence of P elements encoding a polypeptide repressor and that a transgene cluster can repress the expression of a single homologous transgene at a nonallelic position. Implications for models of transposable element silencing are discussed.     

Activation of CMV promoter-controlled glycosyltransferase and β -galactosidase glycogenes by butyrate, tricostatin A, and 5-Aza-2′-deoxycytidine

Cytomegalovirus (CMV) immediate early promoter is a powerful promoter frequently used for driving the expression of transgenes in mammalian cells. However, this promoter gradually becomes silenced in stably transfected cells. We employed Chinese Hamster Ovary (CHO) and human pancreatic cancer (Panc 1) cells stably tansfected with three glycogenes driven by a CMV promoter to study the activation of silenced glycogenes. We found that butyrate, tricostatin A (TSA), and 5-aza-2-deoxycytidine (5-Aza-dC) can activate these CMV-driven glycogenes. The increase in mRNA and protein of a glycogene occurred 8–10 h after butyrate treatment, suggesting an indirect effect of butyrate in the activation of the transgene. The enhanced expression of the trangenes by butyrate and TSA, known inhibitors of histone deacetylase, was independent of the transgene or cell type. However, the transgene can be activated by these two agents in only a fraction of the cells derived from a single clone, suggesting that inactivation of histone deacetylase can only partially explain silencing of the transgenes. Combination treatment of one or both agents with 5-Aza-dC, a known inhibitor of DNA methylase, resulted in a synergistic activation of the transgene, suggesting a cross-talk between histone acetylation and DNA demethylation. Understanding the mechanisms of the inactivation and reactivation of CMV promoter-controlled transgenes should help develop an effective strategy to fully activate the CMV promoter-controlled therapeutic genes silenced by the host cells. Published in 2005.     

Modification of subtelomeric DNA

There is a discrepancy in telomere length as measured by signal intensity of telomere restriction fragments on gels and fluorescence in situ hybridization analysis. This difference has been ascribed to the X-region, a segment of subtelomeric DNA that is resistant to being cut by restriction enzymes. To explore the nature of this region, we analyzed the digestibility of an artificial seeded telomere in HeLa cells as well as the Xp/Yp autosomal telomere in human BJ fibroblasts. We found that there is a substantial fraction of subtelomeric DNA containing restriction sites that is not digested with enzymes such as EcoRI, NlaIII, and SphI. Comparison of methylation-sensitive and -resistant enzymes excluded the possibility of the X-region being maintained by DNA methylation. We show that the X-region represents a variable domain whose size changes with telomere length, and neither non-TTAGGG sequences nor cytidine methylation can adequately explain the size of the X-region.