Varied interactions between proviruses and adjacent host chromatin.

Abstract

Retroviruses integrated at unique locations in the host genome can be expressed at different levels. We have analyzed the preintegration sites of three transcriptionally competent avian endogenous proviruses (evs) to determine whether the various levels of provirus expression correlate with their location in active or inactive regions of chromatin. Our results show that in three of four cell types, the chromatin conformation (as defined by relative nuclease sensitivity) of virus preintegration sites correlates with the level of expression of the resident provirus in ev+ cells: two inactive proviruses (ev-1 and ev-2) reside in nuclease-resistant chromatin domains and one active provirus (ev-3) resides in a nuclease-sensitive domain. Nuclear runoff transcription assays reveal that the preintegration sites of the active and inactive viruses are not transcribed. However, in erythrocytes of 15-day-old chicken embryos (15d RBCs), the structure and activity of the ev-3 provirus is independent of the conformation of its preintegration site. In this cell type, the ev-3 preintegration site is organized in a nuclease-resistant conformation, while the ev-3 provirus is in a nuclease-sensitive conformation and is transcribed. In addition, the nuclease sensitivity of host sequences adjacent to ev-3 is altered in ev-3+ 15dRBCs relative to that found in 15d RBCs that lack ev-3. These data suggest that the relationship between preintegration site structure and retrovirus expression is more complex than previously described.Avian and murine retroviruses can be expressed at different levels after integration into the host cell genome (2,14,21,26,29). In some cases, this variation is not due to genetic differences between the proviruses but instead is dependent on their site of integration (26,29). Analyses of two mouse mammary tumor virus proviruses acquired by exogenous infection in cultured cells indicated that the differential activity of these proviruses is dependent on their location in nuclease-sensitive or nuclease-resistant regions of the host cell genorne (14). An analogous relationship is not evident, however, for murine viruses introduced into the mouse germ line (Mov proviruses;27,28). In the latter case, inherited proviruses are inactive, even when located in transcriptionally active regions of chromatin (4,20). In contrast to Mov proviruses, some of the naturally occurring avian endogenous viruses (evs) are active in their inherited chromosomal location (2, 21). We were therefore interested in the relationship between provirus expression and the structure and activity of virus preintegration sites in this system.The avian endogenous viruses are highly related and stable genetic elements that were introduced into the germ line of chickens during evolution (1,7,24,25,33). Of the three viruses analyzed in this report, ev-1 and ev-2 are normally inactive in their inherited locations (producing less than 1 copy of stable RNA per cell; 2, 21), while ev-3 is active (producing 50 to 100 copies of stable mRNAs per cell; 2, 21...

Transcriptional enhancers act in cis to suppress position-effect variegation.

“…We find that clones expressing ~-geo revert to a silent state at a rate that is different for each clone and presumably a function of the site of integration~ this rate is not correlated with the level of expression in the clone. The behavior of these clones is similar to that reported previously for m a m m a r y tumor viruses (Feinstein et al 1982), endogenous avian proviruses (Conklin and Groudine 1986}, and some plasmid constructs {Davies et al 1982J, and resembles PEV in that each integration site appears to have a different ability to silence a juxtaposed transcriptional unit {Henikoff 1992~ Tartof 19941. Silencing is associated with methylation and loss of DNase I hypersensitivity at the promoter (D.I.K.…”

“…Thus, stably integrated plasmid constructs are subject to differences in expression, apparently caused by factors at the integration site ("position effects") that make direct comparison of different constructs difficult. Because integration may occur randomly into chromatin that varies in its ability to allow transcriptional activity, not all integration events result in an active construct (Davies et al 1982;Feinstein et al 1982;Conklin an Groudine 1986). We hypothesized that if an enhancer made a construct more efficient at creating an active region within a region of inactive chromatin, it would tend to increase the number of sites at which promoter activity could occur after integration.…”

Methylation-Mediated Proviral Silencing Is Associated with MeCP2 Recruitment and Localized Histone H3 Deacetylation

“…the level and stability of expression, the propensity for de novo methylation, and the complement of associated DNA binding factors (13). To avoid the inherent complications associated with such position effects, we modified the Cre recombinasebased targeting system, RMCE (17), to target in vitro methylated DNA into the genome (45; http://stke.sciencemag.org/cgi /content/full/OC_sigtrans;2001/83/pl1).…”

DNA interstrand cross-links promote chromosomal integration of a selected gene in human cells.

“…Integration of the vector DNA into host chromosomes appears to occur through nonhomologous recombination (20,34,37,39). However, genomic insertion of plasmid DNA may actually select chromosomal sites prone to recombination, such as regions with relaxed chromatin structure (1,9). To analyze the possible relationship of damage-enhanced transformation to chromosomal integration, we have posed the following questions: Do interstrand cross-links also cause stimulation of transformation?…”