Transcriptional Interference by Independently Regulated Genes Occurs in Any Relative Arrangement of the Genes and Is Influenced by Chromosomal Integration Position

Abstract

Transcriptional interference is the influence, generally suppressive, of one active transcriptional unit on another unit linked in cis. Its wide occurrence in experimental systems suggests that it may also influence transcription in many loci, but little is known about its precise nature or underlying mechanisms. Here we report a study of the interaction of two nearly identical transcription units juxtaposed in various arrangements. Each reporter gene in the constructs has its own promoter and enhancer and a strong polyadenylation signal. We used recombinase-mediated cassette exchange (RMCE) to insert the constructs into previously tagged genomic sites in cultured cells. This strategy also allows the constructs to be assessed in both orientations with respect to flanking chromatin. In each of the possible arrangements (tandem, divergent, and convergent), the presence of two genes strongly suppresses expression of both genes compared to that of an identical single gene at the same integration site. The suppression is most severe with the convergent arrangement and least severe in total with the divergent arrangement, while the tandem arrangement is most strongly influenced by the integration site and the genes' orientation within the site. These results suggest that transcriptional interference could underlie some position effects and contribute to the regulation of genes in complex loci.

Disruption of p53 by the Viral oncoprotein HPV16-E6 does not Deregulate Chromosomal Homologous Recombination in a Transcriptional Interference-Free Assay System

“…38,[62][63][64] Another aspect that may influence the outcome is transcriptional interference or promoter suppression. 34,35 Interference/suppression between repeated genes has been associated with chromatin condensation and histone deacetylation, as revealed by the inaccessibility of silenced promoters to nucleases or restriction enzymes and by their reactivation through treatment with HDAC inhibitor TSA. 35,65 The protein p53 might be involved in interference/suppression through its ability to both recruit HDAC at promoters and alter chromatin structure.…”

“…This level of interference/suppression is much lower than what has been reported with either transcription units arranged in a head-to-tail tandem fashion or in a convergent orientation of the same viral promoter; as in almost all cell clones (>90%) that had been initially selected for the expression of one gene, the other gene linked in cis was repressed. 34,35 Wt p53 does not suppress spontaneous chromosomal HR. To test whether p53 would impair HR with this combination of promoters, we compared HR rates between p53-positive pCAdir/pCAinv cell clones and p53-negative derivative cell clones.…”

“…34 Placing transcription units under the control of distinct promoters can decrease suppression between convergent units (Villemure et al, unpublished results). We, therefore, placed the HR reporter puromycin acetyltransferase (Puro) gene, which when active encodes resistance to puromycin drug, under control of the strong cellular housekeeping phosphoglycerate kinase (PGK) gene promoter in convergent orientation to the marker hygromycin-drug resistance (Hyg) gene under control of the herpes simplex virus thymidine kinase (HSV-tk) gene promoter.…”

“…Interference/suppression occurs at the transcriptional level, is cis-acting, epigenetic, heritable, and can be associated with alteration of chromatin structure. 34,35 In the present study, we employed a chromosomal assay system in which a HR reporter gene can escape interference/suppression. Recently, we found that in this assay system, the breast cancer susceptibility BRCA2 protein, which interacts with p53 and RAD51, 36 suppresses spontaneous HR but promotes HR following chromosomal DSBs.…”

Factors Binding a Non-classical Cis-element Prevent Heterochromatin Effects on Locus Control Region Activity

“…Because LCR activity appears to favor the gene nearest to it (29 -32), we reasoned that HS6 would be more likely to affect YFP activity than hygromycin resistance. This linearizing scheme also minimizes the likelihood of transcriptional interference by ensuring that the two transcription units would be separated by several kilobases of vector DNA and be in divergent transcriptional orientations (33). These constructs were transfected into NIH3T3 fibroblasts to generate individual stable transfected colonies.…”