Promoter Crosstalk Effects on Gene Expression

Abstract

Closely spaced transcription signals formally assigned to different, neighboring genes can functionally interact both in their authentic genomic context as well as in engineered transgene constellations. To describe these promoter crosstalk effects quantitatively and qualitatively, we used various combinations of inducible and constitutive expression signals linked in cis. Our results demonstrate that such interactions can be bidirectional, making it difficult to unambiguously assign a particular promoter element exclusively to a specific gene. We show that especially for inducible promoters, crosstalk effects can cause a substantial distortion in the expression of proximal genes, challenging established strategies in generating transgenic animal models and tissue culture systems. Furthermore, these findings provide guidelines for the design of transgenic transcription units that, while refractory to interactions with their chromosomal environment, leave the expression programs of neighboring genes largely untouched.

Introduction

After random integration in their host's genome, transcription signals controlling the expression of transgenes frequently deviate from their expected activity and specificity patterns.¹ For example, transgene penetrance and expressivity can be superimposed by a variety of epigenetic control mechanisms.² Beside the specific epigenetic environment, transgene expression is also affected by novel constellations of transcriptional elements at the transgene's integration site. Illustrative examples are enhancer trap assays,3., 4. where exogenous minimal promoters linked to appropriate reporter transgenes are activated by endogenous enhancers close to their site of chromosomal integration.⁵ This approach has been instrumental in identifying developmental or tissue-specific transcription signals.⁶ However, such interactions can also work in the opposite direction. One such example is the dominant influence exerted by exogenous transgenic transcription signals on proximal endogenous proto-oncogenes.7., 8., 9., 10. These can be activated by the integration of enhancers of viral origin, causing cellular transformation. Most likely interactions mechanistically similar to the two scenarios outlined above occur more frequently than suggested by selected events monitored by suitable reporter genes or phenotypic changes in the proliferation potential of cells. For example, such local communication has also been demonstrated between endogenous genes, sometimes without any obvious functional consequences.¹¹ In principle such interactions are not limited to the unidirectional mode as in the examples given. We therefore refer to the postulated bidirectional process as promoter crosstalk. Its experimental analysis requires comparative approaches involving either elaborate schemes of chromosome engineering or the analysis of genes of which at least one can be transcriptionally regulated by experimentally controlled stimuli. Otherwise each of the vicinal genes would almost inevitably be considered to be a separate functional entity.

We systematically evaluated defined proximal arrangements of two “independent” transcription units, one driven by an inducible promoter, the other by a panel of different constitutive promoter/enhancer constructs (note: throughout this manuscript we use the term promoter in a rather broad definition, referring to continuous gene regulatory sequences that can contain enhancer elements beside the core promoter sequence itself). This setting allowed us to determine the contribution of individual transcriptionally active elements to the expression of the two genes. Clearly, they can distort each others activity and do not function as autonomous functional units, increasing the complexity of strategies to accomplish predictable transgene expression on the cellular or organismal level.