Enhancers increase the probability but not the level of gene expression.

Abstract

We have studied enhancer function in transient and stable expression assays in mammalian cells by using systems that distinguish expressing from The mechanism by which enhancers activate transcription remains a subject for speculation. One proposed mode of enhancer action is to increase the rate of transcription from a linked promoter (Fig. 1A). This "rate" model is based on nuclear run-on assays of populations of cells transiently transfected with plasmid constructs, which found that more nascent transcripts were synthesized by cells if the transfected construct contained the simian virus 40 (SV40) enhancer (1, 2).The interpretation of this result as an increase in the rate of transcription in every transfected nucleus, or an increase in the density of RNA polymerase on each template (3, 4), underlies the assessment of much subsequent work on transcriptional control. A "probability" model of enhancer action (Fig. 1B) has been suggested by experiments that have examined single cells, rather than populations, expressing transfected constructs (5-7). These experiments revealed that enhancers increase the number of expressing cells but not the level of expression per expressing cell.The results of the nuclear run-on experiments cited above are actually consistent with both models, since either an increase in the number of expressing cells in the population or an increase in the polymerase density per individual template would yield an increase in nascent transcripts in the total cell population. However, only the probability model is consistent with the single cell experiments.We have made a detailed examination of the enhancer effect on expression of a linked reporter gene using both transient and stable expression assays. These experiments were designed to directly test the two models by distinguishing and separating expressing from nonexpressing transfected cells. We find that in both transient and stable assays a linked enhancer increases the number of cells actively expressing a reporter but not the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.A "Rate" B "Probability"

Herpesvirus saimiri-based gene delivery vectors maintain heterologous expression throughout mouse embryonic stem cell differentiation in vitro

“…In these experiments, expression from a simple enhancer-promoter-reporter gene combination randomly integrated as a single copy gene into the host genome was examined on the single cell level. 24,25 It was found that in the absence of selection the stability of expression, not the expression level, was differentially modulated by chromatin-mediated genomic position effects. Expression was progressively silenced after multiple rounds of cell division.…”

“…Such a stochastic behaviour of gene expression driven by simple enhancer-promoter combinations has been observed in other systems and can be molecularly explained by a differential stability of transcription factor complexes. 24,25 The second model would predict that expression would not only be silenced, but would also be switched on again. We therefore studied the develop-a c b 32 …”

Variegation of retroviral vector gene expression in myeloid cells

“…This observation is in favor of the binary model by which enhancers act by increasing the probability of a transgene being active, but not the level of expression per template. 39 This model predicts that, at a defined integration site, expression would be more stable, but not higher, in the presence of an active enhancer. 40 In this context, the results obtained by the analysis of the transduced PLB985 clones indicate that the MoMuLV LTR is much less efficient than the FMEV LTR in counteracting the suppression imposed on provirus gene expression in the hematopoietic cellular environment.…”

Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels

“…As the number of mRNA molecules increases, so does the predictability of the number of proteins synthesized per cell. Likewise, if the number of enhancer and transcription activator molecules in a cell is low, large variation in mRNA copy number is expected (Walters et al 1995;McAdams and Arkin 1997;Fiering et al 2000;Elowitz et al 2002;Raser and O'Shea 2004). These effects may account for the large cell-to-cell variations observed in isogenic cell populations (Levsky and Singer 2003;Peixoto et al 2004).…”