Why do some genes seem to respond in a ‘digital’, on/off manner to a graded signal, while others produce an ‘analog’, graded response?
A new study in Current Biology suggests that the DNA-binding properties of transcription factors (TFs) can exert a strong influence on the response patterns of gene networks.
Many cellular processes operate in an “analog” regime in which the magnitude of the response is precisely tailored to the intensity of the stimulus. In order to maintain the coherence of such responses, the cell must provide for proportional expression of multiple target genes across a wide dynamic range of induction states.
Cell–cell signaling pathways, such as the pheromone response system used for mating in yeast, as well as developmental patterning pathways in animals and plants, often incorporate negative feedback mechanisms (feedback loops) to produce measured transcriptional responses, in proportion to the stimulus.
In this study, genetically engineered yeast expressing a constitutively active form of Msn2 under the control of a hormone-inducible promoter were used to examine transcriptional output from the gene.
By measuring the activity of Msn2-regulated promoters (driving Yellow Fluorescent Protein reporters), they obtained an idea of the relationship between TF activity and gene expression in vivo.
In many contexts, cells respond to stimuli with decisive commitment to a phenotypic state. It is usually assumed that genes that drive this transition exist in just two alternative functional states, active and inactive, and that the switch between these two states occurs decisively in a narrow regime of transcription factor concentration. In addition to making decisive choices, cells and organisms also need to continuously adjust to the demands of their environment. Systems that are responsible for homeostasis or graded developmental processes may need to operate in an “analog” regime where a response is tailored to the exact intensity of the stimulus in order to prevent deleterious over- or under-reactions.
After failing to observe a great deal of cooperativity, the authors suggest that the low afﬁnity of Msn2 for its DNA binding motifs in vitro, as well as the abundance of Msn2 motifs in the yeast genome, together reduce the level of occupancy of Msn2 at its target promoters, via weak TF–DNA interactions and the proteins’ sequestration by surplus genomic binding sites.
They tested this hypothesis by making targeted substitutions to the Msn2 DNA-binding domain, altering its DNA-binding speciﬁcity. The mutant Msn2 showed higher in vitro binding afﬁnity for its new preferred sequence motif than the wild-type protein has for its binding site. This newly preferred binding site is rare in the yeast genome, hence results in non-linear, saturating (but non-sigmoidal) transcriptional output [yellow fluorescence].
…While our studies were focused on the steady-state input/output relationship between Msn2 and its target genes, the linearity we uncovered has important implications for the dynamic operation of the system. This aspect needs to be considered in the context of the PKA regulatory system, which produces dynamic changes in Msn2 activity in response to stress. Low-affinity interactions of Msn2 with DNA induce rapid (subsecond) binding and unbinding of Msn2 to its response elements, allowing for rapid dynamic control of gene expression if the rate-limiting step for promoter activation is transcription factor binding.
In addition to enabling precise tuning of gene expression to the state of the environment, this strategy ensures colinear activation of target genes, allowing for stoichiometric expression of large groups of genes without extensive promoter tuning. Furthermore, such a strategy enables precise modulation of the activity of any given promoter by addition of binding sites without altering the qualitative relationship between different genes in a regulon. This feature renders a given regulon highly “evolvable.”
…As a result, we anticipate this strategy to be a recurring feature of many systems where homeostatic regulation is important.
Stewart-Ornstein et al. (2013) Msn2 Coordinates a Stoichiometric Gene Expression Program. Current Biology, 23(23) 2336–2345