Dissecting quantitative, analogue, antisense-mediated transcriptional control

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One of the most fundamental questions in molecular biology is how quantitative gene expression is achieved. Traditionally, such regulation is ascribed to sequence specific transcription factors that bind to regulatory DNA elements. According to the concentration of the transcription factors, gene expression can then be quantitatively up or down regulated. While in many systems, such regulation undoubtedly occurs, it has become abundantly clear in recent years that this paradigm is fundamentally incomplete. This is especially so in eukaryotes where transcription has to occur in the context of chromatin. There is now substantial data showing that quantitative regulation of transcription comes from modulation of the local chromatin environment of a gene. One possibility is that alteration of the chromatin environment could permit so-called kinetic regulation by, for example, altering the ability of RNA polymerase to progress through a gene. A further important feature of eukaryotic genes is non-coding transcription, often in an anti-sense direction. Whole genome studies have clearly shown that such transcription is pervasive, yet its role in regulating gene expression is obscure and hotly debated. Moreover, answering such mechanistic questions is difficult at the level of whole genomes. It is therefore imperative to focus on specific target loci, thoroughly dissect their mechanisms of regulation, and then leverage this knowledge to properly interpret whole genome datasets. We propose to implement this plan of action at a plant gene called FLC. FLC functions as a repressor of the transition to flowering, and as such is a gene whose expression is under tight, quantitative control. Our preliminary data indicates that FLC expression in warm temperature conditions is controlled in an analogue fashion, like a molecular dimmer switch. This is in contrast to its behaviour after exposure to prolonged cold where expression at individual loci is switched off permanently in an all or nothing digital fashion. Our goal here is to understand how analogue control is quantitatively achieved through a mechanism that appears to function through non-coding antisense transcription. Answering this question at a deep level will require a fusion of advanced multidisciplinary techniques from molecular biology and imaging to mathematical modelling, but will allow us to get to the mechanistic heart of quantitative transcriptional control.