Dissecting quantitative, analogue, antisense-mediated transcriptional control

分子生物学中最基本的问题之一是如何实现定量基因表达。传统上，这种调控归因于序列特异性转录因子与调控DNA元件的结合。根据转录因子的浓度，可以定量地上调或下调基因表达。虽然在许多系统中，这种监管无疑是存在的，但近年来已经非常清楚，这种范式从根本上说是不完整的。这在真核生物中尤其如此，因为转录必须在染色质的背景下发生。现在有大量的数据表明，转录的定量调控来自于基因的局部染色质环境的调节。一种可能性是，染色质环境的改变可能允许所谓的动力学调节，例如，通过改变RNA聚合酶在基因中前进的能力。真核生物基因的另一个重要特征是非编码转录，通常在反义方向上。全基因组研究清楚地表明，这种转录是普遍存在的，但它在调节基因表达方面的作用尚不清楚，而且争论激烈。此外，在整个基因组的水平上回答这样的机械问题是困难的。因此，必须关注特定的靶位点，彻底剖析其调控机制，然后利用这些知识来正确解释全基因组数据集。我们建议在一个叫做FLC的植物基因上实施这个行动计划。FLC作为一种向开花过渡的抑制因子，因此是一种表达受到严格定量控制的基因。我们的初步数据表明，在温暖的温度条件下，FLC的表达以类似的方式控制，就像分子调光开关一样。这与它在长时间暴露于寒冷环境后的行为形成对比，在这种情况下，单个基因座的表达以全有或全无的数字方式永久关闭。我们的目标是了解模拟控制是如何通过非编码反义转录的机制定量实现的。在深层次上回答这个问题需要从分子生物学和成像到数学建模的先进多学科技术的融合，但这将使我们能够达到定量转录控制的机制核心。

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.