The evolutionary origin of DNA accessibility of a new cis-regulatory activity

动物形态的进化在很大程度上是由发育基因表达转变为新的表达模式推动的。这些变化通常由调控变化所支配，例如获得新的顺式调节元件(CRE)或修改控制基因转录的现有CRE。这些DNA序列在一定距离上调节特定基因转录的地点、时间和水平，适应控制其活性的转录因子(TF)的结合。然而，要想发挥作用，Cres还需要是可访问的，即不被核小体覆盖，不像基因组序列的大多数。因此，新的Cre的出现需要两个步骤，即获得Tf结合位点和获得其序列的可及性。本研究探讨了新Cre进化出现过程中这两个步骤之间的关系。我们专注于一个明确的进化转变，在一些果蝇物种中，翅膀顶端获得了一点深色色素，这涉及到着色基因黄色的一种新的Cre的出现，Spot Cre。使用这组苍蝇中代表祖先无斑点状态或衍生斑点状态的不同物种，我们将检查包含该元素的区域的活动和可及性。特别是，我们将从赋予CRE空间活动的信息中解开DNA可及性的起源。在第一个项目中，我们将使用ATAC-SEQ描述果蝇蛹翅膀上Spot Cre可及性的进化和发育起源。我们将进一步绘制调控这种可及性的决定因素图，并测试我们在之前的基因筛查中发现的染色质因子三胸可能的直接控制。在第二个独立的项目中，我们将确定Spot Cre的功能边界，以询问它们与该区域的可及性如何相关。尽管DNA可及性结合染色质标记经常被用作增强子边界的替代，但没有关于两者之间实际关系的定量数据。我们将使用定制的定量成像系统来精确绘制机翼中CRE的空间活动图。这一图谱将揭示自然选择下DNA的确切片段，从而代表完整的Cre进化单位。总之，这一提议将阐明Cre活性与可及性之间的关系，以及重要的是这种可及性的进化起源，这是出现新的调节活动的关键一步。

The evolution of animal forms is driven to a large extent by changes in the expression of developmental genes into new expression patterns. These changes are often governed by regulatory changes, such as the gain of new cis-regulatory elements (CREs) or the modification of existing CREs controlling the transcription of a gene. These DNA sequences, modulating at a distance the places, times and levels at which particular genes are transcribed, accomodate the binding of transcription factors (TFs) controlling their activity. To be active, however, CREs also need to be accessible, i.e. no covered by nucleosomes, unlike the majority of the genome sequence. The emergence of a new CRE therefore takes 2 steps, the acquisition of TF binding sites, and the gain of accessibility to its sequence.This proposal examines the relationship between these 2 steps during the evolutionary emergence of a new CRE. We focus on a defined evolutionary transition, the gain of a spot of dark pigment at the tip of the wings in some Drosophila species, that involves the emergence of a new CRE of the pigmentation gene yellow, the spot CRE. Using different species from this group of flies that represent either the ancestral unspotted state, or the derived spotted state, we will examine the activity and accessibility of the region containing this element. In particular, we will disentangle the origin of DNA accessibility from the information imparting the spatial activity of the CRE.In a first project, we will describe the evolutionary and developmental origin of the spot CRE accessibility using ATAC-seq on Drosophila pupal wings. We will further map the determinants regulating this accessibility, and test the possible direct control by the chromatin factor trithorax, that we identified in a previous genetic screen.In a second, independent project, we will determine the functional boundaries of the spot CRE to ask how they relate to the accessibility of the region. Although DNA accessibility combined to chromatin marks is often used as a proxy for enhancer boundaries, there is no quantitative data on the actual relationship between both. We will use a custom quantitative imaging system to precisely map spatial CRE activity in the wings. This mapping will reveal the exact segment of DNA under natural selection and thereby representing the full spot CRE evolutionary unit.Altogether, this proposal will shed light on the relationship between CRE activity and accessibility, and importantly on the evolutionary origin of this accessibility, a key step in the emergence of a new regulatory activity.