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向新的表达模式的表达变化所驱动的。这些变化通常受到调控变化的控制，例如新的顺式调控元件（cre）的获得或控制基因转录的现有cre的修饰。这些DNA序列在一定距离内调节特定基因转录的地点、时间和水平，适应转录因子（tf）的结合，控制它们的活性。然而，与大多数基因组序列不同，cre还需要易于获取，即不被核小体覆盖。因此，新CRE的出现需要两个步骤，获得TF结合位点，获得其序列的可及性。本文研究了在新CRE的进化过程中这两个步骤之间的关系。我们关注的是一个明确的进化转变，在一些果蝇物种的翅膀尖端获得了一个深色色素斑点，这涉及到色素沉着基因黄色的新CRE的出现，斑点CRE。使用来自这组果蝇的不同物种，代表祖先的无斑点状态或衍生的斑点状态，我们将检查包含该元素的区域的活性和可及性。特别是，我们将把DNA可及性的起源与传递CRE空间活动的信息分开。在第一个项目中，我们将利用果蝇蛹翅膀上的ATAC-seq描述斑点CRE可达性的进化和发育起源。我们将进一步绘制调节这种可及性的决定因素，并测试我们在之前的遗传筛选中发现的染色质因子三胸可能的直接控制。在第二个独立项目中，我们将确定现场CRE的功能边界，并询问它们与该地区的可达性之间的关系。虽然结合染色质标记的DNA可接近性通常被用作增强子边界的代理，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空间CRE活动。该图谱将揭示自然选择下DNA的确切片段，从而代表完整的点CRE进化单位。总之，这一建议将阐明CRE活性与可及性之间的关系，重要的是阐明这种可及性的进化起源，这是新调控活动出现的关键一步。

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.