Dll and Sp1 ChIP-seq data from Drosophila third instar leg imaginal discs

Animal limb development relies on the establishment of organizing centers, which govern limb outgrowth and patterning by regulating the spatial and temporal expression of secreted signaling molecules. On a molecular level the establishment of organizing centers occurs via cis regulatory modules (CRMs), also known as transcriptional enhancers, that integrate upstream temporal and spatial inputs. We elucidated the mechanism that governs the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. We find that EGFR activation occurs by sequential activation of the EGFR ligand Vein (Vn) and the EGFR ligand-processing protease Rhomboid (Rho), each through single CRMs. These CRMs integrate in a distinct manner inputs from the Wingless (Wg) and Decapentaplegic (Dpp) signaling pathways, and from the leg selector transcription factors Distal-less (Dll) and Sp1. Elimination of the vn (vnE) and rho (rhoE) EOC enhancers eliminates the expression of these genes in the center of the leg imaginal discs, respectively. A vnE rhoE double deficiency, but not single deletions, demonstrates an absolute requirement of these CRMs for specifying the most distal, but not more proximal, leg fates. In addition, the cis-regulatory logic of vnE and rhoE transcends the leg EOC developmental program, because genomic regions with similar inputs, based on predicted and genome-wide binding by the transcription factors that establish the EOC, faithfully predicts novel CRMs active in the distal leg. The combinatorial input of Wg, Dpp, Dll and Sp1 at these CRMs reveals a molecular signature for coordinating gene expression in the Drosophila leg that might be analogous to many other multi-cellular systems. Triplicate pools of 100 yw and 100 recombineered Sp1-GFP (BAC) L3 wandering larvae were used to perform independent chromatin IPs. The Sp1-GFP (BAC) is a GFP-tagged Sp1 in BAC clone CH321-64M02 inserted in landing site VK00033 (gift from Dr. Rebecca Spokony). All 6 leg discs from each larva were used as material for each IP. Chromatin from the yw larvae pools was immuno-precipitated with goat anti-Dll antibody (sc-15858, Santa Cruz Biotechnology, 1.5 µg/ml for IP) while chromatin from the Sp1-GFP larvae pools was immuno-precipitated with rabbit anti-GFP antibody (ab290, Abcam, 1∶300 dilution for IP). DNA from non-immunoprecipitated 10% chromatin input was isolated from each pool as reference control.

动物肢体发育依赖于组织中心的建立：这类组织中心通过调控分泌型信号分子的时空表达模式，控制肢体的向外生长与形态建成。在分子层面，组织中心的建立经由顺式调控模块（cis regulatory modules, CRMs，又称转录增强子）完成，该类模块可整合上游的时空调控信号输入。
我们阐明了黑腹果蝇（Drosophila melanogaster）腿部发育过程中表皮生长因子受体（EGFR）组织中心（EOC）的建立机制。研究发现，EGFR的激活通过依次激活其配体脉纹蛋白（Vein, Vn）与配体加工蛋白酶菱形蛋白（Rhomboid, Rho）实现，二者各自依赖单个顺式调控模块。
这些顺式调控模块以独特的方式整合了来自无翅信号通路（Wingless, Wg）、骨形态发生蛋白样信号通路（Decapentaplegic, Dpp）的调控信号，以及腿部选择转录因子远端缺失蛋白（Distal-less, Dll）与Sp1的输入信号。
分别敲除vn的EOC增强子vnE与rho的EOC增强子rhoE，可消除这两个基因在腿部成虫盘中心的表达。vnE与rhoE的双缺失（而非单基因缺失）实验证实，这些CRMs是指定腿部最远端（而非更近端）细胞命运的绝对必需因子。
此外，vnE与rhoE的顺式调控逻辑并不局限于腿部EOC的发育程序：基于建立EOC的转录因子的预测结合位点与全基因组结合数据，具有类似调控输入的基因组区域，可精准预测出在远端腿部发挥功能的新型CRMs。Wg、Dpp、Dll与Sp1在这些CRMs上的组合调控输入，揭示了一种协调果蝇腿部基因表达的分子特征，该特征可能在众多其他多细胞生物系统中具有保守性。
本研究设置三组生物学重复样本池，每池包含100只yw品系幼虫与100只经同源重组构建的Sp1-GFP（细菌人工染色体，BAC）三龄游走幼虫，用于独立的染色质免疫沉淀（ChIP）实验。其中Sp1-GFP（BAC）是指将绿色荧光蛋白（GFP）标签整合到细菌人工染色体克隆CH321-64M02的Sp1基因上，该重组克隆被插入到插入位点VK00033，由Rebecca Spokony博士惠赠。
每只幼虫的全部6个腿部成虫盘均被用作单次ChIP实验的材料。yw品系幼虫样本池的染色质使用山羊抗Dll抗体（sc-15858, Santa Cruz Biotechnology，IP使用浓度为1.5 µg/ml）进行免疫沉淀；而Sp1-GFP幼虫样本池的染色质则使用兔抗GFP抗体（ab290, Abcam，IP稀释比例为1∶300）进行免疫沉淀。从每个样本池中提取未经免疫沉淀的10%染色质输入组分作为参考对照。