Synthetic Hunchback P2 Sequences for “Dissecting the sharp response of a canonical developmental enhancer reveals multiple sources of cooperativity”

This file contains sequences of synthetic versions of the Hunchback P2 enhancer, computationally designed to contain only Bicoid binding sites and various amounts of flanking sequence. These synthetic enhancers drive expression in the anterior of the Drosophila blastoderm embryo. To create SynHbP2, SiteOut was used to remove motifs of interest (Estrada et al., 2016a). First the sequence of WThbP2 was scrambled to remove known motifs of TFs (Bcd, Cad, dStat, D, Gt, Hb, Kni, Kr, Nub, Tll, Zld) involved in AP patterning of the blastoderm embryo. Then Bcd TFBS were restored to their native locations and the resultant sequence checked for any newly created motifs around the restored Bcd TFBS using PATSER (http://stormo.wustl.edu/software.html). The binding motifs are from FlyFactorSurvey (http://pgfe.umassmed.edu/ffs/; Noyes et al., 2008b), and a pseudocount of 0.1 and a GC (guanine and cytosine) content of 0.406 when generating position weight matrices from these count matrices was used.

本文件包含驼背P2增强子（Hunchback P2 enhancer）的合成序列变体，此类序列经计算机设计，仅包含Bicoid结合位点（Bicoid binding site）与不同长度的侧翼序列。这类合成增强子可驱动果蝇囊胚胚胎前部的基因表达。为构建SynHbP2，研究人员使用SiteOut工具移除目标转录因子基序（Estrada等，2016a）。首先将野生型驼背P2（WThbP2）的序列进行随机打乱，以去除参与囊胚胚胎前后轴模式建成的各类转录因子（包括Bcd、Cad、dStat、D、Gt、Hb、Kni、Kr、Nub、Tll、Zld）的已知结合基序。随后将Bcd转录因子结合位点恢复至其原始位置，并通过PATSER软件（http://stormo.wustl.edu/software.html）检测恢复的Bcd结合位点周边是否存在新生成的结合基序。本次研究使用的结合基序源自FlyFactorSurvey数据库（http://pgfe.umassmed.edu/ffs/；Noyes等，2008b），在从计数矩阵生成位置权重矩阵（position weight matrix, PWM）时，采用了0.1的伪计数（pseudocount）与0.406的GC（鸟嘌呤与胞嘧啶）含量参数。