A test of the pioneer factor hypothesis using ectopic liver gene activation [ATACseq]

The Pioneer Factor Hypothesis (PFH) states that pioneer factors (PFs) are a subclass of transcription factors (TFs) that bind to and open inaccessible sites and then recruit non-pioneer factors (nonPFs) that activate batteries of silent genes. We tested the PFH by expressing the endodermal PF FoxA1 and nonPF Hnf4a in K562 lymphoblast cells. While co-expression of FoxA1 and Hnf4a activated a burst of endoderm-specific gene expression, we found no evidence for functional distinction between these two TFs. When expressed independently, both TFs bound and opened inaccessible sites, activated endodermal genes, and “pioneered” for each other, although FoxA1 required fewer copies of its motif to bind at inaccessible sites. A subset of targets required both TFs, but the mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results we propose an alternative to the PFH where “pioneer activity” depends not on the existence of discrete TF subclasses, but on TF binding affinity and genomic context. To measure accessibility, we treated clonally derived K562 cell lines that were previously transduced with lentiviral vectors carrying doxycycline-inducible FoxA1, Hnf4a, or both FoxA1-Hnf4a with +/- 0.5µg/ml doxycycline for 24 hours in duplicate. We input 2e5 cells/sample into the OMNI-ATAC protocol (Corces et al. 2017) and isolate 5e4 nuclei/sample for tagmentation and library preparation. We size selected the libraries with AmpureXP beads and then sequenced them libraries on an Illumina NextSeq 500 using 2x75 paired-end reads.

先驱因子假说（Pioneer Factor Hypothesis, PFH）提出，先驱因子（pioneer factors, PFs）属于转录因子（transcription factors, TFs）的一个亚类，能够结合并打开染色质不可接近位点，随后招募非先驱因子（non-pioneer factors, nonPFs）以激活成簇的沉默基因。本研究通过在K562淋巴母细胞中表达内胚层来源的叉头框A1（FoxA1，PF）与肝细胞核因子4α（Hnf4a，nonPF），对PFH进行了验证。尽管FoxA1与Hnf4a共表达可触发内胚层特异性基因的爆发式表达，但本研究未发现这两种TF存在功能差异的证据。当单独表达时，两种TF均可结合并打开染色质不可接近位点，激活内胚层相关基因，且彼此均可发挥"先驱活性"；不过FoxA1仅需更少的基序（motif）拷贝数即可在染色质不可接近位点上完成结合。部分靶基因同时需要两种TF发挥作用，但这些靶位点上的作用模式并不符合PFH所预测的顺序性激活机制。基于上述结果，本研究提出了PFH的替代假说："先驱活性"并非取决于转录因子亚类的固有划分，而是由转录因子的结合亲和力与基因组背景共同决定。为检测染色质可及性，本研究对前期通过慢病毒载体转导、携带多西环素（doxycycline）诱导型FoxA1、Hnf4a或FoxA1-Hnf4a双基因的克隆源性K562细胞系，分别添加或不添加0.5μg/ml多西环素处理24小时，设置生物学重复两份。每个样本取2×10^5个细胞，采用OMNI-ATAC实验流程（Corces等，2017）进行处理，分离5×10^4个细胞核用于转座酶标签化与文库构建。使用AmpureXP磁珠对文库进行片段大小筛选，随后在Illumina NextSeq 500测序平台上采用2×75双端读长进行测序。