Genetic determinants and epigenetic effects of pioneer factor binding [ChIP-seq]

Transcription factors (TFs) are the core drivers of gene regulatory networks that control developmental transitions and a complete understanding of how they access, alter and maintain specific gene expression patterns remains an important goal. To begin a systematic dissection of the molecular components that either enable or constrain TF activity, we investigated the genomic occupancy of two distinct TFs, the pioneer factor FOXA2 and the pluripotency-associated factor OCT4 (POU5F1), in both endogenous and ectopic settings. We find that, while stable binding of FOXA2 is highly cell type specific and similar to what is observed for most TFs including OCT4, pioneer activity can be distinguished by notable sampling of additional loci that are occupied in alternative lineages. In our ectopic system, FOXA2 binding can be selectively stabilized at previously sampled sites by co-expressing the lineage specific regulator GATA4. Alternatively, we observe minimal influence of chromatin state on discrete, stabilized binding choices for FOXA2 but a strong bias towards open chromatin for ectopic OCT4 targets. Finally, we demonstrate that FOXA2 binding and nucleosome remodeling at silent loci can occur when the cell cycle is halted in G1, but surprisingly subsequent changes in DNA methylation require DNA replication. Taken together, our results provide several new molecular insights that contribute to our basic understanding of gene regulation and pave the way for a more rational use of ectopic TFs for cellular reprogramming. Overall design: In order to understand the binding dynamics of a pioneer transcription factor and its underlying chromatin environment, we performed high through-put sequencing for FOXA1 ChIP along with several histone marks. Cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature, and quenched with 125mM glycine at room temperature. Chips were performed as previously described(Gifford et al., 2013) by isolating nuclei and shearing DNA to 200-600 basepair fragments using Branson sonicator.Antibody incubation with chromatin was performed overnight. ~10 million cells were used per FOXA2 ChIP with 1ug of antibody/ million cells. ~1 million cells were used for each histone ChIP. Following an overnight incubation, antibody-protein complexes were isolated using Protein G/A beads (Life Technologies) and sequencing libraries were generated. Libraries were generated as previously described (Mikkelsen et al., 2010, Gifford et al., 2013) and Libraries were sequenced on a HiSeq 2500 at 11pmol. Please note that the processed data ''RPKM_Table.txt'' was generated from all ATAC-seq and ChIP-seq studies (GSE90453, GSE90454, GSE92490, GSE92491) and is linked to the GSE90454 records. The file MatchingSampleTable.xlsx includes a key to the column headers in RPKM_Table.txt and the sample titles from all the above-listed ATAC-seq and ChIP-seq studies. The RPKM table includes re-analyzed data and these samples are listed in Re-AnalysisTable.xlsx. Links to the re-analyzed samples can be found below.转录因子（Transcription factors, TFs）是调控发育转变的基因调控网络的核心驱动因子，完整阐明其如何结合、调控并维持特定基因表达模式，仍是当前重要的研究目标。 为系统解析能够调控或限制TF活性的分子组分，我们在内源与异位表达环境下，对两种不同的转录因子——先驱因子FOXA2与多能性相关因子OCT4（POU5F1）——的基因组结合位点开展了研究。研究发现，尽管FOXA2的稳定结合具有高度细胞类型特异性，与包括OCT4在内的多数转录因子一致，但其先驱活性可通过显著靶向其他谱系中已被占据的额外基因组位点加以区分。在异位表达系统中，共表达谱系特异性调控因子GATA4可选择性稳定FOXA2在先前已靶向位点的结合。与之相对，我们观察到染色质状态对FOXA2离散且稳定的结合选择几乎无影响，但异位表达的OCT4靶标则强烈偏向开放染色质区域。最后我们证实，当细胞周期停滞于G1期时，沉默位点处的FOXA2结合与核小体重塑均可发生，但令人意外的是，后续DNA甲基化的改变则依赖于DNA复制。综上，我们的研究结果为基因调控的基础认知提供了多项新的分子层面见解，并为更合理地利用异位转录因子进行细胞重编程铺平了道路。 整体实验设计：为解析先驱转录因子的结合动力学及其伴随的染色质环境特征，我们针对FOXA1染色质免疫共沉淀（ChIP）及多种组蛋白修饰标记开展了高通量测序。实验中，细胞于室温下用1%甲醛交联10分钟，随后用125mM甘氨酸终止交联反应。染色质免疫共沉淀实验参照既往报道（Gifford等，2013）开展：先分离细胞核，再用Branson超声破碎仪将DNA剪切为200~600 bp的片段。染色质与抗体的孵育过程于过夜完成。每份FOXA2 ChIP实验使用约1000万个细胞，按每百万细胞对应1μg抗体的比例添加抗体；每份组蛋白ChIP实验则使用约100万个细胞。过夜孵育后，使用Protein G/A磁珠（Life Technologies）分离抗体-蛋白复合物，并构建测序文库。测序文库的构建参照既往报道（Mikkelsen等，2010；Gifford等，2013），随后在HiSeq 2500测序平台上以11 pmol的上机量进行测序。 请注意，处理后数据"RPKM_Table.txt"源自所有ATAC测序（ATAC-seq）与ChIP测序（ChIP-seq）研究（GSE90453、GSE90454、GSE92490、GSE92491），并与GSE90454的记录相关联。文件"MatchingSampleTable.xlsx"包含了"RPKM_Table.txt"的列标题索引，以及上述所有ATAC-seq与ChIP-seq研究的样本名称。RPKM表格包含重新分析的数据，这些样本列于"Re-AnalysisTable.xlsx"中。重新分析样本的链接可参见下文。