Transcription factors (TFs) control cellular processes by binding specific DNA motifs to modulate gene expression. Motif enrichment analysis of regulatory regions can identify direct and indirect TF binding sites. Here, we created a glossary of 108 non-redundant TF-8mer “modules” of shared specificity for 671 metazoan TFs from publicly available and new universal protein binding microarray data. Analysis of 239 ENCODE TF chromatin immunoprecipitation sequencing datasets and associated RNA sequencing profiles suggest the 8mer modules are more precise than position weight matrices in identifying indirect binding motifs and their associated tethering TFs. We also developed GENRE (genomically equivalent negative regions), a tunable tool for construction of matched genomic background sequences for analysis of regulatory regions. GENRE outperformed four state-of-the-art approaches to background sequence construction. We used our TF-8mer glossary and GENRE in the analysis of the indirect binding motifs for the co-occurrence of tethering factors, suggesting novel TF-TF interactions. We anticipate that these tools will aid in elucidating tissue-specific gene-regulatory programs.

转录因子 (TF) 通过结合特定 DNA 基序来调节基因表达，从而控制细胞过程。调节区域的基序富集分析可以识别直接和间接的 TF 结合位点。在这里，我们根据公开的和新的通用蛋白质结合微阵列数据创建了一个包含 108 个非冗余 TF-8mer“模块”的词汇表，这些“模块”对 671 个后生动物 TF 具有共享特异性。对 239 ENCODE TF 染色质免疫沉淀测序数据集和相关 RNA 测序图谱的分析表明，8mer 模块在识别间接结合基序及其相关束缚 TF 方面比位置权重矩阵更精确。我们还开发了 GENRE（基因组等效阴性区域），这是一种可调工具，用于构建匹配的基因组背景序列以分析调控区域。 GENRE 的性能优于四种最先进的背景序列构建方法。我们使用 TF-8mer 术语表和 GENRE 来分析束缚因子共现的间接结合基序，表明新的 TF-TF 相互作用。我们预计这些工具将有助于阐明组织特异性基因调控程序。