During development, transcription factors select distinct gene programs, providing the necessary regulatory complexity for temporal and tissue-specific gene expression. How related factors retain specificity, especially when they recognize the same DNA motifs, is not understood. We address this paradox using basic helix-loop-helix (bHLH) transcription factors ASCL1, ASCL2, and MYOD1, crucial mediators of lineage specification. In vivo, these factors recognize the same DNA motifs, yet bind largely different genomic sites and regulate distinct transcriptional programs. This suggests that their ability to identify regulatory targets is defined either by the cellular environment of the partially defined lineages in which they are endogenously expressed, or by intrinsic properties of the factors themselves. To distinguish between these mechanisms, we directly compared the chromatin binding properties of this subset of bHLH factors when ectopically expressed in embryonic stem cells, presenting them with a common chromatin landscape and cellular components. We find that these factors retain distinct binding sites; thus, specificity of binding is an intrinsic property not requiring a restricted landscape or lineage-specific cofactors. Although the ASCL factors and MYOD1 have some distinct DNA motif preference, it is not sufficient to explain the extent of the differential binding. All three factors can bind inaccessible chromatin and induce changes in chromatin accessibility and H3K27ac. A reiterated pattern of DNA binding motifs is uniquely enriched in inaccessible chromatin at sites bound by these bHLH factors. These combined properties define a subclass of lineage-specific bHLH factors and provide context for their central roles in development and disease.

在发育过程中，转录因子选择不同的基因程序，从而为时间和组织特异性基因表达提供必要的调节复杂性。相关因素如何保持特异性，尤其是当它们识别相同的DNA模体时，尚不清楚。我们使用基本的螺旋-环-螺旋（bHLH）转录因子ASCL1，ASCL2和MYOD1（谱系规范的关键介体）解决了这一悖论。在体内，这些因子识别相同的DNA基序，但结合了大大不同的基因组位点并调节不同的转录程序。这表明它们鉴定调控靶标的能力是由内源表达它们的部分定义谱系的细胞环境或因子本身的固有性质所决定的。为了区分这些机制，我们在胚胎干细胞中异位表达时直接比较了该bHLH因子子集的染色质结合特性，为它们提供了常见的染色质分布和细胞成分。我们发现这些因素保留了独特的结合位点。因此，结合的特异性是不需要限制景观或谱系特异性辅因子的内在特性。尽管ASCL因子和MYOD1具有一些不同的DNA基序偏好，但不足以解释差异结合的程度。所有这三个因素均可结合难以获得的染色质，并诱导染色质可及性和H3K27ac发生变化。重复的DNA结合基序模式在这些bHLH因子结合的位点上独特地富集了难以接近的染色质。