DelRosso Nicole, Suzuki Peter H, Griffith Daniel, Lotthammer Jeffrey M, Novak Borna, Kocalar Selin, Sheth Maya U, Holehouse Alex S, Bintu Lacramioara, Fordyce Polly
Biophysics Program, Stanford University, Stanford, CA, USA.
Department of Bioengineering, Stanford University, Stanford, CA, USA.
bioRxiv. 2024 Aug 20:2024.08.19.608698. doi: 10.1101/2024.08.19.608698.
Sequence-specific activation by transcription factors is essential for gene regulation. Key to this are activation domains, which often fall within disordered regions of transcription factors and recruit co-activators to initiate transcription. These interactions are difficult to characterize via most experimental techniques because they are typically weak and transient. Consequently, we know very little about whether these interactions are promiscuous or specific, the mechanisms of binding, and how these interactions tune the strength of gene activation. To address these questions, we developed a microfluidic platform for expression and purification of hundreds of activation domains in parallel followed by direct measurement of co-activator binding affinities (STAMMPPING, for Simultaneous Trapping of Affinity Measurements via a Microfluidic Protein-Protein INteraction Generator). By applying STAMMPPING to quantify direct interactions between eight co-activators and 204 human activation domains (>1,500 s), we provide the first quantitative map of these interactions and reveal 334 novel binding pairs. We find that the metazoan-specific co-activator P300 directly binds >100 activation domains, potentially explaining its widespread recruitment across the genome to influence transcriptional activation. Despite sharing similar molecular properties ( enrichment of negative and hydrophobic residues), activation domains utilize distinct biophysical properties to recruit certain co-activator domains. Co-activator domain affinity and occupancy are well-predicted by analytical models that account for multivalency, and affinities quantitatively predict activation in cells with an ultrasensitive response. Not only do our results demonstrate the ability to measure affinities between even weak protein-protein interactions in high throughput, but they also provide a necessary resource of over 1,500 activation domain/co-activator affinities which lays the foundation for understanding the molecular basis of transcriptional activation.
转录因子的序列特异性激活对于基因调控至关重要。关键在于激活结构域，其通常位于转录因子的无序区域内，并招募共激活因子以启动转录。这些相互作用很难通过大多数实验技术来表征，因为它们通常很弱且是瞬时的。因此，我们对于这些相互作用是杂乱的还是特异的、结合机制以及这些相互作用如何调节基因激活强度知之甚少。为了解决这些问题，我们开发了一种微流控平台，用于并行表达和纯化数百个激活结构域，随后直接测量共激活因子的结合亲和力（STAMMPPING，即通过微流控蛋白质-蛋白质相互作用发生器同时捕获亲和力测量）。通过应用STAMMPPING来量化八种共激活因子与204个人类激活结构域之间的直接相互作用（>1500次），我们提供了这些相互作用的首张定量图谱，并揭示了334个新的结合对。我们发现后生动物特异性共激活因子P300直接结合>100个激活结构域，这可能解释了它在全基因组中广泛募集以影响转录激活的现象。尽管激活结构域具有相似的分子特性（富含负电荷和疏水残基），但它们利用不同的生物物理特性来募集某些共激活因子结构域。共激活因子结构域的亲和力和占有率可以通过考虑多价性的分析模型得到很好的预测，并且亲和力可以定量预测细胞中的超敏反应激活情况。我们的结果不仅证明了高通量测量甚至是弱蛋白质-蛋白质相互作用之间亲和力的能力，而且还提供了超过1500个激活结构域/共激活因子亲和力的必要资源，为理解转录激活的分子基础奠定了基础。
