Transcription Factor Condensates Mediate Clustering of MET Regulon and Enhancement in Gene Expression [MeDIP]

Some transcription factors (TFs) can form liquid-liquid phase separated (LLPS) condensates. However, the function of these TF condensates in 3D genome organization and gene regulation remains elusive. In response to methionine (met) starvation in budding yeast, Met4 and a few sequence-specific co-activators, including Met32, induce a set of genes involved in met biosynthesis. Here, we show that the endogenous Met4 and Met32 form puncta-like structures that significantly overlap in yeast nuclei upon met depletion. Recombinant Met4 and Met32 form mixed droplets with LLPS properties in vitro. In relation to chromatin, Met4 puncta co-localize with target genes, and at least a subset of these target genes are clustered in 3D in a Met4-dependent manner. A MET3pr-GFP reporter inserted near several native Met4 binding sites becomes co-localized with Met4 puncta and displays enhanced transcriptional activity. A Met4 variant with a partial truncation of an intrinsically disordered region (IDR) shows less puncta formation, and this mutant selectively reduces the reporter activity near Met4 binding sites to the basal level. Overall, these results support a model where Met4 and co-activators form condensates to bring multiple target genes into a vicinity with higher local TF concentrations, which facilitates a strong response to met depletion (-met). MeDip of MTAC strains with MET13, MET6 as VP in MET active (-met) and inactive (+met) conditions with +/- IAA for Met4 depletion.

部分转录因子（transcription factors, TFs）可形成液液相分离（liquid-liquid phase separated, LLPS）凝聚体。然而，这类转录因子凝聚体在三维基因组组织与基因调控中的功能仍未明确。在出芽酵母遭遇甲硫氨酸（methionine, met）饥饿时，Met4与包括Met32在内的数种序列特异性共激活因子，可诱导一系列参与甲硫氨酸生物合成的基因表达。本研究发现，在甲硫氨酸耗尽条件下，内源性Met4与Met32可在酵母细胞核内形成存在显著重叠的点状结构。体外实验中，重组Met4与Met32可形成具备液液相分离特性的混合液滴。就染色质而言，Met4点状结构可与靶基因共定位，且此类靶基因中至少有一部分以Met4依赖的方式在三维空间中发生聚集。将MET3pr-GFP报告基因插入至数个内源性Met4结合位点附近后，该报告基因可与Met4点状结构发生共定位，且转录活性显著增强。对Met4的内在无序区域（intrinsically disordered region, IDR）进行部分截短的突变体，其点状结构形成能力显著减弱，且该突变体可选择性地将Met4结合位点附近的报告基因活性降至基础水平。综上，本研究结果支持如下模型：Met4与共激活因子通过形成凝聚体，将多个靶基因聚集至局部转录因子浓度更高的区域，从而助力细胞对甲硫氨酸耗尽（-met）产生强烈应答。针对处于甲硫氨酸激活（-met）与失活（+met）状态的MTAC菌株，以MET13、MET6作为VP，在添加/不添加吲哚-3-乙酸（IAA）以耗尽Met4的条件下开展MeDIP实验。