在急性早幼粒细胞白血病(APL)中,早幼粒细胞白血病-维甲酸受体α(PML/RARα)融合蛋白破坏PML核体(NBs),导致微斑的形成。然而,我们的理解,主要从形态学观察中学到的,缺乏对PML/RARα介导的微斑形成及其在APL白血病发生中的作用的机制的了解。这项研究提供了证据,揭示了液-液相分离(LLPS)是PML/RARα介导的微斑形成的关键机制。包含大部分PML和较小的RARα段的固有无序区域促进了该过程。我们证明了含溴结构域的蛋白4(BRD4)在PML/RARα介导的缩合物中的共组装,不同于野生型PML形成的NB。在没有PML/RARα的情况下,PMLNB和BRD4puncta作为两个独立的阶段存在,但是PML/RARα的存在会破坏PMLNB,并将PML和BRD4重新分布到一个不同的阶段,形成PML/RARα组装的微斑。全基因组分析揭示了PML/RARα诱导的BRD4在基因组中的再分布,与超增强子和宽启动子(SEBP)优先结合。机械上,BRD4被PML/RARα募集到核冷凝物中,促进BRD4染色质结合以发挥APL存活所必需的转录激活。通过化学抑制(1,6-己二醇)干扰LLPS可显着降低PML/RARα和BRD4的染色质共占有率,从而减弱其靶基因激活。最后,在原发性APL患者样本中的一系列实验验证证实了PML/RARα通过冷凝物形成微斑,招募BRD4共同组装冷凝物,并共同占据SEBP地区。我们的发现阐明了生物物理,病态,和PML/RARα组装的微斑的转录动力学,强调BRD4在介导使PML/RARα启动APL的转录激活中的重要性。
In acute promyelocytic leukemia (APL), the promyelocytic leukemia-retinoic acid receptor alpha (PML/RARα) fusion protein destroys PML nuclear bodies (NBs), leading to the formation of microspeckles. However, our understanding, largely learned from morphological observations, lacks insight into the mechanisms behind PML/RARα-mediated microspeckle formation and its role in APL leukemogenesis. This study presents evidence uncovering liquid-liquid phase separation (LLPS) as a key mechanism in the formation of PML/RARα-mediated microspeckles. This process is facilitated by the intrinsically disordered region containing a large portion of PML and a smaller segment of RARα. We demonstrate the coassembly of bromodomain-containing protein 4 (BRD4) within PML/RARα-mediated condensates, differing from wild-type PML-formed NBs. In the absence of PML/RARα, PML NBs and BRD4 puncta exist as two independent phases, but the presence of PML/RARα disrupts PML NBs and redistributes PML and BRD4 into a distinct phase, forming PML/RARα-assembled microspeckles. Genome-wide profiling reveals a PML/RARα-induced BRD4 redistribution across the genome, with preferential binding to super-enhancers and broad-promoters (SEBPs). Mechanistically, BRD4 is recruited by PML/RARα into nuclear condensates, facilitating BRD4 chromatin binding to exert transcriptional activation essential for APL survival. Perturbing LLPS through chemical inhibition (1, 6-hexanediol) significantly reduces chromatin co-occupancy of PML/RARα and BRD4, attenuating their target gene activation. Finally, a series of experimental validations in primary APL patient samples confirm that PML/RARα forms microspeckles through condensates, recruits BRD4 to coassemble condensates, and co-occupies SEBP regions. Our findings elucidate the biophysical, pathological, and transcriptional dynamics of PML/RARα-assembled microspeckles, underscoring the importance of BRD4 in mediating transcriptional activation that enables PML/RARα to initiate APL.