IPMK physically binds to the SWI/SNF complex and modulates BRG1 occupancy

Inositol polyphosphate multikinase (IPMK), a key enzyme in the inositol polyphosphate (IP) metabolism, is a pleiotropic signaling factor involved in major biological events, including transcriptional control. In the yeast, IPMK and its IP products promote the activity of the chromatin-remodeling complex SWI/SNF, which plays a critical role in gene expression by regulating chromatin accessibility. However, the direct link between IPMK and chromatin remodelers remains unclear, raising a question on how IPMK contributes to the transcriptional regulation in mammals. By employing unbiased screening approaches and in vivo/in vitro immunoprecipitations, here we demonstrated that mammalian IPMK physically interacts with the SWI/SNF complex by directly binding to SMARCB1, BRG1, and SMARCC1. Furthermore, we identified the specific domains required for the IPMK-SMARCB1 binding. Notably, using the CUT&RUN and ATAC-seq assays, we discovered that IPMK co-localizes with BRG1 and regulates BRG1 localization as well as BRG1-mediated chromatin accessibility in a genome-wide manner in mouse embryonic stem cells. Finally, our mRNA-seq analyses revealed that IPMK and SMARCB1 regulate the expression of a common gene set, validating a functional link between IPMK and the SWI/SNF complex. Together, these findings show that IPMK regulates promoter-targeting of the SWI/SNF complex and thereby contributes to SWI/SNF-meditated chromatin accessibility, transcription, and differentiation. For RNA-seq experiment: Strand-specific mRNA-Seq of Control (EgfpKD) and IpmkKD in mouse embryonic stem cells (mESCs) and mouse embryonic fibroblasts (MEFs). For ATAC-seq experiment: Genome-wide analysis of chromatin accessibility upon IPMK depletion in mESCs. For CUT&RUN experiment: Genome-wide distributions of BRG1 upon IPMK depletion (siRNA-mediated and shRNA-mediated) in mESCs.

肌醇多磷酸多激酶（Inositol polyphosphate multikinase, IPMK）是肌醇多磷酸（inositol polyphosphate, IP）代谢通路中的关键酶，亦是一类参与转录调控等多种重要生物学事件的多效性信号因子。在酵母中，IPMK及其IP代谢产物可促进染色质重塑复合物SWI/SNF的活性，该复合物通过调控染色质可及性在基因表达中发挥关键作用。然而，IPMK与染色质重塑因子之间的直接关联仍不明确，这引发了一个科学问题：IPMK在哺乳动物体内如何参与转录调控？
本研究采用无偏筛选方法及体内/体外免疫沉淀实验，证实哺乳动物IPMK可通过直接结合SMARCB1、BRG1与SMARCC1，与SWI/SNF复合物发生物理相互作用。此外，本研究还鉴定了IPMK与SMARCB1结合所需的特定结构域。值得注意的是，借助切割-释放测序（CUT&RUN）与转座酶可及性测序（ATAC-seq）实验，本研究在小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）中开展全基因组水平分析，发现IPMK可与BRG1共定位，并以基因组规模调控BRG1的定位以及BRG1介导的染色质可及性。最后，本研究的信使RNA测序（mRNA-seq）分析显示，IPMK与SMARCB1可共同调控一组基因的表达，验证了IPMK与SWI/SNF复合物之间存在功能关联。综上，本研究结果表明，IPMK可调控SWI/SNF复合物的启动子靶向定位，进而参与SWI/SNF介导的染色质可及性调控、基因转录及细胞分化过程。
mRNA测序实验：针对小鼠胚胎干细胞（mESCs）与小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）中的对照组（EgfpKD）与IpmkKD样本，开展链特异性mRNA测序。
转座酶可及性测序实验：在小鼠胚胎干细胞中，对IPMK敲低后的染色质可及性进行全基因组分析。
切割-释放测序实验：在小鼠胚胎干细胞中，分析经小干扰RNA（siRNA）介导及短发夹RNA（shRNA）介导的IPMK敲低后，BRG1的全基因组分布情况。