Rate Limiting Enzymes in Nucleotide Metabolism Synchronize Nucleotide Biosynthesis and Chromatin Formation [ATAC-Seq]

Fundamental cellular processes including replication, transcription, and repair rely on both an adequate nucleotide supply and sufficient availability of new histones. Chromatin disassembly and reassembly is crucial to maintain genome stability, and is regulated by the orderly engagement of histones with a series of histone chaperones that guide newly synthesized histones from ribosomes to DNA. Although the synthesis of nucleotides and the histone proteins are the two major biosynthetic processes to complete the formation of chromatin, our knowledge about the coordination of these processes is limited. Phosphoribosyl pyrophosphate synthetases (PRPSs) catalyze the rate-limiting step in the nucleotide biosynthesis pathway. PRPS enzymes form a complex with PRPS-associated proteins (PRPSAPs). In the present study, we discover that PRPS-PRPSAP enzyme complex are part of the histone chaperone network involving HSP70/90, NASP, HAT1/RBBP7 and importin-4 to regulate chromatin assembly. We show PRPS enzymes are essential not only for nucleotide biogenesis, but together with PRPSAP also play a key role in the heterodimerization of H3-H4 and posttranslational modification of nascent histones, early steps in the process of histone maturation. Depletion of PRPS proteins leads to limited histone availability and therefore to impaired chromatin assembly. Our discovery bridges nucleotide metabolism and chromatin regulation and provides first evidence on how nucleotide biogenesis and chromatin dynamics work in synchrony. ATAC seq-To evaluate the nucleosome positioning by measuring the fragment size, in response to dTAG47 mediated depletion of PRPS1, for 24 h in HEK293T cells.

包括复制、转录与修复在内的核心细胞过程，均依赖于充足的核苷酸供给与新合成组蛋白的有效供应。染色质的解聚与重建对于维持基因组稳定性至关重要，其调控依赖于组蛋白与一系列组蛋白伴侣（histone chaperone）的有序结合——这类伴侣可引导新合成的组蛋白从核糖体转运至DNA分子。
尽管核苷酸与组蛋白的合成是完成染色质组装的两大核心生物合成过程，但目前学界对这两类过程的协同调控机制认知仍十分有限。磷酸核糖焦磷酸合成酶（Phosphoribosyl pyrophosphate synthetases, PRPSs）催化核苷酸生物合成通路中的限速步骤。PRPS酶可与PRPS关联蛋白（PRPS-associated proteins, PRPSAPs）形成复合物。
本研究发现，PRPS-PRPSAP酶复合物属于组蛋白伴侣网络的一员，该网络包含HSP70/90、NASP、HAT1/RBBP7以及importin-4，可调控染色质组装过程。本研究证实，PRPS酶不仅对核苷酸生物合成不可或缺，还可与PRPSAP协同作用，在H3-H4异二聚体形成以及新生组蛋白的翻译后修饰（posttranslational modification）——组蛋白成熟过程的早期步骤——中发挥关键作用。
PRPS蛋白耗竭会导致组蛋白可获得性受限，进而造成染色质组装功能受损。本研究发现首次搭建了核苷酸代谢与染色质调控之间的关联桥梁，并为核苷酸生物合成与染色质动态过程的协同调控机制提供了首个实验证据。
ATAC测序（ATAC seq）：通过检测片段大小以评估核小体定位情况，分析HEK293T细胞经dTAG47介导的PRPS1蛋白耗竭24小时后的细胞响应。