As the major cell precursors in osteogenesis, mesenchymal stem cells (MSCs) are indispensable for bone homeostasis and development. However, the primary mechanisms regulating osteogenic differentiation are controversial. Composed of multiple constituent enhancers, super enhancers (SEs) are powerful cis-regulatory elements that identify genes that ensure sequential differentiation. The present study demonstrated that SEs were indispensable for MSC osteogenesis and involved in osteoporosis development. Through integrated analysis, we identified the most common SE-targeted and osteoporosis-related osteogenic gene, ZBTB16. ZBTB16, positively regulated by SEs, promoted MSC osteogenesis but was expressed at lower levels in osteoporosis. Mechanistically, SEs recruited bromodomain containing 4 (BRD4) at the site of ZBTB16, which then bound to RNA polymerase II-associated protein 2 (RPAP2) that transported RNA polymerase II (POL II) into the nucleus. The subsequent synergistic regulation of POL II carboxyterminal domain (CTD) phosphorylation by BRD4 and RPAP2 initiated ZBTB16 transcriptional elongation, which facilitated MSC osteogenesis via the key osteogenic transcription factor SP7. Bone-targeting ZBTB16 overexpression had a therapeutic effect on the decreased bone density and remodeling capacity of Brd4^fl/fl Prx1-cre mice and osteoporosis (OP) models. Therefore, our study shows that SEs orchestrate the osteogenesis of MSCs by targeting ZBTB16 expression, which provides an attractive focus and therapeutic target for osteoporosis.

作为成骨过程中的主要细胞前体，间充质干细胞（MSC）对于骨稳态和发育至关重要。然而，调节成骨分化的主要机制存在争议。超级增强子 (SE) 由多种增强子组成，是强大的顺式调控元件，可识别确保顺序分化的基因。目前的研究表明，SE 对于 MSC 成骨是不可或缺的，并参与骨质疏松症的发展。通过综合分析，我们确定了最常见的SE靶向且与骨质疏松相关的成骨基因ZBTB16。ZBTB16受到 SE 的正向调节，促进 MSC 成骨，但在骨质疏松症中表达水平较低。从机制上讲，SEs 在ZBTB16位点招募了含溴结构域 4 (BRD4) ，然后与 RNA 聚合酶 II 相关蛋白 2 (RPAP2) 结合，将 RNA 聚合酶 II (POL II) 转运到细胞核中。随后 BRD4 和 RPAP2 对 POL II 羧基末端结构域 (CTD) 磷酸化的协同调节启动了ZBTB16转录延伸，从而通过关键的成骨转录因子 SP7 促进 MSC 成骨。骨靶向ZBTB16过表达对Brd4 ^fl/fl Prx1降低的骨密度和重塑能力有治疗作用-cre小鼠和骨质疏松症（OP）模型。因此，我们的研究表明SEs通过靶向ZBTB16表达来协调MSCs的成骨，这为骨质疏松症提供了一个有吸引力的焦点和治疗靶点。