Smad4-p65 interactions drive BMP-mediated protection against inflammatory cell death [ChIP-Seq]

Inflammation-induced osteoblast death undermines bone homeostasis and can aggravate bone-destructive diseases, yet how pro-survival BMP signaling intersects with inflammatory TNF–NF-?B signaling remains poorly defined. Here we show that BMP4 protects mouse osteoblasts from TNF-a–triggered apoptosis and cytotoxic death and that this cytoprotection requires both Smad4 and NF-?B p65. Integrative epigenomic profiling (ChIP-seq/ATAC-seq/RNA-seq) reveals extensive Smad4–p65 co-occupancy and identifies Samd9l as a prominent TNF-a–inducible gene that is selectively repressed by BMP4. Two Smad4/p65-bound distal elements physically contact the Samd9l promoter, exhibit TNF-responsive enhancer activity, and are required for Samd9l induction, as demonstrated by 4C-seq and CRISPR/dCas9-based chromatin closing/opening. Functionally, Samd9l depletion attenuates TNF-a–driven caspase activation and cytotoxicity in osteoblasts and enhances BMP2-driven ectopic bone formation in vivo. Cross-species mapping suggests that the two mouse regulatory elements converge into a single promoter-proximal composite element at the human SAMD9L locus, supporting a conserved inflammatory control node. Together, these findings define a BMP4–Smad4 mechanism that reshapes NF-?B enhancer outputs to limit osteoblast death and nominate the SAMD9L regulatory circuit as a therapeutic entry point for inflammatory bone loss. Overall design: Stimulus-dependent ChIP-seq profiling of p65 and Smad4 occupancy in murine osteoblastic cells

炎症诱导的成骨细胞（osteoblast）死亡会破坏骨稳态（bone homeostasis）并加重骨破坏性疾病，但促生存的骨形态发生蛋白（BMP）信号通路与炎症性肿瘤坏死因子-核因子κB（TNF–NF-κB）信号通路的互作机制仍尚不明确。本研究发现，BMP4可保护小鼠成骨细胞免受肿瘤坏死因子α（TNF-α）诱导的细胞凋亡及毒性死亡，且该细胞保护作用同时依赖Smad4与NF-κB p65。整合表观基因组分析（ChIP-seq/ATAC-seq/RNA-seq）揭示了大量Smad4与p65的共结合区域，并鉴定出Samd9l是受TNF-α显著诱导、且可被BMP4选择性抑制的基因。两个被Smad4/p65结合的远端调控元件可与Samd9l启动子发生物理相互作用，展现出TNF-α响应的增强子活性，且是Samd9l诱导所必需的，该结论通过4C-seq以及基于CRISPR/dCas9的染色质闭合/开放实验得到验证。功能实验表明，敲低Samd9l可减弱成骨细胞中TNF-α介导的半胱天冬酶（caspase）激活及毒性死亡，并在体内增强BMP2诱导的异位骨形成。跨物种比对分析显示，小鼠的这两个调控元件在人类SAMD9L基因座上汇聚为一个单一的启动子近端复合元件，印证了该调控节点的进化保守性。综上，本研究阐明了BMP4-Smad4通路通过重塑NF-κB增强子活性以限制成骨细胞死亡的分子机制，并将SAMD9L调控环路定为炎症性骨丢失的潜在治疗靶点。实验整体设计：对小鼠成骨细胞中p65与Smad4的结合情况开展刺激依赖型ChIP-seq分析。