p63 是一种具有内在先锋因子活性和多效性功能的转录因子。通过经典、SMAD 和非经典 MAP 激酶 (MAPK) 途径的激活和协同作用来转化生长因子 β (TGFβ) 信号传导，从而引发抗肿瘤和促肿瘤发生特性，包括细胞干性和侵袭性。 TGFβ 激活癌细胞中的 ΔNp63 转录程序；然而，TGFβ 和 p63 在揭示肿瘤进展过程中的表观遗传景观（允许染色质可及性和基因转录）方面的联系尚未报道。小分子抑制剂，包括蛋白激酶抑制剂和 RNA 沉默，提供了功能丧失分析。利用癌细胞中的球体形成测定、染色质免疫沉淀和 mRNA 表达测定来获得机制证据。质谱分析与免疫共沉淀分析相结合，揭示了新型 p63 相互作用因子及其参与 p63 依赖性转录。乳腺癌细胞的球形形成能力在 TGFβ 刺激后增强，在 ΔNp63 耗尽后显着降低。通过 p38 MAPK 信号传导激活 TGFβ 信号传导诱导 ΔNp63 Ser 66/68 磷酸化，从而产生具有增强 DNA 结合特性的稳定 ΔNp63 蛋白。 TGFβ 刺激改变了 H3K27ac 和 H3K27me3 组蛋白修饰标记的比例，表明 H3K27ac 更高，并增加了 p300 乙酰转移酶向染色质的募集。通过沉默 ΔNp63 的表达，TGFβ 对染色质重塑的影响被消除。 H3K27me3 的抑制揭示了 TGFβ 作为引导 ΔNp63 到达 TGFβ/SMAD 基因位点的上游信号的重要作用，以及 ΔNp63 在将组蛋白修饰酶（如 p300）招募到这些基因组区域、调节染色质方面不可或缺的作用可及性和基因转录。从机制上讲，TGFβ通过SMAD激活诱导ΔNp63从NURD或NCOR/SMRT组蛋白脱乙酰化复合物上解离，同时促进ΔNp63-p300复合物的组装，影响组蛋白乙酰化水平和ΔNp63依赖性转录的结果。ΔNp63被磷酸化并招募通过 TGFβ 连接到 TGFβ/SMAD/ΔNp63 基因位点，促进与干性和细胞侵袭相关的靶基因的染色质可及性和转录。© 2024。作者。

p63 is a transcription factor with intrinsic pioneer factor activity and pleiotropic functions. Transforming growth factor β (TGFβ) signaling via activation and cooperative action of canonical, SMAD, and non-canonical, MAP-kinase (MAPK) pathways, elicits both anti- and pro-tumorigenic properties, including cell stemness and invasiveness. TGFβ activates the ΔNp63 transcriptional program in cancer cells; however, the link between TGFβ and p63 in unmasking the epigenetic landscape during tumor progression allowing chromatin accessibility and gene transcription, is not yet reported.Small molecule inhibitors, including protein kinase inhibitors and RNA-silencing, provided loss of function analyses. Sphere formation assays in cancer cells, chromatin immunoprecipitation and mRNA expression assays were utilized in order to gain mechanistic evidence. Mass spectrometry analysis coupled to co-immunoprecipitation assays revealed novel p63 interactors and their involvement in p63-dependent transcription.The sphere-forming capacity of breast cancer cells was enhanced upon TGFβ stimulation and significantly decreased upon ΔNp63 depletion. Activation of TGFβ signaling via p38 MAPK signaling induced ΔNp63 phosphorylation at Ser 66/68 resulting in stabilized ΔNp63 protein with enhanced DNA binding properties. TGFβ stimulation altered the ratio of H3K27ac and H3K27me3 histone modification marks, pointing towards higher H3K27ac and increased p300 acetyltransferase recruitment to chromatin. By silencing the expression of ΔNp63, the TGFβ effect on chromatin remodeling was abrogated. Inhibition of H3K27me3, revealed the important role of TGFβ as the upstream signal for guiding ΔNp63 to the TGFβ/SMAD gene loci, as well as the indispensable role of ΔNp63 in recruiting histone modifying enzymes, such as p300, to these genomic regions, regulating chromatin accessibility and gene transcription. Mechanistically, TGFβ through SMAD activation induced dissociation of ΔNp63 from NURD or NCOR/SMRT histone deacetylation complexes, while promoted the assembly of ΔNp63-p300 complexes, affecting the levels of histone acetylation and the outcome of ΔNp63-dependent transcription.ΔNp63, phosphorylated and recruited by TGFβ to the TGFβ/SMAD/ΔNp63 gene loci, promotes chromatin accessibility and transcription of target genes related to stemness and cell invasion.© 2024. The Author(s).