Irf6 wildtype and knock out ChIP-seq

The integrity of the mammalian epidermis is essential for organism survival, and it depends on a balance of proliferation and differentiation in the resident stem cell population. The kinase Ripk4 and the transcription factor Irf6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft tissue fusions, resulting in neonatal lethality. However, the mechanism by which these genes control epidermal differentiation in vivo is unknown. By generating various mouse knock-out and knock-in strains we demonstrate that in vivo the role of Ripk4 in development is dependent on its kinase activity, Ripk4 and Irf6 function cell autonomously in the epidermis,Ripk4 and Irf6 lie on a linear pathway and phosphorylation of Irf6 on Serine413 and Serine424 is essential to prime it for activation. This priming then allows Ripk4 to phosphorylate Irf6 on Serine90, which ensures Irf6 activation. We then use RNA-seq, ChIP-seq and ATAC-seq analysis to define the global transcriptional targets of Irf6 in epidermal differentiation. Collectively, our results explain how Ripk4 activates Irf6, and how this pathway ensures epidermal differentiation and a functional barrier. This is crucial for understanding the etiology of developmental syndromes that are characterized by orofacial, skin and genital abnormalities. ChIP-seq of 3 histone modifications (H3K4me3, H3K27ac, H3K27me3)was performed on the following genotypes in two replicates: Irf6 wildtype and Irf6 knock out. We had one pooled input sample as control.

哺乳动物表皮的完整性对生物体存活至关重要，其依赖于驻留干细胞群的增殖与分化动态平衡。激酶Ripk4与转录因子Irf6在人类严重发育综合征中发生突变，缺失这两类基因的小鼠会出现表皮过度增殖和软组织融合，最终导致新生小鼠致死。然而，这两类基因在体内调控表皮分化的具体机制仍不明确。

本研究通过构建多种基因敲除（knock-out）与敲入（knock-in）小鼠品系，证实Ripk4在发育过程中的功能依赖于其激酶活性；Ripk4与Irf6在表皮组织中以细胞自主方式发挥调控作用；二者处于同一线性通路中，且Irf6在丝氨酸413（Ser413）与丝氨酸424（Ser424）位点的磷酸化对其预活化至关重要。该预激活过程可使Ripk4能够对Irf6的丝氨酸90（Ser90）位点进行磷酸化，从而确保Irf6的完全活化。

随后，我们通过RNA测序（RNA-seq）、染色质免疫沉淀测序（ChIP-seq）以及转座酶可及性测序（ATAC-seq）分析，明确了Irf6在表皮分化过程中的全局转录靶标。综上，本研究阐明了Ripk4活化Irf6的分子机制，以及该通路如何保障表皮分化与功能屏障的建立，这对于理解以颅面、皮肤和生殖器异常为特征的发育综合征的病因学具有关键意义。

我们针对以下两种基因型的样本（各设置两个生物学重复）开展了3种组蛋白修饰（H3K4me3、H3K27ac、H3K27me3）的ChIP-seq检测：Irf6野生型与Irf6基因敲除型，并设置一份混合输入样本作为对照。