Irf1-and Egr1-activated transcription plays a key role in macrophage polarization: A multiomics sequencing study with partial validation
Irf1-and Egr1-activated transcription plays a key role in macrophage polarization: A multiomics sequencing study with partial validation
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DOI：
10.1016/j.intimp.2021.108072
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发表时间：
2021-08-16
影响因子：
5.6
通讯作者：
Zhang, Shi
中科院分区：
文献类型：
--
作者：
Chu, Yan-Biao;Li, Jun;Zhang, Shi
背景：巨噬细胞极化在各种临床疾病的发病机制和解决中具有因果作用。DNA结合转录因子（DNA binding transcription factors，TF）是基因转录过程中的重要因子。更好地了解调节巨噬细胞极化的TF可以提供新的治疗靶点。研究方法：利用IFN-γ（50 ng/mL）或IL 4（20 ng/mL）刺激来自小鼠的骨髓衍生的巨噬细胞24 h，分别用于M1和M2极化的巨噬细胞模型构建。首先，进行ATAC-seq（具有高通量测序的靶向可降解染色质的测定）和基序分析以鉴定参与M1和M2巨噬细胞极化的潜在转录因子（TF）。其次，通过RNA-seq鉴定必需的TF，然后比较它们在M0极化和M1/M2极化的巨噬细胞中的表达。此外，利用RNA-seq（鉴定的TF的siRNA敲低）、ChIP-seq和ATAC-seq的多组学分析来探索TF调节的分子网络。通过GO和KEGG分析阐明了TF调控的分子网络的主要功能。最后，通过流式细胞术、ELISA和qPCR验证前5个TF调节基因。高通量测序和qPCR的截止值分别为FDR < 0.05和P < 0.05。结果如下：与M0巨噬细胞相比，在M1和M2巨噬细胞极化期间，ATAC-seq分别鉴定出10，771和4，848个峰（FDR < 0.05）。分别鉴定了50个和62个TF结合基序用于参与M1和M2巨噬细胞极化的TF。M1和M2巨噬细胞中最显著高度表达的TF通过RNA-seq鉴定为Irf 1和Egr 1，LogFC值分别为3.2和2.8。多组学分析进一步发现，Irf 1调控90个基因的转录，Egr 1调控116个基因的转录。Irf 1调控的分子网络在M1巨噬细胞的炎症反应和病毒防御中起关键作用，116个Egr 1调控的基因包括细胞增殖和细胞增殖基因。验证实验表明，IFN-γ诱导的siIrf 1-BMDMs中Gbp 5、Nos 2、CD 86、Cxcl 10和Cxcl 5表达显著下调，IL 4诱导的siEgr 1-BMDMs中Itgax、Nipal 1、Bhlhe 40、CD 206和Ffar 4表达显著下调（P < 0.05）。结论：通过表观遗传测序和RNA-seq的多组学分析与部分验证，目前的研究发现，Irf 1和Egr 1诱导的转录分别在M1和M2巨噬细胞极化中起关键作用。
Background: Macrophage polarization has a causal role in the pathogenesis and resolution of various clinical diseases. DNA-binding transcription factors (TFs) have been identified as essential factors during gene transcription. Better insight into the TFs that regulate macrophage polarization could provide novel therapeutic targets. Methods: IFN-gamma (50 ng/mL) or IL4 (20 ng/mL) was utilized to stimulate bone marrow-derived macrophages from mice for 24 h for M1-and M2-polarized macrophage model construction, respectively. First, ATAC-seq (Assay for Targeting Accessible-Chromatin with high throughout sequencing) and motif analysis were conducted to identify potential transcription factors (TFs) involved in M1 and M2 macrophage polarization. Second, essential TFs were identified through RNA-seq, after which, their expression was compared between M0-polarized and M1/M2polarized macrophages. Furthermore, a multiomic analysis of RNA-seq (siRNA knock down of the identified TFs), ChIP-seq and ATAC-seq was utilized to explore the TF-regulated molecular network. GO and KEGG analyses were used to expound the main functions of the TF-regulated molecular network. Finally, the top 5 TF-regulated genes were validated through flow cytometry, ELISA and qPCR. The cut-off values for high-throughput sequencing and qPCR were FDR < 0.05 and P < 0.05, respectively. Results: Compared with M0 macrophages, 10,771 and 4,848 peaks were identified by ATAC-seq during M1 and M2 macrophage polarization, respectively (FDR < 0.05). Fifty and 62 TF binding motifs were identified for the TFs that participate in M1 and M2 macrophage polarization, respectively. The most significantly highly expressed TFs in M1 and M2 macrophages were identified by RNA-seq as Irf1 and Egr1, with LogFC values of 3.2 and 2.8, respectively. Multiomic analyses further found that Irf1 regulated the transcription of 90 genes and that Egr1 regulated the transcription of 116 genes. The Irf1-regulated molecular network played a key role in the inflammatory response and viral defence of M1 macrophages, and 116 Egr1-regulated genes included antiinflammatory and cell proliferation genes. Validation experiments indicated that IFN-gamma-induced Gbp5, Nos2, CD86, Cxcl10 and Cxcl5 expression was significantly downregulated in siIrf1-BMDMs, and IL4-induced Itgax, Nipal1, Bhlhe40, CD206 and Ffar4 expression was significantly downregulated in siEgr1-BMDMs (P < 0.05). Conclusions: Through multiomic analyses of epigenetic sequencing and RNA-seq with partial validation, the current study found that Irf1-and Egr1-induced transcription plays key roles in M1 and M2 macrophage polarization, respectively.