整合 ATAC-seq 和 RNA-seq 分析揭示了 SLE 中的转录调控网络。
Integrated analysis of ATAC-seq and RNA-seq reveals the transcriptional regulation network in SLE.
机构信息
Department of Dermatology, Hunan Key Laboratory of Medical Epigenomics, The Second Xiangya Hospital of Central South University, Changsha, China; Research Unit of Key Technologies of Immune-Related Skin Diseases Diagnosis and Treatment, Chinese Academy of Medical Sciences, Changsha, China.
Department of Dermatology, Hunan Key Laboratory of Medical Epigenomics, The Second Xiangya Hospital of Central South University, Changsha, China; Research Unit of Key Technologies of Immune-Related Skin Diseases Diagnosis and Treatment, Chinese Academy of Medical Sciences, Changsha, China; Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, China.
出版信息
Int Immunopharmacol. 2023 Mar;116:109803. doi: 10.1016/j.intimp.2023.109803. Epub 2023 Feb 2.
BACKGROUND
CD4 T cells have a vital role in the pathogenesis of systemic lupus erythematosus (SLE), abnormal gene expression in CD4 T cells partly accounting for dysfunctional CD4T cells. However, the underying regulatory mechanisms of abnormal gene expression in CD4 T cells derived from SLE patients are not fully understood.
METHODS
The peripheral blood CD4 T cells were acquired from 4 SLE patients and 4 matched healthy controls. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was conducted to screen differentially accessible chromatin regions between SLE and normals, and motif prediction was used to identify potentially key transcription factors (TFs) involved in CD4T dysfunction. RNA sequencing (RNA-seq) was performed to screen differentially expressed genes in SLE CD4T cells. ATAC-seq and RNA-seq were integrated to further analyze the relationship between chromatin accessibility and gene expression. KEGG pathway enrichment analysis was to determine enriched pathways of interactions between all predicted TFs and differentially expressed genes (DEGs). Meanwhile, the expression changes of target genes followed by siRNA knockdown of the predicted TF were experimentally verified by qPCR. Finally, the H3K27ac modification levels of immune-related genes with open chromatin and up-regulated expression in SLE CD4T cells was detected by ChIP-qPCR.
RESULTS
We identified 3067 differentially accessible regions (DARs) and 1292 DEGs. TF prediction and functional enrichment analyses showed the TF-gene interaction networks were enriched predominantly in T helper 17 (Th17) cell differentiation, the cell cycle and some signaling pathways. Top 5 TFs were predicted based on overlapping genes between the DAR-related genes and the DEGs: ZNF770, THAP11, ZBTB14, ETV1, POU3F1. Validation experiments indicated that the expression of TRIM25, CD163, BST2, IFIT5, IFITM3, OASL, TBX21, IL15RA and IL12RB2 was significantly downregulated in CD4Tcells with ZNF770 knockdown. H3K27ac showed significantly higher levels in the promoter regions of KLF4 and MX2 in SLE CD4 T cells.
CONCLUSION
These DARs associated with this disease may become targets for future treatment of SLE.
背景
CD4 T 细胞在系统性红斑狼疮（SLE）的发病机制中起着至关重要的作用，CD4 T 细胞的异常基因表达部分解释了功能失调的 CD4 T 细胞。然而，SLE 患者来源的 CD4 T 细胞中异常基因表达的潜在调节机制尚不完全清楚。
方法
从 4 名 SLE 患者和 4 名匹配的健康对照中获得外周血 CD4 T 细胞。使用测序（ATAC-seq）进行转座酶可及染色质分析，以筛选 SLE 和正常之间差异可及染色质区域，并进行基序预测以鉴定参与 CD4T 功能障碍的潜在关键转录因子（TF）。进行 RNA 测序（RNA-seq）以筛选 SLE CD4T 细胞中的差异表达基因。将 ATAC-seq 和 RNA-seq 整合在一起，以进一步分析染色质可及性与基因表达之间的关系。KEGG 途径富集分析用于确定所有预测的 TF 和差异表达基因（DEGs）之间相互作用的富集途径。同时，通过 qPCR 实验验证了靶向基因在预测的 TF 敲低后的表达变化。最后，通过 ChIP-qPCR 检测 SLE CD4T 细胞中具有开放染色质和上调表达的免疫相关基因的 H3K27ac 修饰水平。
结果
我们鉴定了 3067 个差异可及区域（DAR）和 1292 个差异表达基因。TF 预测和功能富集分析表明，TF-基因相互作用网络主要富集在辅助性 T 细胞 17（Th17）细胞分化、细胞周期和一些信号通路中。基于 DAR 相关基因和差异表达基因之间的重叠基因，预测了前 5 个 TF：ZNF770、THAP11、ZBTB14、ETV1、POU3F1。验证实验表明，在 ZNF770 敲低的 CD4T 细胞中，TRIM25、CD163、BST2、IFIT5、IFITM3、OASL、TBX21、IL15RA 和 IL12RB2 的表达显著下调。在 SLE CD4 T 细胞中，KLF4 和 MX2 的启动子区域的 H3K27ac 水平明显升高。
结论
这些与疾病相关的 DAR 可能成为 SLE 未来治疗的靶点。
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