Integrated analysis of ATAC-seq and RNA-seq reveals the transcriptional regulation network in SLE

Abstract

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 CD4⁺T 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 CD4⁺T dysfunction. RNA sequencing (RNA-seq) was performed to screen differentially expressed genes in SLE CD4⁺T 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 CD4⁺T 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 CD4⁺Tcells 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.

Keywords: ATAC-seq; Promoter accessibility; RNA-seq; Systemic lupus erythematosus; Transcription factors.