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

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.

3D genome alterations in T cells associated with disease activity of systemic lupus erythematosus.

OBJECTIVES: Three-dimensional (3D) genome alterations can dysregulate gene expression by rewiring physical interactions within chromosomes in a tissue-specific or cell-specific manner and lead to diseases. We aimed to elucidate the 3D genome structure and its role in gene expression networks dysregulated in systemic lupus erythematosus (SLE). METHODS: We performed Hi-C experiments using CD4+ T cells from 7 patients with SLE and 5 age-matched and sex-matched healthy controls (HCs) combined with RNA sequencing analysis. Further integrative analyses, including transcription factor motif enrichment, SPI1 knockdown and histone modifications (H3K27ac, H3K4me1, H3K4me3), were performed for altered loop-associated gene loci in SLE. RESULTS: We deciphered the 3D chromosome organisation in T cells of patients with SLE and found it was clearly distinct from that of HCs and closely associated with the disease activity of SLE. Importantly, we identified loops within chromosomes associated with the disease activity of SLE and differentially expressed genes and found some key histone modifications close to these loops. Moreover, we demonstrated the contribution of the transcription factor SPI1, whose motif is located in the altered loop in SLE, to the overexpression of interferon pathway gene. In addition, we identified the potential influences of genetic variations in 3D genome alterations in SLE. CONCLUSIONS: Our results highlight the 3D genome structure alterations associated with SLE development and provide a foundation for future interrogation of the relationships between chromosome structure and gene expression control in SLE.

LncRNA IL21-AS1 interacts with hnRNPU protein to promote IL21 overexpression and aberrant differentiation of Tfh cells in systemic lupus erythematosus.

BACKGROUND: The aberrant differentiation of T follicular helper (Tfh) cells plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). However, the mechanism of regulating Tfh cells differentiation remains unclear. Long noncoding RNAs (lncRNAs) act as important regulators in the processes of innate and adaptive immune response. Whether lncRNAs are involved in regulating Tfh cell differentiation and autoimmune responses need to be further identified. METHODS: The characters and functions of human IL21-AS1 and its mouse homologous lncRNA (mIl21-AS) were investigated by a series of biochemical assays and cell transfection assay. mIl21-AS1 regulating humoral immune response in vivo was explored by keyhole limpet haemocyanin (KLH) and chronic graft versus host disease (cGVHD) model. RESULTS: Human IL21-AS1 and its mouse homologous lncRNA (mIl21-AS) were identified and cloned. We uncovered that IL21-AS1 was highly expressed in CD4+ T cells of SLE patients and Tfh cells, which promoted differentiation of Tfh cells. Mechanistically, IL21-AS1 bound heterogeneous nuclear ribonucleoprotein U and recruited acetyltransferases CREB-binding protein to the promoter of IL21, leading to the transcriptional activation of IL21 and Tfh cells differentiation through increasing Histone H3 acetylation level on IL21 promoter. Moreover, Tfh proportion and antibodies production were significantly increased in mIl21-AS knock-in mice immunized with KLH. mIl21-AS1 overexpression also exacerbated the lupus-like phenotype in cGVHD mice model. CONCLUSIONS: Our results demonstrate that IL21-AS1 activates IL21 transcription via epigenetic mechanism to promote germinal centre response, adding insight into the molecular regulation of autoimmune pathogenesis and providing a novel target for SLE treatment.