Abstract
Backgrounds: Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease associated with a wide range of clinical features involving different organs and the prognosis is also highly variable. IKZF2, which encodes Helios, is a Krüppel-like zinc finger transcription factor belonging to the Ikaros family. This group of transcription factors is integral to the regulation of the immune system. Furthermore, IKZF2 has been identified as a risk locus for SLE. Given that the expression of Helios is largely restricted to T-cells, and dysregulation of T cell function is an important factor for SLE pathogenesis, I sought to unravel the biological mechanism for the association by leveraging ChIP-Seq data for Helios in Jurkat T-cells. This study employed a multi-dimensional approach to delve into the intricacies of Helios in Jurkat T cells. The research integrated genome-wide identification of Helios binding sites, functional annotation of these sites, and their intersection with gene expression data, offering a comprehensive view of its regulatory network. Furthermore, the investigation extended beyond Jurkat T cells, exploring Helios binding sites and target genes across various T cell subtypes and their potential implications in SLE.Methods: Using a multi-faceted bioinformatics approach, I aimed to investigate the regulatory role of the transcription factor Helios in Jurkat T cells. The research encompassed the comprehensive identification of Helios binding sites and functional annotation of these binding sites using multi-omic datasets from either publicly available sources or accessed through collaboration. Using these resources I also went on to explore the binding sites and target genes of the transcription factor Helios in various T cell subtypes. I predicted Helios binding sites across CD4+ naïve T cells, Th1 cells, and Th17 cells by combing its binding sites in Jurkat T cells and DNase I hypersensitivity data in these cell types. Subsequently, I identified target genes associated with these binding sites using pre-established promoter annotation and enhancer annotation data and conducted gene set enrichment analysis to reveal their role in SLE.
Results: The extensive analysis of Helios in T cell regulation and its impact on SLE has yielded significant insights. A genome-wide chip-seq analysis in Jurkat T cells identified 5,068 binding sites of Helios, indicating its extensive role in gene regulatory mechanisms within T cells. Notably, a substantial proportion of these sites are located in promoter regions and distal intergenic areas, suggesting Helios's involvement in both direct transcription initiation and long-range gene regulation. ChromHMM annotation further revealed the majority of these sites are associated with active promoters and enhancers. The study also observed variable overlaps of Helios binding sites with SNPs in different diseases, with significant associations in Crohn's Disease, Multiple Sclerosis, and Rheumatoid Arthritis, highlighting its potential role in these conditions. Differential gene expression analysis following Helios knockdown in Jurkat T cells showed significant changes in 1,072 genes, with pathway analysis revealing enrichment in crucial processes like cholesterol biosynthesis, apoptosis, and T cell receptor regulation. Utilizing the MCODE plugin in Cytoscape, ten distinct biological modules were identified, encompassing a range of functions from epigenetic modulation to immune responses. A total of 56 priority target genes of Helios in Jurkat T cells were identified by intersecting the genes annotated from Helios binding sites in Jurkat T cells with the differentially expressed genes (DEGs) observed following Helios knockdown in the same cell type. This list includes several key genes that have been previously implicated in the development of SLE, such as IRF4, PRKCB, and CD9.
Significant changes (adjusted p value <0.05) in the expression of Helios in specific T-cell subtypes in SLE patients compared to healthy controls are observed, with Th1 cells showing the most substantial difference (log (FC) =1.96 using with our analysis based on a Japanese bulk RNA-seq dataset that encompasses data from various immune cell types across both healthy controls and SLE patients. In order to observe the function of Helios in different T cell subtypes, we employed a comprehensive bioinformatics approach to predict Helios transcription factor binding sites and their target genes in various T cell subtypes. Our focus was primarily on Th1, naïve CD4+, and Th17 cell types due to the availability of relevant datasets for DNase I hypersensitivity hotspots and pre-established annotation data in these specific subtypes.
Gene Set Enrichment Analysis revealed that the predicted target genes were enriched at the "top" of the list of differentially expressed genes when comparing healthy controls and SLE patients in both Th1 and Th17 cells (with a significance threshold set at p-value <0.05). I identified 190 Helios’ predicted target genes in Th1 cells and 70 Helios’ predicted target genes in Th17 cells that were found to be centrally contributed to differentially expressed genes (DEGs) between healthy controls and SLE patients. Utilizing protein-protein interaction (PPI) networks and plugins like MCODE and Cytohubba in Cytoscape, I pinpointed key hub genes associated with SLE in these cell types. Specifically, CCNA2, MRPL58, and CXCR6 emerged as hub genes in Th1 cells, while IRF4 and CBFB were identified in Th17 cells. Additionally, seven genes – IFI6, FKBP5, TRIB1, PRDM1, TRAT1, LINC00426, and CCND3 – were found to be core predicted target genes of Helios in both Th1 and Th17 cells within the DEGs between healthy controls and SLE patients. Further analysis of the expression changes and correlation with IKZF2 expression in these 12 genes revealed that IFI6 in both Th1 and Th17 cells, as well as CCNA2 in Th1 cells, are particularly significant. These genes not only exhibit considerable changes in expression but also show a notable correlation with IKZF2, underscoring their importance as target genes of Helios and their potential role in SLE pathogenesis in these T cell subsets.
Conclusion: This study elucidates the role of the transcription factor Helios in T cell regulation and its implications in SLE. The study revealed that Helios exhibits a predilection for binding sites near transcription start sites and active promoter regions, indicating its pivotal role in the regulation of gene expression. Functional annotation disclosed Helios' involvement in biological processes such as sterol biosynthesis and cholesterol metabolism, epigenetic modification and immune responses. the study explored changes in Helios expression across various T cell subtypes, particularly highlighting Th1 cells as the most affected. Additionally, gene Set Enrichment Analysis showed that Helios' predicted target genes were significantly enriched among differentially expressed genes in Th1 and Th17 cells in SLE patients, indicating its potential role in the development of this autoimmune disease within these cell types. Through protein-protein interaction network analysis, hub target genes of Helios related to SLE in each cell type were identified.
| Date of Award | 1 Aug 2024 |
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| Original language | English |
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| Supervisor | Deborah Cunninghame Graham (Supervisor) & Timothy Vyse (Supervisor) |