FULL Dee Mon Video Enhancer V1.9.3
Filis Cianciotta
Despite strong evidence that human genetic variants affect the expression of many key transcription factors involved in autoimmune diseases, establishing biological links between non-coding risk variants and the gene targets they regulate remains a considerable challenge. Here, we combine genetic, epigenomic, and CRISPR activation approaches to screen for functional variants that regulate IRF8 expression. We demonstrate that the locus containing rs2280381 is a cell-type-specific enhancer for IRF8 that spatially interacts with the IRF8 promoter. Further, rs2280381 mediates IRF8 expression through enhancer RNA AC092723.1, which recruits TET1 to the IRF8 promoter regulating IRF8 expression by affecting methylation levels. The alleles of rs2280381 modulate PU.1 binding and chromatin state to regulate AC092723.1 and IRF8 expression differentially. Our work illustrates an integrative strategy to define functional genetic variants that regulate the expression of critical genes in autoimmune diseases and decipher the mechanisms underlying the dysregulation of IRF8 expression mediated by lupus risk variants.
We propose a strategy to decipher the genetic regulatory mechanisms of TF expression in diseases using IRF8 as an example to fill this gap. Several genetic studies have nominated IRF8 as an important autoimmune disease risk gene23,24,25,26,27,28,29,30. Consistent with this notion, the function of IRF8 is associated with a variety of autoimmune-related phenotypes, such as immune cell development, inflammatory cytokine production, regulation of IFN-stimulated gene (ISG) expression31,32,33. Despite the substantial heritability of the IRF8 locus, the functional variants, causative genes, and underlying gene regulatory mechanisms involved in autoimmune disease are largely unknown. Here, we combine genetic data, epigenomic analysis, CRISPR activation (CRISPRa) screen, CRISPR-mediated knockout and 3D chromatin structure analysis to identify functional variants in the IRF8 locus. We demonstrate that rs2280381 is likely a causal variant that regulates IRF8 expression through modulating enhancer RNA (eRNA) expression and cell-type-specific enhancer-promoter loop interactions. In addition, eRNA interacts with TET1, which binds to the IRF8 promoter and regulates its methylation levels to regulate IRF8 expression. In particular, the rs2280381 allele differentially affects TF occupancy and chromatin status to fine-tune IRF8 expression, thereby contributing to disease pathogenesis.
CRISPR/Cas9-mediated deletion is a widely used tool to study enhancer function. To directly assess the regulatory function of the rs2280381-containing region, we generated cell clones with an 138-bp deletion at the rs2280381 locus using CRISPR/Cas9 technology in U-937 cells (Fig. 2A, B). These clones underwent the same procedure but with the wildtype genotype as a negative control. As expected, deletion of the fragment containing rs2280381 resulted in a significant reduction in IRF8 expression, both at the mRNA and protein level (Fig. 2C, D). In addition, we examined the enhancer-tagged signal in this region by analyzing the publicly available H3K27ac ChIP-seq data and ATAC-seq data in U-937 cells43. We found that this chromatin region was open and highly modified by the H3K27ac marker (Fig. 2E), further confirmed by FAIRE-qPCR and ChIP-qPCR (Supplementary Fig. 2A, B). Together, these data suggest that the region containing rs2280381 is a functional enhancer that regulates IRF8 expression in U-937 cells.
Distal enhancers typically form enhancer-promoter loops that affect the expression of target genes. To test whether such a linkage exists between the IRF8 promoter and the rs2280381 enhancer, we performed circularized chromosome conformation capture sequencing (4C-seq) to detect looping interactions of the IRF8 promoter within this region. This assay revealed a circular physical interaction between the rs2280381 enhancer and the IRF8 promoter (Fig. 2F). Furthermore, this observation was further confirmed based on the rs2280381 view point (Fig. 2G). Consistent with these observations in U-937 cells, we also observed communication between the IRF8 promoter and the rs2280381 locus in primary monocytes (Supplementary Fig. 2C).
Since lncRNA expression levels are usually tissue- or cell-specific, we first investigated the abundance of AC092723.1 in different human immune cell subpopulations. Consistent with the chromatin landscape of this region, AC092723.1 was highly expressed in human CD14+ monocytes (Fig. 3F). This was also validated by public RNA-sequencing data from different immune cell subpopulations (Supplementary Fig. 6G). In addition, we examined the intracellular localization of AC092723.1 in primary monocytes and U-937 cells by cell fractionation and RT-qPCR. We observed that AC092723.1 was predominantly distributed in the nuclear fraction (Fig. 3G and Supplementary Fig. 6H), similar to most regulatory lncRNAs. To directly assess the regulatory function of AC092723.1, we knocked down this lncRNA by antisense oligonucleotides (ASOs) and tested the expression of IRF8. As shown in Fig. 3H, I and Supplementary Fig. 6I, J, knockdown of AC092723.1 significantly reduced IRF8 expression in both primary monocytes (Fig. 3H, I) and U-937 cells (Supplementary Fig. 6I, J). In contrast, knockdown of IRF8 with siRNA did not reduce the expression of AC092723.1 (Fig. 3J, K and Supplementary Fig. 6K, L). We further confirmed this result by deleting part of the AC092723.1 region in U-937 cells with CRISPR/Cas9-mediated fragment deletion (Supplementary Fig. 6M and Fig. 3L). In conclusion, these data provide direct evidence that the rs2280381 enhancer governs the expression of eRNA AC092723.1 to regulate the expression of IRF8.
Having demonstrated the allele-specific regulatory ability of rs2280381, we next sought to explore the mechanisms behind it. Genetic variation is often associated with different enhancer activities, which is thought to be an important mechanism for allele-specific regulation of gene expression by SNP alleles. To test whether different rs2280381 alleles can alter the chromatin state, we first analyzed the allelic distribution of H3K27ac at the rs2280381 site through the MARIO pipeline55 using public ChIP-seq data. We found that rs2280381 showed a strong bias in the direction of non-risk allele for H3K27ac signaling (Fig. 5D), and H3K27ac allele-specific ChIP-qPCR further confirmed these results in rs2280381 heterozygous cell line (Supplementary Fig. 8J). We also examined the chromatin accessibility of the rs2280381 alleles by FAIRE allele-specific qPCR. Consistent with the histone modified allele bias, enhancers carrying the C allele exhibited more FAIRE signal than those carrying the T allele (Fig. 5E), indicating that the C allele has higher chromatin accessibility than the T allele.
Enhancers have been suggested as effective therapeutic targets for disease interventions because targeting enhancers may facilitate precise treatment due to the cell-type specificity of enhancers11,64,65,66. For example, erythroid-specific enhancer of BCL11A by CRISPR-Cas9 editing restores γ-globin synthesis for treating sickle cell disease11. Thus, the discovery of disease-critical enhancers would provide a valuable therapeutic target for disease treatment. In the present study, using CRISPR-Cas9-mediated deletion, we edited the rs2280381-containing region in different cell lines and different immune cell subpopulations. We found that the rs2280381-containing region acts as a distal and cell-type-specific enhancer to regulate IRF8 expression, suggesting that the rs2280381 enhancer has the potential to be a future SLE therapeutic target. In this way, deciphering the functional gene variants associated with autoimmune disease will help develop new therapeutic approaches.
Gene expression is controlled by regulatory elements, including distal enhancers and the proximal promoter67,68. Distal enhancers interact spatially with promoter regions to regulate the expression of target genes69. We performed a 4C-seq assay that validated a promoter-enhancer loop between the IRF8 promoter and the rs2280381-containing region, which further supports the regulatory function of the rs2280381-containing region. Interestingly, we also observed associations between the IRF8 promoter locus and various other genomic regions (Supplementary Data 10), some of which contain autoimmune disease-associated genetic variants and the regulatory functions have been validated in CRISPRa screening assays. However, the functions of most regions that exist interactions with IRF8 promoter remain unknown. Dissecting the role of these regulatory elements will likely help us understand the complete picture of IRF8 transcriptional regulation.
Most individual SNPs have only a tiny effect on gene expression or disease-associated phenotypes. Several studies have found that genetic variants within multiple enhancers of a gene can synergistically regulate gene expression, thereby amplifying these individual minor effects75. In our study, in addition to the region containing rs2280381, we found several other regions containing genetic variants also increase IRF8 expression in CRISPRa screening assays. These data suggest that combinations of functionally independent genetic variants may be an important risk factor for disease. To fully unravel the mechanisms of genetic-mediated disease risk, the synergistic effects of multiple genetic variants should be emphasized in future studies.
Allele-dependent TF binding is a major factor contributing to allelic expression differences. Using DAPA-MS data, ChIP-qPCR and ChIP-Seq data, we identified PU.1 as a critical TF that binds to the rs2280381 locus. There are differences in the binding of PU.1 to the rs2280381 non-risk allele and risk allele, which may lead to different regulatory functions of the risk and non-risk allele. PU.1 is the key TF that elicits monocyte-specific enhancer of a key linear-determining transcription factor (LDTF)56 and the binding of PU.1 to the rs2280381 locus may contribute to the establishment of cell-type-specific enhancer at this locus. In addition, we observed different chromatin states for the risk and non-risk alleles, as reflected by the higher H3K27ac enrichment and chromatin accessibility of the non-risk C allele compared to the T risk allele. Collectively, these observations shed light on the possible mechanisms of rs2280381 risk allele-mediated disease risk. Notably, our DAPA experiments also revealed binding of several other proteins to rs2280381 (Supplementary Data 9), including LDTFs and chromatin regulators. Whether these factors contribute to the allele-specific regulation of rs2280381 on IRF8 expression, and if so, which mechanism they use to direct the cell-type-specific enhancer activation at this site still will be the subject of future studies.
aa06259810