Three-Dimensional Chromosomal Landscape Revealing miR-146a Dysfunctional Enhancer in Lupus and Establishing a CRISPR-Mediated Approach to Inhibit the Interferon Pathway.

OBJECTIVE: The diminished expression of microRNA-146a (miR-146a) in systemic lupus erythematosus (SLE) contributes to the aberrant activation of the interferon pathway. Despite its significance, the underlying mechanism driving this reduced expression remains elusive. Considering the integral role of enhancers in steering gene expression, our study seeks to pinpoint the SLE-affected enhancers responsible for modulating miR-146a expression. Additionally, we aim to elucidate the mechanisms by which these enhancers influence the contribution of miR-146a to the activation of the interferon pathway. METHODS: Circular chromosome conformation capture sequencing and epigenomic profiles were applied to identify candidate enhancers of miR-146a. CRISPR activation was performed to screen functional enhancers. Differential analysis of chromatin accessibility was used to identify SLE-dysregulated enhancers, and the mechanism underlying enhancer dysfunction was investigated by analyzing transcription factor binding. The therapeutic value of a lupus-related enhancer was further evaluated by targeting it in the peripheral blood mononuclear cells (PBMCs) of patients with SLE through a CRISPR activation approach. RESULTS: We identified shared and cell-specific enhancers of miR-146a in distinct immune cells. An enhancer 32.5 kb downstream of miR-146a possesses less accessibility in SLE, and its chromatin openness was negatively correlated with SLE disease activity. Moreover, CCAAT/enhancer binding protein α, a down-regulated transcription factor in patients with SLE, binds to the 32.5-kb enhancer and induces the epigenomic change of this locus. Furthermore, CRISPR-based activation of this enhancer in SLE PBMCs could inhibit the activity of interferon pathway. CONCLUSION: Our work defines a promising target for SLE intervention. We adopted integrative approaches to define cell-specific and functional enhancers of the SLE critical gene and investigated the mechanism underlying its dysregulation mediated by a lupus-related enhancer.

Identification of Two Subsets of Subcompartment A1 Associated with High Transcriptional Activity and Frequent Loop Extrusion.

Three-dimensional genome organization has been increasingly recognized as an important determinant of the precise regulation of gene expression in mammalian cells, yet the relationship between gene transcriptional activity and spatial subcompartment positioning is still not fully comprehended. Here, we first utilized genome-wide Hi-C data to infer eight types of subcompartment (labeled A1, A2, A3, A4, B1, B2, B3, and B4) in mouse embryonic stem cells and four primary differentiated cell types, including thymocytes, macrophages, neural progenitor cells, and cortical neurons. Transitions of subcompartments may confer gene expression changes in different cell types. Intriguingly, we identified two subsets of subcompartments defined by higher gene density and characterized by strongly looped contact domains, named common A1 and variable A1, respectively. We revealed that common A1, which includes highly expressed genes and abundant housekeeping genes, shows a ~2-fold higher gene density than the variable A1, where cell type-specific genes are significantly enriched. Thus, our study supports a model in which both types of genomic loci with constitutive and regulatory high transcriptional activity can drive the subcompartment A1 formation. Special chromatin subcompartment arrangement and intradomain interactions may, in turn, contribute to maintaining proper levels of gene expression, especially for regulatory non-housekeeping genes.

Integrative Functional Genomics Identifies Systemic Lupus Erythematosus Causal Genetic Variant in the IRF5 Risk Locus.

OBJECTIVE: IRF5 plays a crucial role in the development of lupus. Genome-wide association studies have identified several systemic lupus erythematosus (SLE) risk single-nucleotide polymorphisms (SNPs) enriched in the IRF5 locus. However, no comprehensive genome editing-based functional analysis exists to establish a direct link between these variants and altered IRF5 expression, particularly for enhancer variants. This study was undertaken to dissect the regulatory function and mechanisms of SLE IRF5 enhancer risk variants and to explore the utilization of clustered regularly interspaced short palindromic repeat interference (CRISPRi) to regulate the expression of disease risk gene to intervene in the disease. METHODS: Epigenomic profiles and expression quantitative trait locus analysis were applied to prioritize putative functional variants in the IRF5 locus. CRISPR-mediated deletion, activation, and interference were performed to investigate the genetic function of rs4728142. Allele-specific chromatin immunoprecipitation-quantitative polymerase chain reaction and allele-specific formaldehyde-assisted isolation of regulatory element-quantitative polymerase chain reaction were used to decipher the mechanism of alleles differentially regulating IRF5 expression. The CRISPRi approach was used to evaluate the intervention effect in monocytes from SLE patients. RESULTS: SLE risk SNP rs4728142 was located in an enhancer region, indicating a disease-related regulatory function, and risk allele rs4728142-A was closely associated with increased IRF5 expression. We demonstrated that an rs4728142-containing region could act as an enhancer to regulate the expression of IRF5. Moreover, rs4728142 affected the binding affinity of zinc finger and BTB domain-containing protein 3 (ZBTB3), a transcription factor involved in regulation. Furthermore, in monocytes from SLE patients, CRISPR-based interference with the regulation of this enhancer attenuated the production of disease-associated cytokines. CONCLUSION: These results demonstrate that the rs4728142-A allele increases the SLE risk by affecting ZBTB3 binding, chromatin status, and regulating IRF5 expression, establishing a biologic link between genetic variation and lupus pathogenesis.