Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS

Abstract

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. VIDEO ABSTRACT.

Keywords: GWAS; cell type; epigenomics; genetic risk; multiple sclerosis; oligodendrocytes; outside variants; population genetics; remyelination; transcriptional pause release.

Figure 1.. Cell-Type Specificity of Intralocus Regulatory Elements Enables Prediction of Pathogenic Cell Type of MS Risk Loci

(A) Diagram of approach to identify pathogenic cell type of individual GWAS risk loci. Outside variants physically interact with the same target gene as a GWAS variant. A two-tiered stratification approach is used to determine the impact of outside variants on disease risk (left). Genetic risk barcode is composed of outside-variant sites that significantly alter clinical risk compared to sites where outside variants have no contribution to risk (center). Cell types with H3K27ac molecular activity that is concordant with the genetic risk barcode are identified as likely pathogenic (right). (B) Example GWAS SNP rs13333054, highlighted in gray, physically interacts with IRF8 and EMC8 in activated CD4⁺ T cells (purple) and monocytes (blue). Arcs represent physical interactions identified via promoter capture Hi-C. Highlighted in red is an exemplar outside-variant site that significantly alters risk and is enriched for H3K4me1/H3K27ac and Hi-C activity in monocytes, but not in T cells. (C) GWAS SNPs overlap with H3K27ac peaks and are row ordered based on lineage-specific clusters (far right). Cell-type columns are highlighted if GWAS and/or LD SNPs (r² > 0.8) overlap H3K27ac peaks identified in each cell type (left, top). MS GWAS results for genome-wide chromatin activity enrichment via Variant Set Enrichment (VSE) and LD score regression (LDSR) for MS (left, bottom). Pathogenic cell types identified by outside-variant approach are shown. For each GWAS locus (row), the identified cell types are highlighted (right). Clusters of GWAS loci, as defined by the outside-variant predictions, were identified via similarity of cell type and lineage specificity (far right). (D) Comparison of lineage-specific clusters identified by an outside-variant approach to the heritability of 28 other disorders via LDSR. Heritability enrichment of six other autoimmune disorders within each lineage-specific cluster is shown (left). The top six traits with the strongest heritability enrichment in loci identified to act in the CNS for MS are shown (right). The multi-test-corrected significance threshold is denoted by a dashed line. See also Figures S1, S2, and S3 and Tables S1, S2, and S3.

Figure 2.. Oligodendrocyte-Specific Regulation of Transcriptional Pause Release Factors Is Associated with MS Risk

(A) HEXIM1/2 locus outside variant results (top, Manhattan plot). H3K27ac enrichment across multiple cell types, including three biological replicates of oligodendrocytes (bottom). Highlighted in red are exemplar outside-variant sites that significantly alter risk and are enriched for H3K27ac in oligodendrocytes. (B) Aggregate plot of oligodendrocyte H3k27ac enrichment at intralocus outside-variant sites at the HEXIM1/2 locus. H3k27ac signal aggregate across sites that alter risk (solid line) versus sites that do not alter risk are shown for three ChIP biological replicates. *kruskal-Wallis one-sided p < 2E-6 for each oligodendrocyte ChIP. (C) Same as in (A), but for the BRD3 locus. (D) Same as in (B), but for the BRD3 locus. (E) HEXIM1 gene expression measured by qRT-PCR in an NPC model following transfection of Cas9 and sgRNA targeting outside variant sites (sg1-sg4) or HEXIM1 TSS. Log₂ fold change for two biological replicates shown for each guide compared to cells transfected with Cas9 without a gRNA. (F) Same as in (E), but for the BRD3 locus. (G) MS heritability enrichment estimated for transcriptional pausing gene sets derived from Reactome (n = 200) and Gene Ontology (n = 100) using LDSR. The multi-test-corrected significance threshold is denoted by a dashed line. See also Figure S3 and Tables S1, S3, and S4.

Figure 3.. Inhibition of Transcriptional Pause Release Blocks Oligodendrocyte Maturation

(A) A library of 3,141 bioactive compounds was screened at 2 μM to identify inhibitors of development of mouse MBP-positive oligodendrocytes. Data represent log₂ fold change from standard conditions promoting development (T3; dotted line). Baseline maintenance conditions of OPCs are indicated by the fibroblast growth factor (FGF) dotted line. Select compounds known to induce development are highlighted in yellow. The 2% of compounds that best prevented oligodendrocyte development are black, while dual BET bromodomain inhibitors are highlighted in blue. (B) Quantification of MBP with varying culture conditions. The inhibitory action of dual BET bromodomain inhibitor JQ1 is limited to the racemic form and active (S)- stereoisomer, indicating the effect is likely on-target. Each point represents the average of five fields from a single well. ns, p > 0.05; ****p < 0.0001 by Dunnett’s multiple comparisons test. (C) Dose response of the effect of (S)-JQ1 on formation of MBP-positive oligodendrocytes. JQ1 inhibits oligodendrocyte maturation with an IC₅₀ of 105 nM (dotted line). (D) Representative immunostaining of mouse OPCs grown in oligodendrocyte-promoting conditions and either inactive (R)-JQ1 or active (S)-JQ1 for 3 days or for 6 days with (S)-JQ1 washout at 3 days. OLIG2 (red) marks cells of the oligodendrocyte lineage, while MBP (green) indicates mature oligodendrocytes. Scale bars indicate 50 μm. (E) Representative immunostaining of mouse OPCs co-cultured with DRG neurons for 7 days in the presence of either inactive (R)-JQ1 or active (S)-JQ1 or for 10 days with (S)-JQ1 washout at 3 days. Neurofilament (NF) is shown in red and MBP in green. Scale bars indicate 100 μm. See also Figures S4 and S5.

Figure 4.. Dysregulation of Hexim1 Alters Oligodendrocyte Maturation

(A) qRT-PCR showing CRISPRa-mediated increase in Hexim1 gene expression relative to a non-targeting control (NTC) and normalized to endogenous control Rpl13a (n = 4 technical replicates, mean ± SEM). (B) Fold change of the percentage of oligodendrocytes (MBP/DAPI) after transduction of CRISPRa with guide targeting Hexim1 relative to percentage of oligodendrocytes (MBP/DAPI) after transduction of CRISPRa-NTC (n = 14 independent wells; mean ± SEM; *p < 0.002 by Wilcoxon test). (C) Representative images of oligodendrocyte formation from CRISPRa-NTC, CRISPRa-Hexiim1 OPCs after maturation for 3 days. Myelinating oligodendrocytes are immunostained with anti-MBP (green) and cell nuclei are stained with DAPI (blue). Scale bars indicate 100 μm. (D) qRT-PCR showing a CRISPRi-mediated decrease in Hexim1 gene expression relative to an NTC and normalized to endogenous control Rpl13a (n = 4 technical replicates; mean ± SEM). (E) Fold change of the percentage of oligodendrocytes (MBP/DAPI) after transduction of CRISPRi with guide targeting Hexim1 relative to percentage of oligodendrocytes (MBP/DAPI) after transduction of CRISPRi-NTC (n = 23 independent wells; mean ± SEM; **p < 0.0001 by Wilcoxon test). Same as in (C), but for CRISPRi-NTC and CRISPRi-Hexim1 representative images See also Table S3.

Figure 5.. Transcriptional Elongation Factors Are Dysregulated in MS Patient White Matter

(A) Microarray expression (RMA normalized) of elongation factors in patient white matter (WM). HEXIM1, HEXIM2, and BRD3 are significantly dysregulated in WM lesions compared to control tissue by Dunnett’s multiple comparisons test. (B) Representative immunostaining of PLP1 and HEXIM1 in control and MS patient tissue sections. Lesions are identified by PLP1-low regions in MS images. Scale bars indicate 50 μm. (C) Quantification of HEXIM1/PLP1 double-positive cells in control tissue as compared to primary- and secondary-progressive MS. Each plotted point represents quantification of a single 0.07-mm² field. Co-localization significantly increases in both disease states by Dunnett’s multiple comparisons test, ns, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001. See also Table S4.