Volume 169, Issue 1, Pages 72-84.e13 (March 2017) Human Epistatic Interaction Controls IL7R Splicing and Increases Multiple Sclerosis Risk  Gaddiel Galarza-Muñoz, Farren B.S. Briggs, Irina Evsyukova, Geraldine Schott-Lerner, Edward M. Kennedy, Tinashe Nyanhete, Liuyang Wang, Laura Bergamaschi, Steven G. Widen, Georgia D. Tomaras, Dennis C. Ko, Shelton S. Bradrick, Lisa F. Barcellos, Simon G. Gregory, Mariano A. Garcia-Blanco  Cell  Volume 169, Issue 1, Pages 72-84.e13 (March 2017) DOI: 10.1016/j.cell.2017.03.007 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 DDX39B Regulates Alternative Splicing of IL7R Exon 6 (A–D) Knockdown of DDX39B in HeLa cells using two independent DDX39B siRNAs (DDX_3 and DDX_4) and a non-silencing control siRNA (NSC). (A) Western blot analysis illustrating depletion of DDX39B. (B–C) RT-PCR analysis of IL7R exon 6 splicing (+E6, exon included; −E6, exon skipped) in transcripts from a reporter minigene (B) or the endogenous gene (C). (D) Quantification of sIL7R secretion by ELISA. (E–F) Rescue experiments with HeLa cell lines stably expressing siRNA-resistant DDX39B trans-gene, either wild-type (WT) (E) or helicase mutant, D199A (F), under the control of the tetracycline operator. Top panels illustrate DDX39B western blot analysis, whereas lower panels show RT-PCR analysis of endogenous IL7R transcripts. In all panels the data is shown as mean ± s.d., and statistical significance was assessed using student’s t test (∗∗∗p ≤ 0.0005; ∗∗p ≤ 0.005; ∗p ≤ 0.05). See also Figures S2 and S3. Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Variants within or adjacent to the DDX39B Locus Strongly Associated with MS Risk Each diamond represents a variant analyzed, and the color of the diamonds indicates no association (gray), marginal association (yellow), or strong association (red) with MS risk. The variant rs2523506 is indicated with a larger diamond (see Figure 3). Black- and blue-dotted lines indicate thresholds for marginal (p ≤ 1.0 × 10−2) and strong (p ≤ 5.0 × 10−8) association, respectively. The location of the four genes present in this region of chromosome 6 is illustrated at the bottom. See also Figure S4 and Table S3. Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 The DDX39B 5′ UTR Variant rs2523506 Displays Allele-Specific DDX39B Protein Expression (A) Schematic representation of the DDX39B gene (black), spliced mRNA isoforms (red), and location of MS-associated variants rs2523506, rs2523512, and rs2516478 (asterisks). (B) RT-qPCR quantification of DDX39B mRNA levels in human PBMCs (left), African (YRI/ESN, middle), and European (IBS, right) LCLs stratified by rs2523506 genotype. Each symbol represents cells from one individual, and red lines indicate median and interquartile range for each group. Samples sizes were: PBMC, CC = 32, AC = 31, AA = 23; YRI/ESN, CC = 12, AC = 11, AA = 2; and IBS, CC = 12, AC = 12, AA = 3. (C) Western blot analysis of DDX39B protein abundance in African (YRI/ESN, left) and European (IBS, right) LCLs stratified by rs2523506 genotype. Panels in (C) were assembled with different portions of the same gel. Statistical significance of all measurements was assessed using student’s t test (two-sided; ∗∗∗p ≤ 0.0005; ∗∗p ≤ 0.005; ∗p ≤ 0.05). See also Figures S5 and S6. Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 The Risk Allele of rs2523506 Reduces Translational Efficiency Mediated by DDX39B 5′ UTRs (A) Schematic representation of the different DDX39B 5′ UTR luciferase reporters, which differ by alternative 3′ss in exon 2 and the single nucleotide change at rs2523506 (C/A). (B) Measurements of translational efficiency in transfected HeLa cells. RNA levels and luciferase activity were measured by RT-qPCR and dual luciferase assays, respectively. Translational efficiency was determined by dividing luciferase activity by RNA levels. Statistical significance was assessed using student’s t test (two-sided; ∗∗p ≤ 0.005; ∗p ≤ 0.05). Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Functional Interaction between DDX39B and IL7R Exon 6 Variants The interaction between DDX39B and IL7R was functionally tested in DDX39B-depleted HeLa cells using IL7R splicing reporters carrying either the risk C allele (IL7R-C; left) or the protective T allele (IL7R-T; right) of rs6897932. RT-PCR analysis of exon 6 splicing (mean ± s.d.) reveals higher exon 6 skipping when the levels of DDX39B are reduced in the context of the risk C allele than of the protective T allele of rs6897932 (student’s t test, two-sided; ∗∗p ≤ 0.005). Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 DDX39B Regulates IL7R Exon 6 Splicing in Primary CD4+ T Cells (A and B) Knockdown of DDX39B in primary CD4+ T cells from six donors via lentiviral transduction with two independent shRNA against DDX39B (sh3 and sh5) and a non-targeting control shRNA (NTC). Donors are grouped by IL7R genotype, IL7R-CC (A) and IL7R-CT (B), and each panel illustrates DDX39B western blot analysis (top) and RT-PCR analysis of IL7R exon 6 splicing (bottom) for each donor individually. In each panel, the plots on the right show the average of % exon 6 skipping for each IL7R genotype (mean ± s.d., student’s t test: ∗∗p ≤ 0.005; ∗p ≤ 0.05). See also Figure S7. Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S1 Regulation of IL7R exon 6 Splicing in HeLa Cells and Jurkat T Cells, Related to Figure 1 and Table S1 IL7R reporters carrying the alternative alleles of rs6897932 (C or T) or mutation of the critical exonic splicing enhancer 2 (ΔESE2) were transiently transfected into HeLa (left) or Jurkat (right) cells and exon 6 splicing was determined by RT-PCR as in Figure 1. The data is presented as mean ± s.d. Statistical significance was assessed using Student’s t test (two-sided; ∗∗∗p ≤ 0.0005; ∗∗p ≤ 0.005; ∗p ≤ 0.05). Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S2 DDX39B Requires Intact ESE2 to Regulate IL7R exon 6 Splicing, Related to Figure 1 Ectopic expression of siRNA-resistant DDX39B trans-gene in DDX39B-depleted cells cannot rescue splicing of IL7R in transcripts from a reporter mutant of ESE2 (IL7R-ΔESE2). The experiment was performed as in Figure 1 followed by transfection of IL7R-ΔESE2 reporter. (A) Western blot analysis of DDX39B. (B) RT-PCR analysis of exon 6 skipping in transcripts from the IL7R-ΔESE2 reporter (+E6 = exon 6 included; −E6 = exon 6 skipped). The data is presented as mean ± s.d. and statistical significance was determined using Student’s t test (two-sided; ∗∗∗p ≤ 0.0005; ∗∗p ≤ 0.005; ∗p ≤ 0.05). Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S3 Knockdown of Additional RNA Helicases Has a Small or No Effect on IL7R exon 6 Splicing, Related to Figure 1 Knockdown of RNA helicases DDX5, DDX23 and DDX17 was carried out in HeLa cells using three independent siRNAs against each target and a control siRNA (NSC). (A) Quantification of knockdown by RT-qPCR (normalized to GAPDH). (B) RT-PCR analysis of IL7R exon 6 splicing in endogenous transcripts as in Figure 1C (+E6 = exon 6 included; −E6 = exon 6 skipped). The data is shown as mean ± s.d., and statistical significance was assessed using Student’s t test (two-sided; ∗∗∗p ≤ 0.0005; ∗∗p ≤ 0.005; ∗p ≤ 0.05). Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S4 Regional Plot Showing MS-Associated Variants within the DDX39B Locus Adjusted for All Known MHC Risk Variants, Related to Figure 2 and Table S3 Variants were adjusted for population stratification, cohort of origin, the 10 known HLA risk variants (HLA-adjusted model) and the non-HLA risk variant rs2516489 (MHC-adjusted model). Each diamond represents a variant analyzed: yellow diamonds passed the threshold for statistical significance (p ≤ 0.05; black dotted line), whereas gray diamonds did not. The location of the four genes present in this region of chromosome 6 is shown at the bottom. Notably, rs2523506 and several other DDX39B variants showed a modest but significant association independent of rs2516489 and all other HLA risk variants. In healthy controls from each cohort, rs2516489 was in strong LD (r2 > 0.9) with several of the DDX39B associated variants, which explains the attenuated association after correction for rs2516489. Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S5 Relative Abundance of DDX39B Protein in Lymphoblastoid Cell Lines (LCLs), Related to Figure 3 (A and B) We mined DDX39B protein levels in a large proteomic database where the relative protein abundance for 5,953 genes was determined by mass spectrometry in 95 LCLs derived from mainly European (CEU) and African (YRI) individuals (Wu et al., 2013). We extracted the relative DDX39B protein levels for YRI (A) and CEU (B) LCLs and stratified those by the genotypes of rs2523506 [data was only available for LCLs homozygous for the protective allele (CC) and heterozygous (AC)]. Sample sizes were as follow: YRI (CC n = 24; AC n = 7) and CEU (CC n = 34; AC n = 16). Each symbol represents an individual LCL, whereas the red lines indicate median and interquartile range. Statistical significance was determined using Student’s t test (two-sided; ∗p ≤ 0.05). Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S6 SNP rs2523506 Does Not Affect Expression of Genes in the Vicinity of DDX39B and Knockdown of These Genes Does Not Affect IL7R exon 6 Splicing, Related to Figures 1 and 3 (A) RT-qPCR quantification of genes in the vicinity of DDX39B (MICB, DDX39B, ATP6V1G2, NFKBIL1, LTA, TNF and LTB) in YRI LCLs stratified by rs2523506 genotype (sample sizes: CC = 12, AC = 11, AA = 2). Expression of an additional gene (MCCD1) was not detected. Each symbol represents the average of triplicate measurements from one individual, and red lines indicate median and interquartile range for each group. (B and C) Knockdown of DDX39B vicinal genes that were expressed in HeLa cells (MICB, NFKBIL1, LTA and TNF). The three other genes (MCCD1, ATP6V1G2 and LTB) are not expressed in HeLa cells and thus were not analyzed. (B) RT-qPCR quantification of mRNA knockdown (normalized to GAPDH). (C) RT-PCR analysis of IL7R exon 6 splicing as in Figure 1C (+E6 = exon 6 included; −E6 = exon 6 skipped). Plots in B and C are shown as mean ± s.d. Statistical significance was assessed using Student’s t test (two-sided: ∗∗∗p ≤ 0.0005; ∗∗p ≤ 0.005; ∗p ≤ 0.05). Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure S7 Quantification of sIL7R in Primary CD4+ T Cells, Related to Figure 6 (A) Quantification of sIL7R secretion by ELISA in control (NTC) and DDX39B-depleted (sh3 and sh5) primary CD4+ T cells from Figure 6. The results are shown grouped by IL7R genotypes: IL7R-CC (top) and IL7R-CT (bottom). (B) Correlation of sIL7R levels with relative abundance of IL7R transcripts that skip exon 6 (IL7R −E6) for each shRNA. The relative abundance of IL7R −E6 transcripts was extrapolated from % exon 6 skipping and RT-qPCR quantification of overall abundance of IL7R transcripts for each condition. Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell 2017 169, 72-84.e13DOI: (10.1016/j.cell.2017.03.007) Copyright © 2017 Elsevier Inc. Terms and Conditions