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X-linked genes exhibit miR6891-5p-regulated skewing in Sjögren’s syndrome

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Abstract

Many autoimmune diseases exhibit a strikingly increased prevalence in females, with primary Sjögren’s syndrome (pSS) being the most female-predominant example. However, the molecular basis underlying the female-bias in pSS remains elusive. To address this knowledge gap, we performed genome-wide, allele-specific profiling of minor salivary gland-derived mesenchymal stromal cells (MSCs) from pSS patients and control subjects, and detected major differences in the regulation of X-linked genes. In control female MSCs, X-linked genes were expressed from both paternal and maternal X chromosomes with a median paternal ratio of ~ 0.5. However, in pSS female MSCs, X-linked genes exhibited preferential expression from one of the two X chromosomes. Concomitantly, pSS MSCs showed decrease in XIST levels and reorganization of H3K27me3+ foci in the nucleus. Moreover, the HLA-locus-expressed miRNA miR6891-5p was decreased in pSS MSCs. miR6891-5p inhibition in control MSCs caused XIST dysregulation, ectopic silencing, and allelic skewing. Allelic skewing was accompanied by the mislocation of protein products encoded by the skewed genes, which was recapitulated by XIST and miR6891-5p disruption in control MSCs. Our data reveal X skewing as a molecular hallmark of pSS and highlight the importance of restoring X-chromosomal allelic balance for pSS treatment.

Key messages

  • X-linked genes exhibit skewing in primary Sjögren’s syndrome (pSS).

  • X skewing in pSS associates with alterations in H3K27me3 deposition.

  • pSS MSCs show decreased levels of miR6891-5p, a HLA-expressed miRNA.

  • miR6891-5p inhibition causes H3K27me3 dysregulation and allelic skewing.

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Data availability

Access to data supporting this study is restricted due to the protection of participant confidentiality. Data can be accessed upon request with permission of the third party and approval of UW-Madison and Institutional Review Board.

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Funding

SSM has received research support from the Clinical and Translational Science Award through the NIH National Center for Advancing Translational Sciences, grant UL1TR002373 and KL2TR002374. JG is funded by the NIH National Institute of Diabetes and Digestive and Kidney Diseases award R01 DK109508. YL has received research support from the NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases grant K01 AR073340 and Wisconsin Partnership Program New Investigator Program.

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Authors and Affiliations

  1. Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI, 53706, USA

    Teressa M. Shaw, Adam Pagenkopf, Diana M. Carp, Xueer Qiu & Yun Liang

  2. Department of Medicine, University of California, San Diego, CA, USA

    Wei Zhang

  3. Division of Rheumatology, Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA

    Sara S. McCoy, Shivani Garg & Maxwell H. Parker

  4. Department of Pathology and Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    Robert H. Scofield

  5. Department of Medicine, Carbone Comprehensive Cancer Center, University of Wisconsin, WI, Madison, USA

    Jacques Galipeau

  6. Present address: Bristol Myers Squibb, New York, NY, USA

    Wei Zhang

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  1. Teressa M. Shaw
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Correspondence to Yun Liang.

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Supplementary Information

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Supplementary Table. Table of skewed X-linked genes in pSS.

X-linked genes skewed in each pSS subject are listed with gene symbol, Ensemble ID and minor allele frequency (MAF). (XLSX 18 KB)

Supplementary file2 (DOCX 41 KB)

Supplementary Fig. 1. Demographic and clinical profiles of control and pSS subjects.

Demographic and clinical information of control and pSS subjects are shown, including age, sex, race, group, SSA status, focus score, ANA, rheumatoid factor, C3, C4, organ involvement, risk factor for lymphoma, children, postmenopausal, contraceptive, hormone replacement, and surgical menopause. Supplementary Fig. 2. Venn Diagram of skewed, X-linked genes in pSS subjects. The number of overlapping and non-overlapping skewed, X-linked genes in the four pSS subjects (SS1, SS2, SS3 and SS4) are shown by the Venn Diagram. Supplementary Fig. 3. Stability of allele-specific expression and DNA allelic ratio of skewed expressors. (a) Allele-specific qPCR of TSPAN6 transcripts in control (N) and pSS (SS), passage 1-4 MSCs (n=4 independent subjects each), showing stability in allele-specific expression. Similar (i.e., non-drifting) results have been obtained for CHST7, ZDHHC9 and BCOR. (b) DNA allelic ratio of TSPAN6, CHST7 and ZDHCC9 in control (N) and pSS (SS) MSCs by allele-specific qPCR of DNA, showing ~1:1 ratio of the two alleles with no evidence of aneuploidy. Data shown were from n=3 replicates from one subject, and result was confirmed in samples from an independent subject with skewed expression of given target. Mean ± sem. Supplementary Fig. 4. Allele-specific expression analysis of XIST. Allele-specific qPCR of XIST in control (N) and pSS (SS) MSCs, showing lack of skewing in at least two of the four pSS MSCs. Supplementary Fig. 5. Skewing in whole minor salivary glands in pSS and in blood and skin of SLE. (a) Allele-specific qPCR for representative skewed expressors (TSPAN6, CHST7, ZDHHC9) and BCOR as a control, showing skewed expression of X-linked genes in pSS but not control whole salivary glands (n=4 pSS, n=4 control, independent subjects each, female). N, control. SS, pSS. WG, whole minor salivary gland. (b) Allele-specific qPCR for TSPAN6, CHST7, ZDHHC9 and BCOR, showing skewing of TSPAN6 and BCOR in SLE but not control blood cells (n=6 SLE, n=5 control, independent subjects each, female). N, control. SLE, systemic lupus erythematosus. PBMC, peripheral blood mononuclear cells. (c) Allele-specific qPCR for TSPAN6, CHST7, ZDHHC9 and BCOR, showing skewing of TSPAN6 and BCOR in SLE but not control skin (n=5 SLE, n=2 control, independent subjects each, female). N, control. SLE, systemic lupus erythematosus. Supplementary Fig. 6. Allele-specific expression of select genes on the X chromosome. (a) Allele-specific expression of immune-associated genes in each control (N) or pSS (SS) sample with skewed expression indicated in yellow (MAF < 0.35), showing lack of pSS-associated skewing for these genes. Grey boxes show missing data (no detected common het-SNPs) from RNA-Seq. (b) Allele-specific expression of common variants in each control (N) or pSS (SS) sample with skewed expression indicated in yellow (MAF < 0.35), showing pSS-associated skewing. Grey boxes show missing data (no detected common het-SNPs) from RNA-Seq. (c) Gene length for genes listed in (A, immune-associated) or (B, genes with common variants), showing no significant difference in length between the two groups. (d) Total RNA expression levels for genes listed in (A, immune-associated) or (B, genes with common variants) in control (N) or pSS (SS) MSCs, showing no significant difference in RNA expression levels between the two groups. Supplementary Fig. 7. Skewed expressors associate with histone modification and chromatin organization functions. (a) Network analysis of skewed expressors, showing the three biological networks formed by these genes. (b) Biological process enrichment analysis, showing histone H2A acetylation, protein acylation and chromatin organization as the top biological processes enriched in skewed expressors (FDR < 0.05). (c) Cellular component analysis of skewed expressors, showing enrichment of the NuA4 histone acetylatransferase complex, Swr1 complex and histone deacetylase complex (FDR < 0.05). Supplementary Fig. 8. Protein descriptions for the skewed expressor network. Proteins in the three biological networks formed by skewed expressors, as shown in Supplementary Fig. 5, are described with cluster information, protein name and functional description. Supplementary Fig. 9. Expression levels of skewed expressors in pSS and control MSCs. (a) qPCR of indicated genes (APOO, CHST7, PJA1, TSPAN6, TCEAL4, MORF4L2, SLC25A43, GRIA3, ZDHCC9, AIFM1, MOSPD1, LDOC1, IDS, FLNA) from pSS (SS) and control (N) MSCs, showing comparable expression levels in control and pSS MSCs (n=4 independent subjects each). Mean ± sem. (b) Western blot analysis of indicated proteins (TSPAN6, CHST7 and control GAPDH) in pSS (SS) and control (N) MSCs (4-20% gradient gel, loading equal amounts of protein amounts; markers indicated on gel), showing comparable expression levels in control and pSS MSCs. Supplementary Fig. 10. BCOR localization in MSCs. Immunostaining of BCOR and DAPI in control (N) and pSS (SS) MSCs, showing comparable localization in the two groups (images representative of > 100 cells in four independent subjects each). Supplementary Fig. 11. Copy number analysis in control and pSS MSCs. DNA copy number analysis of genes on the X chromosome and autosomes (chromosome 12 and chromosome 2), showing lack of altered ploidy in pSS MSCs (SS) compared to control (N) (n=4 independent subjects each). Mean ± sem. Supplementary Fig. 12. XIST decrease leads to disease-associated mislocalization of skewed expressors. (a) qPCR of XIST and LINE1 (L1) levels upon scrambled (scr siRNA) or XIST knockdown (XIST siRNA) in control MSCs, showing decrease in XIST upon XIST knockdown. Data shown were from n=3 replicates from one subject, and result was confirmed in samples from two independent subjects. Mean ± sem, * P < 0.05, Student’s t-test, two tailed. Line under asterisk indicates groups compared. (b) Immunostaining of TSPAN6 and DAPI in control MSCs with scrambled (scr siRNA) or XIST knockdown (XIST siRNA), showing ectopic TSPAN6+ foci upon XIST knockdown (pointed by arrow; images representative of > 100 cells from three independent subjects). (c) Immunostaining of CHST7 and DAPI in control MSCs with scrambled (scr siRNA) or XIST knockdown (XIST siRNA), showing loss of CHST7+ foci upon XIST knockdown (pointed by arrow; > 100 cells from three independent subjects). Supplementary Fig. 13. Knockdown efficiency of CTCF siRNA and specificity of miR6891-5p inhibition. (a) qPCR of CTCF levels (left two bars) and miR-3135B levels (right two bars) upon control or miR6891-5p inhibition (n=4 independent subjects). (b) qPCR of CTCF levels upon scrambled or CTCF knockdown in control MSCs, showing decrease in CTCF upon CTCF knockdown (n=4 independent subjects). Mean ± sem, * P < 0.05, Student’s t-test, two tailed. Line under asterisk indicates groups compared. Supplementary Fig. 14. miR6891-5p inhibition leads to disease-associated mislocalization of skewed genes. (a) Immunostaining of TSPAN6 and DAPI in control or miR6891-5p-inhibited MSCs, showing ectopic TSPAN6+ foci upon miR6891-5p inhibition (pointed by arrow; > 100 cells from three independent subjects). (b) Immunostaining of CHST7 and DAPI in control or miR6891-5p-inhibited MSCs, showing loss of CHST7+ foci upon miR6891-5p inhibition (pointed by arrow; > 100 cells from three independent subjects). (c) Allele-specific ChIP of IgG, H3K27me3 and H3K36me3 on BCOR in MSCs with control (Scr siRNA), XIST knockdown (XIST siRNA) or miR6891-5p inhibition (miR6891-5p inh), showing that XIST knockdown or miR6891-5p inhibition does not alter epigenetic state of non-skewed expressors. Data shown were from n=3 replicates from one subject, and result was confirmed in samples from an independent subject. Mean ± sem. Supplementary Fig. 15. pSS MSCs exhibit inflammatory differentiation. (a) qPCR of IL6 in control (N) and pSS (SS) MSCs during differentiation, showing comparable expression levels of IL6 in control and pSS MSCs. (b) qPCR of IL4 in control (N) and pSS (SS) MSCs during differentiation, showing deficiency in IL4 upregulation upon pSS MSC differentiation. (c, d) qPCR of IL4 (c) and IL1B (d) in MSCs with control, XIST knockdown (XIST Ri) or miR6891 inhibition (miR6891-5p inh), showing deficiency in IL4 upregulation during pSS MSC differentiation upon XIST and miR6891 disruption. n=3 independent subjects each. Mean ± sem, * P < 0.05, Student’s t-test, two tailed. Line under asterisk indicates groups compared. (PDF 5174 KB)

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Shaw, T.M., Zhang, W., McCoy, S.S. et al. X-linked genes exhibit miR6891-5p-regulated skewing in Sjögren’s syndrome. J Mol Med 100, 1253–1265 (2022). https://doi.org/10.1007/s00109-022-02205-3

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  • DOI: https://doi.org/10.1007/s00109-022-02205-3

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