The Y chromosome as a regulatory element shaping immune cell transcriptomes and susceptibility to autoimmune disease - PubMed (original) (raw)

The Y chromosome as a regulatory element shaping immune cell transcriptomes and susceptibility to autoimmune disease

Laure K Case et al. Genome Res. 2013 Sep.

Abstract

Understanding the DNA elements that constitute and control the regulatory genome is critical for the appropriate therapeutic management of complex diseases. Here, using chromosome Y (ChrY) consomic mouse strains on the C57BL/6J (B6) background, we show that susceptibility to two diverse animal models of autoimmune disease, experimental allergic encephalomyelitis (EAE) and experimental myocarditis, correlates with the natural variation in copy number of Sly and Rbmy multicopy ChrY genes. On the B6 background, ChrY possesses gene regulatory properties that impact genome-wide gene expression in pathogenic CD4(+) T cells. Using a ChrY consomic strain on the SJL background, we discovered a preference for ChrY-mediated gene regulation in macrophages, the immune cell subset underlying the EAE sexual dimorphism in SJL mice, rather than CD4(+) T cells. Importantly, in both genetic backgrounds, an inverse correlation exists between the number of Sly and Rbmy ChrY gene copies and the number of significantly up-regulated genes in immune cells, thereby supporting a link between copy number variation of Sly and Rbmy with the ChrY genetic element exerting regulatory properties. Additionally, we show that ChrY polymorphism can determine the sexual dimorphism in EAE and myocarditis. In humans, an analysis of the CD4(+) T cell transcriptome from male multiple sclerosis patients versus healthy controls provides further evidence for an evolutionarily conserved mechanism of gene regulation by ChrY. Thus, as in Drosophila, these data establish the mammalian ChrY as a member of the regulatory genome due to its ability to epigenetically regulate genome-wide gene expression in immune cells.

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Figures

Figure 1.

Figure 1.

Natural genetic variation in ChrY mediates susceptibility to autoimmune disease. (A) Male mice from the B6–ChrY consomic strains were immunized with MOG35–55 using the 2× protocol and the clinical score was monitored over 30 d. The consomic strain represented by each line is color-coded with the bar graph in B. Wild-type B6 male are represented by black circles. The significance of the differences in disease course among the strains was determined by two-way ANOVA (interaction [F = 1.18; DFn = 319; DFd = 7770; P = 0.02], day post-injection [F = 132.81; DFn = 29; DFd = 7770; P < 0.0001], and strain [F = 23.53; DFn = 11; DFd = 7770; P < 0.0001]). (B) The degree of EAE susceptibility among the B6–ChrY consomic mice was calculated by subtracting the clinical disease score (CDS) of B6 from the CDS of each strain. The _x_-axis indicates the strain origin of the Y chromosome. F = 2.58, P ≤ 0.0009. A heterogeneity test was used between cohorts of mice with no significant differences detected. Thus, data were pooled from three independent experiments and the total number of animals analyzed in A and B are indicated in Supplemental Table S1. (C) Immunohistopathology of the brain and spinal cord from B6–ChrY consomic strains immunized using the 2× protocol. Only the total histopathology score is shown. Significance determined by one-way ANOVA. (**) P ≤ 0.01. (D) Male mice from wild-type B6 and four consomic lines were infected with 50 PFU CVB3 and their hearts were evaluated for myocarditis. Heart score of B6 was subtracted from the heart score for each consomic. Labeling on the _x_-axis represents the strain donating ChrY to B6. Results are representative of three individual experiments. n ≥ 5 mice per strain. Significance determined by one-way ANOVA and Dunnett's multiple comparison test. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001. (E) Linear regression analysis of heart score and heart viral titers reveals the absence of a significant relationship between the regressed variables. _Left x_-axis (orange linear regression line and orange circles) corresponds to the B6–ChrY consomic data combined from each of the four tested strains. F = 0.72, P = 0.41. _Right x_-axis (black linear regression line and black squares) corresponds to the WT B6 data. F = 0.43, P = 0.52. (F) Correlation analysis of serum testosterone with the CDS for EAE (r = 0.36; P = 0.27). (G) Functionally significant Sry polymorphisms are discordant with ChrY-mediated differences in disease susceptibility. Data from B were grouped according to their functional Sry polymorphisms.

Figure 2.

Figure 2.

Sly and Rbmy gene copy number correlates with EAE and CVB3 susceptibility among B6–ChrY consomic strains. (A) Gene copy number for each of the consomic strains relative to B6. n ≥ 5 mice per strain. (B) Correlation analysis of gene copy number with the CDS for EAE. (C) Correlation analysis of gene copy number with myocarditis heart score. The _x_-axis represents the strain origin of ChrY.

Figure 3.

Figure 3.

ChrY-mediated regulatory variation in genome-wide gene expression influences the activation profile of CD4+ T cells. (A) Venn diagram representing the number of genes passing an FDR < 0.05 between B6–ChrYSJL and B6 CD4+ T cells. MGI GO Term Finder was used to identify enriched biological processes associated with each gene list, examples of which are shown. (B) Heat map from quantitative PCR array specific for mouse epigenetic chromatin remodeling factors on three biological replicates of purified CD4+ T cells from B6 and B6–ChrYSJL. Red represents up-regulated genes and green down-regulated genes. Degree of change indicated by intensity of color. (C) IPA TCR signaling pathway with the molecules differentially regulated between B6–ChrYSJL and B6 CD4+ T cells highlighted in orange. (D) Purified CD4+ T cells from B6–ChrYSJL and B6 lymph nodes were incubated with anti-CD3 and anti-CD28 monoclonal antibodies and the supernatants were tested for IL2, IFNG, IL17A, TNF, and IL6 production after 24, 48, and 72 h by ELISA. Data are representative of three independent experiments. Significance determined by two-way ANOVA followed by Bonferroni post-hoc multiple comparison test. (*) P ≤ 0.05; (****) P ≤ 0.0001.

Figure 4.

Figure 4.

ChrY preferentially regulates the transcriptome of macrophages compared with CD4+ T cells in the SJL genetic background. Venn diagram representing the number of genes passing an FDR < 0.05 between SJL–ChrYB10.S and SJL. (A) CD4+ T cells and (B) macrophages. MGI GO Term Finder was used to identify enriched biological processes associated with each gene list, examples of which are shown. (C) Graph representing the percentage of up-regulated genes in the ChrY consomic strain vs. the parental in relation to the averaged copy number variation for Sly and Rbmy. (D) A Venn diagram representing the genes differentially regulated between the human and B6 mouse CD4+ T-cell microarrays, 440 of which are shared between the two species. MGI GO term analysis on these shared terms identified an enrichment of genes involved in gene regulatory processes.

Figure 5.

Figure 5.

Genetic variation in ChrY modifies the sexual dimorphism in myocarditis and leads to a sex bias in EAE severity. (A) Male and female mice from WT B6 and male mice from four B6–ChrY consomic strains were infected with 50 PFU CVB3 and their hearts were evaluated for myocarditis. Heart score of female B6 was subtracted from the heart score for male WT B6 and each B6–ChrY consomic strain. Labeling on the _x_-axis represents the strain donating ChrY to B6. Results are representative of three individual experiments. n ≥ 5 mice per strain. Significance determined by one-way ANOVA and Dunnett's multiple comparison test. (*) P ≤ 0.05; (****) P ≤ 0.0001. (B) Female B6 and male B6–ChrY consomic mice were immunized with MOG35–55 using the 2× protocol and the clinical score was monitored over 30 d. The significance of the differences in disease course among the strains was determined by two-way ANOVA (interaction [F = 1.18; DFn = 319; DFd = 7770; P = 0.02], day post-injection [F = 132.8; DFn = 29; DFd = 7770; P < 0.0001], and strain [_F_ = 23.5; DFn = 11; DFd = 7770; _P_ < 0.0001]). (_C_) The significance of the differences in disease course between B6 female mice and each B6–ChrY consomic line was determined as described in Supplemental Figure S2 and grouped accordingly. The significance of the differences in sexual dimorphism among the groups was determined by comparing the best-fit values for the F < M and F > M grouping against the F = M group and by two-way ANOVA (interaction [F = 1.14; DFn = 348; DFd = 8880; P = 0.04], day post-injection [F = 155.5; DFn = 29; DFd = 8880; P < 0.0001], and strain [F = 22.58; DFn = 12; DFd = 8880; P < 0.0001]) followed by Holm-Sidak corrected post-hoc multiple comparisons. (*) P ≤ 0.05; (****) P ≤ 0.0001.

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