Parent-of-origin differences in DNA methylation of X chromosome genes in T lymphocytes - PubMed (original) (raw)
Parent-of-origin differences in DNA methylation of X chromosome genes in T lymphocytes
Lisa C Golden et al. Proc Natl Acad Sci U S A. 2019.
Abstract
Many autoimmune diseases are more frequent in females than in males in humans and their mouse models, and sex differences in immune responses have been shown. Despite extensive studies of sex hormones, mechanisms underlying these sex differences remain unclear. Here, we focused on sex chromosomes using the "four core genotypes" model in C57BL/6 mice and discovered that the transcriptomes of both autoantigen and anti-CD3/CD28 stimulated CD4+ T lymphocytes showed higher expression of a cluster of 5 X genes when derived from XY as compared to XX mice. We next determined if higher expression of an X gene in XY compared to XX could be due to parent-of-origin differences in DNA methylation of the X chromosome. We found a global increase in DNA methylation on the X chromosome of paternal as compared to maternal origin. Since DNA methylation usually suppresses gene expression, this result was consistent with higher expression of X genes in XY cells because XY cells always express from the maternal X chromosome. In addition, gene expression analysis of F1 hybrid mice from CAST × FVB reciprocal crosses showed preferential gene expression from the maternal X compared to paternal X chromosome, revealing that these parent-of-origin effects are not strain-specific. SJL mice also showed a parent-of-origin effect on DNA methylation and X gene expression; however, which X genes were affected differed from those in C57BL/6. Together, this demonstrates how parent-of-origin differences in DNA methylation of the X chromosome can lead to sex differences in gene expression during immune responses.
Keywords: X chromosome; autoimmunity; global DNA methylation; parental imprinting; sex differences.
Conflict of interest statement
The authors declare no competing interest.
Figures
Fig. 1.
CD4+ T lymphocytes from XX and XY− mice have distinct transcriptomes. RNA from autoantigen- (A_–_D) and anti–CD3/CD28-stimulated (E_–_H) CD4+ T lymphocytes from C57BL/6 FCG mice was analyzed by RNA-Seq. (A) PCA of autoantigen-stimulated CD4+ T lymphocyte transcriptomes showed separation of XX (n = 6) and XY− (n = 6). (B) Volcano plots showed the distribution of differentially expressed autosomal (black) and X (red) genes between XX and XY−. A cluster of 5 X genes had higher expression in XY− than XX. (C) PCA of autoantigen-stimulated CD4+ T lymphocyte transcriptomes showed separation of XX_Sry_ (n = 5) and XY−Sry (n = 5). (D) Volcano plot showed higher expression of the 5 X gene cluster in XY−Sry compared to XX_Sry_. (E) PCA of anti–CD3/CD28-stimulated CD4+ T lymphocyte transcriptomes showed separation between XX (n = 4) versus XY− (n = 5) (F) Volcano plot showed higher expression of the 5 X gene cluster in XY− than XX. (G) PCA of anti–CD3/CD28-stimulated CD4+ T lymphocyte transcriptomes showed separation of XX_Sry_ (n = 5) and XY−Sry (n = 5). (H) Volcano plot showed higher expression of the 5 X gene cluster in XY−Sry compared to XX_Sry_. False discovery rate (FDR) < 0.1 was used as the threshold for significance (green line); any gene above this line was considered significantly different (FDR was calculated using R package edgeR).
Fig. 2.
XY− has higher expression of a cluster of 5 X genes compared to XX. RNA and protein expression were measured in autoantigen-stimulated CD4+ T lymphocytes from XX and XY− mice immunized with autoantigen. (A_–_E) RNA expression of (A) Msl3, (B) Prps2, (C) Hccs, (D) Tmsb4x, and (E) Tlr7 was measured by quantitative RT-PCR. XY− (n = 10 or 11) had higher RNA expression than XX (n = 10 or 11) for all 5 genes (****P < 0.0001, ***P < 0.0002). (F_–_H) CD4+ T lymphocytes were analyzed for TLR7 protein expression by flow cytometry. See
SI Appendix, Fig. S2
for gating strategy. (F) Representative flow plots for TLR7 expression in XX versus XY−. (G) XY− mice (n = 16) had a higher percentage of TLR7 expressing CD4+ T lymphocytes than XX (n = 16) (**P = 0.0017). (H) XY− mice (n = 16) had higher mean fluorescence intensity (MFI) of TLR7 expression in CD4+ T lymphocytes than XX (n = 16) (****P < 0.0001). (I–P) The same analyses in A–H were performed in XX_Sry_ and XY−Sry gonadal males. (I) Msl3, (J) Prps2, (K) Hccs, (L) Tmsb4x, and (M) Tlr7 RNA expression; XX_Sry n_ = 10 to 12, XY−Sry n = 10 to 12 (*P < 0.045, ****P < 0.0001). (N) Representative flow plots for TLR7 expression in XX_Sry_ versus XY−Sry. (O) TLR7% expression; XX_Sry n_ = 17, XY−Sry n = 14 (****P < 0.0001). (P) TLR7 MFI; XX_Sry n_ = 17, XY−Sry n = 14 (****P < 0.0001). All data are representative of 2 replicate experiments. Error bars represent SD. P values were calculated by Mann–Whitney U test.
Fig. 3.
A monosomic X mouse model to directly study parent-of-origin differences in DNA methylation. (A) Diagrams of the sex chromosomes used in the breeding of XY*x (XO) mice. The Y* chromosome is a rearranged Y chromosome with a translocated X chromosome centromere and a modified X PAR (see ref. for details). The Y*x chromosome is produced from recombination of the X and Y* chromosomes. It is an X chromosome with a massive deletion of about 99% of genes, leaving the PAR and about 8 non-PAR genes (32). (B) Representative metaphase spread of XY*x (XO) chromosomes. An XY*x animal has 39 normal chromosomes, plus 1 small “spec” representing the Y*x chromosome (Inset). (C) Female XmO mice were generated by crossing wild-type XX females and XY* males. (D) Female XpO mice were generated by crossing XmO females with wild-type XY males. (E) Xist expresses in XX, but not in XO. RT-PCR was performed on RNA from ear tissue. The expression of B2m was used as the internal control. (F) DNA methylation of Tlr7 was analyzed using targeted bisulfite sequencing in CD4+ T lymphocytes of XmO (n = 5) and XpO (n = 5) SJL mice 12 d after immunization with autoantigen. The DNA methylation on Xm and Xp is shown as the percentages of methylation at CpG sites of a CpG island upstream of the Tlr7 TSS. Xp had more methylation than Xm at each site analyzed (*P < 0.0120, **P < 0.0008, Mann–Whitney U test). Error bars represent SEM.
Fig. 4.
Genes on the Xp have more DNA methylation in CpG islands than Xm. XmO and XpO female mice were immunized with autoantigen for 12 d. DNA from CD4+ T lymphocytes isolated from lymph nodes was analyzed by bisulfite sequencing to generate the whole methylome of XpO (n = 4) and XmO (n = 5) SJL mice. Yellow spots indicate CpG sites in CpG islands that had DNA methylation in >50% of reads on average. Black dots were mapped based on all gene locations from the Ensemble genome browser (
uswest.ensembl.org/index.html
) and form the chromosome shapes. (A) The Xp chromosome showed greater accumulation of CpG island DNA methylation compared to autosomes (P < 2.2 × 10−16, Fisher’s exact test). (B) The Xm chromosome did not have accumulation of DNA methylation compared to autosomes (P = 1, Fisher’s exact test). Comparing Xp (A) and Xm (B), the number of methylated CpG islands was significantly higher on Xp than on Xm (P < 2.2 × 10−16, Fisher’s exact test). No difference in DNA methylation was observed for autosomal genes between XpO and XmO (P = 0.4258, Fisher’s exact test). (C and D) Gene expression data from F1 hybrid mice derived from CAST/EiJ × FVB/NJ and FVB/NJ × CAST/EiJ reciprocal crosses was analyzed to show parent-of-origin differences in X gene expression in multiple tissues (a: embryonic day [E] 16.5 liver, b: E16.5 brain, c: E16.5 heart, d: day-3 tongue, e: day-3 brain, f: adult brain, g: adult liver, h: adult heart, i: adult lung, j: adult spleen). White dots represent genes on the X chromosome with higher expression from Xp (C) or from Xm (D). There were many more X genes with higher expression from Xm compared to Xp across tissues.
Fig. 5.
Overarching hypothesis that sex differences in the immune system are due to the balance between parental imprinting of X genes that do not escape X inactivation (red stars) and X dosage effects of X genes that do escape X inactivation (blue triangles), which can be modulated by an effect of sex hormones. Males (XY) have 1 X chromosome with maternal imprinting (higher expression). On the other hand, females (XX) have 2 X chromosomes, 1 with maternal and 1 with paternal imprinting. Due to random X inactivation in females, the Xm is inactivated in half of the cells, while Xp is inactivated in the other half, creating a mosaic of cells expressing genes from either Xm (higher expression) or Xp (lower expression). Additionally, some genes escape X inactivation (Kdm6a for example), expressing from both X chromosomes and creating an X dosage effect with higher expression in XX versus XY. Together, this results in higher expression of parentally imprinted genes in XY and higher expression of X escapee genes in XX. These sex chromosome complement effects are not mutually exclusive of sex hormone effects, both organizational effects during development and activational effects during adulthood.
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