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. 2018 Feb 16;7:e31159. doi: 10.7554/eLife.31159

Figure 4. Estrogen Activates the Ire1α-Xbp1 Branch of the UPR.

(A) Unsupervised hierarchical clustering of genes with log2 fold change larger than one identified by RNA-seq. Top 25 genes for each cluster are shown. # indicates Ern1, which encodes Ire1α. (B) GSEA was performed between oil- and E2-treated HSCs. Gene sets significantly enriched in E2-treated HSCs are shown. (C) Pair wise GSEA was performed to test the enrichment of UPR-related genes in wild-type or ERα-deficient HSCs after either oil- or E2-treatment (left). E2-treated wild-type HSCs had enrichment of UPR genes, as indicated by the cumulative enrichment score (right). (D) Fragments per kilobase of exon per million fragments mapped (FPKM) of Ern1 transcript was increased in HSCs upon E2 treatment, and this increase was dependent on ERα. **p<0.01 by two-way ANOVA. (E) Quantitative PCR assays confirmed that the induction of Ire1α in HSCs, as well as in MPPs, upon E2 treatment was dependent on ERα (n = 3, two independent experiments). *p<0.05 by two-way ANOVA. (F) Xbp1 splicing was determined by a two-color Taqman assay. Xbp1 splicing was induced in HSCs and MPPs upon E2 treatment in an ERα-dependent manner. The ratio between spliced (Xbp1s) to unspliced (Xbp1u) Xbp1 transcript is shown (n = 4, two independent experiments). *p<0.05 by two-way ANOVA. (G) Xbp1 splicing was also determined by flow cytometry using the ERAI strain. Grey histogram represents the background signal from ERAI- HSCs. **p<0.01; and ***p<0.001 by Student’s t-test. (H) Immunoblotting was performed using HSC/MPP (CD48-/lowLSK), progenitor cells (CD48+LSK), and WBM cells. Both Ire1α and Xbp1s protein levels were increased by E2 treatment in immature cells. All data represent mean ±standard deviation.

Figure 4.

Figure 4—figure supplement 1. E2 treatment increases the expression of megakaryocyte-related genes and chaperones in HSCs.

Figure 4—figure supplement 1.

(A) Ire1α mRNA expression levels were quantified in LSK cells cultured in vitro with vehicle or varying concentrations of E2. (B–D) We CRISPR-edited LSK cells for Ire1α or control Rosa26, and assessed the relationships between Ire1α and Myc targets genes identified in the GSEA (Figure 4B), using the condition described in (A). Myc targets Tipin (B), Tyms (C), and Ccna2 (D) were induced by E2 dependently on Ire1α. Pole3 (E), Hmga1 (F), Aimp2 (G), and Cdkn2c (H) were induced in E2 treated cells, but deletion of Ire1α did not significantly affect the expression. *p<0.05, two-way ANOVA. (I) FPKMs for megakaryocyte-related genes in Oil and E2 treated WT and ERα-deficient HSCs. *p<0.05; **p<0.01; ***p<0.001 by two-way ANOVA. (J) Heatmap of RPKMs from RNA Seq data. Genes represented in the heatmap were genes discovered to be enriched in the GSEA Hallmark UPR geneset. Data represents fold change from Oil treated HSCs. (K) Average expression values of genes encoding chaperones or foldases as determined by qPCR. Data represents fold change from Oil treated HSCs.
Figure 4—figure supplement 2. Ire1α induction in HSCs is not a function of increased HSC proliferation.

Figure 4—figure supplement 2.

(A) Representative plot used to fractionate CD48-/low LSKs based on CD150 levels. (B) Ire1α mRNA levels were quantified in CD150HI, LO, and NEG CD48-/low LSKs. Ire1α was potently induced equally amongst all three populations. (C) Expression levels of the UPR constituents CHOP, PERK, and Atf6 were assessed in Oil and E2 treated WT and ERα-deficient HSCs by qPCR. Neither treatment caused significant changes in the expression of CHOP, PERK, and Atf6 (accessed by ANOVA). (D) Puromycin incorporation assay. Male mice were treated with oil or E2 for one week. One hour prior to collection, mice were injected with puromycin and puromycin incorporation into each population was analyzed by flow cytometry. (E) Expression levels of Ire1α, PERK, Atf6, and CHOP in G-CSF treated animals were analyzed by qPCR. Male C57BL/6 mice were injected with 4 mg of cyclophosphamide and then with G-CSF (5 μg/day) for 3 or 6 days, and analyzed the following day (n = 3). Control mice were injected with PBS. Since the numbers of HSCs in the spleen of control PBS-injected mice are low, gene expression changes in spleen HSCs were compared to bone marrow HSCs of PBS-injected mice. Although the expression levels of Atf6 and CHOP were increased in mobilized spleen HSCs at day 6, expression levels of Ire1α did not change. (F) Ire1α expression levels in HSCs and WBM from mice treated with varying doses of poly I:C. Although poly I:C increased Ire1α expression in WBM cells, it did not significantly changes the expression in HSCs (G) Expression levels of chaperones was assessed in activated HSCs. HSCs were collected from G-CSF treated mice (upper panel) and poly I:C treated mice (lower panel) and chaperone mRNA levels were measured via qPCR. Whereas a few chaperones were induced, the global UPR network was not increased as extensively as E2 treatment. *p<0.05, Students t test.