ERα promotes murine hematopoietic regeneration through the Ire1α-mediated unfolded protein response

Activation of the unfolded protein response (UPR) sustains protein homeostasis (proteostasis) and plays a fundamental role in tissue maintenance and longevity of organisms. Long-range control of UPR activation has been demonstrated in invertebrates, but such mechanisms in mammals remain elusive. Here, we show that the female sex hormone estrogen regulates the UPR in hematopoietic stem cells (HSCs). Estrogen treatment increases the capacity of HSCs to regenerate the hematopoietic system upon transplantation and accelerates regeneration after irradiation. We found that estrogen signals through estrogen receptor α (ERα) expressed in hematopoietic cells to activate the protective Ire1α-Xbp1 branch of the UPR. Further, ERα-mediated activation of the Ire1α-Xbp1 pathway confers HSCs with resistance against proteotoxic stress and promotes regeneration. Our findings reveal a systemic mechanism through which HSC function is augmented for hematopoietic regeneration.


Sample-size estimation
• You should state whether an appropriate sample size was computed when the study was being designed • You should state the statistical method of sample size computation and any required assumptions • If no explicit power analysis was used, you should describe how you decided what sample (replicate) size (number) to use Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission:

Replicates
• You should report how often each experiment was performed • You should include a definition of biological versus technical replication • The data obtained should be provided and sufficient information should be provided to indicate the number of independent biological and/or technical replicates • If you encountered any outliers, you should describe how these were handled • Criteria for exclusion/inclusion of data should be clearly stated • High-throughput sequence data should be uploaded before submission, with a private link for reviewers provided (these are available from both GEO and ArrayExpress) Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: For experiments that involved collecting sample materials from multiple mice (Fig 1a,b, 4e,f,g, 5f, S4a,b,c,d,f, S5a,b), three independent replicates were obtained to allow statistical inference while using a justifiable number of animals. For other experiments, we aimed for 4-6 biological replicates per group. For most experiments, given a standard deviation of approximately 30% (based on previous experiments), this sample size allowed us to determine changes of 55% in the mean with a power of 80% in two-tailed t tests (alpha = 0.05). In some instances, larger sample sizes were obtained by collecting observations from mice in previous experiments (Fig 1e-j).

Statistical reporting
• Statistical analysis methods should be described and justified • Raw data should be presented in figures whenever informative to do so (typically when N per group is less than 10) • For each experiment, you should identify the statistical tests used, exact values of N, definitions of center, methods of multiple test correction, and dispersion and precision measures (e.g., mean, median, SD, SEM, confidence intervals; and, for the major substantive results, a measure of effect size (e.g., Pearson's r, Cohen's d) • Report exact p-values wherever possible alongside the summary statistics and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05.
Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: (For large datasets, or papers with a very large number of statistical tests, you may upload a single table file with tests, Ns, etc., with reference to sections in the manuscript.)

Group allocation
• Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Statistical analysis methods are described in each figure legends, as well as in the "Quantification and Statistical Analysis" section of the Materials and Methods. Specifically, unless indicated otherwise in the figure legends, analyses were always carried out in comparison to the control group (Oil, DMSO, etc). When comparing means from groups with unequal sample sizes, Mann Whitney tests were used for the comparison of two groups (Fig S5d), and Kruskal Wallis tests with Dunn's multiple comparison tests were used for comparing three or more groups (Fig 6e). Otherwise, two-tailed t-tests were used for the comparison of two groups (Fig. 1c, 2a-c, 3a-d, 5a, and Supplementary Fig S2f-g, i, S3a, S4b,f) and one-and two-way ANOVAs for comparisons involving more than two groups or more than two independent variables (Figs 1a-b, e-j, 2d-f, 3d-g, 4d-g, 5d-e, 6b,d and Supplementary Fig S1d, S2b-e, h, S3b-i, S4c-g, S5c-d) with Bonferroni corrections performed for multiple testing. Analysis of survival curves was performed using a log-rank test (Fig 2g-h). This is described in the "Quantification and Statistical Analysis" section of Materials and Methods. All mice were age and sex matched for each experiment. Unless indicated otherwise only male mice were used, and littermate controls were used for ERaKO studies. Distribution of mice was not formally randomized, and no experiments were blinded.
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