The negative autoregulatory motif Negative autoregulation is a frequent motif in biological networks. Computational and experimental approaches in simple transcriptional networks in E.Coli suggested that it has two principal effects: conferring resistance to random fluctuations, and accelerating the response to a stimulus [32,33,34,35,56,57]. To our knowledge, the functional role of negative autoregulation within higher-level intercellular networks had not been tested experimentally. Our work suggests that the negative regulatory motif may exert similar ‘logic’ in higher-level networks, helping to maintain both stability and a fast stress response of tissue progenitors. Supporting Information Figure S1 Associated with Fig 1: Inhibition of Fas with Fas:Fc decreases EryA death and increases erythropoietic rate. MyD882/2 mice (C57BL/6 background) were each injected intraperitoneally with 100 mg human purified Fas:Fc chimeric protein (BD Biosciences), or with an equal volume of saline. (A) Flow-cytometric histogram of Annexin V binding of spleen EryA cells, showing decreased apoptosis 48 hours following injection of Fas:Fc. (B) Summary of Annexin V binding in two independent experiments, at 48 hours and at 72 hours post-injection. Data points correspond to individual mice. (C) Summary of reticulocyte count (red blood cells younger than 24 hr, identifiable by their cytoplasmic RNA, which is absent in older red cells) in the same mice/experiments as in panel (B). Fas:Fc caused an increase in reticulocytes, reflecting increased erythropoietic rate. (TIF) Figure S2 Associated with Fig 5: Increased variance in lpr/gld erythroid progenitor subsets. (A) Frequency distribution histo- grams for EryA, in male and female gld-Rag12/2 and matched Rag12/2 controls. The coefficient of variation for each group is shown. Purple line is the corresponding normal distribution curve. Same data set as in Fig 3D. A similar analysis for the lpr-Rag12/2 mice is shown in Figure 3E. (B) Coefficient of variation (CV) for subsets ProE, EryA–C and retics in male or female lpr-Rag12/2 or and gld-Rag12/2 mice and corresponding Rag12/2 controls. The References 1. Ebert BL, Bunn HF (1999) Regulation of the erythropoietin gene. Blood 94: 1864–1877. 2. Semenza GL (2009) Involvement of oxygen-sensing pathways in physiologic and pathologic erythropoiesis. Blood 114: 2015–2019. 3. Haase VH (2010) Hypoxic regulation of erythropoiesis and iron metabolism. Am J Physiol Renal Physiol 299: F1–13. 4. D’Andrea AD, Lodish HF, Wong GG (1989) Expression cloning of the murine erythropoietin receptor. Cell 57: 277–285. 5. Broudy VC, Lin NL, Priestely GV, Nocka K, Wolf NS (1996) Interaction of Stem Cell Factor and its receptor c-kit mediates lodgement and acute expansion of hematopoietic cells in the murine spleen. blood 88: 75–81. 6. von Lindern M, Schmidt U, Beug H (2004) Control of erythropoiesis by erythropoietin and stem cell factor: a novel role for Bruton’s tyrosine kinase. Cell Cycle 3: 876–879. 7. von Lindern M, Zauner W, Mellitzer G, Steinlein P, Fritsch G, et al. (1999) The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro. Blood 94: 550–559. 8. Bauer A, Tronche F, Wessely O, Kellendonk C, Reichardt HM, et al. (1999) The glucocorticoid receptor is required for stress erythropoiesis. Genes Dev 13: 2996–3002. 9. Palis J (2008) Ontogeny of erythropoiesis. Curr Opin Hematol 15: 155–161. 10. Stephenson JR, Axelrad AA, McLeod DL, Shreeve MM (1971) Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proc Natl Acad Sci USA 68: 1542–1546. 11. Fawcett DW, Jensh RP (1997) Hemopoiesis. In: Fawcett DW, Jensh RP, eds. Bloom & Fawcett: Concise Histology. NY, NY: Chapman & Hall. pp 84–93. Fas Stabilizes and Accelerates Erythropoiesis difference in CV between control and lpr/gld is significant at p = 0.017 (paired t test, treating the CV as a standard statistical variable). (TIF) Figure S3 Associated with Fig 6: Delayed response to Epo injection in lpr-Rag12/2 mice. Female mice were injected with 10 U of Epo subcutaneously. Hematocrit, ProE and EryA progenitors were measured on day 3 post injection. *p,0.05 (two-tailed t test, unequal variance). Basal hematocrit values are for lpr-Rag12/2 females. (TIF) Table S1 Associated with Fig 3: Complete blood counts (CBC) for the indicated mouse strains. Number of mice used for each strain in each experiment is indicated in parentheses. HCT = hematocrit. HGB = hemoglobin. RBC = red blood cells. MCV = mean corpuscular volume. WBC = white blood cells. PLT = platelets. All data are mean 6sem. t test (unequal variance) p values are indicated. (TIF) Supplementary Materials and Methods. Regulation of the EryA progenitor pool by Fas. Text S1 (DOCX) Text S2 (DOCX) Acknowledgments We thank the UMass flow cytometry core: Richard Konz, Ted Giehl, Barbara Gosselin, Yuehua Gu and Tammy Krupoch; Dr. Douglas Green, St. Jude Children’s Research Hospital, for discussion; Dr. Egil Lien at Umass Medical School for providing MyD882/2 mice and Dr. Stephen Baker for help with statistical analyses. The authors also thank Ramona Pop, Daniel Hidalgo, Ermelinda Porpiglia, and Jeffrey Shearstone for comments on the manuscript. Author Contributions Conceived and designed the experiments: MK MS. Performed the experiments: MK YL KH. Analyzed the data: MK MS. Wrote the paper: MK MS. 12. Liu Y, Pop R, Sadegh C, Brugnara C, Haase VH, et al. (2006) Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo. Blood 108: 123–133. 13. Gregory CJ, McCulloch EA, Till JE (1975) Transient erythropoietic spleen colonies: effects of erythropoietin in normal and genetically anemic W/Wv mice. J Cell Physiol 86: 1–8. 14. Hara H, Ogawa M (1976) Erthropoietic precursors in mice with phenylhydra- zine-induced anemia. Am J Hematol 1: 453–458. 15. Koury MJ, Bondurant MC (1990) Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells. Science 248: 378–381. 16. Papayannopoulou T, Finch CA (1972) On the in vivo action of erythropoietin: a quantitative analysis. J Clin Invest 51: 1179–1185. 17. Koury MJ, Sawyer ST, Brandt SJ (2002) New insights into erythropoiesis. Curr Opin Hematol 9: 93–100. 18. Rhodes MM, Kopsombut P, Bondurant MC, Price JO, Koury MJ (2005) Bcl-xL prevents apoptosis of late-stage erythroblasts but does not mediate the anti- apoptotic effect of erythropoietin. Blood. 19. Silva M, Benito A, Sanz C, Prosper F, Ekhterae D, et al. (1999) Erythropoietin can induce expression of bcl-xL through Stat5 in erythropoietin-dependent progenitor cell lines. J Biol Chem 274: 25855–25861. 20. Socolovsky M, Fallon AEJ, Wang S, Brugnara C, Lodish HF (1999) Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice: a direct role for Stat5 in bcl-XL induction. Cell 98: 181–191. 21. Menon MP, Karur V, Bogacheva O, Bogachev O, Cuetara B, et al. (2006) Signals for stress erythropoiesis are integrated via an erythropoietin receptor- phosphotyrosine-343-Stat5 axis. J Clin Invest 116: 683–694. 22. Wood AD, Chen E, Donaldson IJ, Hattangadi S, Burke KA, et al. (2009) ID1 promotes expansion and survival of primary erythroid cells and is a target of JAK2V617F-STAT5 signaling. Blood 114: 1820–1830. PLoS ONE | www.plosone.org 13 July 2011 | Volume 6 | Issue 7 | e21192