Purification and characterization of BFU-E and CFU-E cells from fetal murine liver We chose to purify erythroid progenitors from mouse E14.5–15.5 fetal liver (FL) rather than bone marrow (BM) since the concentration of these cells is higher in FL, and since ~90% of these cells are erythroid. Although peripheral blood contains BFU-E cells, it was not used since very few CFU-E cells are found in the circulation.1 We assayed levels of enrichment of BFU-E and CFU-E cells by colony forming assays (Fig. S1). Throughout the experiments in this paper we scored BFU-E colonies as late (small)–BFU-Es if they consisted of 5–20 clusters, or early (large)–BFU-Es if they consisted of more than 20 clusters (Fig. S1). Since some BFU-E cells formed clusters of CFU-E colonies (multi–CFU-E) already day 3 (Fig. S1), only cells forming a single CFU-E colony were scored as a CFU-E colony-forming cell.

The first step towards enrichment of BFU-E and CFU-E cells was to remove more mature blood cells from single cell suspensions of fetal livers by magnetic column depletion. We stained fetal liver cells with a panel of biotin labeled anti-lineage (Lin) antibodies (Ter119, B220, Mac-1, CD3 and Gr-1), followed by anti-biotin tetramer and magnetic colloid labeling. All positive cells were depleted using magnetic columns. As anticipated from previous studies the Lin− fraction contains mostly CFU-Es and BFU-Es, but also non-erythroid (CFU-G/M/Mk) and multi-potent (GEMM) colony forming cells (Table S1).2 As suggested by previous studies on mouse BM progenitor cells,3,4 further depletion using Sca-1 removed early multipotent GEMM colony forming cells, and reaction with CD16/32 (FcgR), CD41 and CD34 antibodies depleted myeloid colony-forming cells (CFU-G/M/Mk). The number of erythroid colony-forming cells was unaffected (Table S1), resulting in a very pure fetal liver erythroid progenitor (FLEP) cell population. Although only 50–70% of FLEP cells formed colonies, 99% of colonies that did form were BFU-Es or CFU-Es (Table S1). Thus FLEP cells are a mix of BFU-E and CFU-E cells with possible contamination of dead, apoptotic, or other non–colony-forming cells.

In order to further purify and separate populations of BFU-E and CFU-E cells from FLEP cells, we used flow cytometry (Fig. 2). Since BFU-E and CFU-E cells both express c-kit,4 and since CFU-E cells in BM express high levels of the receptor (CD71) 4 and mature erythroid cells high levels of CD24a,5 we hypothesized that the level of CD71 or CD24a expression could be used to separate CFU-E and BFU-E cells from c-kit+ FLEP cells.

Based on the fact that ≈15% of FLEP cells formed BFU-E colonies, we sorted the fractions of the 10% lowest CD71 (or CD24a) expressing c-kit+ FLEP cells. Similarly, since ≈60% of FLEP cells formed CFU-E colonies we hoped to obtain pure single–CFU-E forming cells by sorting the 20% highest CD71 (or CD24a) expressing c-kit+ FLEP cells. Indeed, Table S2 shows that this is the case. As judged by colony-forming assays BFU-E and CFU-E–forming cells could be separated to high purities using either antibodies against CD71 or CD24 antigen, with a slightly lower purity using CD24a than CD71 (Table S2). The high purity of BFU-Es enriched by CD71 is further shown in the single cell culture experiments (Fig. S2, panels C and D), which demonstrate that in presence of Dex more than 75% of BFU-E cells generate more than 100 erythroblasts (while more than 10% do not survive the sorting procedure). Since a CFU-E will not divide more than 6 times (and cannot generate 100 erythroblasts) more than 75% of the cells in this sorted population are earlier progenitors than CFU-E cells.

In methyl-cellulose assays the different 10%low and 20%high sorted cell populations demonstrated a total colony-forming efficiency (sum of all colonies formed divided by the 1000 input cells) ranging from 40–70%.

The fact that 100% of cells do not form colonies could be explained by a contamination of viable but non–colony-forming cells, or more likely by decreased cell viability caused by the extensive cell manipulation involved in fetal liver harvest, magnetic depletion, cell sorting, and the colony- forming assay procedure. The later explanation is supported by the single cell culture experiments (Fig. S2) and the homogenous morphology of cells within each sorted cell population (Fig. 2). The purities of BFU-E and CFU-E populations can therefore be estimated as the ratio of the number of BFU-E or CFU-E colonies to the total number of colonies formed in the presence of Dex. The BFU-E purity would then be 86% using CD71 only and 94% using both CD71 and CD24a (37% and 59% respectively of total cells being early (large) BFU-E– forming cells). Importantly, this highly enriched BFU-E population contains <1% CFU-G/M/Mk and no GEMM cells. To our knowledge these populations represent the purest separation and enrichment BFU-E and CFU-E cells ever described, although there are several reports describing the isolation of CFU-E 4,6 and BFU-E cells.7,8 Table S1. Efficient fetal liver erythroid progenitor (FLEP) enrichment using only magnetic depletion Embryonic day 14.5–15.5 mouse fetal livers were resuspended and stained with a lineage- negative (Lin−) cocktail containing biotin conjugated antibodies against murine Ter119 (erythroid), B220 (B cell), Mac-1 (monocyte/granulocyte), CD3 (T cell) and Gr-1 (granulocyte). Biotin conjugated antibodies against CD32/16 (myeloid), Sca-1 (multi-potent progenitors), CD41 (megakaryocyte) and CD34 (myeloid) were sequentially added to the Lin− cocktail to increase the purity of erythroid progenitor cells. The negatively enriched cell populations were subjected to colony forming assays under two different conditions. A) 1000 cells were plated in methylcellulose medium containing 10U EPO/ mL with and without 100nM Dex. CFU-E colonies were counted 3 days later. We were particularly careful to not score a multi–CFU-E colony as several CFU-E colonies (Fig. S1). B) 1000 cells were plated in methylcellulose medium containing 10U EPO/ mL, 50ng SCF/mL, 20ng mIL-3/mL and 20ng IL-6/mL with and without 100nM Dex. These cells were cultured 8–9 days before late BFU-E, early BFU-E, CFU- G/M/Mk, and GEMM colonies were scored. BFU-Es consisting of a cluster of only 5–20 CFU- Es were scored as late, while larger BFU-Es were scored as early (Fig. S1). 2,7-fluorenediamine was used to stain hemoglobin-containing colonies in the plates.9 The table shows the number of colonies per 1000 plated cells. Standard deviation between experiments is shown in parenthesis.

Table S2. Separation of BFU-E and CFU-E cells by expression of CD71, CD24a or both CD71 and CD24a C-kit positive BFU-E and CFU-E cells were sorted from FLEP cells based on expression of CD71, CD24a or both CD71 and CD24a. In all experiments the BFU-E fraction was the subset of c-kit+ FLEP cells with the 10% lowest expression of CD71 and/or CD24a, while the CFU-E fraction was the subset with 20% highest expression of CD71 and/or CD24a. Sorted BFU-E and CFU-E populations were subjected to colony forming assays under different conditions.

A fraction of the cells were plated in methylcellulose medium containing 10U EPO/ mL with and without 100nM Dex. The number of CFU-E colonies (± standard deviation) is shown. The sorted “BFU-E” populations mostly formed small CFU-E clusters rather than single CFU-E colonies in these assays. These CFU-E clusters are formed by precursors to CFU-Es and were scored separately as multi-CFU-Es. The number of “multi-CFU-Es” is not shown since these cells likely represent the same cells as those forming small BFU-E colonies. The potential of the sorted cells to form large BFU-E, small BFU-E, CFU-G/M/Mk, and GEMM colonies (± standard deviation) was determined by a second assay in methyl cellulose containing 10U Epo, 20nM IL-3, 20nM IL-6 and 50nM SCF per mL, and was scored at day 8–9. Addition of Dex significantly increases the number of large (early) BFU-E colonies (* p-value < 0.05, using students t-test). Using both CD71 and CD24a resulted in the most pure BFU-E enrichment.

To determine the effect of DMOG, colony assays of sorted BFU-E (CD71andCD24a10% low) cells were also performed in the presence of 333 µM DMOG. In agreement with the finding that DMOG enhances BFU-E self-renewal BFU-E colonies were larger with a more immature appearance in the presence of DMOG and Dex. Therefore, in the presence of DMOG and Dex most BFU-E colonies were scored as “large BFU-Es.”

Table S3. The 83 expressed more than 2-fold higher in BFU-E cells cultured with 100nM Dexamethasone The 83 upregulated genes are listed with relative expression values (RPKM). The score in motif prediction analysis for each is listed. In the column listing HIF1 targets predicted by Whole Genome rVISTA we include the PubMed ID of publications describing five genes (e.g. Egln3 = PubMed ID 15823097) 10 as bona fide HIF1 targets, even though they were not predicted as such by Whole Genome rVISTA.

Table S4. Expression of selected genes in BFU-E, CFU-E, and Ter119+ erythroblasts compared to BFU-E cells cultured 4 hours with or without Dexamethasone and DMOG Next generation mRNA-Seq was performed on freshly isolated BFU-E, CFU-E, and Ter119+ erythroblasts from mouse fetal liver, as well as from BFU-E (CD7110%low) cells cultured 4 hours with and without Dex; and BFU-E (CD24a/CD7110%low) cells cultured 4 hours with and without or both Dex and DMOG. Relative expression values (RPKM) are listed for a group of selected genes. The first group of genes is related to the actions of DMOG and HIF1α. While Hif1a is well expressed in BFU-E cells, Hif2a is not expressed in BFU-E or any other erythroid cells under any of the conditions studied. HIF prolyl hydroxylases (Phd1-3) are expressed at significantly higher levels than are collagen prolyl hydroxylases (P4ha1-3). The negative regulator of HIF signaling Hif3 is upregulated by Dex and the prolyl hydroxylase Phd1 is upregulated by both Dex and DMOG in BFU-E cells. The next group of genes encodes cell surface used as markers in FACS analysis. The high expression of CD71 and CD24a in fresh CFU-Es compared to BFU-Es confirms the rationale for our sorting methods. Also kit expression is higher in BFU-E than CFU-E cells , and increases in cultured BFU-E cells after Dex and DMOG stimulation. The third group of genes shows expression of transcription factors that again confirms the identity of the cells. The expression levels of Nr3c1 (GCR), Myc, Gata2, PU.1, and Stat5a/b are higher in BFU-E than in CFU-E cells.

Table S5. Overlap of genes upregulated >50% by Dex, DMOG and both Dex and DMOG in BFU-E cells The table shows the individual genes depicted in Fig. 4. Out of 9,636 genes highly expressed in BFU-E cells, 98 (37+5+23+33) genes are upregulated by DMOG; 190 (5+48+23+114) genes are upregulated by Dex; and 273 (33+23+114+103) genes are upregulated by a combination of Dex and DMOG.

Table S6. Overlap of genes downregulated >50% by Dex, DMOG and both Dex and DMOG in BFU-E cells The table shows the individual genes depicted in Fig. 4. Out 9,636 highly expressed genes, 45 (16+6+16+7), genes are downregulated by DMOG; 275 (6+16+151+102) genes are downregulated by Dex and 262 (7+16+102+137) genes are downregulated by a combination of Dex and DMOG.

Table S7. Promoter region motif enrichement analysis of genes 2-fold upregulated in 6 cell types The table shows results from the same analysis as in Fig. S2 but on other cell types than BFU-E cells.11–16 The table shows the result of motif enrichment analysis of genes whose expression is increased more than 2-fold in response to Dex in six previously published datasets. The ten most significantly enriched transcription factor binding motifs in the 5,000 bp upstream “promoter” regions as determined by Whole Genome rVISTA is shown for each dataset. There was significant enrichment of HIF1 binding sites in upregulated genes from three datasets, but at lower significe than in BFU-E cells. Importantly the software did not detect significant enrichment of GRE elements in 5 of the datasets and in the one dataset with GRE enrichment the significance was only 1e-3.4. The p value is calculated based on the number of hits in the submitted genomic regions for each motif, compared to the number of conserved hits in the genome. The average base distributions within the called hits for each enriched motif are shown below the table.

* Enrichment for GRE elements but the p-value is very low: −log10(p-value) = 3.4. ** No enrichment for HIF1 binding sites. ‡ Gene expression data generated with mRNA-sequencing. The rest were determined using microarray. † Liver samples were isolated from GR-null and wild-type adult mice 3 h after treatment with dexamethasone (200 µg/ 100 g body weight).

Figure S1. Dexamethasone promotes formation of larger BFU-E colonies (A) When scoring BFU-E colonies at day 8–9 we score the large BFU-Es with more than 20 CFU-E clusters “early BFU-Es” and smaller BFU-Es with 5–20 CFU-E clusters “late BFU-Es”. Representative early and late BFU-E colonies from cultures with and without 100nM Dex are shown. When Dex was added to the medium early BFU-Es were generally larger and contained more CFU-E clusters. (B) Three day CFU-E colony assays were performed in methylcellulose medium containing 10U EPO/ mL, with and without 100nM Dex. Dex does not affect the size of single CFU-E colonies. Micrographs were acquired by SPOT Advanced v3.5.2 software, using an inverted Nikon Eclipse TS100 microscope, equipped with a RT Monochrome camera. A Nikon 10×/0.25 lens was used in A and a Nikon 20×/0.40 lens was used in (B). Scales were derived from pictures of a 0.01mm objective micrometer.

Figure S2. Early BFU-E progenitor daughter cells perform 6 additional cell divisions in response to Dex (A) Sorted BFU-E (CD7110% low) cells were cultured in SFELE medium with different concentrations of Dex. Shown is the fraction of c-kit+ cells after 72 hours. (B) A second Dex dose-response curve was generated based on the total number of BFU-E daughter cell divisions at day 6. (C) 180 single BFU-E cells were sorted into individual wells of 96 well plates containing SFELE medium with 100nM Dex and 180 into medium without Dex. Cell numbers in each clone were estimated under the microscope on Day 8. 80% of sorted BFU-E cells form more than 100 daughter cells in erythroid expansion culture. (D) To determine how Dex affects early–BFU-E cell proliferation, cells from 53 BFU-E clones (50 cultured with Dex and 3 without Dex) that were scored as 1000+ at day 8 were counted using a hemocytometer. While several individual BFU-E cells were able to generate more than 100,000 erythroblasts in presence of 100 nM Dex, the most productive BFU-E cell without Dex generated 4,100 erythroblasts. At the end point of the culture individual BFU-E clones as expected had exclusively formed erythroblasts equivalent to the cells in Fig. 6B (data not shown).

Figure S3. As determined by Whole Genome rVISTA, Dexamethasone enhances expression of genes in BFU-E cells enriched for HIF1 and MYC binding sites in their promoters The figure shows the result of motif enrichment analysis of the 83 genes whose expression in BFU-E cells is increased more than 2-fold in response to Dex. Whole Genome rVISTA recognized 77 of the 83 genes and predicted a very high enrichment of HIF1 and MYC binding sites, conserved from mice to humans, in the 5,000 bp upstream “promoter” regions. The software did not detect significant enrichment of GRE elements. The p value is calculated based on the number of hits in the submitted genomic regions for each motif, compared to the number of conserved hits in the genome. The average base distributions within the called hits for each enriched motif are shown below the table.

Figure S4. The most enriched motif in promoters of genes upregulated by Dex, as determined by WebMOTIFS, is the HIF1 binding motif caCGTGga We used WebMOTIFS to analyze “promoter” regions –2000 to +200 bp of the 83 genes upregulated by Dex. The software did not detect significant enrichment of GRE elements. The most enriched motif caCGTGga, is a perfect HIF1a motif but is also a possible MYC binding motif.

Figure S5. DMOG and Dexamethasone synergize in stimulating BFU-E proliferation This figure, which is is a replot of the data in Fig. 5 clearly illustrates that DMOG and Dex have a synergistic effect on BFU-E proliferation. DMOG enhances the number of erythroblasts formed from a BFU-E progenitor around 2-fold in the absence of DEX, and 8-to-12–fold in the presence of DEX. The effect on BFU-E proliferation is synergistic since DMOG enhances proliferation more than 2-fold in presence of Dex.

Figure S6. FACS and Benzidine-Giemsa staining confirm that more than 85% of expanding cells are erythroid progenitors (A) Linneg, c-kitpos, Sca-1neg mouse bone marrow cells were cultured in SFELE medium with 333 µM DMOG and 100nM Dex. Cells were analyzed by FACS on day 10. 85% of cells are erythroid (CD71 and/or Ter119+), 4% are megakaryocytes (CD41) and 7% are granulocytes or monocytes (Mac-1, GR-1). (B) Fetal liver BFU-E cells were cultured in the same medium as in A. Cells were analyzed by FACS day 11. 91% of cells are erythroid (CD71 and/or Ter119+), 2% are megakaryocytes (CD41) and 2% are granulocytes or monocytes (Mac-1, GR-1). (C) Benzidine-Giemsa staining of cells in A show that approximately 30% are benizidine positive, and the rest are more immature erythroblasts (compare to Fig. 6B). D) Benzidine-Giemsa staining of cells in B show that approximately 30% are benizidine positive, and the rest are more immature erythroblasts (compare to Fig. 6C).

Figure S7. Maximum erythroblast formation from of human peripheral blood erythroid progenitors is synergistically enhanced by DMOG and Dex Human mobilized CD34+ cells were first thawed and cultured in serum free medium supplemented with StemSpan® CC100 cytokine mix containing rhSCF, rhFlt-3, rhIL-3, and rhIL-6 for 5 days. At day 5 days cells were switched to culture in the equivalent of SFELE medium containing rhEpo rhSCF and rhIGF-1, with or without 100 µM DMOG, and with our without 1 nM Dex. Total cell number was counted every other day until cell numbers dropped at day 5+19. More than 95% of cells at the end of the culture have erythroblast morphology (data not shown). DMOG enhances the effect of Dex also in human erythroid progenitors, allowing formation of 10-times more erythroblasts than in cultures with Dex alone graph show the maximum number of erythroblasts obtained from each condition (n=2). Error bars show one standard deviation.

REFERENCES

1. Praloran V, Klausman M, Naud MF, Harousseau JL. Blood erythroid progenitors (CFU-E and BFU-E) in acute lymphoblastic leukemias. Blut. 1989;58:75–78. 2. McKearn JP, McCubrey J, Fagg B. Enrichment of hematopoietic precursor cells and cloning of multipotential B-lymphocyte precursors. Proc Natl Acad Sci U S A. 1985;82:7414–7418. 3. Pronk CJ, Rossi DJ, Mansson R, et al. Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy. Cell Stem Cell. 2007;1:428– 442. 4. Terszowski G, Waskow C, Conradt P, et al. Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E). Blood. 2005;105:1937–1945. 5. Nielsen PJ, Lorenz B, Muller AM, et al. Altered erythrocytes and a leaky block in B-cell development in CD24/HSA-deficient mice. Blood. 1997;89:1058–1067. 6. Nijhof W, Wierenga PK. Isolation and characterization of the erythroid progenitor cell: CFU- E. J Cell Biol. 1983;96:386–392. 7. Heath DS, Axelrad AA, McLeod DL, Shreeve MM. Separation of the erythropoietin- responsive progenitors BFU-E and CFU-E in mouse bone marrow by unit gravity sedimentation. Blood. 1976;47:777–792. 8. Sawada K, Krantz SB, Dai CH, et al. Purification of human blood burst-forming units- erythroid and demonstration of the evolution of erythropoietin receptors. J Cell Physiol. 1990;142:219–230. 9. Kaiho S, Mizuno K. Sensitive assay systems for detection of hemoglobin with 2,7- diaminofluorene: histochemistry and colorimetry for erythrodifferentiation. Anal Biochem. 1985;149:117–120. 10. Pescador N, Cuevas Y, Naranjo S, et al. Identification of a functional hypoxia-responsive element that regulates the expression of the egl nine homologue 3 (egln3/phd3) gene. Biochem J. 2005;390:189–197. 11. Reddy TE, Pauli F, Sprouse RO, et al. Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation. Genome Res. 2009;19:2163–2171. 12. Gupta V, Galante A, Soteropoulos P, Guo S, Wagner BJ. Global gene profiling reveals novel glucocorticoid induced changes in gene expression of human lens epithelial cells. Mol Vis. 2005;11:1018–1040. 13. John S, Johnson TA, Sung MH, et al. Kinetic complexity of the global response to glucocorticoid receptor action. Endocrinology. 2009;150:1766–1774. 14. Pantoja C, Huff JT, Yamamoto KR. Glucocorticoid signaling defines a novel commitment state during adipogenesis in vitro. Mol Biol Cell. 2008;19:4032–4041. 15. Bianchini R, Nocentini G, Krausz LT, et al. Modulation of pro- and antiapoptotic molecules in double-positive (CD4+CD8+) thymocytes following dexamethasone treatment. J Pharmacol Exp Ther. 2006;319:887–897. 16. Wong S, Tan K, Carey KT, Fukushima A, Tiganis T, Cole TJ. Glucocorticoids stimulate hepatic and renal catecholamine inactivation by direct rapid induction of the dopamine sulfotransferase Sult1d1. Endocrinology;151:185–194.

A Colonies per 1000 plated cells, scored day 3

Magnetic depletion antibodies Dex CFU-E Lin- (n=7) - 485 (±77) + 472 (±79) Lin-, CD32/16- (n=2) - 634 (±19) + 560 (±245) Lin-, CD32/16-, Sca-1- (n=3) - 717 (±105) + 791 (±151) Lin-, CD32/16-, Sca-1-, CD41- (n=5) - 734 (±271) + 689 (±212) Lin-, CD32/16-, Sca-1-, CD41-, CD34- (n=4) - 486 (±170) (FLEP cells) + 640 (±148)

B Colonies per 1000 plated cells, scored day 8-9

Magnetic depletion antibodies Dex late BFU-E early BFU-E CFU-G/M/Meg GEMM Lin- (n=7) - 14 (±8) 21 (±9) 38 (±10) 5 (±3) + 53 (±13) 39 (±12) 25 (±5) 7 (±4) Lin-, CD32/16- (n=2) - 12 (±3) 6 (±2) 10 (±1) 3 (±2) + 69 (±14) 37 (±12) 17 (±10) 5 (±2) Lin-, CD32/16-, Sca-1- (n=3) - 17 (±4) 10 (±7) 5 (±3) 0.3 (±0.3) + 110 (±10) 24 (±5) 7 (±3) 0.2 (±0.3) Lin-, CD32/16-, Sca-1-, CD41- (n=5) - 33 (±26) 6 (±4) 4 (±5) 0.3 (±0.4) + 142 (±77) 10 (±4) 9 (±12) 0.3 (±0.4) Lin-, CD32/16-, Sca-1-, CD41-, CD34- (n=4) - 42 (±33) 5 (±2) 2 (±4) 0.3 (±0.5) (FLEP cells) + 141 (±84) 23 (±17) 4 (±6) 0.3 (±0.5)

Table S1

Colonies per 1000 plated cells

Cell population DEX/DMOG CFU-E small BFU-E large BFU-E CFU-G/M/Meg GEMM CD71 10% low - 110 (± 58) 194 (± 40) 66 (± 57) 21 (± 19) 0 (n=6) DEX 66 (± 28) 289 (± 85) * 222 (± 84) *20 (± 21) 0 CD71 20% high - 605 (± 126) 2 (± 5) 000 (n=9) DEX 565 (± 120) 17 (± 31) 000 CD24a 10% low - n/d n/d n/d n/d n/d (n=6) DEX 96 (± 23) 336 (± 27) 178 (± 28) 19 (± 6) 0.8 (± 2) CD24a 20% high - n/d n/d n/d n/d n/d (n=6) DEX 454 (± 19) 15 (± 2) 0.4 (± 2) 0.3 (± 0.3) 0 CD71/CD24a 10% low - 84 (± 20) 337 (± 68) 177 (± 29) 9 (± 8) 0 (n=4) DEX 33 (± 10) 251 (± 40) 412 (± 23) 7 (± 2) 0 DMOG 21 (± 11) 243 (± 30) 164 (± 23) 5 (± 6) 0 DMOG+DEX 7 (± 2) 83 (± 13) 516 (± 63) 6(± 1) 0 CD71 and CD24a 20% high - 518 0.7 0 0 0 (n=1) + 526 29 1 0 0

Table S2 Whole Whole Gene Genome Genome number of Expression of Expression of found in rVista rVista caCGTGgc mRNA in BFU- mRNA in BFU- Expression (MYC/HIF1a) Name of Whole predicted predicted Gene found motifs E after 4h E after 4h Expression level change upregulated Genome HIF1 c-myc in -2000 to 0nM DEX 100nM DEX log2((100nM log2((100nM Gene rVista target* target* WebMOTIFS +200bp (RPKM) (RPKM) DEX)*(0nM DEX)) DEX)/(0nM DEX))2 GENE ID GENE NAME molecular_function;cellular_component;cytoplasm;apoptosis;biological_process;nega Ddit4 YES YES YES YES 0 2.39 20.23 5.59 3.08 74747 DNA-damage-inducible transcript 4 tive regulation of signal transduction NO (but confirmed by iron ion binding;nucleus;cytoplasm;apoptosis;oxidoreductase activity;oxidoreductase PubMed ID: activity, acting on single donors with incorporation of molecular oxygen, incorporation Egln3 YES 15823097) - YES 0 2.05 16.47 5.08 3.00 112407 EGL nine homolog 3 (C. elegans) of two atoms of oxygen;oxidoreductase activity, acting on paired donors, with incor nucleic acid binding;DNA binding;intracellular;nucleus;transcription;regulation of Zfp1 YES - - YES 1 0.73 5.67 2.05 2.96 22640 finger 1 transcription, DNA-dependent;zinc ion binding;metal ion binding

molecular_function;guanyl-nucleotide exchange factor activity;Rho guanyl-nucleotide Rho guanine nucleotide exchange factor exchange factor activity;cellular_component;intracellular;cytoplasm;intracellular Arhgef3 YES - YES YES 0 2.20 16.21 5.15 2.88 71704 (GEF) 3 signaling cascade;biological_process;regulation of Rho protein signal transduction

0610040J01Rik YES - YES YES 2 2.06 14.77 4.93 2.84 76261 RIKEN cDNA 0610040J01 gene regulation of protein amino acid phosphorylation;protein binding;cellular_component;intracellular signaling cascade;JAK-STAT cascade;negative regulation of signal transduction;kinase inhibitor activity;cytokine- Socs1 YES - - YES 0 5.08 34.03 7.43 2.74 12703 suppressor of cytokine signaling 1 mediated signaling pathway;modification-depe acid phosphatase activity;iron ion binding;lysosome;immune response;dephosphorylation;hydrolase activity;response to lipopolysaccharide;negative regulation of interleukin-1 beta production;negative Acp5 YES - YES YES 2 3.14 20.40 6.00 2.70 11433 acid phosphatase 5, tartrate resistant regulation of interleukin-12 production;negative regulati

Gsta4 YES - - YES 0 1.49 9.00 3.74 2.60 14860 glutathione S-transferase, alpha 4 glutathione transferase activity;cellular_component;cytoplasm;metabolic process v-maf musculoaponeurotic in utero embryonic development;DNA binding;transcription factor fibrosarcoma oncogene family, protein activity;nucleus;regulation of transcription, DNA-dependent;sequence-specific DNA Maff YES YES YES YES 0 1.02 6.11 2.65 2.58 17133 F (avian) binding;regulation of epidermal cell differentiation NO (but confirmed by nucleotide binding;catalytic activity;6-phosphofructo-2-kinase activity;fructose-2,6- PubMed 6-phosphofructo-2-kinase/fructose-2,6- bisphosphate 2-phosphatase activity;ATP binding;fructose metabolic process;fructose Pfkfb4 YES ID:17143338) YES YES 0 2.22 12.25 4.76 2.47 270198 biphosphatase 4 2,6-bisphosphate metabolic process;transferase activity;hydrolase activity receptor activity;integrin binding;protein binding;cell-substrate junction assembly;cell adhesion;cell-matrix adhesion;integrin-mediated signaling pathway;integrin complex;external side of plasma membrane;integral to membrane;regulation of cell Itgb3 YES - YES YES 2 1.43 7.39 3.41 2.37 16416 integrin beta 3 migration signal transducer activity;protein binding;nucleus;cytoplasm;transcription;regulation of transcription, DNA-dependent;signal transduction;circadian rhythm;response to light stimulus;negative regulation of gene-specific transcription from RNA polymerase Per1 YES YES YES YES 4 4.75 23.34 6.79 2.30 18626 period homolog 1 (Drosophila) II intracellular calcium activated chloride channel activity;integral to plasma Clca1 YES - YES 3 1.80 7.54 3.76 2.07 12722 chloride channel calcium activated 1 membrane;chloride transport serine-type endopeptidase inhibitor activity;cysteine-type endopeptidase inhibitor serine (or cysteine) peptidase inhibitor, activity;cellular_component;nucleus;cytoplasm;apoptosis;immune response;response Serpina3g NO n/a n/a NO n/a 8.65 36.06 8.28 2.06 20715 clade A, member 3G to cytokine stimulus;response to peptide hormone stimulus NO (but integral to plasma membrane;transport;phosphate transport;phosphate confirmed by transmembrane transporter activity;integral to membrane;outer PubMed membrane;regulation of bone mineralization;inorganic diphosphate transmembrane Ank YES ID:19419319) YES YES 1 11.69 48.60 9.15 2.06 11732 progressive ankylosis transporter activity

Frmd4a YES - YES YES 0 7.05 28.77 7.66 2.03 209630 FERM domain containing 4A binding;cytoplasm;cytoskeleton angiogenesis;nucleic acid binding;DNA binding;transcription factor activity;protein binding;intracellular;nucleus;regulation of transcription, DNA-dependent;zinc ion Klf5 YES YES YES YES 3 1.26 4.68 2.56 1.89 12224 Kruppel-like factor 5 binding;microvillus assembly;positive regulation of transcription;metal ion binding nucleus;transcription;cell wall macromolecule catabolic process;regulation of Ncoa7 YES - - YES 2 2.04 7.54 3.94 1.89 211329 nuclear receptor coactivator 7 transcription negative regulation of transcription from RNA polymerase II promoter;trophectodermal cell differentiation;DNA binding;transcription factor activity;protein binding;nucleus;cytoplasm;regulation of transcription, DNA- Hopx YES - YES YES 2 3.77 13.32 5.65 1.82 74318 HOP homeobox dependent;multicellular organismal devel nucleotide binding;protein kinase activity;protein serine/threonine kinase activity;protein binding;ATP binding;protein amino acid Plk3 YES - YES YES 1 1.70 5.75 3.29 1.76 12795 polo-like kinase 3 (Drosophila) phosphorylation;membrane;transferase activity;polo kinase kinase activity calcium ion binding;protein binding;extracellular region;nucleus;;Golgi apparatus;plasma membrane;cellular calcium ion homeostasis;cell adhesion;homophilic cell adhesion;synaptic transmission;integral to membrane;cell Clstn1 YES - YES YES 0 21.47 70.51 10.56 1.72 65945 calsyntenin 1 junction;cell pr

Fkbp5 YES YES YES YES 4 22.79 74.76 10.73 1.71 14229 FK506 binding protein 5 peptidyl-prolyl cis-trans isomerase activity;binding;nucleus;cytoplasm;protein folding G1/S transition of mitotic cell cycle;protein phosphatase type 2A complex;protein polyubiquitination;cell morphogenesis;response to acid;ossification;ovarian follicle development;metanephros development;ureteric bud development;branching Bcl2 YES - YES YES 1 2.00 6.45 3.69 1.69 12043 B-cell leukemia/lymphoma 2 involved in urete

Rcsd1 YES - YES 0 2.53 8.11 4.36 1.68 226594 RCSD domain containing 1 family with sequence similarity 46, Fam46a NO n/a n/a NO n/a 2.01 6.44 3.70 1.68 212943 member A negative regulation of transcription from RNA polymerase II promoter;nucleic acid binding;DNA binding;transcription factor activity;intracellular;nucleus;regulation of transcription, DNA-dependent;zinc ion binding;response to glucose Egr1 YES YES YES YES 0 5.94 18.06 6.75 1.60 13653 early growth response 1 stimulus;transcriptio plasma membrane;transport;ion transport;cation transport;zinc ion transport;zinc ion Slc30a10 YES - - NO n/a 2.34 7.11 4.06 1.60 226781 solute carrier family 30, member 10 binding;cation transmembrane transporter activity;integral to membrane catalytic activity;serine-type endopeptidase activity;extracellular Hp YES - - YES 1 1.51 4.41 2.73 1.55 15439 haptoglobin region;proteolysis;hemoglobin binding G1/S transition of mitotic cell cycle;protein binding;nucleus;cytoplasm;nervous system development;cyclin-dependent protein kinase regulator activity;cell Cables1 YES YES YES YES 1 1.72 4.90 3.07 1.51 63955 CDK5 and Abl enzyme substrate 1 projection;cell division;regulation of cell division;regulation of cell cycle monooxygenase activity;tryptophan 5-monooxygenase activity;iron ion binding;metabolic process;aromatic amino acid family metabolic process;oxidoreductase activity;amino acid binding;oxidoreductase activity, acting on Tph1 YES - YES YES 0 1.93 5.51 3.41 1.51 21990 tryptophan hydroxylase 1 paired donors, with incorporation or r cellular zinc ion homeostasis;nitric oxide mediated signal transduction;zinc ion Mt2 YES - - YES 1 460.94 1306.42 19.20 1.50 17750 metallothionein 2 binding;detoxification of copper ion;metal ion binding extracellular region;Golgi apparatus;protein amino acid glycosylation;sialyltransferase ST3 beta-galactoside alpha-2,3- activity;membrane;integral to membrane;transferase activity, transferring glycosyl St3gal2 YES - - YES 1 8.28 23.29 7.59 1.49 20444 sialyltransferase 2 groups;integral to Golgi membrane DNA binding;protein binding;nucleus;DNA repair;double-strand break repair via nonhomologous end joining;response to DNA damage stimulus;response to ionizing Nhej1 YES - YES YES 0 1.61 4.51 2.86 1.49 75570 nonhomologous end-joining factor 1 radiation;B cell differentiation;T cell differentiation branching involved in ureteric bud morphogenesis;morphogenesis of a polarized epithelium;neural crest cell migration;hair follicle development;receptor binding;integrin binding;protein binding;extracellular region;proteinaceous Lama5 YES - YES NO n/a 1.39 3.90 2.44 1.49 16776 laminin, alpha 5 extracellular matrix;baseme

Wdr21 YES - - YES 1 17.19 48.20 9.69 1.49 73828 DDB1 and CUL4 associated factor 4 peroxisome;peroxisomal membrane;peroxisome organization;integral to Pex11a YES - - YES 3 2.10 5.87 3.62 1.49 18631 peroxisomal biogenesis factor 11 alpha membrane;peroxisome fission;brown fat cell differentiation nucleotide binding;GTPase activity;signal transducer activity;GTP binding;heterotrimeric G-protein complex;protein amino acid ADP-ribosylation;signal guanine nucleotide binding protein, transduction;G-protein coupled receptor protein signaling pathway;guanyl nucleotide Gna14 YES - YES YES 0 1.21 3.37 2.03 1.47 14675 alpha 14 binding AMP deaminase activity;purine base metabolic process;nucleotide metabolic process;purine ribonucleoside monophosphate biosynthetic process;hydrolase adenosine monophosphate deaminase activity;hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in Ampd3 YES - YES YES 2 5.68 15.71 6.48 1.47 11717 3 cyclic amidines family with sequence similarity 46, Fam46c NO n/a n/a NO n/a 5.12 13.90 6.15 1.44 74645 member C transcription factor activity;regulation of transcription, DNA-dependent;anti- Tsc22d3 YES - YES YES 1 7.39 19.83 7.20 1.42 14605 TSC22 domain family, member 3 apoptosis;response to osmotic stress

Tns1 NO n/a n/a NO n/a 7.48 20.01 7.22 1.42 21961 tensin 1 actin binding;focal adhesion;cell-substrate junction assembly;cell migration family with sequence similarity 83, Fam83g YES YES YES YES 3 3.25 8.67 4.82 1.41 69640 member G actin binding;protein binding;nuclear envelope;sarcomere;establishment of nucleus Syne1 YES - YES YES 2 2.79 7.36 4.36 1.40 64009 synaptic nuclear envelope 1 localization response to hypoxia;DNA binding;transcription factor activity;signal transducer hypoxia inducible factor 3, alpha activity;nucleus;cytoplasm;regulation of transcription, DNA-dependent;transcription Hif3a YES YES YES YES 1 39.00 101.46 11.95 1.38 53417 subunit from RNA polymerase II promoter;signal transduction;transcription regulator activity nucleic acid binding;DNA binding;RNA polymerase II transcription factor activity;intracellular;nucleus;transcription from RNA polymerase II promoter;zinc ion binding;regulation of transcription;positive regulation of transcription from RNA Klf13 YES - YES YES 1 11.46 29.57 8.41 1.37 50794 Kruppel-like factor 13 polymerase II p nuclear factor of kappa light protein import into nucleus, translocation;cytoplasm;cytosol;lipopolysaccharide- mediated signaling pathway;negative regulation of NF-kappaB transcription factor polypeptide gene enhancer in B-cells activity;response to muramyl dipeptide;response to lipopolysaccharide;toll-like Nfkbia YES YES YES 0 3.02 7.75 4.55 1.36 18035 inhibitor, alpha receptor 4 sig nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay;nucleic acid binding;DNA binding;RNA binding;nucleus;cytoplasm;zinc ion binding;regulation Zfp36l2 YES YES YES YES 1 64.10 163.74 13.36 1.35 12193 zinc finger protein 36, C3H type-like 2 of mRNA stability;metal ion binding transporter activity;integral to plasma membrane;water transport;water channel activity;integral to membrane;basolateral plasma membrane;pore Aqp3 YES - YES YES 2 2.75 6.98 4.27 1.34 11828 aquaporin 3 complex;transmembrane transport cyclin-dependent protein kinase holoenzyme complex;re-entry into mitotic cell cycle;protein kinase activity;nucleus;nucleoplasm;cytosol;protein amino acid phosphorylation;lactation;cyclin-dependent protein kinase regulator activity;negative Ccnd1 YES YES YES YES 0 2.32 5.86 3.76 1.34 12443 cyclin D1 regulation of skeletal system development;DNA binding;protein binding;nucleus;zinc ion binding;negative regulation of cell proliferation;embryonic pattern zinc finger and BTB domain containing specification;anterior/posterior pattern formation;specific transcriptional repressor Zbtb16 YES YES YES NO n/a 2.49 6.14 3.93 1.30 235320 16 activity;transcriptional rep negative regulation of transcription from RNA polymerase II promoter;chromatin binding;RNA polymerase II transcription factor activity, enhancer transducin-like enhancer of split 4, binding;transcription corepressor activity;protein binding;nucleus;Wnt receptor Tle4 YES YES YES YES 2 6.67 16.27 6.76 1.29 21888 homolog of Drosophila E(spl) signaling pathway nucleotide binding;protein kinase activity;protein tyrosine kinase activity;non- membrane spanning protein tyrosine kinase activity;protein binding;ATP Fes YES YES YES YES 0 1.46 3.48 2.34 1.26 14159 feline sarcoma oncogene binding;cytosol;protein amino acid phosphorylation;transferase activity nucleotide binding;magnesium ion binding;protein kinase activity;protein ribosomal protein S6 kinase serine/threonine kinase activity;ATP binding;spindle;ribosome;protein amino acid Rps6ka1 YES - YES YES 0 16.31 39.02 9.31 1.26 20111 polypeptide 1 phosphorylation;protein kinase cascade;transferase activity GTPase activity;signal transducer activity;heterotrimeric G-protein complex;plasma membrane;signal transduction;G-protein coupled receptor protein signaling guanine nucleotide binding protein (G pathway;activation of phospholipase C activity by G-protein coupled receptor protein Gng13 YES - YES YES 1 2.43 5.76 3.81 1.25 64337 protein), gamma 13 signaling pat Rap guanine nucleotide exchange factor guanyl-nucleotide exchange factor activity;protein binding;intracellular;plasma Rapgef2 NO n/a n/a NO n/a 1.38 3.28 2.18 1.25 76089 (GEF) 2 membrane;signal transduction;small GTPase mediated signal transduction polymerase (RNA) III (DNA directed) Polr3gl YES - YES YES 3 2.51 5.93 3.89 1.24 69870 polypeptide G like ion channel activity;voltage-gated ion channel activity;voltage-gated chloride channel activity;transport;ion transport;chloride transport;membrane;integral to membrane;chloride ion binding;chloride channel complex;transmembrane Clcn2 YES YES YES YES 0 3.74 8.78 5.04 1.23 12724 chloride channel 2 transport;cell differentia

Man2a2 YES - YES 2 15.30 35.22 9.07 1.20 140481 mannosidase 2, alpha 2 mannosidase activity;hydrolase activity, hydrolyzing N-glycosyl compounds

Ier3 YES YES YES YES 1 2.46 5.65 3.80 1.20 15937 immediate early response 3 membrane;integral to membrane

Clint1 YES - YES NO n/a 127.14 285.78 15.15 1.17 216705 clathrin interactor 1 protein binding;cytoplasm;endocytosis;lipid binding;membrane;cytoplasmic vesicle nucleic acid binding;DNA binding;intracellular;nucleus;transcription;embryo implantation;zinc ion binding;transcription regulator activity;regulation of Klf9 YES YES YES YES 1 3.59 8.07 4.86 1.17 16601 Kruppel-like factor 9 transcription;metal ion binding;progesterone receptor signaling pathway RAB3A interacting protein (rabin3)-like guanyl-nucleotide exchange factor activity;protein binding;Rab guanyl-nucleotide Rab3il1 YES YES YES YES 3 2.20 4.94 3.44 1.17 74760 1 exchange factor activity NO (but confirmed by negative regulation of transcription from RNA polymerase II promoter;enzyme PubMed ID: inhibitor activity;protein binding;cytoplasm;protein import into nucleus;response to Txnip YES 18376310) YES YES 1 11.19 24.97 8.13 1.16 56338 thioredoxin interacting protein oxidative stress;cell cycle;platelet-derived growth factor receptor signaling pathway

Slc44a2 YES YES YES YES 1 16.21 35.91 9.19 1.15 68682 solute carrier family 44, member 2 transport;membrane;integral to membrane endosome;endocytosis;zinc ion binding;membrane;integral to membrane;ligase membrane-associated ring finger activity;modification-dependent protein catabolic process;cytoplasmic vesicle;metal 38413 YES - YES YES 1 3.72 8.25 4.94 1.15 320253 (C3HC4) 3 ion binding

Cd59a YES - - YES 1 1.44 3.15 2.18 1.13 12509 CD59a antigen plasma membrane;external side of plasma membrane;anchored to membrane ATPase, Ca++ transporting, plasma nucleotide binding;ATP binding;integral to plasma membrane;transport;ion Atp2b4 YES - - YES 0 20.11 43.89 9.79 1.13 381290 membrane 4 transport;calcium ion transport;integral to membrane;hydrolase activity ruffle;guanyl-nucleotide exchange factor activity;ARF guanyl-nucleotide exchange factor activity;protein binding;phosphatidylinositol-3,4,5-trisphosphate binding;intracellular;cytoplasm;plasma membrane;regulation of ARF protein signal Cyth3 YES - - YES 1 34.55 75.31 11.35 1.12 19159 cytohesin 3 transduction;positiv protein kinase activity;protein kinase inhibitor activity;protein binding;ATP Trib2 YES - YES YES 1 2.81 6.12 4.11 1.12 217410 tribbles homolog 2 (Drosophila) binding;cytoplasm;cytoskeleton;protein amino acid phosphorylation calmodulin binding;cytoplasm;protein kinase cascade;cell junction;cell Nrgn YES - YES YES 0 9.78 21.01 7.68 1.10 64011 neurogranin projection;synapse catalytic activity;phospholipase D activity;metabolic process;membrane;integral to Pld6 YES - - NO n/a 1.70 3.65 2.64 1.10 194908 phospholipase D family, member 6 membrane;hydrolase activity;NAPE-specific phospholipase D activity MOB1, Mps One Binder kinase activator- Mobkl2c YES - YES YES 0 4.07 8.65 5.14 1.09 100465 like 2C (yeast) protein binding;zinc ion binding;kinase activity;metal ion binding

E430018J23Rik YES - - YES 0 1.42 3.02 2.10 1.09 101604 RIKEN cDNA E430018J23 gene nucleus;transcription;zinc ion binding;metal ion binding NO (but metanephros development;ureteric bud development;induction of an organ;protein confirmed by binding;cytoplasm;multicellular organismal development;negative regulation of cell PubMed ID: proliferation;regulation of signal transduction;membrane;negative regulation of Ras Spry1 YES 20054616) - YES 0 4.42 9.31 5.36 1.08 24063 sprouty homolog 1 (Drosophila) GTPase ac peptidyl-prolyl cis-trans isomerase activity;protein folding;membrane;integral to Fkbp11 YES - - YES 0 21.16 44.44 9.88 1.07 66120 FK506 binding protein 11 membrane nucleotide binding;myeloid progenitor cell differentiation;lymphoid progenitor cell differentiation;myeloid leukocyte differentiation;protein kinase activity;protein tyrosine kinase activity;transmembrane receptor protein tyrosine kinase Kit YES - YES YES 5 46.21 97.02 12.13 1.07 16590 kit oncogene activity;receptor family with sequence similarity 55, Fam55c NO n/a n/a NO n/a 5.35 11.20 5.91 1.07 385658 member C leucine rich repeat and fibronectin type Lrfn1 YES - YES YES 1 1.62 3.37 2.45 1.05 80749 III domain containing 1 protein binding;membrane;integral to membrane;cell junction;synapse

Ier2 YES - YES YES 2 22.77 47.27 10.07 1.05 15936 immediate early response 2 molecular_function;cytoplasm;biological_process

Wdr60 YES - - YES 1 3.93 8.14 5.00 1.05 217935 WD repeat domain 60 nucleotide binding;catalytic activity;glutamate-ammonia ligase activity;ATP glutamate-ammonia ligase (glutamine binding;intracellular;cytoplasm;mitochondrion;glutamine biosynthetic Glul YES - - YES 1 53.96 110.54 12.54 1.03 14645 synthetase) process;nitrogen compound metabolic process;response to glucose stimulus urea cycle;acetylglutamate kinase activity;acetyl-CoA:L-glutamate N- acetyltransferase activity;mitochondrion;arginine biosynthetic process;glutamate Nags YES - - YES 1 1.42 2.88 2.04 1.02 217214 N-acetylglutamate synthase metabolic process;acyltransferase activity;cellular amino acid biosynthetic process Gm129 YES YES YES NO n/a 1.56 3.14 2.29 1.01 229599 predicted gene 129

Table S3

After 4 hour culture of CD71 Freshly isolated cells (10% low) BFU-E After 4 hour culture of CD71 and CD24a (10% low) BFU-E

DMOG/HIF1a BFU-E interacting BFU-E BFU-E BFU-E 0nM BFU-E 100nM BFU-E 333uM DMOG genes BFU-E CFU-E Ter119+ 0nM DEX 100nM DEX DEX DEX 333uM DMOG +100nM DEX Arnt2 00 0 00 00 0 0 Hif1a 76 1 12 10 22 22 22 22 Hif1an (Fih1) 16 17 7 15 16 19 22 19 22 Hif1b (Arnt) 14 15 13 11 12 15 14 14 14 Hif2a (Epas1) 00 0 00 00 0 0 Hif3a 59 63 26 39 101 21 64 25 68 Phd1 (Egln3) 80 0 2 16 2 7 19 41 Phd2 (Egln2) 55 54 11 42 53 36 39 35 29 Phd3 (Egln1) 18 21 8 10 18 8 9 14 16 Vhl 76 1 67 88 9 8 P4ha1 46 1 34 6 6 15 16 P4ha2 01 0 01 01 1 2 P4ha3 00 0 00 00 0 0 P4hb 174 152 23 194 214 196 203 221 224 P4htm 21 0 11 00 1 1

BFU-E 333uM Cell surface BFU-E 0nM BFU-E BFU-E 0nM BFU-E 100nM BFU-E 333uM DMOG+100nM protein BFU-E CFU-E Ter119+ DEX 100nM DEX DEX DEX DMOG DEX CD105 (Eng) 31 25 1 11 19 55 6 6 CD150 (Slamf1) 70 0 42 65 5 3 Cd24a 25 192 1547 169 255 120 311 143 300 CD34 13 0 0 22 23 2 3

CD41 (Itga2b) 47 2 5 14 18 21 22 19 21 CD71 (Tfrc) 20 113 436 205 119 266 194 276 205 EpoR 92 219 416 82 97 51 44 51 44 FcgR (Fcgr3) 10 1 01 00 0 0 Kit 185 90 1 46 97 51 76 59 92 Ptprc (B220) 40 0 11 32 2 2 Sca-1 (Ly6a) 00 0 00 00 0 0 BFU-E 333uM Transcription BFU-E 0nM BFU-E BFU-E 0nM BFU-E 100nM BFU-E 333uM DMOG+100nM factors BFU-E CFU-E Ter119+ DEX 100nM DEX DEX DEX DMOG DEX Cited2 22 7 3 24 37 18 25 19 28 Fog1 (Zfpm1) 279 275 338 208 159 76 43 84 43 Gata1 178 178 234 138 142 155 151 144 129 Gata2 88 1 0 25 21 49 24 48 23 Klf1 197 366 523 168 195 88 77 91 72 Mafk 28 37 134 32 39 40 33 39 31 Myc 300 148 4 278 271 236 186 209 161 Nfe2 166 57 619 101 94 153 138 153 135 Nr3c1 25 15 4 11 12 14 15 17 21 Sfpi1 (PU.1) 18 1 2 11 17 13 10 11 10 Sp1 24 20 12 25 21 25 26 24 25 Stat5a 64 39 0 33 42 31 33 34 34 Stat5b 52 39 3 25 28 27 27 29 25 Trp53 184 137 13 135 150 93 84 98 81

Table S4 Table S5 Overlap of genes 50% up-regulated by each condition DEX DMOG DEX+DMOG 23 Abcg4 Abcg4 Abcg4 Cables1 Cables1 Cables1 Ddit4 Ddit4 Ddit4 Depdc6 Depdc6 Depdc6 Dpf1 Dpf1 Dpf1 EG245405 EG245405 EG245405 EG668668 EG668668 EG668668 Egln3 Egln3 Egln3 Fam46c Fam46c Fam46c Fam57a Fam57a Fam57a Gpr114 Gpr114 Gpr114 Gramd3 Gramd3 Gramd3 Il10ra Il10ra Il10ra Immp2l Immp2l Immp2l Lpl Lpl Lpl Mgl1 Mgl1 Mgl1 Mt2 Mt2 Mt2 Ndrg1 Ndrg1 Ndrg1 Npff Npff Npff Ppp1r3b Ppp1r3b Ppp1r3b Slc30a10 Slc30a10 Slc30a10 Spry1 Spry1 Spry1 Tph1 Tph1 Tph1 DEX DMOG DEX+DMOG 5 1110034G24Rik 1110034G24Rik 4933413G19Rik 4933413G19Rik Cox7a1 Cox7a1 Gstm1 Gstm1 Nradd Nradd DEX DMOG DEX+DMOG 114 0610040J01Rik 0610040J01Rik March3 March3 6530418L21Rik 6530418L21Rik Aak1 Aak1 Acp5 Acp5 Akap13 Akap13 Ampd3 Ampd3 Ank Ank Aqp3 Aqp3 Arhgef3 Arhgef3 Arl4a Arl4a Atp2b4 Atp2b4 Atp6v1b2 Atp6v1b2 B3galtl B3galtl B4galt1 B4galt1 B9d1 B9d1 BC048609 BC048609 Bcl2 Bcl2 Camk1d Camk1d Cd24a Cd24a Cd59a Cd59a Clint1 Clint1 Clstn1 Clstn1 Cmpk2 Cmpk2 Cyth3 Cyth3 Cytip Cytip Dnaja4 Dnaja4 EG381438 EG381438 EG630138 EG630138 Enah Enah Fabp5 Fabp5 Fam168a Fam168a Fam46a Fam46a Fes Fes Fkbp5 Fkbp5 Frmd4a Frmd4a Fth1 Fth1 Galc Galc Glipr2 Glipr2 Glul Glul Gna14 Gna14 Gsta4 Gsta4 Gzf1 Gzf1 Havcr2 Havcr2 Hif3a Hif3a Hopx Hopx Ier3 Ier3 Il12rb2 Il12rb2 Il1rl1 Il1rl1 Itgb3 Itgb3 Jak1 Jak1 Kctd12 Kctd12 Kctd14 Kctd14 Klf13 Klf13 Klf9 Klf9 Lama5 Lama5 Leprel1 Leprel1 Maff Maff Maml3 Maml3 Man2a2 Man2a2 Map3k1 Map3k1 Mobkl2c Mobkl2c Mpp1 Mpp1 Mt1 Mt1 Mtap7 Mtap7 Ncoa7 Ncoa7 Nt5e Nt5e P2ry5 P2ry5 Pcp2 Pcp2 Per1 Per1 Pex11a Pex11a Pfkfb4 Pfkfb4 Plxdc2 Plxdc2 Polr3gl Polr3gl Prdx6-rs1 Prdx6-rs1 Prkcq Prkcq Pttg1ip Pttg1ip Rab3il1 Rab3il1 Rasgef1b Rasgef1b Rcsd1 Rcsd1 Rec8 Rec8 Rhag Rhag Rnf19a Rnf19a Saps2 Saps2 Serpina3g Serpina3g Serpine1 Serpine1 Sh2d4a Sh2d4a Sla Sla Slc44a2 Slc44a2 Slco3a1 Slco3a1 Snord87 Snord87 Socs1 Socs1 Srp54a Srp54a St3gal2 St3gal2 St3gal4 St3gal4 St6galnac3 St6galnac3 Syne1 Syne1 Tgm2 Tgm2 Tle4 Tle4 Tnnc1 Tnnc1 Tnnt1 Tnnt1 Tns1 Tns1 Trerf1 Trerf1 Trib2 Trib2 Trim58 Trim58 Tsc22d3 Tsc22d3 Vav3 Vav3 Wdr60 Wdr60 Ywhah Ywhah Zbtb16 Zbtb16 Zfp1 Zfp1 Zfp36l2 Zfp36l2 Znrf1 Znrf1 DEX DMOG DEX+DMOG 33 A630007B06Rik A630007B06Rik Acer2 Acer2 Aig1 Aig1 Ak3l1 Ak3l1 Aldoa Aldoa Aldoc Aldoc Ampd2 Ampd2 Ankrd37 Ankrd37 Bhlhe40 Bhlhe40 Bnip3 Bnip3 Ccng2 Ccng2 Clec2d Clec2d EG433182 EG433182 Egln1 Egln1 Gm129 Gm129 Hk2 Hk2 Hmox1 Hmox1 Kdm3a Kdm3a Letm2 Letm2 LOC100039707 LOC100039707 Narf Narf P4ha1 P4ha1 Paqr7 Paqr7 Pf4 Pf4 Pgk1 Pgk1 Pkm2 Pkm2 Plekha2 Plekha2 Plod2 Plod2 Pnrc1 Pnrc1 Rlf Rlf Slc16a3 Slc16a3 Slc2a1 Slc2a1 Smtnl2 Smtnl2 DEX DMOG DEX+DMOG 48 4833442J19Rik 4930422I07Rik 4930579E17Rik 4931406C07Rik AI662250 Acrbp Acvr1b Adrbk2 Afap1l1 Alox5ap Aqp1 Aqp11 Arl4d Atg10 Birc3 Bpgm Ccnd1 Cd55 Dock10 Fam115a Fam62b Fam83g Gngt2 Gpr124 Gstk1 Hpcal1 Kcnj5 Klc4 Klf7 Myl6b Mylip Nlrp6 Pabpc4 Pak4 Phf13 Pnmt Rapgef2 Rnf128 Slfn2 Snora69 Spa17 St3gal1 Tmem131 Trpv2 Txnip Wdr21 Wnk4 Zfp358 DEX DMOG DEX+DMOG 37 Ager Apoc1 Car9 Cd248 Fam69b Fbxo44 Fhl1 Gapdh Gpr19 Hbb-y Hs1bp3 LOC100040259 Mfap1b Mfsd6 Ndufb4 Necab3 Nefh Odf3b Pgam1 Ppox Ppp1r13l Rasgrp4 Rasl11a Reep1 Rpl29 S100a11 Sec61g Slc2a8 Slc35f2 Snx16 Tmem106c Tmem120b Tmem150 Tnni1 Ttc30a1 Vegfa Zfp184 DEX DMOG DEX+DMOG 103 1110002B05Rik 1810022K09Rik 1810027O10Rik 4831426I19Rik Actg1 Ankrd46 Anks1 Anxa2 Apoa1 Atp5k Atp7a Calcrl Calr3 Cd34 Cited2 Clcn2 Cradd Ctsf Dynlt1 E2f1 EG621697 EG625054 EG664849 Eif4ebp3 Fam100b Fam3a Fam55c Fkbp11 Gfod1 Golt1b Gpr146 Gys1 H2-Ab1 Hmga2 Hspa12b Hspb11 Imp3 Kit Klhl23 Krt10 Lancl3 Magoh Map1lc3a Mbp Mthfs Mxd4 Nav1 Ndufb2 Ndufb3 Nr1i3 Nr3c1 Nrgn Nudt6 Obfc1 Obfc2a Olfm1 Paox Pcmtd2 Pcx Pdcd4 Pfkfb3 Pfkp Pgm2 Pld6 Ppm1e Pygl Rad51l1 Rfx2 Rpl24 Rpl35a Rps17 Rps6ka1 Rwdd2a S100a13 Sec24d Sema6b Sepx1 Shfm1 Siae Slc35c1 Slc35d3 Slmo1 Snord22 Snrpd3 Snx10 Snx14 St5 Stc2 Tacc1 Tbca Tgfbr2 Them4 Tmem158 Tnfrsf13c Tomm6 Tpmt Tuba1b Usp53 Vamp4 Zfp292 Zfp395 Zfp711 Zhx3 Table S6 Overlap of genes 50% down- regulated by each condition DEX DMOG DEX+DMOG 16 Acmsd Acmsd Acmsd Adssl1 Adssl1 Adssl1 Angptl2 Angptl2 Angptl2 Apobec2 Apobec2 Apobec2 Espn Espn Espn Hist1h2bj Hist1h2bj Hist1h2bj Hist1h4b Hist1h4b Hist1h4b Hist1h4d Hist1h4d Hist1h4d Hist2h4 Hist2h4 Hist2h4 Lars2 Lars2 Lars2 Pcbd1 Pcbd1 Pcbd1 Pik3r1 Pik3r1 Pik3r1 Rmrp Rmrp Rmrp Rnu73a Rnu73a Rnu73a Rpph1 Rpph1 Rpph1 Tulp2 Tulp2 Tulp2 DEX DMOG DEX+DMOG 6 2810405K02Rik 2810405K02Rik 2810405K02Rik Senp8 Senp8 Senp8 Snora15 Snora15 Snora15 Trim12 Trim12 Trim12 Zfp119 Zfp119 Zfp119 Zfp397os Zfp397os Zfp397os DEX DMOG DEX+DMOG 102 1300017J02Rik 1300017J02Rik 2810006K23Rik 2810006K23Rik 8430427H17Rik 8430427H17Rik 9130213B05Rik 9130213B05Rik Axl Axl Brp44l Brp44l Btf3l4 Btf3l4 Cd1d1 Cd1d1 Cd300a Cd300a Cdc42ep3 Cdc42ep3 Cdkn1a Cdkn1a Cited4 Cited4 Clec4d Clec4d Cpd Cpd Csf2rb Csf2rb Csf2rb2 Csf2rb2 Csrnp1 Csrnp1 Dnahc8 Dnahc8 Dst Dst EG630579 EG630579 Epb4.9 Epb4.9 Erh Erh F10 F10 Fam109a Fam109a Fam55b Fam55b Fau Fau Fcer1g Fcer1g Fgl2 Fgl2 Fzd5 Fzd5 Gadd45b Gadd45b Gata2 Gata2 Gm1821 Gm1821 Gpr150 Gpr150 Grtp1 Grtp1 Gtrgeo22 Gtrgeo22 Gucy1a3 Gucy1a3 Hbb-b1 Hbb-b1 Hbb-b2 Hbb-b2 Hbb-bh1 Hbb-bh1 Hemgn Hemgn Hsd17b7 Hsd17b7 Icam1 Icam1 Id1 Id1 Jun Jun Klhl36 Klhl36 LOC100040259 LOC100040259 LOC100043225 LOC100043225 LOC218963 LOC218963 LOC547349 LOC547349 Lcp2 Lcp2 Lmo2 Lmo2 Lst1 Lst1 Magmas Magmas Mef2c Mef2c Mrpl53 Mrpl53 Mycn Mycn Nab2 Nab2 OTTMUSG00000021609 OTTMUSG00000021609 OTTMUSG00000022083 OTTMUSG00000022083 Osm Osm P2rx1 P2rx1 Pard6b Pard6b Pcdh7 Pcdh7 Pin4 Pin4 Plekhg5 Plekhg5 Ppard Ppard Ppia Ppia Ppp2r5b Ppp2r5b Prkcdbp Prkcdbp Rab44 Rab44 Rasl11a Rasl11a Rhbdf2 Rhbdf2 Rpl18a Rpl18a Rpl28 Rpl28 Rpl36a Rpl36a Rps23 Rps23 Rps27 Rps27 Rps27a Rps27a Rps29 Rps29 Sft2d1 Sft2d1 Slc37a1 Slc37a1 Slc44a1 Slc44a1 Slc7a11 Slc7a11 Srxn1 Srxn1 Ssh1 Ssh1 Sumo2 Sumo2 Tgm1 Tgm1 Thbs1 Thbs1 Tmc8 Tmc8 Tmcc2 Tmcc2 Tpt1 Tpt1 Tpt1p Tpt1p Traf5 Traf5 Trim10 Trim10 Trim16 Trim16 Tubb1 Tubb1 Ubash3b Ubash3b Vwf Vwf Wdr44 Wdr44 Zfp3 Zfp3 Zfp689 Zfp689 Zfpm1 Zfpm1 DEX DMOG DEX+DMOG 7 Atf7ip Atf7ip Chac1 Chac1 H3f3a H3f3a Hba-a1 Hba-a1 Hba-a2 Hba-a2 Hmgn2 Hmgn2 Xlr4a Xlr4a DEX DMOG DEX+DMOG 151 1110012D08Rik 1200009F10Rik 2210408I21Rik 2900062L11Rik 3830402I07Rik A930041I02Rik AY358078 Aasdhppt Acbd4 Actr6 Adcy9 Akt3 Aldoc Als2cr4 Amacr Angpt1 B3gnt9 BC039632 Bbs4 Bhlhe40 Bloc1s3 Bri3 C1galt1c1 C1qtnf6 C85492 Camsap1l1 Car13 Ccdc102a Ccdc112 Ccdc73 Cd248 Cdc42ep2 Chka Chst11 Cml1 Cno Coq10b Csrp2 Dab2ip Ddef2 Doc2g Dusp3 Dusp5 E130303B06Rik E130306D19Rik EG668158 ENSMUSG00000063754 Eif2c3 Epb4.1l1 Ethe1 F2rl2 F630110N24Rik Fam132b Fam151a Fbxl15 Flt3l Frmd6 Ftl1 Gatsl3 Gca Glp1 Gm561 Gp5 Gpt Hint3 Hist3h2a Hspb6 Ifitm5 Ikzf4 Il1rap Irak2 Junb Ksr1 LOC100039355 Lat Lat2 Lcor Lrrc49 Lsm5 Mast1 Mblac1 Mc5r Mettl10 Mllt3 Mobkl3 Mpp2 Mthfd2l Mxi1 Ndrg2 Ndufb4 Nfyc Nhlrc1 Nrarp Nubpl Nudt21 OTTMUSG00000011097 Osgin1 Palm Pcgf1 Pde1b Pear1 Pf4 Phospho1 Phyh Pi4k2b Pias3 Pik3ap1 Pim3 Plod1 Prkch Prrg4 Ptprc Ptprv Rab3d Rad9b Rhoq Rom1 Rpl39 Rps10 Rps16 S100a11 Sgk3 Shf Sirpa Slc18a2 Slc25a45 Smad7 Snapc3 Snhg10 Socs2 Sox13 Spsb2 Stradb Tifa Tigd5 Tmeff1 Tmem17 Tmem67 Tnfrsf4 Trim23 Trpt1 Ttpa Ttyh2 Ube2s Vangl1 Vkorc1l1 Wdr38 Wdr40b Zdhhc15 Zfp551 Zfp688 DEX DMOG DEX+DMOG 16 1190007I07Rik 449631 4921520G13Rik Bex2 Efcab4b Gm1943 Hyal3 Il18bp Katnal1 Lamp3 Nagk OTTMUSG00000010657 Pcp2 Snora69 Tmem176a Ttc19 DEX DMOG DEX+DMOG 137 1600012F09Rik 1810043H04Rik 2310028H24Rik 2610036L11Rik 6230427J02Rik 6330442E10Rik 9-Sep A230020J21Rik Aacs Adam1a Adrm1 Agfg2 Agk Aim2 Alox5 Ap2m1 Aph1a Arrdc4 Ass1 Atg4a Atp5g2 B3galt6 BC011248 C2cd2l Ccrk Cd74 Cda Cdc42ep4 Cish Cldn13 Coro1a Cox5b Cox7c Dlg4 Dok2 Dusp2 EG432879 EG621100 EG627782 EG668137 ENSMUSG00000044330 ENSMUSG00000061104 Efcab7 Efnb1 Ehd3 Eid3 Ephb6 Fam117a Fam60a Fgfrl1 Foxh1 G6pd2 Gorab Gse1 Gstt1 Guca1b Hist2h2bb Hscb Iffo1 Ifitm1 Il4ra Iscu Isg20 Itgb2 Itpa Ldoc1l Lenep Lig4 Lrrc29 Lyl1 Med31 Mid1ip1 Mrpl23 Naip7 Nat5 Ndn Nicn1 Nkiras2 Nupr1 OTTMUSG00000000426 OTTMUSG00000010671 Omp Phldb1 Pin1 Plaur Pld3 Plvap Plxna3 Pnp2 Ptpn14 Ptpn18 Rab7 Ring1 Rpl12 Rpl18 Rpl27 Rpl38 Rpl5 Rps13 Rps28 Rps7 Rps9 Rpsa Rrm2b Rwdd3 S1pr4 Sh3bp1 Slamf1 Slc22a4 Slc39a3 Slc7a7 Snora70 Snord32a Spn Susd3 Tceb2 Tgfb1 Tmco3 Tmem9 Tnfaip8l2 Tnfsf14 Tspan8 Tufm Ubl4 Unkl Uprt Wdr20a Yif1b Yrdc Zdhhc23 Zfp36l1 Zfp509 Zfp513 Zfp647 Zfp69 Table S7

Over-represented TFBS in 5Kbp upstream of genes up-regulated by DEX treatment Cell type Number of hits in the Total number of hits TFBS -log10(p-value) submitted on genome regions ATF4 139 26282 20.045 FOXP3 185 42427 17.632 A594 lung epithelial FOXO1 181 43570 15.35 carcinoma cell line MYC 117 23703 14.965 (human)* ‡ RFX1 195 48905 14.785 Reddy et al., 2009 NRF2 144 32616 14.353 55 genes up-regulated FOXO4 146 34073 13.558 > 2 fold MEIS1 125 27385 13.55 POU1F1 163 40305 12.998 NFE2 68 11215 12.849

FOXO1 155 43570 5.089 TEF 112 29679 4.884 Lens epithelial cells CDPCR3 18 2306 4.567 (human)** IRF1 107 28928 4.366 Gupta et al., 2005 POU6F1 134 38731 3.995 POU1F1 137 40305 3.751 90 genes up-regulated FOXO4 117 34073 3.465 > 2 fold E2F1DP2 116 34433 3.155 ETS1 81 22501 3.151 XFD1 28 5806 3.0792

ATF4 70 21283 3.584 HFH1 31 7397 3.474 3134 mammary tumor NFE2 35 9153 3.131 cell line (mouse)** MEIS1 69 22446 2.803 John et al., 2008 CMYB 64 20915 2.604 106 genes up-regulated HES1 63 20683 2.530 > 2 fold CREBP1CJUN 13 2584 2.410 BACH1 4 301 2.403 MYC 58 19322 2.255

3T3-L1 pre-adipocyte MINI20 153 30053 16.846 cell line (mouse) LFA1 117 22988 13.106 Pantoja et al., 2008 HIF1 51 6956 11.270

50 genes up-regulated E2F1DP2 125 27745 10.508 > 2 fold MYC 96 19322 10.376 Over-represented TFBS in 5Kbp upstream of genes up-regulated by DEX treatment Cell type Number of hits in the Total number of hits TFBS -log10(p-value) submitted on genome regions STAF 101 20894 10.230 ETS1 91 18391 9.796 BARBIE 125 28841 9.503 CREBATF 75 14222 9.374 ATF3 70 12915 9.284

HFH3 125 32385 13.829 Fetal thymus cells HIF1 33 6956 6.027 (mouse) MYC 59 19322 4.0176 Bianchini et al., 2006 POU3F2 7 772 3.2476 XBP1 15 3312 2.9106 45 genes up-regulated NMYC 43 14488 2.8858 > 2 fold ARNT 10 1797 2.7443 ATF 13 2779 2.7203

CREBATF 123 14222 4.831 FOXO4 216 28117 4.480 Hepatocytes FOXO1 266 35798 4.377 (mouse) CEBP 123 15288 3.573 Wong et al., 2010 ATF 31 2779 3.241 MEIS1 166 22446 2.928 120 genes up-regulated HIF1 61 6956 2.890 > 2 fold† LEF1TCF1 240 34906 2.342 HFH1 61 7397 2.318 TCF4 77 9760 2.293

Promoter region motif enrichement analysis of genes 2-fold up-regulated in 6 cell types

The table shows results from the same analysis as in Figure S2 but on other cell types than BFU-E cells. The table shows the result of motif enrichment analysis of genes whose expression is increased more than 2-fold in response to Dex in six previously published datasets. The ten most significantly enriched transcription factor binding motifs in the 5,000 bp upstream “promoter” regions as determined by Whole Genome rVISTA is shown for each dataset. There was significant enrichment of HIF1 binding sites in upregulated genes from three datasets, but at lower significe than in BFU-E cells. Importantly the software did not detect significant enrichment of GRE elements in 5 of the datasets and in the one dataset with GRE enrichment the significance was only 1e-3.4. The p value is calculated based on the number of hits in the submitted genomic regions for each motif, compared to the number of conserved hits in the genome. The average base distributions within the called hits for each enriched motif are shown below the table.

* Enrichment for GRE elements but the p-value is very low: -log10(p-value) = 3.4.

** No enrichment for HIF1 binding sites.

‡ Gene expression data generated with mRNA-sequencing. The rest were determined using microarray.

† Liver samples were isolated from GR-null and wild-type adult mice 3 h after treatment with dexamethasone (200 µg/ 100 g body weight).

Figure S1

Figure S2

Figure S3

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Figure S5

Figure S6

Figure S7