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Legends to supplemental figures Fig. S1 Mi-2ß is present at active and repressed GATA-1/FOG-1 target genes. Anti-Mi-2ß ChIP in G1E-ER4 cells before and after treatment with estradiol at the repressed GATA-1 target genes Gata2 (A), Myc and Lyl1promoter and Kit (B), and the indicated GATA-1activated genes (C). Top panel: anti-Mi-2ß A301-081A (081), bottom panel: A301-082A (082). Numbers indicate positions in kilobases relative to the transcription start site. The region 224.9 kb upstream of the Kit promoter served as negative control. Note that both Mi-2ß antibodies produced very similar results that are consistent with ChIP experiments in Fig.1. Bars represent results from 5 independent experiments. Error bars denote SEM. * P<0.05. Fig. S2 Anti-HA-ChIP in G1E-ER4 cells stably expressing HA-tagged Mi-2ß before and after treatment with estradiol at the repressed GATA-1 target genes Kit (A), Gata2 (B), Myc and Lyl1 promoter (C) and the indicated GATA-1-activated genes (D). Results are averages of 2-4 independent experiments. Error bars denote SEM. * P<0.05. Fig. S3 NuRD occupies GATA-1/FOG-1-activated genes in megakaryocytes. ChIP using cultured fetal liver-derived megakaryocytes with antibodies against MTA-2, RbAp46 or control IgG, and primers for promoters of indicated genes. Hbb-b1 and a region 3.3 kb upstream of the aIIb gene served as negative controls. Bars represent results from 4-6 independent experiments. Error bars denote SEM. * P<0.05. Fig. S4 Disruption of NuRD binding by FOG-1 abolishes transcriptional repression. A) GSTpulldown experiments using GST alone, GST fused to the first 45 amino acids of FOG-1 (WT) with a single point mutation (K5A) or a triple mutation R3G, R4G, K5A (triple mut) and in vitro translated, 35S labeled MTA-1, MTA-2, RbAp46, or RbAp48. Bottom panel shows Coomassie Blue-stained gel of GST-fusion proteins. B) The N-terminal 45 amino acids of FOG-1 were fused to GAL4 and assayed in transient transfection experiments with a luciferase reporter gene driven by the thymidine kinase promoter and containing 5 GAL4 binding sites. The triple mutation in FOG-1 essentially abolished repressive activity (compare with GAL4 alone). Error bars denote SEM. Fig. S5 NuRD binding is necessary for GATA-1/FOG-1-dependent transcriptional activation. 3T3 cells were treated with control siRNA (siRNA ctr1) or siRNAs against MTA-1, MTA2, and MTA-3 (siRNA/MTA). 24 hours later, constructs expressing GATA-1 and/or FOG-1 were co-transfected with a luciferase reporter gene driven by the IIb promoter. Bars denote averages of 2 independent experiments. Error bars represent SEM. Fig. S6 1 Generation of FOG-1 mutant mice. A) Map of the Zfpm1 locus and targeting vector (see Materials & Methods for details). B) Southern blot showing DNA samples from 8 agouti mice and two control samples (ctr) are from wild type C57/Bl6 mice. Numbers 7 and 8 show samples with a recombined Zfpm1 locus. C) PCR was used to distinguish the wildtype (wt), gene targeted and neo-excised loci. The presence of the FRT site leads to the production of a PCR fragment of 278 base pairs when compared to the un-targeted locus. Half arrows indicate position and orientation of PCR primers. D) Representative PCR analysis from offspring from +/ki,+/ki crosses. Results show mice that are heterozygous (lanes 2, 7, and 8) or homozygous (lane 6) for the gene-targeted neo-excised Zfpm1 locus. E) Anti-FOG-1 Western blots from primary cultured megakaryocytes (MK) or fetal liver erythroid cells (ery) showing that the triple mutation does not impair FOG-1 expression in either lineage. ß-actin served as loading control. Fig. S7 Hematoxylin and eosin stained sections from bone marrow and spleens of +/+ and ki/ki mice. Arrowheads denote megakaryocytes. Note the relative decrease of the white pulp within the spleen of ki/ki mice due to expansion of erythroid cells. Fig. S8 Pronounced anemia in sporadic cases of aged ki/ki mice. A) Peripheral blood mononuclear cells (PBMC) were analyzed by Ter119 and CD71 flow cytometry. Note the increase of CD71+ cells in 2 out of 3 ki/ki animals. B) Increased numbers of reticulocytes in ki/ki mice as determined by thiazole orange staining. C) Expansion of immature erythroid cell populations in ki/ki spleens and bone marrows (BM). D) Increased cell death in ki/ki bone marrow erythroid cells as determined by 7-AAD staining. Fig. S9 Delay in cell cycle progression and increased apoptosis in ki/ki E12.5 fetal liver erythroid precursors. A) Cells were stained using anti-Ter-119, anti-CD71 antibodies followed by permeabilization and labeling with propidium iodide. Note that permeabilization led to loss of detection of terminally differentiated Ter119+CD71- erythroid cells (compare with Fig.S7). The fractions of proerythroblasts (ProE) and basophilic erythroblasts (BasE) in G1/G0 phase were considerably higher in ki/ki mice. (B,C) CD71+ erythroblasts were labeled with AnnexinV and 7-AAD. The percentages of dead cells (7-AAD+) and cells in early apoptosis (AnnexinV+7-AAD-) were significantly increased in ki/ki mice. ki/+ erythroblasts displayed an intermediate phenotype. Error bars represent SEM (n=4). Fig. S10 Normal ploidy of ki/ki MKs. Flow cytometric study of propidium iodide-stained MKs from spleens and bone marrows (upper panels). Results were quantitated and plotted (lower panel). Error bars represent SEM (n=3). *P<0.05. Fig. S11 2 Gene expression analysis of bone marrow-derived Ter119+, CD71+ erythroblasts. Note reduction in Hbb-b1 expression and increased Gata2 levels. Results were plotted as foldchanges compared to +/+ littermates. Error bars denote SEM of three independent experiments except for Myc where n=2. * P<0.01. 3 Supplemental Materials and Methods Generation of mutant mice. A BAC vector containing the whole Zfpm1 gene (129S6/SvEvTACC/Br, RP22-43G10, CHORI) was a gift from Eric Svensson. Plasmids PL451, PL253 and the bacterial stain SW102 (gifts from Neal Copeland) were used according to instructions (http://recombineering.ncifcrf.gov). Briefly, a 17kb genomic DNA fragment containing the 2nd exon of Zfpm1 was retrieved into PL253. To generate the targeting vector, the triple point mutation was produced by overlapping PCR. The mutant FOG-1 sequence and the neo cassette flanked by FRT sequences (in PL451) were amplified by PCR and inserted into PL253 (Fig.S5A). The mutant Zfpm1 sequence contained a SacI restriction site to facilitate genotyping. M represents the initiating methionine of FOG-1 and resides in the second exon. The vector was linearized with XmnI and introduced into TL1 ES cells by electroporation and cells selected in G418 and gangcyclovir. 11 out of 288 doubly resistant clones were identified by Southern blotting to contain the desired recombined Zfpm1 locus. All of these carried the point mutations, as determined by restriction digests with SacI of genomic PCR products. Two clones with a normal karyotype were injected into C57BL/6 blastocysts to generate chimeras. One of these transmitted the mutant allele through the germline. Genotyping for heterozygous mice was performed by Southern blot analysis. The neomycin resistance cassette was removed by mating to a ubiquitous Flp-expressing transgenic mouse (129S4/SvJaeSorGt (ROSA)26Sortm1(FLP1)Dym/J, Jackson Laboratory), and subsequent genotyping was performed by PCR over the remaining FRT site. Genomic DNA was isolated from tails of mice by standard procedures. Histology Bone marrow and spleens of 6-8 week-old mice were fixed in 4% paraformaldehyde overnight at RT. After dehydration through a series of ethanol washes, the tissues were embedded in paraffin. Tissue sections were stained with hematoxylin and eosin. Additional methods. RT-PCR was performed according to standard protocols. Primers PCR primers to generate Zfpm1 probe for Southern blotting: F: CGCGGATCCTTCACATCTGTGCAGTGGGCCTAT R: CCGGAATTCTGCTTCTGGATACAGGCACGATCA Primers for genotyping PCR: F: CGCGCCGAGTGCCAAGCGCGCCGT R: CCCTATCGCCGCACCATCTCGGAT Primers for ChIP Primers for the Kit and Gata2 loci are listed in (Jing et al, 2008) and (Martowicz et al, 2005), respectively. 4 Primers for HS3 of the -globin locus F: CTAGGGACTGAGAGAGGCTGCTT R: ATGGGACCTCTGATAGACACATCTT HS2 of the -globin locus F: AGGGTGTGTGGCCAGATGTT R: ACCCAGATAGCACTGATCACTCAC Hbb-b1 promoter F: CCGAAGCCTGATTCCGTAGA R: TCCTACCTCACCTTATATGCTCTGC Band III (Slc4a1) promoter F: CTGAGCAGTCAAGCCTTAGTTCAC R: CCTGTCCAGTCCCTAAGGTCTTT BandIII (Slc4a1) transcribed region F: GGTGGATGGGAGGGTAATGTG R: CCACTCCCCGCCATAGGT Klf1 promoter F: TCTGCTCAAGGAGGAACAGAGCTA R: GGCTCCCTTTCAGGCATTATCAGA Eraf promoter F: TGCCTGCGTCTCGCTTAGT R: GCTGAGCCCGCCTCATC Eraf transcribed region F: TTAAAGGGTCTGGGCATCATG R: CAACATCTTGGGAGAACGGTC Myc promoter F: GCAGGGCAAGAACACAGTTCA R: TCGCGCGCTAGTCCTTTC Lyl1 promoter F: TCAGCATTGCTTCTTATCAGCC R: CGCAGAGGCCAGAGGATG) IIb (Igta2b) gene proximal promoter region F: GCCATGAGCTCCAGTCTGATAA R: AGCTCTTTCCCTTTCCCTGAA Mpl gene proximal promoter region 5 F: CTGCCAACAGAAGGCTCATG R: CTGTCAGATACAGCCCCACGT Gp9 gene proximal promoter region F: GCCTCCTGGCCCTGACA R: TGTGGCTGCTGCCTGACA Gp1b gene proximal promoter region F: TGGTGGCTAGTAGCTGCAAAGTC R: TTATCAGCTCTCTGCACAGCATTC Pf4 gene proximal promoter region F: GCTGCTGGCCTGCACTTAAG R: GCCACTGGACCCAAAGATAAAG IIb, -3.3 kb upstream region (Negative control) F: AAATAGATGTCAAGTTGGCATAAACCT R: TGCCAGCGTTCAAGTACAAAA CD4, First intron (Negative control) F: GGGGCCAGTTTTAACATTTTC R: GGGCTGATATTTAGGGTTTTTG Primers for RT-PCR: Zfpm1 F: CCTTGCTACCGCAGTCATCA, R: ACCAGATCCCGCAGTCTTTG Gata1 F: GCCCAAGAAGCGAATGATTG R: GTGGTCGTTTGACAGTTAGTGCAT Lyl1 F: CCTGACCTGGACTGACAAACCT R: CACATGGACCCCACGGATA Gata2 F: GCAGAGAAGCAAGGCTCGC R: CAGTTGACACACTCCCGGC Kit F: AGCAATGGCCTCACGAGTTCTA R: CCAGGAAAAGTTTGGCAGGAT Myc 6 F: CAGCTCGCCCAAATCCTGTA R: CGAGTCCGAGGAAGGAGAGA Hbb-b1 F: TTTAACGATGGCCTGAATCACTT R: CAGCACAATCACGATCATATTGC Eraf F: GATCTCCACAGGGATAAAGGAGTTT R: CAGTCATGAACCACAATCACCAT BandIII (SLC4A1) F: TGGAGGCCTGATCCGTGATA R: AGCGCATCGGTGATGTCA Klf1 F: TTCCGGAGAGGACGATGAGA R: AACCTGGAAAGTTTGTAAGGAAAAGA Tal1 F: CGAGCGCTGCTCTATAGCCTT R: TCACCCGGTTGTTGTTGGT Gapdh F: GAAGGTACGGAGTCAACGGATTT R: GAATTTGACCATGGGTGGAAT Primers for the IIb (Mm00439768_m1), Mpl (Mm00440310_m1), Gp9 (Mm007671_g1), Gp1b (Mm0050 677_g1) and Pf4 (Mm00451315_g1) were commercially available from TaqMan Gene Expression Assays (Pang et al, 2006). 7 Supplemental References Jing H, Vakoc CR, Ying L, Mandat S, Wang H, Zheng X, Blobel GA (2008) Exchange of GATA factors mediates transitions in looped chromatin organization at a developmentally regulated gene locus. Mol Cell 29(2): 232-242 Martowicz ML, Grass JA, Boyer ME, Guend H, Bresnick EH (2005) Dynamic GATA factor interplay at a multicomponent regulatory region of the GATA-2 locus. J Biol Chem 280(3): 1724-1732 Pang L, Xue HH, Szalai G, Wang X, Wang Y, Watson DK, Leonard WJ, Blobel GA, Poncz M (2006) Maturation stage-specific regulation of megakaryopoiesis by pointeddomain Ets proteins. Blood 108(7): 2198-2206 8