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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
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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.
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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.
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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
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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
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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).
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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
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