Supplementary
A.
B.
C.
Supplementary Figure 1. Morphology of EML cells transduced with MSCV
vectors. (A) Undifferentiated cells in normal culture medium. EML, EML-GFP, and
EML-HOXB4 cells all had a similar morphology, with large vacuoles and the presence of
cells having a hand mirror shape in regular growth medium. (B) Cells differentiated to
the EPRO stage, maintained in culture medium with GM-CSF. Over 90% of the cells of
each category had numerous azurophilic granules and kidney bean-shaped nuclei. (C)
Cells differentiated to the neutrophil stage of maturation after culture in the presence of
10uM ATRA for 96 hrs. Typical ring-shaped and segmented nuclei are observed.
Wright-Giemsa stain at the 45X magnification.
Lineage Markers
EPRO-EML
EPRO-GFP EPRO-HOXB4
Stem/Progenitor Markers
EPRO-EML
EPRO-GFP EPRO-HOXB4
Supplementary Figure 2. Cell surface immuno-phenotype of cells differentiated to
the EPRO stage of granulocytic maturation. By cell surface immuno-phenotyping,
EPRO-EML, EPRO-GFP and EPRO-HOXB4 cells all had characteristics of granulocytic
cells with similar patterns of expression of Gr-1 and Mac-1 in a high percentage of the
cells. The percentage of cells expressing Sca-1, B220, Sca-1 and CD34 levels is
markedly decreased in all three cell types when compared to EML cells.
Supplementary Figure 3. Southern Blot analysis of DNA from single-cell clones of
EML-GFP and EML-HOXB4 cells. Southern Blot analysis of DNA extracted from
cultures of single-cell clones, digested with EcoRI and hybridized to a GFP probe. This
analysis will reveal a single junction fragment for each integration site. Each single-cell
clone has a different pattern of retroviral insertion sites. There are one to three
integration sites per clone. This analysis helped to exclude the possibility that any of the
phenotypes we observed in the EML-HOXB4 cells was due to a particular position effect
of retroviral insertion and also provided us with true biological triplicates for use in our
microarray studies.
Supplementary Table 1. RNA expression profiling results.
RNA expression profiling results comparing KLS-EML-B4 cells to KLS-EML-GFP
cells. 465 gene transcripts were observed to be differentially expressed by at least 2-fold.
PLEASE SEE ATTACED EXCEL SHEET
Supplementary Table 2. ChIP-chip Results.
ChIP-chip results using Tilescope software revealed that the promoter regions of 1910
genes were bound by HOXB4
PLEASE SEE ATTACED EXCEL SHEET
Supplementary Table 3. This table lists the 71 differentially expressed genes, identified
by microarray analysis in our RNA expression profiling experiments, that were also
observed by ChIP-chip to have DNA binding sites occupied by HOXB4 in their proximal
promoter region. The majority of these target genes (54/71) expressed transcripts that
were up-regulated in KLS-EML-HOXB4 cells. The gene transcripts are ordered
according to their rank in the ChIP-chip data set.
Fold
Difference
HOXB4/GFP
Gene Symbol
8.39
2.50
-2.53
3.65
Gp49a
Rab38
2010301N04Rik
Clec4e
9.82
2.76
2.19
2.13
Laptm4b
AI586015
Arhgap15
Bmp2k
-2.14
Rpl3
4.26
Kazald1
3.90
2.68
2.46
2.45
-2.73
Chchd6
Zfp521
Tm2d2
Fcgr2b
Rad23a
2.17
2.79
2.07
2.45
2.64
Als2
Mfge8
Scap2
Rab19
1500041B16Rik
-2.74
2.52
Orc5l
Stat3
Gene Title
glycoprotein 49 A /// leukocyte
immunoglobulin-like receptor, subfamily
B, member 4
Rab38, member of RAS oncogene family
RIKEN cDNA 2010301N04 gene
C-type lectin domain family 4, member e
lysosomal-associated protein
transmembrane 4B
expressed sequence AI586015
Rho GTPase activating protein 15
BMP2 inducible kinase
ribosomal protein L3 /// similar to 60S
ribosomal protein L3 (J1 protein)
Kazal-type serine peptidase inhibitor
domain 1
coiled-coil-helix-coiled-coil-helix domain
containing 6
zinc finger protein 521
TM2 domain containing 2
Fc receptor, IgG, low affinity IIb
RAD23a homolog (S. cerevisiae)
amyotrophic lateral sclerosis 2 (juvenile)
homolog (human)
milk fat globule-EGF factor 8 protein
src family associated phosphoprotein 2
RAB19, member RAS oncogene family
RIKEN cDNA 1500041B16 gene
origin recognition complex, subunit 5-like
(S. cerevisiae)
signal transducer and activator of
ChIPChip
Rank
(out of
1910)
RNA
Expression
Profiling
Rank (out
of 465)
29
68
88
95
4
169
369
38
96
122
141
162
2
111
257
284
188
331
208
24
328
379
382
399
440
32
125
184
187
381
454
488
510
533
586
272
106
309
185
133
630
654
383
163
3.41
-2.38
2.08
2.50
4.42
2.90
3.25
2.53
Cd69
Clcn3
Il6
Zfp238
Cd34
Grcc10
Arhgap28
AI504432
2.93
Sox4
2.26
2.57
3.45
-4.52
3.83
Phtf2
Prkd2
Gpr171
Tsc22d1
Myct1
2.74
4.49
Ift57
Meis1
-3.21
2.61
Hmbs
4732435N03Rik
2.59
-2.36
2.26
Lat2
Fbxl19
Kifap3
2.04
3.47
-3.31
2.22
P2rx3
Lztfl1
Ifngr1
Gstm1
2.58
2.79
2.58
2.28
3.10
2.68
2.17
Slc39a6
Glipr1
Rasa2
Lrmp
Cpne3
Msrb2
Hes6
transcription 3
CD69 antigen
chloride channel 3
interleukin 6
zinc finger protein 238
CD34 antigen
gene rich cluster, C10 gene
Rho GTPase activating protein 28
expressed sequence AI504432
SRY-box containing gene 4 /// similar to
Transcription factor SOX-4
putative homeodomain transcription factor
2
protein kinase D2
G protein-coupled receptor 171
TSC22 domain family, member 1
myc target 1
intraflagellar transport 57 homolog
(Chlamydomonas)
myeloid ecotropic viral integration site 1
hydroxymethylbilane synthase /// similar
to hydroxymethylbilane synthase ///
similar to hydroxymethylbilane synthase
RIKEN cDNA 4732435N03 gene
linker for activation of T cells family,
member 2
F-box and leucine-rich repeat protein 19
kinesin-associated protein 3
purinergic receptor P2X, ligand-gated ion
channel, 3
leucine zipper transcription factor-like 1
interferon gamma receptor 1
glutathione S-transferase, mu 1
solute carrier family 39 (metal ion
transporter), member 6
GLI pathogenesis-related 1 (glioma)
RAS p21 protein activator 2
lymphoid-restricted membrane protein
copine III
methionine sulfoxide reductase B2
hairy and enhancer of split 6 (Drosophila)
-2.04
2.24
2.09
2.43
-4.33
-2.64
-2.28
2.51
-2.27
-3.20
2.12
C920005C14Rik
Il2rg
Prcp
Coro7
Aqp9
Ctse
Cdr2
Arl6
Dpy19l1
Wrn
Rab27a
RIKEN cDNA C920005C14 gene
interleukin 2 receptor, gamma chain
prolylcarboxypeptidase (angiotensinase C)
coronin 7
aquaporin 9
cathepsin E
cerebellar degeneration-related 2
ADP-ribosylation factor-like 6
dpy-19-like 1 (C. elegans)
Werner syndrome homolog (human)
RAB27A, member RAS oncogene family
663
711
729
733
742
755
761
769
51
352
307
170
22
95
60
159
788
92
789
824
859
904
913
236
146
47
425
34
959
964
116
16
1057
1111
403
136
1151
1158
1248
142
351
234
1254
1286
1296
1305
315
46
405
250
1306
1309
1352
1419
1436
1517
1518
145
107
144
225
73
126
271
1553
1574
1594
1624
1632
1693
1705
1745
1767
1790
1791
321
244
304
190
422
376
346
165
345
402
290
2.11
Rhob
3.73
2.75
7.58
P2ry10
Nrgn
Cldn12
2.76
B3gnt5
2.61
Pik3cg
ras homolog gene family, member B
purinergic receptor P2Y, G-protein
coupled 10
neurogranin
claudin 12
UDP-GlcNAc:betaGal beta-1,3-Nacetylglucosaminyltransferase 5
phosphoinositide-3-kinase, catalytic,
gamma polypeptide
1794
293
1802
1835
1843
37
113
7
1848
110
1908
135
Supplementary Table 4. Only 16 out of the 156 downstream targets of HOXB4
identified by Schiedlmeier et al.17 were also observed to be downstream targets of
HOXB4 in this report. Furthermore, only one direct downstream target, Rasa2,
overlapped in both studies (shown in bold).
Gene Symbol
Ccr2
Etv5
Gadd45a
IL15
Klf3
Narf
Nfkbie
Osbpl5
Ptgs2
Rasa2
Rassf4
Serpina3g
Socs5
Tex9
Tnfaip2
Zfp260
Gene Title
chemokine (C-C motif) receptor 2
ets variant gene 5
growth arrest and DNA-damage-inducible 45 alpha
interleukin 15
Kruppel-like factor 3 (basic)
nuclear prelamin A recognition factor
nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, epsilon
oxysterol binding protein-like 5
prostaglandin-endoperoxide synthase 2
RAS p21 protein activator 2
Ras association (RalGDS/AF-6) domain family 4
serine (or cysteine) peptidase inhibitor, clade A, member 3G
suppressor of cytokine signaling 5
Testis expressed gene 9
tumor necrosis factor, alpha-induced protein 2
zinc finger protein 260 /// similar to Zinc finger protein OZF (Zinc
finger protein 260) (Zfp-260)
Supplementary Material and Methods
ChIP-chip
Formaldehyde cross-linked chromatin was prepared from the three KLS-EML-HOXB4
clones and incubated with the I12 anti-human HOXB4 rat monoclonal antibody
(Developmental Studies Hybridoma Bank, University of Iowa) that had been previously
bound to Dynabeads magnetic beads (Invitrogen) conjugated to sheep-anti-rat IgG2a
antibody. As a control, formaldehyde cross-linked chromatin was prepared from KLSEML-HOX4 clone #4 cells and incubated with the isotype control anti-rat IgG2a
antibody (sc-53740) (Santa Cruz Biotechnology) that had been previously bound to
Dynabeads magnetic beads (Invitrogen) conjugated to sheep-anti-rat IgG2a antibody.
ChIP was performed using the Dynabeads magnet system (Invitrogen). The
immunoprecipitate (IP) was serially washed eight times using the Dynabeads magnet
system (Invitrogen) and supernatants from the serial washings were collected and
represent eluates 1-8 in Figure 5. Western blot analysis of ChIP eluates was performed
using the anti-HOXB4 (N-18) antibody (sc-15001) (Santa Cruz Biotechnology) and antiActin antibody (sc-1616) (Santa Cruz Biotechnology). Blots were visualized with the
HRP-conjugated donkey-anti-goat secondary antibody (sc-2020) (Santa Cruz
Biotechnology). Processing and microarray hybridization of the IP samples isolated
utilizing the I12 anti-rat human HOXB4 antibody or the isotype control anti-rat IgG2a
antibody and control total input DNA samples were carried out by Roche NimbleGen.
NimbleGen microarray surface artifacts were removed using the SmudgeKit package
from J. Emerson et al. (manuscript in preparation). The 2000 highest scoring HOXB4
binding sites were identified using the Tilescope software,1 using quantile normalization,
a window size of 1000, p-value threshold of 2.0, peak interval size of 1000, a feature
length of 500 and the iterative peak identification method. These sites showed an
enrichment over control of 1.77 fold or greater. 1910 of the 2000 sites were close to
RefSeq gene transcription start sites and were used for analysis. The 1000 regions with
the strongest binding were used to perform de novo motif discovery of overrepresented 6,
8 and 10mers on both DNA strands using the Weeder software.2 Tilescope software and
Weeder software analyses were provided by the Yale Center of Excellence in Molecular
Hematology (YCEMH). Quantitative PCR analysis was performed using primers
designed to the corresponding gene promoter regions and are available upon request.
References
1.
Zhang ZD, Rozowsky J, Lam HY, Du J, Snyder M, Gerstein M. Tilescope: online
analysis pipeline for high-density tiling microarray data. Genome Biol. 2007;8:R81.
2.
Pavesi G, Pesole G. Using Weeder for the discovery of conserved transcription
factor binding sites. Curr Protoc Bioinformatics. 2006;Chapter 2:Unit 2 11.