1
2
Supplementary methods
3
Cell Culture
4
KG1 cells were cultured in IMDM (STEMCELL Technologies, Vancouver, BC, Canada)
5
supplemented with 20% (v/v) FBS (Invitrogen, Burlington, ON, Canada) and antibiotics
6
(100U/mL of penicillin and streptomycin). K562, Jurkat and HL60 cells were cultured in
7
RPMI 1640 supplemented with 10% (v/v) FBS and antibiotics. HEK293 (293) cells were
8
cultured in DMEM supplemented with 10% (v/v) FBS and antibiotics. All cell cultures were
9
maintained at 37 °C in 5% CO2.
10
Reverse transcription and real-time PCR
11
Total RNA was isolated using Trizol (Invitrogen) following the manufacturer’s instructions.
12
cDNAs synthesis and Real-time PCR were performed as described previously1.
13
DNase I hypersensitivity assay
14
DNase I was purchased from Worthington Biochemical Corporation (Lakewood, NJ, USA).
15
Nuclei isolation and DNase I digestion were done as described previously2. Nuclei from 106
16
cells were treated with each of the different DNase I concentrations ranging from 3 unit/ml to
17
24 unit/ml for 10 min. The purified DNA was digested with specific NEB restriction enzymes
18
(BamH I, EcoR I, Hind III, Hpa I, Nco I or EcoR V), separated on a 0.8 % agarose gel and
19
subjected to Southern blotting as described2. Probes for Southern blots ranged in size from
20
300 bp to 500 bp, excluded any repeat elements and were from PCR amplified and purified
21
DNA fragments. Detailed sequence information for the probes is available upon request. A
22
particular region’s DNase I hypersensitivity was labeled as “1” if the corresponding band on
23
Southern blot was readily detected by eye. An example was shown at Sfig.1, where MEIS1
1
24
promoter region is defined as 1 in all the cell lines, whereas the distal promoter region at -2
25
kb is defined as 0 in the two MEIS1Lo cell lines (HL60 & Jurkat) but as 1 in the others.
26
compared our data set to the DNase I hypersensitivity data for K562 cells obtained using
27
digital DNase I methodology and made available via the ENCODE project on the UCSC
28
genome
29
similarity of the two patterns suggested that our data obtained using the traditional Southern
30
blot method to profile DNase I hypersensitivity was a reliable representation of the
31
distribution of DHSs.
32
Chromatin immunoprecipitation (ChIP) assay
33
Rabbit polyclonal antibodies against acetylated histone H3 (06-599) and CTCF (07-729) were
34
purchased from Millipore (Billerica, MA, USA). Sybr green PCR kits were from Roche
35
(Indianapolis, IN, USA). ChIP assays were performed as previously described with
36
modifications3. The human GAPDH gene was selected as an internal control. The detailed
37
information of primers used for real-time PCR is available upon request. Real time PCR was
38
performed in the 7900 HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA,
39
USA). All data were expressed as the ratio of the PCR readings of a given primer set over
40
the internal control GAPDH region comparing to the input control.
41
acetylation seen here in K562 cells was consistent with that reported previously in
42
genome-wide studies (Sfig.9).
43
DNA methylation analysis in the MEIS1 locus
44
Genomic DNA was modified with sodium bisulphite using a kit from Zymo Research (Irvine,
45
CA, USA) following the manufacturer’s suggested protocol. Converted DNA was used as
browser
(http://genome.cse.ucsc.edu/cgi-bin/hgTracks)
(Sfig.8).
The
We
strong
The pattern of histone
2
46
template for the following preamplification and amplification as described1. The detailed
47
information of primers used is in Supplementary Table 1.
48
separated on 1% agarose gels. The correct size products were subjected to combined bisulfite
49
restriction analysis (COBRA) or directly cloned into the TOPO-TA cloning vector (Invitrogen,
50
Burlington, ON, USA) as described previously1.
51
Generation of luciferase reporter constructs
52
The MEIS1 promoter construct used in this study was generated through the subcloning of a
53
540 bp fragment from the MEIS1 promoter construct in a pGL3 backbone from our previous
54
study
55
enhancer function, a 300 bp to 350 bp fragment was selected from each candidate region at
56
the peak of histone H3 acetylation, PCR amplified and cloned into the Kpn I / Xho I site of
57
the MEIS1 promoter construct. Since the fragment of E2 and E3 all contain CTCF binding
58
sites, a similar size fragment (labeled as “C” in Figure 2d) was selected from the region at -6
59
kb including a conserved CTCF binding site (Sfig.2) and used as a control for the potential
60
function from binding of CTCF as well as size control for transfection efficiency.
61
Sequences of the primers for the enhancers are in Supplementary Table 2.
62
Cell transfection and luciferase assays
63
Cells were transfected using either Lipofectamine 2000 (for K562 or U937 cell lines) or
64
Lipofectamin LTX (for Jurkat cell line) (Invitrogen, Burlington, ON, USA) according to the
65
manufacturer’s instructions. Cells were then incubated at 37ºC for 36 hr before assaying for
66
luciferase activity using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI,
67
USA). The enhancer candidate firefly luciferase activity was normalized relative to Renilla
1
The resulting products were
into the Bgl II / Hind III sites of the pGL4 firefly luciferase basic vector. To detect
3
68
luciferase activity for each transfection and calculated as fold increase over the MEIS1
69
promoter firefly luciferase vector. All data points were generated from an average of at least
70
three biological repeats.
71
Chromosome Conformation Capture (3C) assay and PCR analysis of the ligation
72
products
73
3C assay was performed following a previously described procedure with some
74
modifications4. To correct for differences in quality and quantity of template, we normalized
75
ligation frequencies between MEIS1 promoter to enhancer candidate site pairs to those
76
detected between two restriction fragments in the human Ercc3 locus. Ercc3 encodes a
77
subunit of the basal transcription factor TFIIH, and we assumed that expression levels and
78
spatial conformation of this gene were similar in all analyzed cell types. We included a
79
control template containing all possible ligation products in equimolar amounts to correct for
80
the PCR amplification efficiency of different primer sets. For this purpose, two bacterial
81
artificial chromosome (BAC) clones spanning the analyzed loci, a 200-kb BAC (BAC Clone
82
#RP11-809P8, BACPAC) and a 213-kb BAC containing human Ercc3 locus (BAC Clone
83
#RP11-313N8, BACPAC Resources, CA, USA) were mixed at equimolar amounts, digested
84
with Hind III and then ligated to produce the control template as described previously4. To
85
increase the sensitivity as well as specificity of the PCR analysis for detecting the ligated
86
products from the 3C assay, we added a preamplification step before the real time PCR with a
87
modified linear amplification mediated PCR protocol5. This step was performed with
88
200-250 ng of DNA sample from 3C assay, 5 l Platinum Taq 10X PCR buffer, 2.5 l 50 mM
89
MgCl2, 1 l 10mM dNTP mixture, 0.5 l Platinum Taq DNA polymerase, 0.5 l of 1 M
4
90
primer mixture of a primer from MEIS1 promoter containing Hind III fragment (primer A)
91
and a primer from ERRC3 locus (primer C) and adjusted with suitable amount of water to a
92
total of 50 l reaction volume. The PCR condition was: 94 oC, 2min; followed by 50 cycles
93
of 94 oC, 30 sec; 58 oC, 30 sec; 72 oC, 1 min step and a final 7 min extension at 72 oC. The
94
products were then diluted with 100 l of TE buffer. 2 l of the products were used as
95
template for the following RT-PCR. A nested primer from the MEIS1 promoter containing
96
Hind III fragment (primer nA) was used with another primer from the enhancer candidate
97
containing Hind III fragment (from primer B1 to B10) in the RT-PCR to detect the frequency
98
of interaction between these two fragments. This was normalized to the frequency of
99
interaction between two Hind III fragments from the ERRC3 locus measured through
100
RT-PCR, where one primer is the nested primer of primer C (primer nC) and another primer
101
D in a different Hind III fragment about 10 kb away.
102
original and the preamplified control template, we determined that the ratio of frequency of
103
interactions between different primers sets was not significantly affected by this
104
preamplification step.
105
PCR due to the usage of a nested primer and also reduced the background problem associated
106
with the previous protocol using the template directly after 3C in the RT-PCR. All data
107
points were generated from an average of four different experiments. The primer information
108
is in Supplementary Table 3.
From the comparison between the
The preamplification step greatly increased the specificity of the
109
110
111
Supplementary Tables
112
5
ST1
Promoter region
E1 region
E2 region
E3 region
Primers used in DNA methylation detection
ProF
AGAGAAAAGAATATTGAAAATAAA
PronR
CTA TCT AAA TTT ATC TTA AAA AAA CCA
ProR
CCC TCT TTA CAA ATA CTA CAC T
E1F
GGAT GAA GGG ATA TTT TTA ATT TT
E1nF
TTT GAG GGT GAG GGT TTA GT
E1R
AACCTCACATCCACAAACAAAAC
E2F
GGATTAAGATATTAGTAGAAGTTGTAA
E2nR
AAC ACA AAC CTA ACT AAT CT
E2R
TAC TTA TAA AAA AAA CCA AAA CCA
E3F
GGT TTT TAT TTT TGT TAG GAT TTT T
E3nF
AGA GTT ATA GGA AAA GAG GAA
E3R
AAC TCC TAA TAC CAT CCA TAC A
113
ST2
Promoter
Control
E1 region
E2 region
E3 region
Primers used for generation of luciferase reporter constructs
ProF
AGATCTGAGAAAAGAATACTGAAAATAAAGCTG
ProR
AAGCTTCTTCTGGTCAGAACACCTCAA
ControlF
CTCGAGTGAGTCTAAGTTGGGTACTGTGG
ControlR
AGATCTATCTTAAGTTTTCAGGTACTTCACTGC
E1F
GGTACCTTGAGGGTGAGGGTTCAGTC
E1R
GGTACCGGCTCCGAGAGGATTTTTC
E2F
GTCGACTGGGATCGACGACTTTGATA
E2R
GGTACCTCGCTTCCCCTAGCCTCT
E3F
AGATCTCCACTGCTTTTCGCTTGTATT
E3R
CTCGAGTGGATAATAAGGGGGTTTCC
114
ST3 Primers used in Chromosome Conformation Capture (3C) assay
Primer A
TTTCTAAACTAGTTGATAGGGACACAAG
Primer nA
AGTCTCGCTGAGAATTCTGTATTTTT
Primer B1
TCACTGTCGAACAGAGGTCAA
Primer B2
CATCTCGGCAGATCAGAGC
Primer B3
TTCATTAAGGCTGGCTCTGG
Primer B4
CTGGTTAAGAGTGGGCTTCG
Primer B5
AAGAAGCGGGGGAAATTCT
Primer B6
TTTAACATGCCATGATTTCAAGA
Primer B7
CGCAGTTGATGCATGAGTTA
Primer B8
TTTGTGGAAAGAGGAGAAATTGA
Primer B9
TTGGAGGTTTGGTCCCTAGTT
Primer B10
AGGAAAGGGAAAGGCTTGAG
Primer C
CAGGCCTTAGTTTGCCTCAG
Primer nC
GTTTGCCTCCCAGACATCAG
Primer D
CCCTGAAATCTCCACCCTCT
6
115
116
Supplementary Figure legends
117
118
Sfig.1. Extended characterization of MEIS1 promoter region consistent with our previous
119
work1, where the MEIS1Lo cell lines display a more epigentically closed status, while both
120
MEIS1Med and MEIS1Hi cell lines display similar open chromatin status at this region.
121
Southern blot detecting DNase I hypersensitivity around MEIS1 promoter region (BamH I
122
digestion, size of mother band 6554 bp, Chr2: 66513385 -66519939, hg18). (b) RT-PCR
123
results from chromatin immunoprecipitation (ChIP) assay with acetylated histone H3 (K9/14)
124
antibody. MEIS1 promoter region has a similar level of acetylated histone as the
125
housekeeping gene GAPDH promoter region in MEIS1Med and MEIS1Hi cell lines, but is
126
weakly acetylated in MEIS1Lo cell lines. (c) DNA methylation status at the CpG island of
127
MEIS1 promoter region through COBRA analysis. There is significantly more methylated
128
DNA at the MEIS1 promoter region in the two MEIS1Lo cell lines: HL60 and Jurkat. (d)
129
Despite the variable MEIS1 expression level, the promoter of MEIS1 has a similar activity (~
130
half of the in the SV40 PGL3 promoter) in the three cell lines tested through the dual
131
luciferase assay.
132
Sfig.2. Mapping of DHSs in the +140 kb region through Southern blot within MEIS1 locus
133
(Nco I digestion, size of mother band 13328 bp, Chr2: 66649340 -66662668, hg18).
134
Sfig.3. Mapping of DHSs in the +10.6 kb region through Southern blot within MEIS1 locus
135
(EcoR V digestion, size of mother band 6302 bp, Chr2: 66523305 -66529607, hg18).
136
Sfig.4. DNA methylation analysis in the three identified enhancer candidate regions. a) Direct
(a)
7
137
cloning after bisulfite treatment and sequencing at the enhancer E1, E2 or E3 regions.
Each
138
line represents an individual clone with red lines representing clones from U937, green from
139
HL60, blue from KG1 and black from K562 cells.
140
CpG site with “open” indicating non methylated while “black filled” indicating methylated.
141
The TCGA sites recognized by Taq1 in the combined bisulfite restriction analysis (COBRA)
142
are highlighted with black arrows, where the methylation status of these sites are good
143
indicators of the whole region.
144
to detect the DNA methylation of these regions. The DNA remain unmethylated where the
145
histone H3 is highly acetylated (marked with “*” in the figure).
146
Sfig.5. A snapshot from http://epigenomegateway.wustl.edu/browser/ with MEIS1 locus at the
147
center, highlighting the representative epigenetic data from CD34+ mobilized peripheral stem
148
cells and mature peripheral blood mononuclear cells (PBMC). Stronger color intensities
149
correspond to stronger signals.
150
Sfig.6. CTCF binding sites analysis within MEIS1 locus. a) A snapshot from UCSC genome
151
browser centered at the MEIS1 locus shown the CTCF ChIP–seq data with insertions of the
152
predicted CTCF binding sites at the -6kb, +10.6kb and +140kb regions, which display high
153
sequence similarities to the primary motif of CTCF consensus sequence shown below.
154
Interestingly, except the predicted CTCF binding sites at -6kb and +140kb regions, rest of the
155
potential CTCF binding regions (ex. +10.6kb) are missing in the zebrafish genome and
156
therefore not conserved between fish and human.
157
additional upstream motif quite similar to those found in the IL-3 insulator 6 that represents a
158
secondary contact site for CTCF, which suggests this region potentially function as an
Every oval shaped circle represents a
b) COBRA assay with TaqI restriction enzymes digestion
The -6kb CTCF binding sites has an
8
159
insulator.
160
Sfig.7. A snapshot from UCSC genome browser (chr2:66,481,696-66,687,735, hg 18) with
161
MEIS1 locus at the center with the candidate enhancer E1, E2 and E3 highlighted. While
162
there is some sequence conservation at the E3 region, the corresponding regions of E1 and E2
163
are missing in zebrafish genome. The detailed evolutionarily conservation analyses of the
164
regions from each candidate enhancer tested in luciferase assay are shown below.
165
Sfig.8. Comparison of our traditional way of detecting DHS through Southern blot analysis to
166
digitalized high through put data within the MEIS1 locus of K562 cells from the UCSC
167
genome browser.
168
Sfig.9. Comparison of our H3 acetylation (K9/14) ChIP-RT PCR data to H3K9 acetylation
169
ChIP-Seq data within the MEIS1 locus of K562 cells from UCSC genome browser.
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Supplementary References
1.
Xiang P, Lo C, Argiropoulos B, Lai CB, Rouhi A, Imren S et al. Identification of E74-like factor 1
(ELF1) as a transcriptional regulator of the Hox cofactor MEIS1. Experimental hematology 2010;
38(9): 798-8, 808 e1-2.
2.
Li Q, Zhang M, Duan Z, Stamatoyannopoulos G. Structural analysis and mapping of DNase I
hypersensitivity of HS5 of the beta-globin locus control region. Genomics 1999; 61(2): 183-93.
3.
Duan ZJ, Fang X, Rohde A, Han H, Stamatoyannopoulos G, Li Q. Developmental specificity of
recruitment of TBP to the TATA box of the human gamma-globin gene. Proceedings of the National
Academy of Sciences of the United States of America 2002; 99(8): 5509-14.
4.
Fang X, Xiang P, Yin W, Stamatoyannopoulos G, Li Q. Cooperativeness of the higher chromatin
structure of the beta-globin locus revealed by the deletion mutations of DNase I hypersensitive site 3 of
the LCR. Journal of molecular biology 2007; 365(1): 31-7.
5.
Schmidt M, Schwarzwaelder K, Bartholomae C, Zaoui K, Ball C, Pilz I et al. High-resolution
insertion-site analysis by linear amplification-mediated PCR (LAM-PCR). Nature methods 2007; 4(12):
9
192
193
194
195
196
197
198
1051-7.
6.
Bowers SR, Mirabella F, Calero-Nieto FJ, Valeaux S, Hadjur S, Baxter EW et al. A conserved insulator
that recruits CTCF and cohesin exists between the closely related but divergently regulated
interleukin-3 and granulocyte-macrophage colony-stimulating factor genes. Molecular and cellular
biology 2009; 29(7): 1682-93.
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