Developmental Cell, Volume 28 Supplemental Information Polycomb Potentiates Meis2 Activation in Midbrain by Mediating Interaction of the Promoter with a Tissue-Specific Enhancer Takashi Kondo, Kyoichi Isono, Kaori Kondo, Takaho A. Endo, Shigeyoshi Itohara, Miguel Vidal, and Haruhiko Koseki Inventory for Supplemental Information There are 3 supplemental figures (Figure S1-3), 1 supplemental table (Table S1). -Figure S1 related to Figure 1. (A) ChIP-seq data at promoter and RBS of Meis2 gene, (B) section planes employed in Figure 1, (C) distribution of distances between promoter/RBS, (D) statistical analysis on the FISH experiments in Figures 1D, 1E and 1F. -Figure S2 related to Figure 2. (A) genomic map of tested fragments for transgenic analysis, (B) summary results for transgenic study, (C) 3C results between promoter and enhancer candidates, (D) ChIP-seq data by H3K4me1 and H3K27ac antibodies at the region surrounding Meis2 locus, (E) statistical analysis on the FISH experiments in Figure 2C and 2D, and (F) Immuno-FISH for PRT/MBE/RING1B. -Figure S3 related to Figure 3. (A) summary for the tripartite interaction between promomter/MBE/RBS in Figures 3B and 3C, (B) summary for association between RING1B and each FISH probe at different stage embryos Figures 3D and 3E, and (C) summary of the RING1B binding sites surrounding genes and several examples of gene loci bound by RING1B. -Table S1 related to Figure 4. (A) summarizes promoter/MBE/RBS association in Figure 4B, and (B) summarizes promoter/MBE/RBS association in Figure 4C.
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Developmental Cell, Volume 28
Supplemental Information
Polycomb Potentiates Meis2 Activation
in Midbrain by Mediating Interaction
of the Promoter with a Tissue-Specific Enhancer
Takashi Kondo, Kyoichi Isono, Kaori Kondo, Takaho A. Endo, Shigeyoshi Itohara, Miguel
Vidal, and Haruhiko Koseki
Inventory for Supplemental Information
There are 3 supplemental figures (Figure S1-3), 1 supplemental table (Table S1).
-Figure S1 related to Figure 1. (A) ChIP-seq data at promoter and RBS of Meis2 gene, (B)
section planes employed in Figure 1, (C) distribution of distances between promoter/RBS, (D)
statistical analysis on the FISH experiments in Figures 1D, 1E and 1F.
-Figure S2 related to Figure 2. (A) genomic map of tested fragments for transgenic analysis, (B)
summary results for transgenic study, (C) 3C results between promoter and enhancer candidates,
(D) ChIP-seq data by H3K4me1 and H3K27ac antibodies at the region surrounding Meis2 locus,
(E) statistical analysis on the FISH experiments in Figure 2C and 2D, and (F) Immuno-FISH for
PRT/MBE/RING1B.
-Figure S3 related to Figure 3. (A) summary for the tripartite interaction between
promomter/MBE/RBS in Figures 3B and 3C, (B) summary for association between RING1B
and each FISH probe at different stage embryos Figures 3D and 3E, and (C) summary of the
RING1B binding sites surrounding genes and several examples of gene loci bound by RING1B.
-Table S1 related to Figure 4. (A) summarizes promoter/MBE/RBS association in Figure 4B, and
(B) summarizes promoter/MBE/RBS association in Figure 4C.
-Supplemental Figure Legends
-Supplemental Experimental Procedures
-Supplemental References
Kondo T. Figure S1
section planes
LM
MB
LGEMGE
CM
A
C
1050Log10(P-value)
PRT/RBS
MB
LGE
MGE
CM
LM
average distance (μm)
0.290
0.344
0.143
0.126
0.137
SD
0.136
0.169
0.143
0.121
0.0956
n
297
209
195
246
226
wt
wt
MB LGE MGE CM LM
MB
LGE
MGE
CM
LM
average distance (μm)
0.293
0.313
0.287
0.253
0.274
SD
0.124
0.126
0.107
0.0998
0.136
n
264
209
223
214
234
R1
Ring1 mut
MB LGE MGE CM LM
D
0 10
250
Distance (µm)
Num
ber o
f pai
red
foci
Cluster 2(50%)
Cluster 1(44%)
Cluster 3(6%)
0.16µm
71.2% 28.8%68.7% 31.3% 72.8% 27.2%MGE LM CM
0 0.75 0 0.75 0 0.75 0 0.750
100
Distance (μm)
Num
bers
LGE12.0% 88.0%
MB14.8% 85.2%
0 0.75
FB
1 2 3 4 5
B
RBS promoter
115920000
Meis2
ES
chr2:115660000 115710000 115870000
FB
MB
LM
2810405F15Rik
4.20 x 10-19.76 x 10-151.19 x 10-147.59 x 10-146.55 x 10-145.87 x 10-1
0
500
P value
TissueExpression
FB+
MB+
LM-
ES-
Rea
ds /
M re
ads
TSS
ES
TSSRING1B
RING1B
RING1B
RING1B
H3K27me3
Human chromosome: chr15 37,110,000-37,710,000 (hg19)
The 3C technique was performed as described previously (Splinter et al., 2004) with slight
modifications. 106 to 5 x 106 cells were used for 3C analysis, and chromatin was digested
with 400U of the EcoRI restriction enzyme. Primers used for 3C are summarized below.
Quantification of ligation products was based on standard curves made using the cloned
fusion fragments for each expected product. Relative values for 3C were further normalized
by 3C values obtained from the results using the ERCC3 allele in each experiment.
Primer used for 3C analysis
primer name sequence promoter gtttgtcccacactccagcccttg
sequence C acggattgaagctggtcgctcttc sequence V gaagagaaaggcgactcagtctc Ercc3-1-r gcttgagcatgacgtcactg Ercc3-2-r gctgagatcaaggatgaggc
SUPPLEMENTAL REFERENCE Atsuta, T., Fujimura, S., Moriya, H., Vidal, M., Akasaka, T., and Koseki, H. (2001). Production of monoclonal antibodies against mammalian Ring1B proteins. Hybridoma 20, 43-46. Chambeyron, S., Da Silva, N.R., Lawson, K.A., and Bickmore, W.A. (2005). Nuclear re-organisation of the Hoxb complex during mouse embryonic development. Development 132, 2215-2223. del Mar Lorente, M., Marcos-Gutierrez, C., Perez, C., Schoorlemmer, J., Ramirez, A., Magin, T., and Vidal, M. (2000). Loss-and gain-of- function mutations show a Polycomb group function for Ring1A in mice. Development 127, 5093-5100. De Napoles, M., Merrnoud, J.E., Wakao, R., Tang, Y.A., Endoh, M., Appanah, R., Nesterova, T.B., Silva, J., Otter, A.P., Vidal, M., Koseki, H., and Brockdorff, N. (2004). Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev. Cell 7, 663-676. Isono, K., Endo, T.A., Ku, M., Yamada, D., Suzuki, R., Sharif, J., Ishikura, T., Toyoda, T., Bernstein, B.E., and Koseki, H. (2013). SAM domain polymerization links subnuclear clustering of PRC1 to gene silencing. Dev. Cell 26, 565-577. Kondo, T., and Duboule, D. (1999). Breaking colinearity in the mouse HoxD complex. Cell 97, 407-417. Sasaki, H. and Hogan, B.L.M. (1996). Enhancer analysis of HNF-3b gene: regulatory elements for node/notochord and floor plate are independent and consist of multiple sub-elements. Genes to Cells 1, 59-72. Splinter, E., Grosveld, F., and de Laat, W. (2004). 3C technology: analyzing the spatial
organization of genomic loci in vivo. Methods Enzymol. 375, 493-507. Tanemura, K., Ogura, A., Cheong, C., Gotoh, H., Matsumoto, K., Sato, E., Hayashi, Y., Lee, H.W., and Kondo, T., (2005). Dynamic rearrangement of telomeres during spermatogenesis in mice. Dev. Biol. 281, 196-207.