1. Volume 28, Issue 1, Pages 94-101 (January 2014)
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, Haruhiko Koseki 
Developmental Cell 
Volume 28, Issue 1, Pages (January 2014)
DOI: /j.devcel
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2. Developmental Cell 2014 28, 94-101DOI: (10.1016/j.devcel.2013.11.021)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Role of RING1 in Meis2 Transcription and Regulation of the Promoter-RBS Interaction
(A) Meis2 expression at 8.5, 9.5, and 11.5 dpc.
(B) RING1B ChIP-seq data (GSE48464) over 10 Mb of the mouse genome (mm9) around Meis2 in FB, MB, and LM at 11.5 dpc. RING1B binds to the promoter and RBS regions of Meis2 (indicated by open blue and red triangles, respectively). An enlarged view of Meis2 is shown along with the positions of the coding (yellow boxes) and noncoding (blue boxes) regions of exons. CpG islands and genomic fragments used for FISH probes (indicated as promoter and RBS) are indicted below the panels.
(C) Meis2 expression in 11.5 dpc wild-type (WT) and Ring1A−/−:Ring1Bflox/flox:ERT2Cre+/− (Ring1 mut) embryos treated with tamoxifen at 8.5 dpc. Note the considerable downregulation of Meis2 in MB (indicated by open boxes) of Ring1 mutants compared with the WT.
(D) Immuno-FISH analyses for the topology of promoter (PRT; blue), RBS (red), and RING1B (green) foci in MB, LGE, MGE, CM, and LM of 11.5 dpc WT (top panels). In Ring1 mutants (bottom panels), RBS (red) does not colocalize with the promoter (green) in any of the tissues tested. For the analysis of Ring1 mutants, we chose cells that do not express RING1B. Fluorescent signals from each channel in the region indicated by dotted boxes are shown in the insets.
(E) Bar graph summary for (D). Bars represent the ratio of paired foci in close proximity (<0.16 μm) versus the total number of paired foci measured. Blue-green and light-green bars indicate WT and Ring1 mutants (Ring1 mut), respectively. The p values by chi-square test between WT versus Ring1 mutant are indicated above the graph; p values < 10−3 are denoted by red font throughout this study.
See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 2
Identification of the MBE of Meis2 and Its RING1-Dependent Association with the Promoter in 11.5 dpc MB
(A) Schematic representations of genomic structures of human (top) and mouse (bottom) Meis2. Filled and open boxes on the lines represent MBE and RBS, respectively, in both human and mouse. The position of the human BAC clone (RP11-991D13) used for transgenic analysis is shown below the human Mies2. The positions of the fosmid clones used to generate FISH probes for promoter, MBE, and RBS of mouse Meis2 are indicated below the mouse gene.
(B) BAC and conventional transgenic approaches to identify the MBE of Meis2. BAC transgenic mice harboring RP11-991D13 expressed GFP (indicated by open boxes) in MB, whereas those with RP11-991D13ΔMBE did not. The mouse MBE fragment drives MB expression of LacZ (indicated by an open box) in MBEhsp68-LacZ transgenic mice. Results of the transgenic analyses are summarized in the table, in which numbers of mice harboring full-length transgenic DNA (D), the expression of the transgene (E), and MB expression (M) are indicated.
(C) 3D-FISH analyses of the promoter (green)-MBE (red) interaction in WT (upper panels) and Ring1 mutant (lower panels). Fluorescent signals from each channel in the regions indicated by dotted boxes are shown in insets.
(D) Bar graph summary for Figure 2C. The bar graph represents the ratio of paired foci in close proximity (<0.16 μm) versus the total number of paired foci measured. Blue and green bars indicate WT and Ring1 mutants (Ring1 mut), respectively.
(E) Binding of H3K27ac and RING1B in 11.5 dpc MB of WT and Ring1 mutant (Ring1 mut) revealed by ChIP-quantitative PCR (ChIP-qPCR) analysis. The positions of the respective primer pairs are indicated below the schematic representation of the mouse Meis2 gene. The primer sequences can be found in Supplemental Experimental Procedures. Three randomly selected regions (Co1, Co3, and Co5) were used as negative controls. Note that RING1B binding to the promoter region is considerably lower than that to RBS. The level of H3K27ac is equivalent between WT and Ring1 mutants at the MBE. Error bars indicate the SDs of three independent ChIP-qPCR results.
See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 3
The Promoter-MBE Association Is Preceded by a Promoter-MBE-RBS Tripartite Interaction in MB
(A) Meis2 expression in 6.5 dpc, 16–20 somite stage, and 22–26 somite stage embryos. Meis2 expression in MB starts around the 21–22 somite stage. Section planes for FISH are indicated by lines.
(B) Tricolor FISH images showing the topological transition of the promoter (PRT) (blue), MBE (red), and RBS (green) in 6.5 dpc epiblasts and MB of 16–20 and 22–26 somite stage embryos. Fluorescent signals from each channel in the regions indicated by dotted boxes are shown in insets.
(C) Bar graph summary of the results in (B). The ratio of paired foci in close proximity (<0.16 μm) versus the total number of paired foci measured is shown. Promoter-MBE and promoter-RBS interactions are indicated by dark- and light-blue bars, respectively.
(D) DNA topology of promoter-MBE (left panels) and promoter-RBS (right panels), and their association with RING1B speckles in MB of 16–20 somite stage (top panels) and 22–26 somite stage (bottom panels) embryos as revealed by immuno-FISH.
(E) Graphic summary of the results in (D). The frequency of colocalization of RING1B foci with the promoter (PRT), MBE, or RBS at each stage is shown. Note that RBS constitutively associates with the RING1B foci.
See also Figure S3.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 4
The Promoter-MBE-RBS Tripartite Interaction Occurs in a RING1-Dependent Manner
(A) Experimental schemes for tamoxifen injection. In the first experiment (experiment B), tamoxifen was injected intraperitoneally into pregnant females harboring Ring1A−/−:Ring1Bflox/flox:ERT2Cre+/− embryos at 8.0 dpc, and embryos were harvested and analyzed at 9.0 dpc (15–20 somite stage). In the second set of experiments (experiments C and D), tamoxifen was injected at 8.5 dpc, at a time before initiation of Meis2 transcription (as in the animals shown in Figures 1 and 2), or at 9.5 dpc, after initiation of Meis2 expression. The embryos were harvested and analyzed at 11.5 dpc.
(B) Tripartite interaction of promoter (PRT; blue), MBE (red), and RBS (green) in MB of 18 somite stage embryos requires RING1. Fluorescent signals from each channel in the regions indicated by dotted boxes are shown in insets. The bar graph summary of these results shows the ratio of paired foci in close proximity (<0.16 μm) versus the total number of paired foci measured. The frequencies of promoter-MBE and promoter-RBS interactions are indicated by dark- and light-blue bars, respectively. In this experiment, embryos lacking ERT2Cre were used as a control.
(C) Dissociation of promoter (PRT), MBE, and RBS in 11.5 dpc MB induced by tamoxifen injection at 8.5 dpc, but not 9.5 dpc. Fluorescent signals from each channel in the regions indicated by dotted boxes are shown in insets. A bar graph summary for these results is also shown. The ratio of paired foci in close proximity (<0.16 μm) versus the total number of paired foci measured is shown as bar graphs. Promoter-MBE and promoter-RBS interactions are indicated by dark- and light-blue bars, respectively.
(D) Comparison of Meis2 expression in WT and tamoxifen-injected mice (inj) at 8.5 or 9.5 dpc and analyzed at 11.5 dpc. The MB region is indicated by open boxes.
(E) Schematic summary showing the topological transition of promoter (PRT), MBE, and RBS that occurs during MB development to mediate transcriptional activation of Meis2. Promoter, MBE, and RBS regions are indicated by blue, red, and green lines, respectively, and other unannotated genomic regions are indicated as a black line. The RING1B speckle is indicated by a filled yellow circle. Promoter-RBS and promoter-MBE interactions are shown by blue and pink bidirectional arrows, respectively.
See also Tables S1A and S1B.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions