1. Volume 21, Issue 8, Pages 2160-2170 (November 2017)
Kdm2b Regulates Somatic Reprogramming through Variant PRC1 Complex-Dependent Function 
Zhiwei Zhou, Xuejie Yang, Jiangping He, Jing Liu, Fang Wu, Shengyong Yu, Yuting Liu, Runxia Lin, He Liu, Yuanbin Cui, Chunhua Zhou, Xiaoshan Wang, Jian Wu, Shangtao Cao, Lin Guo, Lihui Lin, Tao Wang, Xiaozhong Peng, Boqing Qiang, Andrew P. Hutchins, Duanqing Pei, Jiekai Chen 
Cell Reports 
Volume 21, Issue 8, Pages (November 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 2160-2170DOI: (10.1016/j.celrep.2017.10.091)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Kdm2b Promotes Robust Reprogramming through Variant KDM2B-PRC1.1
(A) Effect of Kdm2b and Kdm2a on Oct4-mediated iPSC generation in vitamin-C-containing iCD1 medium. The number of GFP+ colonies was counted at day 20 (n = 3 independent experiments each with 2 technical replicates). Error bar is SEM, ∗∗∗p <
(B) Representative images of iPSC colonies generated with Oct4/Kdm2b or Oct4/Kdm2a-induced reprogramming in systems with DR(DsRed) as a control. Images were captured at day 20 (scale bars, 250 μm).
(C) Left: scheme depicting the generation of Kdm2b mutants. Right: all the mutants lack the ability to enhance Oct4-induced reprogramming, unlike wild-type control. DR as a control (n = 3 biological replicates each with 2 technical replicates). Error bar is SEM, ∗∗∗p <
(D) Knocking down the KDM2B-PRC1 members, Pcgf1, Skp1a, with Pcgf6 as control by shRNAs impairs Oct4/Kdm2b-induced reprogramming. The number of GFP+ colonies were counted at day 20 (n = 3 biological replicates each with 2 technical replicates). Error bar is SEM, ∗∗p < 0.01, ∗∗∗p <
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4. Figure 2 Gene Regulation of Kdm2b during Reprogramming
(A) Heatmap representation of KDM2B, CGI (CpG islands), and H2AK119ub occupancy at all annotated gene promoters (10 kb flanking TSSs of Refseq genes) in Oct4/Kdm2b-induced reprogramming at day 14.
(B) Left: RNA-seq gene expression profile (from three independent replicates) in Oct4/DR, Oct4/Kdm2a, or Oct4/Kdm2b-induced reprogramming at day 14. Genes upregulated (groups I, II, and III) or downregulated (groups IV, V, and VI) constitute two distinct clusters. Middle: the black/white panel shows the presence (black) or absence (white) of KDM2B binding sites at the TSS. Right: moving window average plot of the density of KDM2B binding. The black dashed line is the mean, and the dashed red line represents 1 SD from the mean. KDM2B is more likely to be bound in the vicinity of genes that are upregulated when Kdm2b is overexpressed.
(C) The observed and expected Kdm2b target genes for each cluster. The p value was calculated by two-sided Fisher exact test.
(D) Examples genome views of the occupancy of H2AK119ub, H3K27me3, and H3K36me2 in Oct4/DR, Oct4/Kdm2a, or Oct4/Kdm2b-induced reprogramming at day 14. The corresponding expression change (RNA-seq) and the locations of the CpG islands and genes are shown.
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5. Figure 3
Kdm2b Promotes Reprogramming through the Early Activation of Pluripotent Genes in the Manner of Co-overexpression with Oct4
(A) Principal component (PC) analysis indicates the transitions that occur during reprogramming. PC2 roughly corresponds to MEF to ESCs acquisition. Percentage explained variance is indicated.
(B) qPCR analysis for the indicated pluripotent genes at day 6. Data are the mean from two independent experiments.
(C) Effect of Oct4 on Kdm2b-induced reprogramming with DR as control. The number of GFP+ colonies were counted at day 20 (n = 3 independent experiments each with 2 technical replicates). Error bar is SEM, ∗∗∗p < 0.001).
(D) qPCR analysis for the indicated pluripotent genes at day 6. Data are the mean from two independent experiments.
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6. Figure 4
BMP4 Abolishes Kdm2b’s Reprogramming Promoting Function Specifically
(A) Effect of BMP4 in Oct4/DR or Oct4/Kdm2b-induced reprogramming (n = 3 biological replicates each with 2 technical replicates, error bar is SEM, ∗∗p < 0.01, ∗∗∗p < 0.001).
(B) Western blot analysis of KDM2B-3xFLAG protein level as in (A).
(C) Effect of BMP4 on indicated factors co-transfected with Oct4 during reprogramming; the number of GFP+ colonies were counted at day 20.
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7. Figure 5
BMP4-SMAD1 Pathway Attenuates PRC1.1 Occupation and H2AK119 Ubiquitination
(A) Examples genome views of the occupancy of KDM2B, H2AK119ub with (+) or without (–) BMP4 treatment in Oct4/Kdm2b-induced reprogramming at day 14. The corresponding expression change (RNA-seq) and the locations of the CpG islands and genes are shown.
(B) Heatmap showing KDM2B and H2AK119ub occupancy ±10 kb around the TSSs of all PRC1.1 target genes with or without BMP4 treatment.
(C) The average pileup KDM2B and H2AK119ub signals as in (B).
(D) Left: CoIP detection of interaction between KDM2B and SMAD1 with or without BMP4 treatment. Anti-SMAD1 IP (immunoprecipitation) of KDM2B-3FLAG expressed in platE cells with or without BMP4 treatment for 2 hr, followed by immunoblot using antibodies against the indicated proteins. Right: western blot analysis of p-SMAD1 and SMAD1 protein with GAPDH as control.
(E) Western blot analysis for KDM2B, RYBP, and RING1B in nuclear extract (NE) or chromatin-bound nuclear extract (CBNE) of platE cells ectopically expressing KDM2B-3Flag with or without BMP4 treatment. Ponceau staining for histone is as control.
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8. Figure 6
Mesendoderm Factors Are Released from PRC1.1’s Regulation, Resulting in Inhibition of Reprogramming
(A) Heatmap of RNA-seq data showing the expression level of genes that are specifically upregulated by BMP4 in Oct4/Kdm2b-induced reprogramming at day 14. Two independent data are shown.
(B) Venn diagram showing the common genes targets of low expressed PRC1.1 target genes and genes upregulated during reprogramming with Oct4/Kdm2b+BMP4 at day 14.
(C) GO analysis of the 395 genes as in (B).
(D) qPCR analysis of mesendodermal genes expression.
(E) Immunofluorescence (IF) analysis of SOX17 in reprogramming samples with or without BMP4 treatment (scale bar, 250 μm). IF was performed at day 14 after co-transfection of Oct4 and Kdm2b with BMP4 treatment.
(F) Sox17 substantially inhibits Oct4/Kdm2b-induced reprogramming. The number of GFP+ colonies were counted at day 20. Data are from 3 biological replicates each with 2 technical replicates. Error bars are SEM. ∗∗∗p value <0.001.
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