Volume 64, Issue 5, Pages 913-925 (December 2016) Cooperative Action between SALL4A and TET Proteins in Stepwise Oxidation of 5- Methylcytosine  Jun Xiong, Zhuqiang Zhang, Jiayu Chen, Hua Huang, Yali Xu, Xiaojun Ding, Yong Zheng, Ryuichi Nishinakamura, Guo-Liang Xu, Hailin Wang, She Chen, Shaorong Gao, Bing Zhu  Molecular Cell  Volume 64, Issue 5, Pages 913-925 (December 2016) DOI: 10.1016/j.molcel.2016.10.013 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 64, 913-925DOI: (10.1016/j.molcel.2016.10.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 SALL1 and SALL4 Preferentially Associate with 5hmC In Vitro (A) Scatter plot of a SILAC-assisted DNA pull-down assay to identify 5hmC-associated proteins. (B) Enrichment values of 5hmC binders and other related proteins. (C) Schematic representation of SALL1 and SALL4 protein structures. Zinc finger motifs are represented by black rectangles. Mutation sites in Figure 1F are indicated with red arrows. (D) EMSA showing the binding of recombinant truncates of SALL1 and SALL4 proteins containing individual ZFCs to indicated DNA probes. (E) EMSA competition assay with excess amounts of unlabeled DNA containing various cytosine modifications. (F) EMSA showing the binding of SALL1-ZFC3 and SALL4-ZFC1 mutant proteins to 5hmC-containing DNA. Mutant proteins were added with increasing concentrations compared with wild-type proteins (one time, three times, and ten times, respectively). See also Figure S1 and Table S1. Molecular Cell 2016 64, 913-925DOI: (10.1016/j.molcel.2016.10.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 SALL1 and SALL4A Occupy Enhancers in Mouse ESCs (A) Venn diagram showing a large overlap between the SALL1 and SALL4A peaks. (B) Genome browser tracks representing the occupancy of SALL1, SALL4A, NANOG, OCT4, and P300 at two representative enhancers. (C) Genome-wide distribution of SALL4A peaks at promoters (±2 kb around TSSs) and other genomic regions. (D) Pie chart showing the distance distribution of SALL4A peaks to the nearest TSSs. (E) Profiles of normalized read density (RP10M) of H3K4me3, P300, H3K4me1, and H3K27ac around SALL4A peaks distal to TSSs. Right y-axis is dedicated to present the normalized read density of P300. RP10M, reads per 10 million reads. (F) Heat map representing the co-occupancy of SALL1 and SALL4A in the mouse genome, which are enriched for P300, H3K4me1, H3K27ac, TET1, and TET2, but not H3K4me3 peaks. All SALL4A peaks were sorted according to their distance to the nearest TSSs. See also Figure S2. Molecular Cell 2016 64, 913-925DOI: (10.1016/j.molcel.2016.10.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Genomic Distribution of SALL4A Largely Depends on TET Enzymes (A) Profiles of normalized read density of TET1 and TET2 around the SALL4A peaks in TT2 ESCs. (B) Genome browser tracks representing the occupancy changes of SALL4A in Tet1 or Tet2 knockdown TT2 cells. (C) Heat map representing occupancy changes of SALL4A peaks, which are categorized into three groups, TET1-independent, intermediate, and TET1-dependent, in TT2 ESCs treated with shRNA against Tet1. The numbers of peaks in each group are indicated. FE, fold enrichment. (D) Heat map representing occupancy changes of SALL4A peaks, which are categorized into three groups, TET2-independent, intermediate, and TET2-dependent, in TT2 ESCs treated with shRNA against Tet2. The numbers of peaks in each group are indicated. (E) Venn diagram showing the overlap between TET1-dependent (TET1-dep.) and TET2-dependent (TET2-dep.) SALLA4 peaks. See also Figure S3. Molecular Cell 2016 64, 913-925DOI: (10.1016/j.molcel.2016.10.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 5hmC Is Further Oxidized at SALL1/SALL4A-Binding Regions in Mouse ESCs (A) 5hmC profiles around SALL1 and SALL4A peaks in TT2 ESCs. (B) 5hmC profiles at enhancers with (w/) or without (w/o) SALL4A peaks. (C) 5caC profiles around SALL1 and SALL4A peaks. (D) 5caC profiles at enhancers with or without SALL4A peaks. (E) 5fC profiles around TET1-dependent or -independent (TET1-indep.) SALL4A peaks. (F) Genome browser tracks representing the DNA modification status at the same SALL4A-binding sites as in Figure 3B. See also Figure S4. Molecular Cell 2016 64, 913-925DOI: (10.1016/j.molcel.2016.10.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 SALL4A Facilitates Further Oxidation of 5hmC at Its Binding Regions (A) Changes of normalized read density of 5hmC and 5mC around SALL1 and SALL4A peaks between WT and Sall DKO ESCs. The original SALL1 and SALL4A peaks were identified by independent ChIP-seq experiments with Sall WT ESCs. (B) Changes of 5hmC profiles around TET1-dependent and -independent SALL4A peaks in WT and Sall DKO ESCs. (C) Changes of 5hmC profiles around TET2-dependent and -independent (TET2-indep.) SALL4A peaks in WT and Sall DKO ESCs. (D) Changes of 5hmC profiles around SALL4A and SALL1 peaks in Sall4 KO and Sall1 KO ESCs, respectively. The SALL1 peaks were determined in TT2 ESCs, and the original SALL4A peaks were identified by an independent ChIP-seq experiment with Sall4 Het ESCs. (E) 5caC profiles around SALL4A peaks in Sall4 Het and KO ESCs with TDG depletion. (F) Genome browser tracks representing the changes of DNA modification status upon loss of the Sall4 gene at genomic loci as in Figure 3B. (G) Scatter plot showing the correlation between 5caC levels in Sall4 Het ESCs depleted of TDG with 5hmC changes upon Sall4 knockout. (H) 5hmC profiles around TET1-dependent SALL4A peaks in Sall4 Het, Sall4 KO, and Sall4 KO ESCs expressing exogenous wild-type (KO + WT) or C420A mutant (KO + Mut#2) SALL4A proteins. (I) Left, heat map representing ChIP-seq signals of ectopically expressed wild-type and mutant SALL4A in Sall4 KO ESCs. Right, box plot showing the fold enrichments of mutant SALL4A ChIP-seq signals were not significantly different from that of wild-type SALL4A in Sall4 KO ESCs. Whiskers indicate the range of data points which is no more than 1.5 times of the IQR (interquartile range) from the box. See also Figure S5. Molecular Cell 2016 64, 913-925DOI: (10.1016/j.molcel.2016.10.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 SALL4A Cooperates with TET2 to Stimulate Further Oxidation of 5hmC at Its Binding Sites (A) TET1 (upper) and TET2 (lower) profiles at enhancers with or without SALL4A peaks. (B) Heat map representing the changes of 5hmC, TET1, and TET2 at the original SALL4A peaks upon deletion of the Sall4 gene. All peaks were ranked by the changes of 5hmC level, and then were categorized into four groups, which are indicated by the colored bars on the right. (C and D) Changes of normalized read density of TET2 (C) and TET1 (D) around the original SALL4A peaks upon deletion of the Sall4 gene. The groups were colored the same as in (B). (E and F) Changes of 5hmC profiles around SALL4A peaks in mouse ESCs treated with shRNA against Tet2 (E) or Tet1 (F) (Huang et al., 2014). The four groups were colored the same as in (B). (G) 5caC profiles around SALL4A peaks in Sall4 Het ESCs. All SALL4A peaks were ranked and classified into five groups according to the strength of TET2 occupancy. The averaged 5caC level of each group in Sall4 Het cells with Tdg knockdown was plotted. (H) 5hmC profiles around SALL4A peaks in mouse ESCs treated with shRNA against Tet2 using data published previously by Rao’s lab (Huang et al., 2014). The four groups were colored the same as in (B). (I) Genome browser tracks representing the changes of 5hmC, TET1, and TET2 upon loss of the Sall4 gene at genomic loci as in Figure 3B. See also Figure S6. Molecular Cell 2016 64, 913-925DOI: (10.1016/j.molcel.2016.10.013) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Role of SALL4A in Transcription Regulation (A) Hierarchical clustering and heat map for gene expression profiles for Sall4 Het, Sall4 KO, and Sall4 KO with reintroduced wild-type (KO + WT) or mutant (C420A) SALL4A (KO + Mut). For Sall4 Het and KO ESCs, RNA-seq experiments were performed in duplicates. For Sall4 KO ESCs with reintroduced SALL4A-C420A mutant, two independent cell clones (Mut#2 and Mut#5; Figure S2C) were analyzed. (B) Box plots showing the fold changes of SALL4A target gene expression. The red dash lines indicate 2-fold changes. Target genes associated with the top 2,000 SALL4A peaks with the highest 5hmC increase (left) or the least 5hmC change (right) upon deletion of the Sall4 gene were selected. Ratio of variances (RoVs) among treatments is indicated. (C) Model of SALL4A-mediated stimulation of 5hmC oxidation at TET1-dependent SALL4A-occupied enhancers. See also Figure S7. Molecular Cell 2016 64, 913-925DOI: (10.1016/j.molcel.2016.10.013) Copyright © 2016 Elsevier Inc. Terms and Conditions