1. Volume 5, Issue 2, Pages 471-481 (October 2013)
An Extensive Network of TET2-Targeting MicroRNAs Regulates Malignant Hematopoiesis 
Jijun Cheng, Shangqin Guo, Suning Chen, Stephen J. Mastriano, Chaochun Liu, Ana C. D’Alessio, Eriona Hysolli, Yanwen Guo, Hong Yao, Cynthia M. Megyola, Dan Li, Jun Liu, Wen Pan, Christine A. Roden, Xiao-Ling Zhou, Kartoosh Heydari, Jianjun Chen, In-Hyun Park, Ye Ding, Yi Zhang, Jun Lu 
Cell Reports 
Volume 5, Issue 2, Pages (October 2013)
DOI: /j.celrep
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2. Cell Reports 2013 5, 471-481DOI: (10.1016/j.celrep.2013.08.050)
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3. Figure 1
A 3′ UTR Reporter Screen Identifies Candidate TET2-Targeting miRNAs
(A) A schematic shows the high-throughput screen in which ∼460 human miRNA expression constructs were assayed one by one with human or mouse TET2 3′ UTR reporters.
(B) Data for a subset of candidate TET2-targeting miRNAs identified through the screen, as well as the nontargeting miR-128b, are shown. Normalized luciferase activities are plotted with the red line and with 1 representing averaged luciferase activities of controls. miRNAs that belong to the same family are boxed. Error bars represent SD (n = 4).
See also Figures S1 and S2 and Tables S1 and S6.
Cell Reports 2013 5, DOI: ( /j.celrep )
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4. Figure 2
TET2-Targeting miRNAs Regulate TET2 Expression and Cellular 5hmC Levels
(A) BaF3 cells were transduced with control vector (Ctrl) or indicated miRNAs, including miR-128b, which does not target TET2. TET2 and HSP90 protein levels were determined by western blot. Representative data are shown out of two to three repeats. Quantification of TET2 protein level is indicated, after normalization to HSP90.
(B) Quantification of the effect of indicted miRNAs on TET2 protein or RNA levels in BaF3 cells is shown. Data were quantified from three repeats.
(C) Primary mouse bone marrow cells were transduced with indicated miRNAs. Representative western blots are shown out of two (miR-125a) and three (miR-29b) experiments.
(D) Genomic DNA from BaF3 cells transduced with Ctrl or indicated miRNAs or a TET2 overexpression vector (TET2OE) was analyzed for 5hmC levels using dot blot assay. Blot was stained with methylene blue to control for loading. Normalized 5hmC levels are indicated. Representative blot is shown out of two to three repeats. Note that the separated 5hmC images were from the same exposure of the same blot and the same for methylene blue.
(E) Quantification of 5hmC data (n = 3) is presented.
(F) TET2 cDNA or Ctrl was coexpressed with indicated miRNAs. Cellular 5hmC was assayed with dot blot. Representative data are shown out of three repeats.
(G) Quantification of data in (F) (n = 3) is shown. Indicated statistical significance (by an asterisk [∗]) was evaluated in comparison to Ctrl+Ctrl.
(H) BaF3 cells were transduced with a Ctrl vector, or sponges that inhibit the miR-29 family, miR-26 family, or miR-125 family. TET2 and HSP90 protein levels were determined by western blot. Representative data are shown from three repeats.
(I) Quantification of western data in (H) (n = 3) is presented.
(J) Cellular 5hmC levels from BaF3 cells transduced with Ctrl or indicated miRNA sponges were determined by dot blot analysis. Representative data are shown from three repeats.
(K) Quantification of 5hmC levels for (J), after normalizing with methylene blue (n = 3), is shown.
Error bars represent SD. ∗p < 0.05.
See also Figures S3 and S4 and Table S2.
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5. Figure 3
Some TET2-Targeting miRNAs Regulate Additional TET Family Members
(A) Human TET1 luciferase reporter was analyzed with indicated miRNAs, with the red line representing Ctrl levels (n = 4).
(B) Mouse TET3 luciferase reporter was analyzed with indicated miRNAs, with the red line representing Ctrl levels (n = 4).
(C) TET3 RNA levels in K562 cells transduced with indicated Ctrl or miRNAs were determined by qRT-PCR (n = 3).
(D) TET3 protein levels were analyzed in K562 cells transduced with Ctrl or indicated miRNAs by western blot. Representative blots are shown out of two repeated experiments.
All error bars represent SD. ∗p < 0.05.
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6. Figure 4 TET2-Targeting miRNAs Induce Abnormal Hematopoiesis In Vivo
(A and B) Wild-type bone marrow cells were transduced with Ctrl or indicated miRNAs, and transplanted into recipient mice, with GFP-labeling transduced cells. Peripheral blood was analyzed in recipients at the indicated time points, with each dot representing one recipient. (A) Myeloid bias index (frequency ratios of (%GFP+Mac1+/%GFP+Mac1−)/(%GFP−Mac1+/%GFP−Mac1−) was calculated to reflect the biased presence of myeloid cells in transduced population. (B) Normalized GFP ratios were also calculated (by taking the ratio of GFP+/GFP− cells and normalized to the average at 3.5 weeks) to reflect hematopoietic expansion. Numbers of mice per group are indicated in parentheses. The short horizontal bars represent median levels.
(C and D) Representative flow cytometry plots of recipients at ∼7 weeks posttransplantation (C) or 3.5 weeks posttransplantation (D) show myeloid marker Mac1 and GFP.
(E) Splenomegaly in miR-29b recipients is shown. A representative image is shown on the left, with pooled spleen weight data shown on the right (n = 4 for Ctrl and n = 5 for 29b).
(F) Bone marrow cells from Ctrl or miR-29b recipients were analyzed for granulocyte (Ly6G+Ly6C−) and monocytes (Ly6G−Ly6C+). Representative flow cytometry plots are shown, after gating on transduced myeloid cell populations (GFP+Mac1+ population). Note the increased monocytic frequency in miR-29b-transduced cells.
Error bars represent SDs. ∗p < 0.05.
See also Figure S5.
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7. Figure 5
TET2 Expression Rescues Malignant Phenotypes by Oncogenic miRNAs
Bone marrow cells were transduced with Ctrl or miR-125a or miR-29b in combination with a cDNA Ctrl vector or TET2. The same number of sorted transduced cells was transplanted into each recipient in each experiment.
(A and B) For miR-125a/TET2 rescue, GFP+ percentage and myeloid bias index at 3.5 weeks posttransplantation are shown, with each dot representing one recipient.
(C) Representative flow cytometry plots for (A) and (B) are presented.
(D) Bone marrow cells from miR-125a+Ctrl or miR-125a+TET2 recipients were analyzed for granulocyte (Ly6G+Ly6C−) and monocytes (Ly6G−Ly6C+). Representative flow cytometry plots are shown, after gating on transduced myeloid cell populations (GFP+Mac1+ population). Note that the monocytic bias in the miR-125a+Ctrl recipient was largely corrected by TET2 expression.
(E and F) For miR-29b/TET2 rescue, GFP+ percentage and myeloid bias index at 3.5 weeks posttransplantation are shown, with each dot representing one recipient.
(G) Representative flow cytometry plots for (E) and (F) are shown. The short horizontal bars represent median levels. The p values are indicated.
See also Figure S5.
Cell Reports 2013 5, DOI: ( /j.celrep )
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8. Figure 6
Preferential Overexpression of TET2-Targeting miRNAs in TET2-Wild-Type AML
The expression of TET2-targeting miRNAs was measured in a cohort of 67 cytogenetically normal AMLs, among which 16 samples were TET2 mutant. Expression data were normalized by subtracting cohort median and dividing by median average deviation to reflect outlier expression patterns. The data for miR-125b-5p (A), miR-29b-3p (B), miR-29c-3p (C), miR-101-3p (D), and miR-7-5p (E) are plotted, with higher bars indicating higher expression. The p values reflect the probability of observing more frequent higher expression for the indicated miRNA in TET2-wild-type AMLs than TET2 mutant AMLs. The specific cutoff applied for these p values is examining the top 33% samples with the highest expression of the indicated miRNA. See also Figure S6 and Tables S3, S4, and S5.
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9. Figure S1
3′ UTR Reporter Screen, Data Comparison, and Validation, Related to Figure 1
(A) Normalized luciferase activity data from the screen are shown for human TET2 3′UTR and mouse TET2 3′UTR. Each dot represents the mean of assays from one miRNA construct, with data from ∼460 constructs sorted from low to high. Red lines reflect the control levels. Error bars represent standard deviations. N = 4.
(B) Histograms of the screen results showing the distribution of the mean of normalized luciferase activity of each miRNA-UTR pair.
(C) A Venn diagram is shown to illustrate comparison of the screen results with TargetScan and mirSVR predictions. In this figure, to score as a screen hit, a threshold of > 25% luciferase activity repression was applied to miRNA-3′UTR pairs. miRNA cluster constructs were not considered in this calculation.
(D) Independent luciferase reporter assays were carried out to validate the screen data for the indicated miRNAs. Error bars represent standard deviation. Red lines represent control levels. N = 4. See also Table S6.
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10. Figure S2
TET2-Targeting miRNAs Regulate TET2 3′ UTR through Direct Interaction, Related to Figure 1
Mouse TET2 3′UTR was fragmented (frag) as illustrated, and mutagenesis was carried out to disrupt putative miRNA binding sites for miR-29 family (A), miR-26 family (B), miR-101 family (C), and miR-125 family (D). Schematics of fragmentation and mutations are shown on the left, whereas normalized luciferase activities assayed in the presence of the corresponding miRNAs are shown on the right, with red lines representing control levels. Error bars represent standard deviation. N = 4. ∗ indicates significant p values of < 0.05.
Cell Reports 2013 5, DOI: ( /j.celrep )
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11. Figure S3
TET2 Antibody Validation and Endogenous TET2 Regulation by TET2-Targeting miRNAs, Related to Figure 2
(A) The antibody detects full length TET2 protein in both human K562 and mouse BaF3 cells with Western blot analysis. Protein ladder (in kD) is shown on the left. HSP90 is shown as a control.
(B) Left: TET2 levels in BaF3 cells or BaF3 cells with murine TET2 cDNA overexpression were determined by Western blot analysis. Right: TET2 levels in BaF3 cells treated with control shRNA or shRNA against TET2 were determined by Western blot. Representative blots are shown out of two repeats. Note that the antibody detects the full length TET2 band.
(C) K562 human hematopoietic cells were transduced with control vector (ctrl) or indicated miRNAs. miR-128b, which does not target TET2, was used as an additional control. Tet2 and HSP90 protein levels were determined by Western blot. Representative data are shown out of two repeats. Quantification of Tet2 protein level is indicated, after normalization to HSP90.
(D) Genomic DNA from K562 cells transduced with ctrl or indicated miRNAs or a TET2 overexpression vector (TET2OE) was analyzed for 5hmC levels using dot blot assays. Blot was stained with methylene blue to control for loading. Normalized 5hmC levels are indicated. Data are representative of two experiments. See also Table S2.
Cell Reports 2013 5, DOI: ( /j.celrep )
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12. Figure S4
Expression Levels of TET2-Targeting miRNAs, Related to Figure 2
(A) U6 small RNA was assessed as a proper control for quantitative RT-PCR analysis. Ct cycles of U6 were determined from reverse-transcription products from 1.33 ng of total RNA which were derived from BaF3 and K562 cells, mouse bone marrow Mac1+ cells, and from a panel of human AML samples (names starting with NKAML). Note that similar Ct cycles were obtained across different sample types. N = 3 (triplicate PCR).
(B) The amplification efficiency of quantitative PCR primers was determined for the indicated miRNA or control gene by serial dilution of reverse-transcription products. Data represent mean of duplicate determination. A slope < −1 in the plots means a lower efficiency than the ideal 2-fold per cycle amplification.
(C–F) The overexpression of the indicated miRNAs were determined in BaF3 cells, K562 cells, or in FACS-sorted Mac1+GFP+ cells (indicated as BM) alongside human AML specimens. For experimental cell types, expression data from control (Ctrl) or miRNA transduced populations are shown. Data were corrected for primer amplification efficiency. N = 3.
(G) The expression level of miR-22 was determined in 293T cells transfected with a control or miR-22 expression construct. All error bars represent standard deviation. ∗p < See also Table S2.
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13. Figure S5
In Vivo Phenotypes of TET2-Targeting miRNAs, Related to Figures 4 and 5
Wild-type bone marrow cells were transduced with control (ctrl) or indicated miRNAs, and transplanted into recipient mice, with GFP labeling transduced cells.
(A and B) Peripheral blood was analyzed in recipients at the indicated time points, with each dot representing one recipient. (A) Myeloid bias index (frequency ratios of (GFP+Mac1+/GFP+Mac1-)/(GFP-Mac1+/GFP-Mac1-)) was calculated to reflect biased presence of myeloid cells in transduced population. (B) Normalized GFP ratios were also calculated (by taking the ratio of GFP+/GFP- cells and normalized to the average at 3.5 weeks) to reflect hematopoietic expansion. Numbers of mice per group are indicated in parentheses. The short horizontal bars represent median levels. ∗p < 0.05; ∗∗p < 0.01.
(C) Representative flow cytometry plots are shown for ctrl, miR-29c, miR-33, miR-520d at ∼7 weeks post-transplantation or ∼12 weeks post-transplantation. For miR-767 and miR-153-2, the outlier mice with GFP+% expansion are shown.
(D) A table summarizing the observations of TET2-targeting miRNAs in vivo with regard to myeloid differentiation bias and hematopoietic expansion. miR-144 was used as a negative control.
(E) The levels of 5hmC were determined in bone marrow cells isolated from in vivo models of miR-125a, miR-29b or control (Ctrl) transduced bone marrow transplant recipients. Specifically, Mac1+GFP+ bone marrow cells were FACS-sorted and analyzed. Methylene blue (the blue blot) was used as a loading control. Quantification of 5hmC levels is shown. Note that the separated images for miR-125a and control were from the same exposure of the same blot.
(F) Wild-type bone marrow cells were transduced with a control (ctrl) vector, miR-125a or miR29b with a GFP maker, in combination with a cDNA ctrl vector or TET2 (with a puromycin resistance marker). 500 transduced cells with both vectors were purified and plated for colony formation assay. Primary colony numbers (1°) were quantified and the cultures were replated to quantify secondary colony (2°). Data are shown in log scale. N = 3. Error bars represent standard deviations.
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14. Figure S6
Expression Analysis of TET2-Targeting miRNAs in Acute Myeloid Leukemia, Related to Figure 6
The expression of TET2-targeting miRNAs was measured in a cohort of 67 cytogenetically normal AMLs, among which 16 samples were TET2 mutant (same experiment as in Figure 6). Expression data were normalized by subtracting cohort median and dividing by median average deviation to reflect outlier expression patterns. The data for miR-30e-5p (A), miR-520a-5p (B), miR-202-5p (C) and miR-26-5p (D) were plotted, with higher bars indicating higher expression. ∗ P value was calculated using a cutoff of the top 25% samples with the highest expression of the indicated miRNA. (E) Expression of miR-125b, miR-29b, miR-29c, miR-101 and miR-7 were plotted in grouped bars for each of the 67 AMLs, with each bar group representing one sample. Note that overexpression pattern of these TET2-targeting miRNAs is largely non-overlapping. (F) The expression of miR-125b and miR-99a from the 67 AML samples were plotted, with each dot representing one sample. Note the correlation between the two miRNAs. See also Tables S3, S4, and S5.
Cell Reports 2013 5, DOI: ( /j.celrep )
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