An evolutionarily conserved PTEN-C/EBPα-CTNNA1 axis controls myeloid development and transformation by Chun-Tang Fu, Kang-Yong Zhu, Jian-Qing Mi, Yuan-Fang Liu, Susan T. Murray, Yan-Fang Fu, Chun-Guang Ren, Zhi-Wei Dong, Yi-Jie Liu, Mei Dong, Yi Jin, Yi Chen, Min Deng, Wu Zhang, Bin Chen, Peter Breslin, Sai-Juan Chen, Zhu Chen, Michael W. Becker, Jiang Zhu, Ji-Wang Zhang, and Ting Xi Liu Blood Volume 115(23):4715-4724 June 10, 2010 ©2010 by American Society of Hematology

Elevated H3K27 trimethylation levels in the proximal promoter of the retained CTNNA1 allele. Elevated H3K27 trimethylation levels in the proximal promoter of the retained CTNNA1 allele. (A) A diagram of the human CTNNA1 genomic locus and the regions analyzed by ChIP. TSS indicates transcriptional start site. (B) ChIP analyses of histone modifications with the indicated antibodies in HL-60 and NB4 leukemia cell lines. (C-D) Semiquantitative RT-PCR and ChIP analyses of CTNNA1 expression and histone modifications in HL-60 cells treated with TSA for the indicated time. (E) Semiquantitative RT-PCR and ChIP analyses of CTNNA1 expression and H3K27me3 in HL-60 cells treated with DAC for the indicated times. (F) ChIP analyses of CTNNA1 promoter for H3K27me3 enrichment in FACS-sorted LICs (CD34+CD38−CD123+lineage−) from patient samples with or without del(5q). Enrichment of H3K27me3 was calculated by: [ChIP-P1E1(LICs)/INPUT-P1E1(LICs)]/[ChIP-P1E1(HL-60)/INPUT-P1E1(HL-60)]. ● represents case V with del(5q), which expresses a normal amount of CTNNA1.7 Chun-Tang Fu et al. Blood 2010;115:4715-4724 ©2010 by American Society of Hematology

PRC2 suppresses CTNNA1 expression through H3K27 trimethylation. PRC2 suppresses CTNNA1 expression through H3K27 trimethylation. (A) HL-60 cells were treated with 0.6μM TSA for 0 and 12 hours. ChIP analyses were performed with the indicated antibodies. (B) Western blot analyses of EZH2, SUZ12, and α-catenin proteins in HL-60 cells treated with 0.4μM TSA for the indicated times. (C-D) HL-60 cells were infected with retroviral constructs generating SUZ12- and EZH2-specific shRNA. Knockdown of SUZ12 (C) and EZH2 (D), as well as levels of CTNNA1 expression, were determined by both real-time quantitative RT-PCR and Western blot analysis. (E) HL-60 cells were infected with retroviral SUZ12 shRNA, and ChIP analyses were performed with the indicated antibodies at the UP1, P1E1, and I1E2 sites, or at the promoter of the GAPDH gene. Knockdown of SUZ12 resulted in a massive reduction of the repressive H3K27me3, with concomitant increase of the activating H3K4me3 marked specifically at the P1E1 site. Chun-Tang Fu et al. Blood 2010;115:4715-4724 ©2010 by American Society of Hematology

Involvement of C/EBPα isoforms in PRC2-mediated H3K27me3 and CTNNA1 expression from the retained allele. Involvement of C/EBPα isoforms in PRC2-mediated H3K27me3 and CTNNA1 expression from the retained allele. (A) ChIP analyses were performed with the indicated antibodies in the HL-60, NB4, and KG-1 leukemia cell lines. Note that the C/EBPα (N) antibody recognizes only the p42 wild-type full-length C/EBPα protein, whereas C/EBPα (C1) and C/EBPα (C2) recognize both p42 and p30 C/EBPα. (B) Western blot analysis of p42, p30 C/EBPα, and α-catenin proteins in HL-60, NB4, and KG-1 leukemia cells. Note that the ratio of p42/p30 is significantly reduced only in HL-60 cells. (C) HL-60, NB4, and KG-1 cells were treated with 10nM rapamycin for 96 hours followed by Western blot analyses with the indicated antibodies. (D) ChIP analyses were performed with the indicated antibodies in HL-60, NB4, and KG-1 cells treated with 10nM rapamycin for 96 hours. (E-H) Effects of p42 and p30 C/EBPα overexpression on CTNNA1 levels. Retrovirally mediated overexpression of p42 and p30 C/EBPα (E) in HL-60 cells as determined by Western blot analysis (F). The levels of CTNNA1 transcripts and α-catenin protein were determined by real-time quantitative RT-PCR (G) and Western blot analysis (H), respectively. Chun-Tang Fu et al. Blood 2010;115:4715-4724 ©2010 by American Society of Hematology

PTEN acts upstream to determine the p42/p30 C/EBPα ratio and CTNNA1 expression. PTEN acts upstream to determine the p42/p30 C/EBPα ratio and CTNNA1 expression. (A) Western blot analysis of PTEN protein levels in HL-60, NB4, and KG-1 cells. (B) Effects of lentivirally mediated overexpression of PTEN on α-catenin protein levels in HL-60 and NB4 cells as determined by Western blot analysis. (C) Effects of lentivirus-mediated overexpression of PTEN on H3K27me3 modification at the P1E1 site. (D-E) Western blot and quantitative real-time RT-PCR analyses of murine C/ebpα protein and Ctnna1 transcripts in MNCs and FACS-sorted Lin−c-Kit+ myeloid progenitors from Pten−/− knockout mice,15 respectively. The Arabic numerals at the bottom of panel D denote the p42/p30 ratio. (F) FACS-sorted c-Kit+ myeloid progenitors from Pten−/− knockout mice were infected with MSCV-IRES-GFP or MSCV-p42 C/ebpα-IRES-GFP. FACS-GFP positive cells were serially plated on methylcellulose and colonies were counted (n = 3). (G) FACS-sorted c-Kit+ myeloid progenitors from wild-type mice were infected with MSCV-IRES-GFP or MSCV-p30 C/ebpα-IRES-GFP. FACS-sorted GFP+ cells were serially plated on methylcellulose and colonies were counted (n = 3). Chun-Tang Fu et al. Blood 2010;115:4715-4724 ©2010 by American Society of Hematology

An evolutionarily conserved function of Ptenb regulates C/ebpα and α-catenin levels and prevents myelodysplasia in zebrafish embryos. An evolutionarily conserved function of Ptenb regulates C/ebpα and α-catenin levels and prevents myelodysplasia in zebrafish embryos. (A) Western blot analysis of C/ebpα and α-catenin proteins in embryos injected with the indicated morpholino oligonucleotides at 22 hpf. Note that the polyclonal antibody used detected only zebrafish wild-type p38-kDa C/ebpα protein. (B-D) Morphology of embryos and appearance of EGFP+ myeloid progenitors in Tg(zpu.1:EGFP) transgenic embryos injected with the indicated morpholinos at 22 hpf. hb indicates hindbrain; and s, somite. (E-G) EGFP+ myeloid progenitors in Tg(zpu.1:EGFP) transgenic embryos injected with the indicated morpholinos at 28 hpf. hb indicates hindbrain; and s, somite. All embryos are shown in lateral view with the head to the left. (H-I) Whole-mount mRNA in situ hybridization analysis of l-plastin expression in control and ptenb-deficient embryos at 22 hpf. Chun-Tang Fu et al. Blood 2010;115:4715-4724 ©2010 by American Society of Hematology

Frame-shift mutations are exclusively detected in primary LICs with low CTNNA1 transcripts. Frame-shift mutations are exclusively detected in primary LICs with low CTNNA1 transcripts. (A) Quantitative real-time RT-PCR analysis of CTNNA1 transcripts in FACS-sorted LICs (CD34+CD38−CD123+Lin−) from MDS or AML patients. The down-regulation of CTNNA1 transcripts was arbitrarily defined as 60% reduction (dashed line) compared with normal HSCs and the control NB4 cell line. (B-C) Summary of frame-shift mutations detected in the PTEN (B) and CEBPA (C) genes. Note that these mutations were only identified in 6 of 8 patient's LICs expressing low levels of CTNNA1 transcripts. (D) Western blot analysis with protein lysates extracted from the MNCs of patients expressing low CTNNA1 and carrying frame-shift mutations in the PTEN gene (P-1, P-24, and P-26). Note the reduction of the p42/p30 ratio in patients P-1, P-24, and P-26 who had frame-shift mutations in the PTEN gene. #Wild-type PTEN protein that is probably expressed from the normal blood cells or blasts within the heterogeneous MNC mixture. (E) Western blot analysis with protein lysates extracted from MNCs of patient expressing low CTNNA1 and carrying N-terminal frame-shift mutations in the CEBPA gene (P-27). (F) Western blot analysis with protein lysates extracted from MNCs of patients expressing appropriate levels of CTNNA1 and carrying no mutations in either PTEN or CEBPA (P-25 and P-6). (G) The MNCs derived from patient P-1 were treated with rapamycin at the indicated concentrations for 5 days. Western blot analysis showed a dose-dependent increase in the p40/p30 C/EBPα ratio (left) and a coincident up-regulation of CTNNA1 transcripts as determined by quantitative real-time RT-PCR analysis (right). The Arabic numbers at the bottom of panels D through G denote the p42/p30 ratio. (H) ChIP analyses with the indicated antibodies in primary MNCs of patients expressing low (P-1, P-24, and P-35) and appropriate CTNNA1 levels (P-7, P-17, and P-22). Chun-Tang Fu et al. Blood 2010;115:4715-4724 ©2010 by American Society of Hematology