Physical and functional link of the leukemia-associated factors AML1 and PML by Lan Anh Nguyen, Pier Paolo Pandolfi, Yukiko Aikawa, Yusuke Tagata, Misao Ohki, and Issay Kitabayashi Blood Volume 105(1):292-300 January 1, 2005 ©2005 by American Society of Hematology

PML I is a part of the AML1 complex. PML I is a part of the AML1 complex. (A) Purification of the AML1b complex. The complexes were purified from cell lysates prepared from L-G infectants carrying empty vector (mock), stably expressing FLAG-tagged AML1a (AML1a, a splicing variant lacking the C-terminal transcriptional regulatory domain), and FLAG-tagged full-length AML1b proteins (AML1b). The complexes were immunoprecipitated on anti-FLAG antibody-conjugated agarose, and the bound materials were eluted with the epitope peptide. Proteins were resolved by SDS-PAGE and visualized by silver staining. The proteins were identified by mass analysis. (B) The amino acid sequences of peptides derived from the 125-kDa protein specific to the fraction purified from the FLAG-AML1b expressing cells. The protein was identified as PML (accession number NM178087). (C) Detection of PML protein in the AML1 complex. The purified AML1 complexes were analyzed by immunoblot with anti-mouse PML antibody (rabbit polyclonal, Wang et al, 1998). (D) Interaction of endogenous AML1 and PML proteins. Cell lysates were prepared from 5 × 107 K562 cells and then were immunoprecipitated with anti-human PML monoclonal antibody 1B9 (MBL) or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-mouse AML1 rabbit polyclonal antibody. Lan Anh Nguyen et al. Blood 2005;105:292-300 ©2005 by American Society of Hematology

PML I isoform specifically interacts with AML1b. PML I isoform specifically interacts with AML1b. (A) Schematic representation of the PML protein and genomic structure of various PML transcripts. The PML protein contains the RBCC motif that consists of the RING finger domain (RING), the 2 B boxes (B1 and B2), and the helical coiled-coil region. The proline rich region (P) and serine-proline-rich region (S-P) have been thought to contain the putative phosphorylation sites.44 SUMOylation sites at lysines 65, 160, and 49056 are indicated. The PML gene consists of 9 exons (shaded boxes) with exon 7 divided into exons 7a and 7b, and the introns being retained in several transcripts as a consequence of alternative splicing (black boxes).54 The nomenclature for PML isoforms proposed by Jensen55 was used in this study. The alternative usage of 3′ exons generates multiple transcripts I-VI. In the case of PML III and PML VI, the encoding regions in which frame-shift occurred as a result of the retained intron are indicated by the gray color. The total number of amino acids for each isoform is given on the right side. (B) The interaction with AML1b is specific to the PML I isoform. BOSC23 cells were cotransfected with HA-tagged AML1b and together either with control vector (mock) or FLAG-tagged PML isoforms (I-VI). The expression of AML1 in the lysates of transfectants was detected by immunoblotting using anti-HA (3F10) antibody (top). The lysates of transfectants were immunoprecipitated with anti-FLAG (M2) antibody. The immunoprecipitates were analyzed by immunoblotting using anti-FLAG (M2) (middle) and anti-HA (3F10) antibodies (bottom). (C) Identification of regions of PML I required for interaction with AML1b. BOSC23 cells were cotransfected with FLAG-AML1b and either with control vector (mock) or constructs of PML I as indicated. The expression of PML I in the lysates of transfectants were detected by immunoblotting using anti-HA antibody (3F10) (top). The lysate of transfectants were immunoprecipitated with anti-FLAG (M2) antibody and were analyzed by immunoblotting using anti-FLAG (M2) (middle) and anti-HA (3F10) antibodies (bottom). Lan Anh Nguyen et al. Blood 2005;105:292-300 ©2005 by American Society of Hematology

PML I interacting domain is located at the C-terminal of AML1. PML I interacting domain is located at the C-terminal of AML1. (A) Schematic diagram of the structure of AML1 deletion mutants. The Runt, activation, and inhibition domains are indicated as Runt, AD, and ID, respectively. Nuclear import signal (NLS) and nuclear matrix targeting signal (NMTS) are shown. (B) Identification of regions of AML1b required for interaction with PML I. BOSC23 cells were cotransfected with PML I and control vector (mock) or AML1 constructions as indicated. The expression of PML I in the lysates of transfectants was detected by immunoblotting using anti-HA (3F10) antibody (top). The lysates of transfectants were immunoprecipitated with anti-FLAG (M2) antibody. The immunoprecipitates were analyzed by immunoblotting using anti-FLAG (M2) (middle) and anti-HA (3F10) antibodies (bottom). (C) The Runt domain is dispensable for the interaction of AML 1 with PML I. BOSC23 cells were cotransfected with PML I and full-length AML1b or deletion mutants of AML1 partially lacking Runt domain as indicated. The expression of PML I (top) and AML1 (middle) proteins in the cell lysates was detected with anti-HA antibody and anti-FLAG antibody, respectively. The immunoprecipitates with anti-FLAG antibody were analyzed using anti-HA antibody (lower). Lan Anh Nguyen et al. Blood 2005;105:292-300 ©2005 by American Society of Hematology

PML could interact with 2 other members of RUNX family AML2 and AML3. PML could interact with 2 other members of RUNX family AML2 and AML3. (A) BOSC23 cells were cotransfected with PML I and AMLs as indicated. The expression of AML proteins (upper) and PML I (middle) in the lysate of transfectants was analyzed by immunoblotting and detected with anti-AML antibody28 mixed with anti-AML3 antibody (Upstate Biotechnology, Charlottesville, VA) and anti-FLAG (M2) antibody, respectively. The lysate of transfectants were immunoprecipitated with anti-FLAG (M2) antibody, analyzed by immunoblotting, and probed with a mixture of anti-AML1 antibodies (lower). (B) Comparison of amino acid sequences for the C-terminal of AML1, AML2, and AML3. Identical amino acids are indicated in red. The boxed region corresponds to the PML I interacting domain of AML1. Lan Anh Nguyen et al. Blood 2005;105:292-300 ©2005 by American Society of Hematology

PML I recruits AML1b into nuclear bodies. PML I recruits AML1b into nuclear bodies. (A) SaOS2 cells were transfected with LNCX-FLAG-AML1a, LNCX-FLAG-AML1b, or LNCX-HA-PML I. (B,C) AML1b, not AML1a, is recruited into NBs. SaOS2 cells were cotransfected with LNCX-FLAG-AML1a (B) or LNCX-FLAG-AML1b (C) together with LNCX-HA-PMLI. (D,E) Localization of AML1b and PML I mutants. SaOS2 cells were cotransfected with LNCX-FLAG-AML1b together with either LNCX-HA-PMLI d851 (D) or LNCX-HA-PMLI d592 (E). (F) AML1b was not colocalized with PML IV into NBs. SaOS2 cells were cotransfected with LNCX-FLAG-AML1b and LNCX-HA-PML IV. (G) AML1b is presented in the aggregates formed by PML I 3R. SaOS2 cells were cotransfected with LNCX-FLAG-AML1b and LNCX-HA-PML I 3R. In all experiments, AML1 was stained with anti-human AML rabbit polyclonal antibody, as shown by the green color, and PML was stained with anti-HA antibody (3F10), as shown by the orange color. Merging of the 2 colors results in a yellow signal, indicating the colocalization of 2 proteins. Nuclear staining with DAPI is shown by the blue color. Lan Anh Nguyen et al. Blood 2005;105:292-300 ©2005 by American Society of Hematology

PML I stimulates cooperation of AML1 and p300. PML I stimulates cooperation of AML1 and p300. (A) PML I specifically enhanced AML1-induced transcription. The effect of PML proteins on AML1-dependent transcription was investigated using the reporter gene under the control of mCCP1 promoter. PmL-/- MEF cells were spread in 24-well plates and transfected with 50 ng of reporter gene, 200 ng of pLNCX-AML1b, 30 ng of empty vector or PML isoforms as indicated, and 2 ng of pRL-CMV. (B) PML I stimulates the cooperated transcription activity of AML 1 and p300. PmL-/- MEF cells were spread in 24-well plates and transfected with 50 ng of CCP1-luc, 200 ng of pLNCX-AML1b (lanes 4-9), 250 ng of pCMV-p300 (lanes 6-9), and either 30 ng (lanes 2, 5, 8) or 100 ng (lanes 3, 6, 9) of pLNCX-PML I and 2 ng of pRL-CMV. Luciferase activity was assayed as described in the Experimental Procedure. (C) PML I recruits AML1b and p300 into nuclear bodies. SaOS2 cells were cotransfected with pCMV-p300 (i-iii) together with LNCX-FLAG-AML1b (ii, iii) and LNCX-HA-PMLI (i, iii). p300 stained with anti-p300 rabbit polyclonal antibody (N15) is shown by the green color, and PML (i) stained with anti-HA antibody (3F10) and AML1 (ii, iii) stained with anti-FLAG antibody (M2) are shown by orange color. Merging of the 2 colors results in a yellow signal, indicating the colocalization of 2 proteins. Nuclear staining with DAPI is shown by the blue color. Lan Anh Nguyen et al. Blood 2005;105:292-300 ©2005 by American Society of Hematology

PML I inhibits the proliferation and stimulates the differentiation of murine myeloid progenitor cells in response to G-CSF. PML I inhibits the proliferation and stimulates the differentiation of murine myeloid progenitor cells in response to G-CSF. (A) Expression of PML protein in L-G infectants. Cell lysates were prepared in SDS-PAGE sample buffer and then analyzed by Western blot using anti-HA antibody. Subfraction of PML protein modified by SUMO-1 is indicated. (B) The L-G infectants were cultured in the presence of 5 ng/mL G-CSF. The relative number of viable cells is shown. (C,D) Morphology of L-G infectants exposed to G-CSF for 6 days (C) and 8 days (D) were tested by staining with May-Gruenwald and Giemsa solutions. (E,F) Percentage of L-G infectants differentiated by G-CSF at day 6 (E) and day 8 (F). Immature includes myeloblasts and promyelocytes; intermediate, myelocytes and metamyelocytes; and mature, band cells and segmented neutrophil cells. Lan Anh Nguyen et al. Blood 2005;105:292-300 ©2005 by American Society of Hematology