The growth suppressor promyelocytic leukemia protein (PML) is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL). PML plays a key role in multiple pathways of apoptosis and regulates cell cycle progression. The present study demonstrates that PML represses transcription by functionally and physically interacting with histone deacetylase (HDAC). Transcriptional repression mediated by PML can be inhibited by trichostatin A, a specific inhibitor of HDAC. PML coimmunoprecipitates a significant level of HDAC activity in several cell lines. PML is associated with HDAC in vivo and directly interacts with HDAC in vitro. The fusion protein PML-RAR␣ encoded by the t(15;17) breakpoint interacts with HDAC poorly. PML interacts with all three isoforms of HDAC through specific domains, and its expression deacetylates histone H3 in vivo. Together, the results of our study show that PML modulates histone deacetylation and that loss of this function in APL alters chromatin remodeling and gene expression. This event may contribute to the development of leukemia.The nonrandom chromosomal translocation t(15;17), a cytogenetic hallmark of acute promyelocytic leukemia (APL), fuses the retinoic acid receptor ␣ gene (RAR␣) and the promyelocytic leukemia gene (PML) (8,17,34). The fusion gene PML-RAR␣ encodes a fusion protein that has been shown to interfere with leukemia cell differentiation (25,26) and to cause leukemia in animal models (11,27,32,33). Disruption of PML's growth suppressor function in APL is also believed to play a role in leukemogenesis (51). PML is a nuclear-matrixassociated protein localized in the nucleus in a distinct nuclear speckled pattern designated the PML nuclear body (NB), which is disrupted in the leukemic blasts of APL (14,15,20,75). A significant number (Ͼ90%) of APL patients can be induced to complete clinical remission by high-dose all-transretinoic acid (ATRA) or arsenic trioxide (As 2 O 3 ) therapy (16,59,60,72,74). Retinoic acid (RA) treatment induces differentiation of the leukemic blasts, rapid degradation of the fusion protein PML-RAR␣, and restoration of a normal PML NB (20,75). Recent studies demonstrated that PML-RAR␣ recruits histone deacetylase (HDAC) by directly interacting with the N-CoR-Sin3 complex through the RAR␣ portion of the fusion protein, turning the fusion protein into a strong transcription repressor for RA-responsive genes. Treating APL cells with high-dose ATRA reverses the binding of PML-RAR␣ to the N-CoR-Sin3 corepressor complex and reactivates RA-responsive genes (24,32,45). PML belongs to a family of nuclear proteins consisting of the RING finger motif and two other Cys-His domains designated the B-box motif. The region following is the ␣-helical domain, which is responsible for dimerization (57). PML is the major component of this novel NB, and many proteins associated with PML have been identified. For example, the ubiquitin-like protein modifier SUMO-1 (PIC-1 or sentrin) (7,35,36,53,62), interferon-induced protein ISG20 (23), the im...

The PML tumor suppressor gene is consistently disrupted by t(15;17) in patients with acute promyelocytic leukemia. Promyelocytic leukemia protein (PML) is a multifunctional protein that plays essential roles in cell growth regulation, apoptosis, transcriptional regulation, and genome stability. Our study here shows that PML colocalizes and associates in vivo with the DNA damage response protein TopBP1 in response to ionizing radiation (

The promyelocytic leukemia protein (PML) is a nuclear phosphoprotein with growth-and transformationsuppressing ability. Having previously shown it to be a transcriptional repressor of the epidermal growth factor receptor (EGFR) gene promoter, we have now shown that PML's repression of EGFR transcription is caused by inhibition of EGFR's Sp1-dependent activity. On functional analysis, the repressive effect of PML was mapped to a 150-bp element (the sequences between ؊150 and ؊16, relative to the ATG initiation site) of the promoter. Transient transfection assays with Sp1-negative Drosophila melanogaster SL2 cells showed that the transcription of this region was regulated by Sp1 and that the Sp1-dependent activity of the promoter was suppressed by PML in a dose-dependent manner. Coimmunoprecipitation and mammalian two-hybrid assays demonstrated that PML and Sp1 were associated in vivo. In vitro binding by means of the glutathione S-transferase (GST) pull-down assay, using the full-length and truncated GST-Sp1 proteins and in vitrotranslated PML, showed that PML and Sp1 directly interacted and that the C-terminal (DNA-binding) region of Sp1 and the coiled-coil (dimerization) domain of PML were essential for this interaction. Analysis of the effects of PML on Sp1 DNA binding by electrophoretic mobility shift assay (EMSA) showed that PML could specifically disrupt the binding of Sp1 to DNA. Furthermore, cotransfection of PML specifically repressed Sp1, but not the E2F1-mediated activity of the dihydrofolate reductase promoter. Together, these data suggest that the association of PML and Sp1 represents a novel mechanism for negative regulation of EGFR and other Sp1 target promoters.The promyelocytic leukemia gene, PML, was first identified at the breakpoint of the t(15;17) translocation in acute promyelocytic leukemia (APL) (10,14,19,35,37). PML encodes a nuclear phosphoprotein that functions as a transcriptional regulator (9, 50, 58) and belongs to the RING family of proteins, which share a cysteine-rich motif at the N terminus. This motif is divided into a RING finger (C 3 C 4 zinc binding) motif and two B-box (B1 and B2) motifs (18). This region is followed by a predicted ␣-helical coiled-coil (dimerization) domain, which allows PML to homodimerize and form heterodimer complexes with the APL fusion protein PMLRAR␣ and the promyelocytic leukemia zinc finger (PLZF) protein (37,40). PML localizes to distinct domains in the nucleus called PML nuclear bodies, or PML oncogenic domains (PODs) (16,60). In addition to PML, there are several other POD-associated factors, including SP100, the ubiquitin-like protein PIC1, and the interferon-stimulated 20-kDa gene product called ISG20 (3,6,20). PODs are frequently targeted and/or reorganized by viral proteins, such as the herpes simplex virus type 1 (HSV-1) gene product Vmw110 (17), the adenoviral proteins E1A and E4-ORF3 (8), the Epstein-Barr virus-encoded nuclear antigen EBNA-5 (53), and the human cytomegalovirus major immediate-early proteins IE1 and IE2 (1).PMLRAR␣, which r...

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The growth and transformation suppressor function of promyelocytic leukemia (PML) protein are disrupted in acute promyelocytic leukemia (APL) as a result of its fusion to the RARa gene by t(15;17) translocation. There is signi®cant sequence homology between the dimerization domain of PML and the Fos family of proteins, which imply that PML may be involved in AP-1 activity. Here we show that PML can cooperate with Fos to stimulate its AP-1-mediated transcriptional activity. Cotransfection of PML with GAL4/Fos strongly induced Fos-mediated activation of GAL4-responsive reporters, indicating a functional interaction between Fos and PML in vivo. Deletion analysis of Fos and PML demonstrated that the intact C-terminal domain of Fos (containing the dimerization domain), and the RING-®nger, B1 box and nuclear localization domains of PML are involved in the cooperative activity of Fos and PML. Immunoprecipitation and electrophoretic mobility shift assay showed that PML is associated with the AP-1 complex. PMLRARa was also found to enhance the transcriptional activity of GAL4/Fos. The addition of retinoic acid abrogated the PMLRARa, but not PML-induced stimulation of GAL4/Fos activity in a dose-dependent manner. This study demonstrated that PML is involved in the AP-1 complex and can modulate Fos-mediated transcriptional activity, which may contribute to its growth suppressor function.

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Since the translocation breakpoint t(15;17) (q22;q21) in acute promyelocytic leukemia (APL) occurs within the retinoic acid receptor- alpha (RARA) gene, the expression of many genes normally regulated by RARA may be affected by this translocation. To identify genes that may be aberrantly expressed in APL, a subtraction cDNA library of an APL patient with t(15;17) was constructed. A cDNA, pRD1, specifically expressed in APL was identified. DNA sequence analysis of pRD1 showed that this gene is similar to the DNA sequence of annexin VIII, a gene which encodes a vascular anticoagulant. The annexin VIII gene was assigned to chromosome 10, which indicates that specific expression of this gene in APL is not directly involved in the t(15;17) breakpoint region. We have analyzed the expression of annexin VIII gene in nine t(15;17)-positive APL patients and one APL patient with a chromosome 17q-abnormality. We found that all APL samples expressed high levels of the annexin VIII gene. Expression of the annexin VIII gene in all other leukemias, including acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and acute lymphoblastic leukemia, was undetectable, except in one patient with acute myelogenous leukemia in which a very low level of expression was detected. Annexin VIII is highly expressed in the APL cell line, NB4. Its expression was significantly reduced after 8 hours of all-trans retinoic acid (ATRA) treatment, whereas the expression of RARA increased several-fold within 4 hours postinduction. Thus, increased expression of RARA preceded the downregulation of annexin VIII after ATRA induction, suggesting an inverse relationship between RARA and annexin VIII expression. Since increased expression of the fusion transcript was seen after ATRA induction and an APL without a t(15;17) translocation expressed high levels of annexin VIII, it appears that increased expression of annexin VIII in APL is not related to the fusion transcript. Therefore, dysregulation of the RARA gene may be related to the overexpression of annexin VIII in APL.

Annexin VIII is a calcium-dependent phospholipid-binding protein previously identified as a blood anticoagulant based on in vitro studies. However, the physiologic function of annexin VIII remains unknown. In acute promyelocytic leukemia (APL) the annexin VIII gene is highly expressed, but its expression is undetectable in the blasts of other acute leukemias. In the present investigation, we showed using the APL-derived NB4 cell line that expression of the annexin VIII gene is regulated at the transcription level during induced differentiation by all-trans retinoic acid (ATRA). The half-life of the annexin VIII mRNA is about 5 to 6 hours, as determined by using actinomycin D as a transcription inhibitor. Analysis of the expression of annexin VIII protein in NB4 cells and in APL samples showed a consistent expression of a predominant 36-kD protein and a weak 72-kD protein. After ATRA- induced differentiation of NB4 cells, the annexin VIII protein level reduced gradually, but a detectable level persisted even after 4 days of induction. Because annexin VIII mRNA becomes undetectable after 48 hours of ATRA induction, this result indicates that annexin VIII is a relatively stable protein. A multiple tissue Northern blot analysis was performed, and we found that annexin VIII is normally expressed in the placenta and the lung. Cellular localization of the annexin VIII protein was determined by immunofluorescence staining and subcellular fractionation. These results indicated that annexin VIII is predominantly localized to the plasma membrane. The annexin VIII is neither an extracellular protein nor associated with the cell surface suggesting that it does not play a role in blood coagulation in vivo. The plasma membrane localization and its property as a phospholipase inhibitor suggests that annexin VIII may have a role in the signal transduction pathway in the APL cells.