Anne Dejean

The PML-RARα fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR

We have previously shown that the t(15;17) translocation specifically associated with acute promyelocytic leukemia (APL) fuses the retinoic acid receptor alpha (RAR alpha) locus to an as yet unknown gene, initially called myl and now renamed PML. We report here that this gene product contains a novel zinc finger motif common to several DNA-binding proteins. The PML-RAR alpha mRNA encodes a predicted 106 kd chimeric protein containing most of the PML sequences fused to a large part of RAR alpha, including its DNA- and hormone-binding domains. In transient expression assays, the hybrid protein exhibits altered transactivating properties if compared with the wild-type RAR alpha progenitor. Identical PML-RAR alpha fusion points are found in several patients. These observations suggest that in APL, the t(15;17) translocation generates an RAR mutant that could contribute to leukemogenesis through interference with promyelocytic differentiation.

The Nucleoporin RanBP2 Has SUMO1 E3 Ligase Activity

Posttranslational modification with SUMO1 regulates protein/protein interactions, localization, and stability. SUMOylation requires the E1 enzyme Aos1/Uba2 and the E2 enzyme Ubc9. A family of E3-like factors, PIAS proteins, was discovered recently. Here we show that the nucleoporin RanBP2/Nup358 also has SUMO1 E3-like activity. RanBP2 directly interacts with the E2 enzyme Ubc9 and strongly enhances SUMO1-transfer from Ubc9 to the SUMO1 target Sp100. The E3-like activity is contained within a 33 kDa domain of RanBP2 that lacks RING finger motifs and does not resemble PIAS family proteins. Our findings place SUMOylation at the cytoplasmic filaments of the NPC and suggest that, at least for some substrates, modification and nuclear import are linked events.

Retinoic acid regulates aberrant nuclear localization of PML-RARα in acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) translocation that fuses the retinoic acid receptor alpha (RAR alpha) to a novel gene product, PML. The involvement of RAR alpha is particularly intriguing in view of the efficient therapeutic effect of retinoic acid (RA) in this disease. In this report, we show that PML is specifically localized within a discrete subnuclear compartment corresponding to nuclear bodies recognized by patient autoimmune sera. In APL cells, the PML-RAR alpha hybrid displays an abnormal localization and directs RXR and other nuclear antigens into aberrant structures that are tightly bound to chromatin. This suggests that the hybrid could exert a dominant negative effect by diverting a subset of proteins from their natural sites of action. Interestingly, treatment of APL cells with RA induces a complete relocalization of each of these proteins. We propose that the beneficial role of RA in promoting myeloid differentiation in APL might be related to its ability to restore a normal subnuclear organization.

Conjugation with the ubiquitin‐related modifier SUMO‐1 regulates the partitioning of PML within the nucleus

The PML protein, identified first as part of the oncogenic PML–RARα chimera in acute promyelocytic leukemia (APL), concentrates within discrete subnuclear structures, corresponding to some types of nuclear bodies. These structures are disrupted in APL cells, and retinoic acid (RA) can trigger their reorganization, correlating with its therapeutic effect in this type of leukemia. Recently, arsenic trioxide (As2O3) was identified as a potent antileukemic agent which, similarly to RA, induces complete remissions in APL patients. Here we show that, in APL cells, As2O3 triggers rapid degradation of PML–RARα and provokes the restoration of intact nuclear bodies. In non‐APL cells, the ubiquitin‐like protein SUMO‐1 is covalently attached to a subset of wild‐type PML in a reversible and phosphorylation‐dependent manner. The unmodified form of PML is found in the soluble nucleoplasmic fraction, whereas the SUMO‐1‐polymodified forms of PML are compartmentalized exclusively in the PML nuclear bodies. As2O3 administration strikingly increases the pool of SUMO‐1–PML conjugates that, subsequently, accumulate in enlarged nuclear bodies. In contrast to PML–RARα, the overall amount of PML seems to remain unaltered up to 36 h following As2O3 treatment. These findings indicate that the conjugation of PML with SUMO‐1 modulates its intracellular localization and suggest that post‐translational modification by SUMO‐1 may be more generally involved than previously suspected in the targeting of proteins to distinct subcellular structures. They provide additional evidence that the role of ‘ubiquitin‐like’ post‐translational modification is not limited to a degradation signal.

Nuclear and unclear functions of SUMO

Post-translational modification by the ubiquitin-like SUMO protein is emerging as a defining feature of eukaryotic cells. Sumoylation has crucial roles in the regulatory challenges that face nucleate cells, including the control of nucleocytoplasmic signalling and transport and the faithful replication of a large and complex genome, as well as the regulation of gene expression.

miR-221 overexpression contributes to liver tumorigenesis

MicroRNA (miRNAs) are negative regulators of gene expression and can function as tumor suppressors or oncogenes. Expression patterns of miRNAs and their role in the pathogenesis of hepatocellular carcinoma (HCC) are still poorly understood. We profiled miRNA expression in tissue samples (104 HCC, 90 adjacent cirrhotic livers, 21 normal livers) as well as in 35 HCC cell lines. A set of 12 miRNAs (including miR-21, miR-221/222, miR-34a, miR-519a, miR-93, miR-96, and let-7c) was linked to disease progression from normal liver through cirrhosis to full-blown HCC. miR-221/222, the most up-regulated miRNAs in tumor samples, are shown to target the CDK inhibitor p27 and to enhance cell growth in vitro. Conversely, these activities can be efficiently inhibited by an antagomiR specific for miR-221. In addition, we show, using a mouse model of liver cancer, that miR-221 overexpression stimulates growth of tumorigenic murine hepatic progenitor cells. Finally, we identified DNA damage-inducible transcript 4 (DDIT4), a modulator of mTOR pathway, as a bona fide target of miR-221. Taken together, these data reveal an important contribution for miR-221 in hepatocarcinogenesis and suggest a role for DDIT4 dysregulation in this process. Thus, the use of synthetic inhibitors of miR-221 may prove to be a promising approach to liver cancer treatment.

The SUMO E3 ligase RanBP2 promotes modification of the HDAC4 deacetylase.

Transcriptional repression mediated through histone deacetylation is a critical component of eukaryotic gene regulation. Here we demonstrate that the class II histone deacetylase HDAC4 is covalently modified by the ubiquitin‐related SUMO‐1 modifier. A sumoylation‐deficient point mutant (HDAC4‐K559R) shows a slightly impaired ability to repress transcription as well as reduced histone deacetylase activity. The ability of HDAC4 to self‐aggregate is a prerequisite for proper sumoylation in vivo. Calcium/calmodulin‐dependent protein kinase (CaMK) signalling, which induces nuclear export, abrogates SUMO‐1 modification of HDAC4. Moreover, the modification depends on the presence of an intact nuclear localization signal and is catalysed by the nuclear pore complex (NPC) RanBP2 protein, a factor newly identified as a SUMO E3 ligase. These findings suggest that sumoylation of HDAC4 takes place at the NPC and is coupled to its nuclear import. Finally, modification experiments indicate that the MEF2‐interacting transcription repressor (MITR) as well as HDAC1 and ‐6 are similarly SUMO modified, indicating that sumoylation may be an important regulatory mechanism for the control of transcriptional repression mediated by both class I and II HDACs.

Histone deacetylase associated with mSin3A mediates repression by the acute promyelocytic leukemia-associated PLZF protein

The PLZF gene was identified first by its fusion with the retinoic acid receptor α gene in the t(11;17) translocation associated with a retinoic acid resistant form of acute promyelocytic leukemia (APL). It encodes a krüppel-like zinc finger protein with a POZ domain shared with a subset of regulatory proteins including the BCL6 leukemogenic protein. PLZF, like BCL6, strongly represses transcription initiated from different promoters. Here we show that PLZF associates in vitro and in vivo with the Mad co-repressor mSin3A and the histone deacetylase HDAC1. Two domains in PLZF and the PAH1 structure of mSin3A mediate these interactions. Trichostatin A, a specific inhibitor of histone deacetylases, significantly reduces PLZF repression. These data strongly suggest that, like nuclear receptors and Mad, PLZF represses transcription by recruiting a histone deacetylase through the SMRT-mSin3-HDAC co-repressor complex. We also show that BCL6 associates with HDAC1 indicating that this type of regulation might be common to POZ/Zinc finger proteins involved in human leukemias. This work supports a role for deregulated histone deacetylation in the development of both lymphoid and myeloid neoplasia in human and suggests that targeted histone deacetylase inhibitors may be useful for treatment of certain types of malignancies.

Comprehensive allelotyping of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers in many parts of the world, however the molecular mechanisms underlying liver cell transformation remain obscure. A genome-wide scan of loss of heterozygosity (LOH) in tumors provides a powerful tool to search for genes involved in neoplastic processes. To identify recurrent genetic alterations in liver tumors, we examined DNAs isolated from 120 HCCs and their adjacent non tumorous parts for LOH using a collection of 195 microsatellite markers located roughly every 20 cM throughout 39 autosomal arms. The mean heterozygosity was 73%. Our findings provide additional support that LOH for loci on chromosomal arms 1p, 4q, 6q, 8p, 13q and 16p is significantly elevated in HCC. The highest percentage of LOH is found for a locus in 8p23 (42% of informative csaes). This corresponds to one of the most common genetic abnormalities reported to date in these tumors. In addition, high ratio of LOH (⩾35%) is observed on chromosome arms which had not been implicated in previous studies, notably on 1q, 2q and 9q. No correlation was found between LOH of specific chromosomal regions and etiologic factors such as chronic infections with hepatitis B or C viruses. This first report of an extensive allelotypic analysis of HCC should help in identifying new genes whose loss of function contributes to the development of liver cancer.

Differential expression and ligand regulation of the retinoic acid receptor alpha and beta genes.

Retinoic acid (RA) is a vitamin A derivative that exhibits major effects on biological processes such as cell differentiation and embryo pattern formation. Two human retinoic acid receptors (RAR alpha and beta) have been recently characterized. These receptors are encoded by two genes and their affinities for RA differ, suggesting that these two nuclear receptors may have distinct roles in mediating the varied biological effects of RA. Here we show that RAR alpha and beta differ in the regulation of expression of their mRNAs. Different levels of RAR alpha and beta transcripts were found in the various human tissues analysed. In addition, treatment of human hepatoma cells with RA leads to a rapid 10‐ to 50‐fold increase in RAR beta mRNA levels, whereas RAR alpha mRNA expression is not affected. The induction of RAR beta transcription does not require de novo protein synthesis but is completely abolished by inhibitors of RNA synthesis. Nuclear transcript elongation assays indicate that the mechanism of RAR beta mRNA induction lies at the transcriptional level. These data demonstrate that the RAR beta gene is a primary target for RA. The differences in regulation of RAR gene expression might be a fundamental aspect of retinoid physiology and may prove especially important in the analysis of the morphogenic properties of RA.