The nuclear corepressors recognize distinct nuclear receptor complexes

The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms have the capacity to silence gene expression in the absence of their ligands on target response elements. This active repression is mediated by the ability of the corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to recruit a complex containing histone deacetylase activity. Interestingly, NCoR and SMRT share significant differences in the their two nuclear receptor-interacting domains (IDs), suggesting that they may recruit receptors with different affinities. In addition, the role of the receptor complex bound to a response element has not been fully evaluated in its ability to recruit separate corepressors. We demonstrate in this report that the proximal ID in NCoR and SMRT, which share only 23% homology, allows preferential recognition of nuclear receptors, such that TR prefers to recruit NCoR, and RAR prefers to recruit SMRT, to DNA response elements. However, mutations in the TR found in the syndromes of resistance to thyroid hormone can change the corepressor recruited by changing the complex (homodimer or heterodimer) formed on the TRE. These results demonstrate that the corepressor complex recruited can be both nuclear receptor- and receptor complex-specific.     

Coiled-coil domain of PML is essential for the aberrant dynamics of PML-RARalpha, resulting in sequestration and decreased mobility of SMRT

Promyelocytic leukemia-retinoic acid receptor α (PML-RARα) is the most frequent RARα fusion protein in acute promyelocytic leukemia (APL). Our previous study has demonstrated that, compared with RARα, PML-RARα had reduced intranuclear mobility accompanied with mislocalization. To understand the molecular basis for the altered dynamics of PML-RARα fusion protein, we performed FRAP analysis at a single cell level. Results indicated that three known sumoylation site mutated PML-RARα had same intracellular localization and reduced mobility as wild-type counterpart. The coiled-coil domain of PML is responsible for the aberrant dynamics of PML-RARα. In addition, we revealed that co-repressor SMRT co-localized with PML-RARα, resulting in the immobilization of SMRT while ATRA treatment eliminated their association and reversed the immobile effect of SMRT. Furthermore, co-activator CBP, co-localized with PML-RARα in an ATRA-independent way, was demonstrated as a high dynamic intranuclear molecule. These results would shed new insights for the molecular mechanisms of PML-RARα-associated leukemogenesis.     

Altered nuclear cofactor switching in retinoic-resistant variants of the PML-RARα oncoprotein of acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) results from a reciprocal translocation that fuses the gene for the PML tumor suppressor to that encoding the retinoic acid receptor alpha (RARα). The resulting PML-RARα oncogene product interferes with multiple regulatory pathways associated with myeloid differentiation, including normal PML and RARα functions. The standard treatment for APL includes anthracycline-based chemotherapeutic agents plus the RARα agonist all-trans retinoic acid (ATRA). Relapse, which is often accompanied by ATRA resistance, occurs in an appreciable frequency of treated patients. One potential mechanism suggested by model experiments featuring the selection of ATRA-resistant APL cell lines involves ATRA-resistant versions of the PML-RARα oncogene, where the relevant mutations localize to the RARα ligand-binding domain (LBD). Such mutations may act by compromising agonist binding, but other mechanisms are possible. Here, we studied the molecular consequence of ATRA resistance by use of circular dichroism, protease resistance, and fluorescence anisotropy assays employing peptides derived from the NCOR nuclear corepressor and the ACTR nuclear coactivator. The consequences of the mutations on global structure and cofactor interaction functions were assessed quantitatively, providing insights into the basis of agonist resistance. Attenuated cofactor switching and increased protease resistance represent features of the LBDs of ATRA-resistant PML-RARα, and these properties may be recapitulated in the full-length oncoproteins.     

Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.     

Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.     

Determination of nuclear receptor corepressor interactions with the thyroid hormone receptor

The thyroid hormone receptor (TR) recruits the nuclear corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to target DNA elements in the absence of ligand. While the TR preferentially recruits NCoR, the mechanism remains unclear. The corepressors interact with the TR via interacting domains (IDs) present in their C terminus which contain a conserved motif termed a CoRNR box. Despite their similarity, the corepressor IDs allow for nuclear receptor specificity. Here we demonstrate that NCoR stabilizes the TR homodimer when bound to DNA by preventing its dissociation from thyroid hormone response elements. This suggests that NCoR acts to hold the repression complex in place on target elements. The TR homodimer recruits NCoR through two of its three IDs, one of which is not present in SMRT. This unique ID, N3, contains a CoRNR box but lacks the extended helical motif present in each of the other IDs. Instead, N3 contains an isoleucine just proximal to this motif. This isoleucine is also conserved in N2 but not in the corresponding S2 domain in SMRT. On thyroid hormone response elements and in mammalian cells this residue is critical in both N3 and N2 for high-affinity TR binding. In addition, this residue also controls specificity for the interactions of TR with NCoR. Together these data suggest that the specific recruitment of NCoR by the TR through a unique motif allows for stabilization of the repression complex on target elements.     

The PML-RAR alpha 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.     

Determinants of CoRNR-dependent repression complex assembly on nuclear hormone receptors

Ligand-dependent exchange of coactivators and corepressors is the fundamental regulator of nuclear hormone receptor (NHR) function. The interaction surfaces of coactivators and corepressors are similar but distinct enough to allow the ligand to function as a switch. Multiple NHRs share features that allow corepressor binding, and each of two distinct corepressors (N-CoR and SMRT) contains two similar CoRNR motifs that interact with NHRs. Here we report that the specificity of corepressor-NHR interaction is determined by the individual NHR interacting with specific CoRNR boxes within a preferred corepressor. First, receptors have distinct preferences for CoRNR1 versus CoRNR2. For example, the retinoic acid receptor binds CoRNR1, while RXR interacts almost exclusively with CoRNR2. Second, the NHR preference for N-CoR or SMRT is due to differences in CoRNR1 but not CoRNR2. Moreover, within a single corepressor, affinity for different NHRs is determined by distinct regions flanking CoRNR1. The highly specific determinants of NHR-corepressor interaction and preference suggest that repression is regulated by the permissibility of selected receptor-CoRNR-corepressor combinations. Interestingly, different NHR surfaces contribute to binding of CoRNR1 and CoRNR2, suggesting a model to explain corepressor binding to NHR heterodimers.     

Differential regulation of PML-RARα stability by the ubiquitin ligases SIAH1/SIAH2 and TRIAD1

The ubiquitin proteasome system plays an important role in normal and malignant hematopoiesis and relies on the concerted action of three enzyme families. The E2 ubiquitin conjugase UBCH8 (ubiquitin conjugating enzyme [human] 8) cooperates with the E3 ubiquitin ligases SIAH1 and SIAH2 (seven in absentia homolog 1/2) to mediate the proteasomal degradation of oncoproteins. One such protein is the leukemia fusion protein PML-RARα (promyelocytic leukemia-retinoic acid receptorα) that is associated with acute promyelocytic leukemia. A limited number of UBCH8 interaction partners that participate in the UBCH8-dependent depletion of cancer-relevant proteins are known. We report here that TRIAD1 (two RING fingers and DRIL [double RING finger linked] 1), an E3 ubiquitin ligase relevant for the clonogenic growth of myloid progenitors, binds UBCH8 as well as PML-RARα. Moreover, there is concurrent induction of TRIAD1 and UBCH8 upon combinatorial treatment of acute promyelocytic leukemia cells with the pro-apoptotic epigenetic modulator valproic acid and the differentiation inducing agent all-trans retinoic acid. However, in sharp contrast to SIAH1/SIAH2 and UBCH8, TRIAD1 binding to PML-RARα has no effect on its turnover. In summary, our data exclude TRIAD1 as crucial regulator of the leukemic determinant PML-RARα, but highlight the prominence of the UBCH8/SIAH axis in PML-RARα degradation.