Sp110 localizes to the PML-Sp100 nuclear body and may function as a nuclear hormone receptor transcriptional coactivator.
ABSTRACT: The nuclear body is a multiprotein complex that may have a role in the regulation of gene transcription. This structure is disrupted in a variety of human disorders including acute promyelocytic leukemia and viral infections, suggesting that alterations in the nuclear body may have an important role in the pathogenesis of these diseases. In this study, we identified a cDNA encoding a leukocyte-specific nuclear body component designated Sp110. The N-terminal portion of Sp110 was homologous to two previously characterized components of the nuclear body (Sp100 and Sp140). The C-terminal region of Sp110 was homologous to the transcription intermediary factor 1 (TIF1) family of proteins. High levels of Sp110 mRNA were detected in human peripheral blood leukocytes and spleen but not in other tissues. The levels of Sp110 mRNA and protein in the human promyelocytic leukemia cell line NB4 increased following treatment with all-trans retinoic acid (ATRA), and Sp110 localized to PML-Sp100 nuclear bodies in ATRA-treated NB4 cells. Because of the structural similarities between Sp110 and TIF1 proteins, the effect of Sp110 on gene transcription was examined. An Sp110 DNA-binding domain fusion protein activated transcription of a reporter gene in transfected mammalian cells. In addition, Sp110 produced a marked increase in ATRA-mediated expression of a reporter gene containing a retinoic acid response element. Taken together, the results of this study demonstrate that Sp110 is a member of the Sp100/Sp140 family of nuclear body components and that Sp110 may function as a nuclear hormone receptor transcriptional coactivator. The predominant expression of Sp110 in leukocytes and the enhanced expression of Sp110 in NB4 cells treated with ATRA raise the possibility that Sp110 has a role in inducing differentiation of myeloid cells.
Project description:The SP100 family members comprise a set of closely related genes on chromosome 2q37.1. The widely expressed SP100 and the leukocyte-specific proteins SP110 and SP140 have been associated with transcriptional regulation and various human diseases. Here, we have characterized the SP100 family member SP140L. The genome sequence analysis showed the formation of SP140L gene through rearrangements of the two neighboring genes, SP100 and SP140, during the evolution of higher primates. The SP140L expression is interferon-inducible with high transcript levels in B cells and other peripheral blood mononuclear cells. Subcellularly, SP140L colocalizes with SP100 and SP140 in nuclear structures that are devoid of SP110, PML, or p300 proteins. Similarly to SP100 and SP140 protein, we detected serum autoantibodies to SP140L in patients with primary biliary cirrhosis using luciferase immunoprecipitation system and immunoblotting assays. In conclusion, our results show that SP140L is phylogenetically recent member of SP100 proteins and acts as an autoantigen in primary biliary cirrhosis patients.
Project description:Bright (B cell regulator of IgH transcription) transactivates the immunoglobulin heavy chain (IgH) intronic enhancer, Emicro, by binding to matrix attachment regions (MARs), sites necessary for DNA attachment to the nuclear matrix. Here we report that Bright interacts with the ubiquitous autoantigen Sp100, a component of promyelocytic leukemia nuclear bodies (PML NBs), and with LYSp100B/Sp140, the lymphoid-restricted homolog of Sp100. Both in intact cells and in nuclear matrix preparations, the majority of Bright and Sp100 colocalize within PML NBs. In contrast, Bright colocalizes with only a small fraction of LYSp100B while inducing a redistribution of the majority of LYSp100B from its associated nuclear domains (LANDs) into nucleoplasm and cytoplasm. Sp100 represses the MAR-binding and transactivation activity of Bright. LYSp100B interacts more weakly with Bright but requires significantly higher levels than Sp100 to inhibit MAR binding. However, it strongly stimulates Bright transactivation through E mu. We suggest that Sp100 and LYSp100B interactions with Bright have different consequences for IgH transcription, potentially through differential association of E mu MARs with nuclear matrix- associated PML NBs and LANDs.
Project description:Standard therapy for acute promyelocytic leukaemia (APL) includes retinoic acid (all-trans retinoic acid (ATRA)), which promotes differentiation of promyelocytic blasts. Although co-administration of arsenic trioxide (ATO) with ATRA has emerged as an effective option to treat APL, the molecular basis of this effect remains unclear.Four leukaemia cancer human models (HL60, THP-1, NBR4 and NBR4-R2 cells) were treated either with ATO alone or ATO plus ATRA. Cancer cell survival was monitored by trypan blue exclusion and DEVDase activity assays. Gene and protein expression changes were assessed by RT-PCR and western blot.ATO induced an antioxidant response characterised by Nrf2 nuclear translocation and enhanced transcription of downstream target genes (that is, HO-1, NQO1, GCLM, ferritin). In cells exposed to ATO plus ATRA, the Nrf2 nuclear translocation was prevented and cytotoxicity was enhanced. HO-1 overexpression reversed partially the cytotoxicity by ATRA-ATO in HL60 cells. The inhibitory effects of ATRA on ATO-mediated responses were not observed in either the ATRA-resistant NB4-R2 cells or in NB4 cells pre-incubated with the RAR? antagonist Ro-41-52-53.The augmented cytotoxicity observed in leukaemia cells following combined ATO-ATRA treatment is likely due to inhibition of Nrf2 activity, thus explaining the efficacy of combined ATO-ATRA treatment in the APL therapy.
Project description:<h4>Background</h4>SP110, an interferon-induced nuclear protein, belongs to the SP100/SP140 protein family. Very recently, we showed that SP110b, an SP110 isoform, controls host innate immunity to Mycobacterium tuberculosis infection by regulating nuclear factor-?B (NF-?B) activity. However, it remains unclear how the structure of SP110 relates to its cellular functions. In this study, we provide experimental data illustrating the protein domains that are responsible for its functions.<h4>Methods</h4>We examined the effects of SP110 isoforms and a series of deletion mutants of SP110 on transcriptional regulation by luciferase reporter assays. We also employed confocal microscopy to determine the cellular distributions of enhanced green fluorescent protein-tagged SP110 isoforms and SP110 mutants. In addition, we performed immunoprecipitation and Western blotting analyses to identify the regions of SP110 that are responsible for protein interactions.<h4>Results</h4>Using reporter assays, we first demonstrated that SP110 isoforms have different regulatory effects on NF-?B-mediated transcription, supporting the notion that SP110 isoforms may have distinct cellular functions. Analysis of deletion mutants of SP110 showed that the interaction of the N-terminal fragment (amino acids 1-276) of SP110 with p50, a subunit of NF-?B, in the cytoplasm plays a crucial role in the down-regulation of the p50-driven tumor necrosis factor-? (TNF?) promoter activity in the nucleus, while the middle and C-terminal regions of SP110 localize it to various cellular compartments. Surprisingly, a nucleolar localization signal (NoLS) that contains one monopartite nuclear localization signal (NLS) and one bipartite NLS was identified in the middle region of SP110. The identification of a cryptic NoLS in the SP110 suggests that although this protein forms nuclear speckles in the nucleoplasm, it may be directed into the nucleolus to carry out distinct functions under certain cellular conditions.<h4>Conclusions</h4>The findings from this study elucidating the multidomain structure of the SP110 not only identify functional domains of SP110 that are required for transcriptional regulation, cellular translocation, and protein interactions but also implicate that SP110 has additional functions through its unexpected activity in the nucleolus.
Project description:The decoding of histone post-translational modifications by chromatin-binding modules ("readers") constitutes one major mechanism of epigenetic regulation. Nuclear antigen Sp100 (SPECKLED, 100 kDa), a constitutive component of the promyelocytic leukemia nuclear bodies, plays key roles in intrinsic immunity and transcriptional repression. Sp100C, a splicing isoform specifically up-regulated upon interferon stimulation, harbors a unique tandem plant homeodomain (PHD) finger and bromodomain at its C terminus. Combining structural, quantitative binding, and cellular co-localization studies, we characterized Sp100C PHD finger as an unmethylated histone H3 Lys(4) (H3K4me0) reader that tolerates histone H3 Thr(3) phosphorylation (H3T3ph), histone H3 Lys(9) trimethylation (H3K9me3), and histone H3 Ser(10) phosphorylation (H3S10ph), hallmarks associated with the mitotic chromosome. In contrast, whereas H3K4me0 reader activity is conserved in Sp140, an Sp100C paralog, the multivalent tolerance of H3T3ph, H3K9me3, and H3S10ph was lost for Sp140. The complex structure determined at 2.1 Å revealed a highly coordinated lysine ?-amine recognition sphere formed by an extended N-terminal motif for H3K4me0 readout. Interestingly, reader pocket rigidification by disulfide bond formation enhanced H3K4me0 binding by Sp100C. An additional complex structure solved at 2.7 Å revealed that H3T3ph is recognized by the arginine residue, Arg(713), that is unique to the PHD finger of Sp100C. Consistent with a restrictive cellular role of Sp100C, these results establish a direct chromatin targeting function of Sp100C that may regulate transcriptional gene silencing and promyelocytic leukemia nuclear body-mediated intrinsic immunity in response to interferon stimulation.
Project description:All-trans retinoic acid (ATRA) is one of the first line agents in differentiation therapy for acute promyelocytic leukemia (APL). However, drug resistance is a major problem influencing the efficacy of ATRA. Identification of mechanisms of ATRA resistance are urgenly needed. In the present study, we found that expression of C/EBPα, an important transcription factor for myeloid differentiation, was significantly suppressed in ATRA resistant APL cell line NB4-R1 compared with ATRA sensitive NB4 cells. Moreover, two forms of C/EBPα were unequally suppressed in NB4-R1 cells. Suppression of the full-length form P42 was more pronounced than the truncated form P30. Inhibition of PI3K/Akt/mTOR pathway was also observed in NB4-R1 cells. Moreover, C/EBPα expression was reduced by PI3K inhibitor LY294002 and mTOR inhibitor RAD001 in NB4 cells, suggesting that inactivation of the PI3K/Akt/mTOR pathway was responsible for C/EBPα suppression in APL cells. We restored C/EBPα P42 and P30 by lentivirus vectors in NB4-R1 cells, respectively, and found C/EBPα P42, but not P30, could increase CD11b, CD14, G-CSFR and GM-CSFR expression, which indicated the occurrence of myeloid differentiation. Further upregulating of CD11b expression and differential morphological changes were found in NB4-R1 cells with restored C/EBPα P42 after ATRA treatment. However, CD11b expression and differential morphological changes could not be induced by ATRA in NB4-R1 cells infected with P30 expressing or control vector. Thus, we inferred that ATRA sensitivity of NB4-R1 cells was enhanced by restoration of C/EBPα P42. In addition, we used histone deacetylase inhibitor trichostatin (TSA) to restore C/EBPα expression in NB4-R1 cells. Similar enhancement of myeloid differentiation and cell growth arrest were detected. Together, the present study demonstrated that suppression of C/EBPα P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and ATRA sensitivity of APL cells. Restoring C/EBPα P42 is an attractive approach for differentiation therapy in ATRA resistant APL.
Project description:In acute promyelocytic leukemia (APL), the translocation t(15;17) induces a block at the promyelocytic stage of differentiation in an all-trans-retinoic acid (ATRA)-responsive manner. Here we report that upon treatment with ATRA, t(15;17) cells (NB4) reveal a very rapid increase in protein level and binding activity of C/EBPbeta, a C/EBP family member, which was not observed in an ATRA-resistant NB4 cell line. We further provide evidence that ATRA mediates a direct increase of C/EBPbeta, only in PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha)-expressing cells. In addition, transactivation experiments indicate that the PML-RARA fusion protein, but not PML-RARA mutants defective in transactivation, strongly transactivates the C/EBPbeta promoter. These results suggest that PML-RARA mediates ATRA-induced C/EBPbeta expression. Finally, we demonstrate the importance of C/EBPbeta in granulocytic differentiation. We show that not only does C/EBPbeta induce granulocytic differentiation of non-APL myeloid cell lines independent of addition of ATRA or other cytokines, but also that C/EBPbeta induction is required during ATRA-induced differentiation of APL cells. Taken together, C/EBPbeta is an ATRA-dependent PML-RARA target gene involved in ATRA-induced differentiation of APL cells.
Project description:The multidrug resistance 1 (MDR1) gene product P-glycoprotein (P-gp) is frequently implicated in cross-resistance of tumors to chemotherapeutic drugs. In contrast, acute promyelocytic leukemia (APL) cells do not express MDR1 and are highly sensitive to anthracyclines. The combination of ATRA and the novel histone deacetylase inhibitor (HDACI) depsipeptide (FK228) induced P-gp expression and prevented growth inhibition and apoptosis in NB4 APL cells subsequently exposed to doxorubicin (DOX). ATRA/FK228 treatment after exposure to DOX, however, enhanced apoptosis. Both agents, ATRA or FK228, induced MDR1 mRNA. This effect was significantly enhanced by ATRA/FK228 administered in combination, due in part to increased H4 and H3-Lys9 acetylation of the MDR1 promoter and recruitment of the nuclear transcription factor Y alpha (NFYA) transcription activator to the CCAAT box. Cotreatment with specific P-gp inhibitor PSC833 reversed cytoprotective effects of ATRA/FK228. G1 cell-cycle arrest and p21 mRNA induction were also observed in response to ATRA/FK228, which may restrict DOX-induced apoptosis of cells in G2 phase. These results indicate that epigenetic mechanisms involving NF-YA transcription factor recruitment and histone acetylation are activated by ATRA and HDACI, induce MDR1 in APL cells, and point to the critical importance of mechanism-based sequential therapy in future clinical trials that combine HDAC inhibitors, ATRA, and anthracyclines.
Project description:HOXA cluster antisense RNA 2 (HOXA-AS2) is a long non-coding RNA located between the HOXA3 and HOXA4 genes in the HOXA cluster. Its transcript is expressed in NB4 promyelocytic leukemia cells and human peripheral blood neutrophils, and expression is increased in NB4 cells treated with all trans retinoic acid (ATRA). Knockdown of HOXA-AS2 expression by transduced shRNA decreases the number of viable cells and increases the proportion of apoptotic cells, measured by annexin V binding and by activity and cleavage of caspases-3, -8, and -9. The increase in death of HOXA-AS2 knockdown cells was accompanied by an elevated TNF-related apoptosis-inducing ligand (TRAIL) levels, but ATRA-induced NB4 cells treated with TRAIL did show an increase in HOXA-AS2 expression. These results demonstrate that ATRA induction of HOXA-AS2 suppresses ATRA-induced apoptosis, possibly through a TRAIL-mediated pathway. HOXA-AS2-mediated negative regulation thus contributes to the fine-tuning of apoptosis during ATRA-induced myeloid differentiation in NB4 cells.
Project description:Acute promyelocytic leukemia (APL) is characterized by arrested differentiation of promyelocytes. Patients treated with all-trans retinoic acid (ATRA) alone experience relapse, while patients treated with ATRA and arsenic trioxide (ATO) are often relapse-free. This suggests sustained changes have been elicited by the combination therapy. To understand the lasting effects of the combination therapy, we compared the effects of ATRA and ATO on NB4 and ATRA-resistant NB4-MR2 APL cells during treatment versus post treatment termination. After treatment termination, NB4 cells treated with ATRA or ATO reverted to non-differentiated cells, while combination-treated cells remained terminally differentiated. This effect was diminished in NB4-MR2 cells. This suggests combination treatment induced more permanent changes. Combination treatment induced higher expression of target genes (e.g., transglutaminase 2 and retinoic acid receptor beta), which in NB4 cells was sustained post treatment termination. To determine whether sustained epigenetic changes were responsible, we quantified the enrichment of histone modifications by chromatin immunoprecipitation, and CpG methylation by bisulfite-pyrosequencing. While ATRA and combination treatment induced similar histone acetylation enrichment, combination treatment induced greater demethylation of target genes, which was sustained. Therefore, sustained demethylation of target genes by ATRA and ATO combination treatment is associated with lasting differentiation and gene expression changes.