PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation

Arsenic is a clinically effective treatment for acute promyelocytic leukaemia (APL) in which the promyelocytic leukaemia (PML) protein is fused to retinoic receptor alpha (RARα). PML-RARα is degraded by the proteasome by a SUMO-dependent, ubiquitin-mediated pathway in response to arsenic treatment, curing the disease. Six major PML isoforms are expressed as a result of alternative splicing, each of which encodes a unique C-terminal region. Using a system in which only a single EYFP-linked PML isoform is expressed, we demonstrate that PMLI, PMLII and PMLVI accumulate in the cytoplasm following arsenic treatment, whereas PMLIII, PMLIV and PMLV do not. 3D structured illumination was used to obtain super-resolution images of PML bodies, revealing spherical shells of PML along with associated SUMO. Arsenic treatment results in dramatic isoform-specific changes to PML body ultrastructure. After extended arsenic treatment most PML isoforms are degraded, leaving SUMO at the core of the nuclear bodies. A high-content imaging assay identifies PMLV as the isoform most readily degraded following arsenic treatment, and PMLIV as relatively resistant to degradation. Immunoprecipitation analysis demonstrates that all PML isoforms are modified by SUMO and ubiquitin after arsenic treatment, and by using siRNA, we demonstrate that arsenic-induced degradation of all PML isoforms is dependent on the ubiquitin E3 ligase RNF4. Intriguingly, depletion of RNF4 results in marked accumulation of PMLV, suggesting that this isoform is an optimal substrate for RNF4. Thus the variable C-terminal domain influences the rate and location of degradation of PML isoforms following arsenic treatment

Molecular Cloning and Rare Cleavage Mapping of Human 2P, 6Q, 8Q, 12Q, and 18Q Telomeres

Large terminal fragments of human chromosomes 2p, 6q, 8q, 12q, and 18q were cloned using yeast artificial chromosomes (YACs). RecA-assisted restriction endonuclease (RARE) cleavage analysis of genomic DNA samples from 11 unrelated individuals using YAC-derived probes confirmed the telomeric localizations of the half-YACs studied. The cloned Fragments provide telomeric closure of maps for the respective chromosome arms and will supply the reagents needed for analyzing and sequencing these distal subtelomeric regions

SUMO Pathway Dependent Recruitment of Cellular Repressors to Herpes Simplex Virus Type 1 Genomes

Components of promyelocytic leukaemia (PML) nuclear bodies (ND10) are recruited to sites associated with herpes simplex virus type 1 (HSV-1) genomes soon after they enter the nucleus. This cellular response is linked to intrinsic antiviral resistance and is counteracted by viral regulatory protein ICP0. We report that the SUMO interaction motifs of PML, Sp100 and hDaxx are required for recruitment of these repressive proteins to HSV-1 induced foci, which also contain SUMO conjugates and PIAS2β, a SUMO E3 ligase. SUMO modification of PML and elements of its tripartite motif (TRIM) are also required for recruitment in cells lacking endogenous PML. Mutants of PML isoform I and hDaxx that are not recruited to virus induced foci are unable to reproduce the repression of ICP0 null mutant HSV-1 infection mediated by their wild type counterparts. We conclude that recruitment of ND10 components to sites associated with HSV-1 genomes reflects a cellular defence against invading pathogen DNA that is regulated through the SUMO modification pathway

Functional Interaction of Nuclear Domain 10 and Its Components with Cytomegalovirus after Infections: Cross-Species Host Cells versus Native Cells

Species-specificity is one of the major characteristics of cytomegaloviruses (CMVs) and is the primary reason for the lack of a mouse model for the direct infection of human CMV (HCMV). It has been determined that CMV cross-species infections are blocked at the post-entry level by intrinsic cellular defense mechanisms, but few details are known. It is important to explore how CMVs interact with the subnuclear structure of the cross-species host cell. In our present study, we discovered that nuclear domain 10 (ND10) of human cells was not disrupted by murine CMV (MCMV) and that the ND10 of mouse cells was not disrupted by HCMV, although the ND10-disrupting protein, immediate-early protein 1 (IE1), also colocalized with ND10 in cross-species infections. In addition, we found that the UL131-repaired HCMV strain AD169 (vDW215-BADrUL131) can infect mouse cells to produce immediate-early (IE) and early (E) proteins but that neither DNA replication nor viral particles were detectable in mouse cells. Unrepaired AD169 can express IE1 only in mouse cells. In both HCMV-infected mouse cells and MCMV-infected human cells, the knocking-down of ND10 components (PML, Daxx, and SP100) resulted in significantly increased viral-protein production. Our observations provide evidence to support our hypothesis that ND10 and ND10 components might be important defensive factors against the CMV cross-species infection

Post-GWAS Functional Characterization of Susceptibility Variants for Chronic Lymphocytic Leukemia

Recent genome-wide association studies (GWAS) have identified several gene variants associated with sporadic chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Many of these CLL/SLL susceptibility loci are located in non-coding or intergenic regions, posing a significant challenge to determine their potential functional relevance. Here, we review the literature of all CLL/SLL GWAS and validation studies, and apply eQTL analysis to identify putatively functional SNPs that affect gene expression that may be causal in the pathogenesis of CLL/SLL. We tested 12 independent risk loci for their potential to alter gene expression through cis-acting mechanisms, using publicly available gene expression profiles with matching genotype information. Sixteen SNPs were identified that are linked to differential expression of SP140, a putative tumor suppressor gene previously associated with CLL/SLL. Three additional SNPs were associated with differential expression of DACT3 and GNG8, which are involved in the WNT/β-catenin- and G protein-coupled receptor signaling pathways, respectively, that have been previously implicated in CLL/SLL pathogenesis. Using in silico functional prediction tools, we found that 14 of the 19 significant eQTL SNPs lie in multiple putative regulatory elements, several of which have prior implications in CLL/SLL or other hematological malignancies. Although experimental validation is needed, our study shows that the use of existing GWAS data in combination with eQTL analysis and in silico methods represents a useful starting point to screen for putatively causal SNPs that may be involved in the etiology of CLL/SLL

SUMOylation of the Forkhead Transcription Factor FOXL2 Promotes Its Stabilization/Activation through Transient Recruitment to PML Bodies

International audienceBACKGROUND: FOXL2 is a transcription factor essential for ovarian development and maintenance. It is mutated in the genetic condition called Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES) and in cases of isolated premature ovarian failure. We and others have previously shown that FOXL2 undergoes several post-translational modifications. METHODS AND PRINCIPAL FINDINGS: Here, using cells in culture, we show that interference with FOXL2 SUMOylation leads to a robust inhibition of its transactivation ability, which correlates with a decreased stability. Interestingly, FOXL2 SUMOylation promotes its transient recruitment to subnuclear structures that we demonstrate to be PML (Promyelocytic Leukemia) Nuclear Bodies. Since PML bodies are known to be sites where post-translational modifications of nuclear factors take place, we used tandem mass spectrometry to identify new post-translational modifications of FOXL2. Specifically, we detected four phosphorylated, one sulfated and three acetylated sites. CONCLUSIONS: By analogy with other transcription factors, we propose that PML Nuclear Bodies might transiently recruit FOXL2 to the vicinity of locally concentrated enzymes that could be involved in the post-translational maturation of FOXL2. FOXL2 acetylation, sulfation, phosphorylation as well as other modifications yet to be discovered might alter the transactivation capacity of FOXL2 and/or its stability, thus modulating its global intracellular activity

THE CLONING AND PHYSICAL MAPPING OF THE SUBTELODIMENSIONAL REGIONS OF THE MAN'S CHROMOSOMES

The object of investigation: the subtelodimensional region of the man's chromosome I long shoulder. The purpose of the work: the confirm the origin of the cloned DNA from the body-dimensional region of the man's chromosome. The physical chart of the subbody-dimensional region of the man's chromosome I long shoulder of 260 t.p.n. extent. The polymorphism of the bodymeasure 1q, 2q, 6q, 8p, 8q, 12q, 13q, 14q, 18p and 18q of the man's genome has been examinedAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Differential Functions of Interferon-Upregulated Sp100 Isoforms: Herpes Simplex Virus Type 1 Promoter-Based Immediate-Early Gene Suppression and PML Protection from ICP0-Mediated Degradation▿

Cells have intrinsic defenses against virus infection, acting before the innate or the adaptive immune response. Preexisting antiviral proteins such as PML, Daxx, and Sp100 are stored in specific nuclear domains (ND10). In herpes simplex virus type 1 (HSV-1), the immediate-early protein ICP0 serves as a counterdefense through degradation of the detrimental protein PML. We asked whether interferon (IFN)-upregulated Sp100 is similarly antagonized by ICP0 in normal human fibroblasts by using a selective-knockdown approach. We find that of the four Sp100 isoforms, the three containing a SAND domain block the transcription of HSV-1 proteins ICP0 and ICP4 at the promoter level and that IFN changes the differential splicing of the Sp100 transcript in favor of the inhibitor Sp100C. At the protein level, ICP0 activity does not lead to the hydrolysis of any of the Sp100 isoforms. The SAND domain-containing isoforms are not general inhibitors of viral promoters, as the activity of the major immediate-early cytomegalovirus promoter is not diminished, whereas the long terminal repeat of a retrovirus, like the ICP0 promoter, is strongly inhibited. Since we could not find a specific promoter region in the ICP0 gene that responds to the SAND domain-containing isoforms, we questioned whether Sp100 could act through other antiviral proteins such as PML. We find that all four Sp100 isoforms stabilize ND10 and protect PML from ICP0-based hydrolysis. Loss of either all PML isoforms or all Sp100 isoforms reduces the opposite constituent ND10 protein, suggesting that various interdependent mechanisms of ND10-based proteins inhibit virus infection at the immediate-early level