PML multimerization into nuclear bodies following its oxidation promotes sumoylation and sequestration of partner proteins in these structures.

SummaryGlobal transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis.

Here, we identify fetal bone marrow (BM)-derived CD34hiCD45RAhiCD7+ hematopoietic progenitors as thymus-colonizing cells. This population, virtually absent from the fetal liver (FL), emerges in the BM by development weeks 8-9, where it accumulates throughout the second trimester, to finally decline around birth. Based on phenotypic, molecular, and functional criteria, we demonstrate that CD34hiCD45RAhiCD7+ cells represent the direct precursors of the most immature CD34hiCD1a- fetal thymocytes that follow a similar dynamics pattern during fetal and early postnatal development. Histological analysis of fetal thymuses further reveals that early immigrants predominantly localize in the perivascular areas of the cortex, where they form a lymphostromal complex with thymic epithelial cells (TECs) driving their rapid specification toward the T lineage. Finally, using an ex vivo xenogeneic thymus-colonization assay, we show that BM-derived CD34hiCD45RAhiCD7+ progenitors are selectively recruited into the thymus parenchyma in the absence of exogenous cytokines, where they adopt a definitive T cell fate.

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Myelodysplastic syndromes (MDS) are clonal stem cell hematologic disorders that evolve to acute myeloid leukemia (AML) and thus model multistep leukemogenesis. Activating RAS mutations and overexpression of BCL-2 are prognostic features of MDS/AML transformation. Using NRASD12 and BCL-2, we created two distinct models of MDS and AML, where human (h)BCL-2 is conditionally or constitutively expressed. Our novel transplantable in vivo models show that expression of hBCL-2 in a primitive compartment by mouse mammary tumor virus-long terminal repeat results in a disease resembling human MDS, whereas the myeloid MRP8 promoter induces a disease with characteristics of human AML. Expanded leukemic stem cell (Lin À /Sca-1 + /c-Kit + ) populations and hBCL-2 in the increased RAS-GTP complex within the expanded Sca-1 + compartment are described in both MDS/AML-like diseases. Furthermore, the oncogenic compartmentalizations provide the proapoptotic versus antiapoptotic mechanisms, by activating extracellular signal-regulated kinase and AKT signaling, in determination of the neoplastic phenotype. When hBCL-2 is switched off with doxycycline in the MDS mice, partial reversal of the phenotype was observed with persistence of bone marrow blasts and tissue infiltration as RAS recruits endogenous mouse (m)BCL-2 to remain active, thus demonstrating the role of the complex in the disease. This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex. The colocalization of BCL-2 and RAS in the bone marrow of MDS/AML patients offers targeting either oncogene as a therapeutic strategy. [Cancer Res 2007;67(24):11657-67]

Despite a lack of signaling motifs in their cytoplasmic domain, major histocompatibility complex (MHC) class II molecules trigger a variety of intracellular signals that regulate antigen-presenting cell function. They thus may use associated effector molecules as demonstrated on B cells and dendritic cells. The starting point of this study comes from our previous work, which demonstrated that the ecto-enzyme CD38 is functionally linked to MHC class II molecules. We report that CD38 and human leukocyte antigen-DR (HLA-DR) are functionally and physically associated in lipid rafts microdomains of cellsurface monocytes and that the integrity of these domains is necessary for the HLA-DR and CD38 signaling events. Moreover, we identified the tetraspanin CD9 molecule as a partner of the CD38/HLA-DR complex and demonstrated that HLA-DR, CD38, and CD9 share a common pathway of tyrosine kinase activation in human monocytes. The analysis of conjugate formation between monocytes presenting superantigen and T cells shows the active participation of CD9 and HLA-DR on the monocyte surface. Together, these observations demonstrate the presence of a CD38 and HLA-DR signaling complex within tetraspanin-containing lipid rafts and the functional impact of their molecular partner CD9 in antigen presentation. IntroductionReorganization of proteins at the interface between the T cell and the antigen-presenting cells (APCs) leads to the formation of the immunologic synapse. 1 Once a specific T-cell receptor has recognized the major histocompatibility complex (MHC)-peptide complex, MHC class II-mediated signals influence antigen-presenting function, adhesion, apoptosis, and cytokine production of the APC. 2 While MHC class II molecules do not harbor any known signaling motifs in their cytoplasmic domains, they can act as signal transducers, leading to activation of protein tyrosine kinases, phospholipase C␥, protein kinase C, and mitogen-activated protein kinases pathways. [3][4][5][6][7][8][9] Thus, MHC class II-mediated signals are likely to be at least partially dependent on interactions with other APC surface molecules. They also can be influenced by their compartmentalization into specialized membrane microdomains 10 such as glycosphingolipid-enriched microdomains, also called lipid rafts, 11 which are considered as platforms where signaling events are generated. MHC class II molecules are constitutively present in lipid rafts from murine and human B-cell lines, 12 monocytic cell lines, 13 human monocytes, 14 and tumor cells, 15 and the integrity of these microdomains is necessary for the transmission of MHC class II-mediated signals. 16 We reported that engagement of CD38 or MHC class II molecules on monocytes induced phosphorylation of common cytosolic substrates with marked additive effects between the 2 activation pathways. 17 CD38 plays an important role in activation and adhesion processes of monocytes. 18 CD38 ligation regulates the response to respiratory-burst activators 19 and enhances MHC class II and B7 expression in m...

Neutrophils are increasingly thought to modulate dendritic cell (DC) functions. We investigated the role of the neutrophil-DC partnership in the response to Mycobacterium bovis BCG-the vaccine used against tuberculosis. We compared neutrophil-DC crosstalk in humans and mice, searching for functional differences. In both species, neutrophils captured fluorescent BCG-enhanced green fluorescent protein (EGFP) and were more phagocytic than DC. Non-apoptotic BCG-infected neutrophils clustered with immature DC, establishing intimate contacts with dendrites, at which fluorescent intact bacilli were observed. Physical interactions between neutrophils and DC were required for DC activation. Human BCG-infected DC produced interleukin (IL)-10, an inhibitory cytokine, whereas DC exposed to BCG-infected neutrophils produced low to undetectable amounts of the cytokine. Mouse BCG-infected neutrophils induced sustained IL-2 production by DC. Human DC exposed to BCG-infected neutrophils stimulated recall T cell reactivity from vaccinated donors. Mouse DC infected with recombinant ovalbumin (OVA)-producing BCG (rBCG ova ) elicited proliferation of TCR-OVA-transgenic CD4 and CD8 T cells. Moreover, exposing DC to rBCG ova -infected neutrophils enhanced OVA presentation. Thus, in mice and humans, neutrophils help DC to cross-present BCG antigens to T cells. Our results suggest that this "mØnage à trois" involving neutrophils, DC and T cells plays a major role in the immune response to BCG.

The human T-lymphotropic virus type I oncoprotein Tax is critical for T-cell transformation, acting mainly through nuclear factor kappa B essential modulator (NEMO) binding and subsequent nuclear factor-B activation. Tax localizes to Tax nuclear bodies and to the centrosome and is subjected to ubiquitylation and small ubiquitin-like modifier (SUMO)ylation, which are both necessary for complete transcriptional activation. Using the photoconvertible fluorophore Dendra-2 coupled with live video confocal microscopy, we show for the first time that the same Tax molecule shuttles among Tax nuclear bodies and between these nuclear bodies and the centrosome, depending on its posttranslational modifications. Ubiquitylation targets Tax to nuclear bodies to which NEMO is recruited and subsequently SUMOylated. We also demonstrate that Tax nuclear bodies contain the SUMOylation machinery including SUMO and the SUMO conjugating enzyme Ubc9, strongly suggesting that these nuclear bodies represent sites of active SUMOylation. IntroductionTax, the viral oncoprotein encoded by the pX region of the human T-lymphotropic virus type I (HTLV-I) genome is a key player in the pathogenesis of HTLV-I-associated diseases, mainly adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLVassociated myelopathy. Tax deregulates the cellular machinery at multiple levels leading to the transformation of HTLV-I-infected T lymphocytes, predominantly through the activation of the nuclear factor-B (NF-B) pathway, which regulates key genes implicated in inflammation, apoptosis, and oncogenesis. 1,2 A corner stone in this NF-B activation is Tax binding to the regulatory subunit of the IB kinase (IKK) complex, IKK␥ (also known as NEMO). 3,4 Tax binding to NEMO phosphorylates both IKK␣ and IKK␤ leading to the formation of the IKK complex that phosphorylates the NF-B inhibitors IBs. This leads to the nuclear translocation of the active NF-B dimers and to transcriptional activation of target genes. 5 Tax/NEMO binding can also induce the IKK␣-dependent processing of the NF-B p100 precursor protein to its active p52 form. 6 Tax displays a dual nuclear and cytoplasmic localization with functions that are essential for cellular transformation in each compartment. 7,8 In the nucleus, Tax is predominantly located in RelA-enriched nuclear bodies. 9 In the cytoplasm, a major fraction of Tax lays in perinuclear hotspots colocalizing with the centrosome or microtubule organizing center in close association with the cis-Golgi compartment. 10,11 Posttranslational modification of proteins regulates protein functions by modifying their subcellular localization, stability, and/or network of interaction. We and others have described different forms of Tax posttranslational modifications including phosphorylation, 12,13 acetylation, 14 ubiquitylation, and small ubiquitin-like modifier (SUMO)ylation, 15,16 all of which are implicated in Tax-mediated activation of gene expression. Indeed, we showed that Tax is differentially ubiquitylated by either K-48 ubiquitin...