Resistance of Foxp3+ Regulatory T Cells to Nur77-Induced Apoptosis Promotes Allograft Survival

The NR4A nuclear receptor family member Nur77 (NR4A1) promotes thymocyte apoptosis during negative selection of autoreactive thymocytes, but may also function in mature extrathymic T cells. We studied the effects of over-expression of Nur77 on the apoptosis of murine peripheral T cells, including thymic-derived Foxp3+ regulatory (Treg) cells. Overexpression of Nur77 in the T cell lineage decreased numbers of peripheral CD4 and CD8 T cells by ∼80% compared to wild-type (WT) mice. However, the proportions of Treg cells were markedly increased in the thymus (61% of CD4+Foxp3+ singly positive thymocytes vs. 8% in WT) and secondary lymphoid organs (40–50% of CD4+Foxp3+ T cells vs. 7–8% in WT) of Nur77 transgenic (Nur77Tg) mice, and immunoprecipitation studies showed Nur77 was associated with a recently identified HDAC7/Foxp3 transcriptional complex. Upon activation through the T cell receptor in vitro or in vivo, Nur77Tg T cells showed only marginally decreased proliferation but significantly increased apoptosis. Fully allogeneic cardiac grafts transplanted to Nur77Tg mice survived long-term with well-preserved structure, and recipient splenocytes showed markedly enhanced apoptosis and greatly reduced anti-donor recall responses. Allografts in Nur77Tg recipients had significantly increased expression of multiple Treg-associated genes, including Foxp3, Foxp1, Tip60 and HDAC9. Allograft rejection was restored by CD25 monoclonal antibody therapy, indicating that allograft acceptance was dependent upon Treg function in Nur77Tg recipients. These data show that compared to conventional CD4 and CD8 T cells, Foxp3+ Tregs are relatively resistant to Nur77-mediated apoptosis, and that tipping the balance between the numbers of Tregs and responder T cells in the early period post-transplantation can determine the fate of the allograft. Hence, induced expression of Nur77 might be a novel means to achieve long-term allograft survival

HDAC7 Is a Repressor of Myeloid Genes Whose Downregulation Is Required for Transdifferentiation of Pre-B Cells into Macrophages

B lymphopoiesis is the result of several cell-commitment, lineage-choice, and differentiation processes. Every differentiation step is characterized by the activation of a new, lineage-specific, genetic program and the extinction of the previous one. To date, the central role of specific transcription factors in positively regulating these distinct differentiation processes to acquire a B cell-specific genetic program is well established. However, the existence of specific transcriptional repressors responsible for the silencing of lineage inappropriate genes remains elusive. Here we addressed the molecular mechanism behind repression of non-lymphoid genes in B cells. We report that the histone deacetylase HDAC7 was highly expressed in pre-B cells but dramatically down-regulated during cellular lineage conversion to macrophages. Microarray analysis demonstrated that HDAC7 re-expression interfered with the acquisition of the gene transcriptional program characteristic of macrophages during cell transdifferentiation; the presence of HDAC7 blocked the induction of key genes for macrophage function, such as immune, inflammatory, and defense response, cellular response to infections, positive regulation of cytokines production, and phagocytosis. Moreover, re-introduction of HDAC7 suppressed crucial functions of macrophages, such as the ability to phagocytose bacteria and to respond to endotoxin by expressing major pro-inflammatory cytokines. To gain insight into the molecular mechanisms mediating HDAC7 repression in pre-B cells, we undertook co-immunoprecipitation and chromatin immunoprecipitation experimental approaches. We found that HDAC7 specifically interacted with the transcription factor MEF2C in pre-B cells and was recruited to MEF2 binding sites located at the promoters of genes critical for macrophage function. Thus, in B cells HDAC7 is a transcriptional repressor of undesirable genes. Our findings uncover a novel role for HDAC7 in maintaining the identity of a particular cell type by silencing lineage-inappropriate genes

Unraveling biogeographical patterns and environmental drivers of soil fungal diversity at the French national scale

The fungal kingdom is among the most diversified kingdoms on Earth, with estimations of up to 12 million species. However, it remains poorly understood, with only 150 000 fungal species currently described. Given the major ecological role of fungi in ecosystem functioning, these numbers stress the importance of investigating fungal diversity description across different ecosystem types. Here, we explored the spatial distribution of the soil fungal diversity on a broad geographical scale, using the French Soil Quality Monitoring Network that covers the whole French territory (2171 soils sampled along a systematic grid). Fungal alpha diversity was assessed directly from soil DNA using a meta-barcoding approach by targeting the 18S rDNA gene. The total accumulated fungal diversity across France included 136 219 operational taxonomic units (OTUs), i.e., about 1 % of worldwide soil fungal diversity (based on a maximum diversity estimate of 12 million) for a territory representing only 0.3 % of the terrestrial surface on Earth. Based on this dataset, the first extensive map of fungal alpha diversity was drawn and showed a heterogeneous and spatially structured distribution in large biogeographical patterns of 231 km radius for richness (Hill diversity of order 0) and smaller patterns of 36 km radius for dominant fungi (Hill diversity of order 2). As related to other environmental parameters, the spatial distribution of fungal diversity (Hill numbers based on different orders of diversity) was mainly influenced by local filters such as soil characteristics and land management and also by global filters such as climate conditions with various relative influences. Interestingly, cropped soils exhibited the highest pool of fungal diversity relative to forest and vineyard soils. To complement this, soil fungal OTU network interactions were calculated for the different land uses across France. They varied hugely and showed a loss of 75 % of the complexity in crop systems and grasslands compared to forests and up to 83 % in vineyard systems. Overall, our study revealed that a nationwide survey with a high spatial-resolution approach is relevant for deeply investigating the spatial distribution and determinism of soil fungal diversity. Our findings provide novel insights for a better understanding of soil fungal ecology across the 18S rDNA gene and upgrade biodiversity conservation policies by supplying representative repositories dedicated to soil fungi.</p

Phosphorylation Regulates SIRT1 Function

BACKGROUND: SIR2 is an NAD(+)-dependent deacetylase [1]-[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo[9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported. METHODOLOGY/PRINCIPAL FINDINGS: We identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD(+)-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540) disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13]. CONCLUSIONS/SIGNIFICANCE: Pharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]-[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify phosphorylation by cell cycle dependent kinases as a major mechanism controlling the level and function of this sirtuin and complement recent reports of factors that inhibit [17], [18] and activate [19] SIRT1 by protein-protein interactions

Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames.

Complex oncoviruses contain, in addition to the classical retroviral genes (gag, pol, and env), a region (X) located between the envelope sequences and the 3' long terminal repeat. The X region contains two genes, tax and rex, whose protein products are involved in transcriptional and posttranscriptional regulation of viral expression. In addition to these activators, the bovine leukemia virus (BLV) and the human T-cell leukemia virus (HTLV) contain alternative open reading frames (R3 and G4 for BLV; p30, p13, and p12 for HTLV). As a virus/animal model for HTLV-induced leukemogenesis, BLV provirus can be injected intradermally into sheep, where it induced B-lymphocyte transformation. Deletion of the R3 and G4 sequences from an infectious and tumorigenic BLV provirus greatly impaired the in vivo propagation of the viruses as demonstrated by DNA polymerase chain reaction, RNA blots, structural-protein ELISA, and immunofluorescence analysis. Our results show that the alternative open reading frames are required for maintaining high virus loads during the course of persistent infection in vivo. Thus, R3 and G4 are candidates for antiviral drug development. Furthermore, viruses with a deletion in these sequences should be tested as live attenuated vaccines

Both wild-type and strongly attenuated bovine leukemia viruses protect peripheral blood mononuclear cells from apoptosis.

Bovine leukemia virus (BLV) and the human T-cell leukemia viruses belong to the same subfamily of oncoviruses. Although much attention has focused on the mechanisms of cell proliferation and transformation by these viruses, experiments on the apoptotic process have yielded conflicting data in in vitro cell culture. Experimental infection of sheep with BLV proviruses offers the opportunity to analyze apoptosis in vivo. Here, we show that BLV-infected peripheral mononuclear cells, cultivated ex vivo, are protected from spontaneous programmed cell death. Moreover, the virus is able to specifically interfere with the apoptotic program of infected B lymphocytes. Strongly attenuated mutant proviruses that harbor deletions in the G4 and/or R3 genes also decrease the global susceptibility to apoptosis at levels similar to those obtained with the wild-type virus. In addition, cell culture supernatants from wild-type and mutant viruses can prevent uninfected cells from undergoing programmed cell death. These observations demonstrate that the R3 and G4 genes are not required to maintain both direct and indirect protection against apoptosis. They also imply that the level of programmed cell death observed ex vivo is independent of the amounts of proviruses in the animals. The failure of these cells to undergo apoptosis might be related to the pathogenesis induced by BLV