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1.  FOXP3 controls an miR-146/NFκB negative feedback loop that inhibits apoptosis in breast cancer cells 
Cancer research  2015;75(8):1703-1713.
FOXP3 functions not only as the master regulator in regulatory T cells but also as an X-linked tumor suppressor. The tumor suppressive activity of FOXP3 has been observed in tumor initiation, but its role during tumor progression remains controversial. Moreover, the mechanism of FOXP3-mediated tumor suppressive activity remains largely unknown. Using chromatin immunoprecipitation sequencing, we identified a series of potential FOXP3-targeted microRNAs (miRs) in MCF7 cells. Notably, FOXP3 significantly induced the expression of miR-146a/b. In vitro, FOXP3-induced miR-146a/b prevented tumor cell proliferation and enhanced apoptosis. Functional analyses in vitro and in vivo revealed that FOXP3-induced miR-146a/b negatively regulate NF-κB activation by inhibiting the expression of IRAK1 and TRAF6. In chromatin immunoprecipitation assays, FOXP3 directly bound the promoter region of miR-146a but not of miR-146b, and FOXP3 interacted directly with NF-κB p65 to regulate an miR-146-NF-κB negative feedback regulation loop in normal breast epithelial and tumor cells, as demonstrated with luciferase reporter assays. Although FOXP3 significantly inhibited breast tumor growth and migration in vitro and metastasis in vivo, FOXP3-induced miR-146a/b contributed only to the inhibition of breast tumor growth. These data suggest that miR-146a/b contribute to FOXP3-mediated tumor suppression during tumor growth by triggering apoptosis. The identification of a FOXP3-miR-146-NF-κB axis provides an underlying mechanism for disruption of miR-146 family member expression and constitutive NF-κB activation in breast cancer cells. Linking the tumor suppressor function of FOXP3 to NF-κB activation reveals a potential therapeutic approach for cancers with FOXP3 defects.
PMCID: PMC4706751  PMID: 25712342
FOXP3; microRNA; NF-κB; breast cancer
2.  An mTORC1-Mdm2-Drosha Axis for miRNA Biogenesis in Response to Glucose- and Nutrient-deprivation 
Molecular cell  2015;57(4):708-720.
mTOR senses nutrient and energy status to regulate cell survival and metabolism in response to environmental changes. Surprisingly, targeted mutation of Tsc1, a negative regulator of mTORC1, caused a broad reduction in miRNAs due to Drosha degradation. Conversely, targeted mutation of Raptor, an essential component of mTORC 1, increased miRNA biogenesis. mTOR activation increased expression of Mdm2, which is hereby identified as the necessary and sufficient ubiquitin E3 ligase for Drosha. Drosha was induced by nutrient and energy deprivation and conferred resistance to glucose deprivation. Using a high throughput screen of a miRNA library, we identified 4 miRNAs that were necessary and sufficient to protect cells against glucose deprivation-induced apoptosis. These miRNA was regulated by glucose through the mTORC1-MDM2- Drosha axis. Taken together, our data reveal an mTOR-Mdm2-Drosha pathway in mammalian cells that broadly regulates miRNA biogenesis as a response to alteration in cellular environment.
PMCID: PMC4511160  PMID: 25639470
3.  A Critical Role for Regulated Wnt-Myc Pathway in Naïve T cells Survival 
Wnt signaling is involved in T cell development, activation, and differentiation. However, the role for Wnt signaling in mature naïve T cells has not been investigated. Here, we report that activation of Wnt signaling in T cell lineages by deletion of the Apc gene causes spontaneous T cell activation and severe T lymphopenia. The lymphopenia is the result of rapid apoptosis of newly exported, mature T cells in the periphery and is not due to defects in thymocyte development or emigration. Using chimera mice consisting of both WT and Apc-deficient T cells, we found that loss of naïve T cells is due to T-cell intrinsic dysregulation of Wnt signaling. Since Apc deletion causes over-expression of the Wnt target gene cMyc, we generated mice with combined deletion of the cMyc gene. Since combined deletion of cMyc and Apc attenuated T cell loss, cMyc over-expression is partially responsible for spontaneous T cell apoptosis and lymphopenia. Cumulatively, our data reveals a missing link between Wnt signaling and survival of naïve T cells.
PMCID: PMC4272883  PMID: 25429066
4.  Siglec-G/10 in self–nonself discrimination of innate and adaptive immunity 
Glycobiology  2014;24(9):800-806.
Siglec-G/10 is broadly expressed on B cells, dendritic cells and macrophage subsets. It binds strongly to CD24, a small glycosyl-phosphatidylinositol-anchored sialoprotein, in a sialylation-dependent manner. Targeted mutation of Siglecg dramatically elevates the level of natural IgM antibodies and its producer, B1 B cells. Incorporation of Siglec-G ligands to both T-dependent and T-independent immunogens reduces antibody production and induces B-cell tolerance to subsequent antigen challenges. By interacting with CD24, Siglec-G suppresses inflammatory responses to danger (damage)-associated molecular patterns, such as heat-shock proteins and high mobility group protein 1, but not to Toll-like receptor ligands. By a CD24-independent mechanism, Siglec-G has been shown to associate with Cbl to cause degradation of retinoic acid-inducible gene 1 and reduce production of type I interferon in response to RNA virus infection. The negative regulation by Siglec-G/10 may provide a mechanism for the host to discriminate between infectious nonself and noninfectious self, as envisioned by the late Dr. Charles A. Janeway.
PMCID: PMC4116048  PMID: 24996822
DAMP; PAMP; self–nonself; Siglec-G
5.  Intracellular CD24 Disrupts the ARF-NPM Interaction and Enables Mutational and Viral Oncogene-mediated p53 Inactivation 
Nature communications  2015;6:5909.
CD24 is over-expressed in nearly 70% human cancers while TP53 is the most frequently mutated tumor suppressor gene that functions in a context-dependent manner. Here we show that both targeted mutation and shRNA silencing of CD24 retard the growth, progression, and metastasis of prostate cancer. CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2, and decrease levels of p53 and the p53 target p21/CDKN1A. CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen. In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels. These data provide a general mechanism for functional inactivation of ARF and reveal an important cellular context for genetic and viral inactivation of TP53.
PMCID: PMC4300525  PMID: 25600590
ARF; p53; CD24; tumor suppression
6.  FOXP3 as X-linked Tumor Suppressor 
Discovery medicine  2010;10(53):322-328.
The FOXP3 gene was initially identified because its mutation caused lethal autoimmune diseases in mouse and human. Mice with heterozygous mutation of Foxp3 succumb to mammary tumor spontaneously, while those with prostate-specific deletion develop prostate intraepithelial neoplasia. Somatic mutations, deletion and epigenetic inactivation of FOXP3 are widespread among human breast and prostate cancers. Unlike autosomal tumor suppressor genes that were usually inactivated by mutations in both alleles, X-linked FOXP3 mutations in cancer samples are usually heterozygous. The unique inheritance suggests a new approach to reactivation FOXP3 for cancer therapy.
PMCID: PMC4500105  PMID: 21034673
FOXP3; Tumor suppressor gene; Human cancer; X-linked gene; X-chromosome inactivation
7.  Deletion of CD24 impairs development of heat shock protein gp96 -driven autoimmune disease through expansion of myeloid derived suppressor cells 
CD24 binds to and suppresses inflammation triggered by danger associated molecular patterns (DAMPS) such as heat-shock proteins (HSPs) and HMGB1. Paradoxically, CD24 has been shown to enhance autoimmune disease. Here we attempt to reconcile this paradox by deletion of CD24 (24KO) in a lupus-like disease model driven by forced expression of HSP gp96 at the cell surface (tm). As expected, tm24KO mice showed increased CD11c+ DC activation coupled to a significant increase in DC-specific IL-12 production compared to tm mice. However, tm24KO mice showed less CD4 T cell activation and peripheral inflammatory cytokine production in comparison to tm mice. We characterized an enhanced immune suppressive milieu in tm24KO mice distinguished by increased TGF-β and greater Treg suppressive capacity. We found greater absolute numbers of MDSCs in tm24KO mice and showed that the Ly6C+ MDSC subset had greater suppressive capacity from tm24KO mice. Deletion of CD24 in tm mice led to diminished lupus-like pathology as evidenced by anti-nuclear antibody deposition and glomerulonephritis. Finally, we show that expanded MDSC populations were mediated by increased free HMGB1 in tm24KO mice. Thus, the deletion of CD24 in an HSP-driven model of autoimmunity led to the unexpected development of Treg and MDSC populations that augmented immune tolerance. Further study of these populations as possible negative regulators of inflammation in the context of autoimmunity is warranted.
PMCID: PMC4185284  PMID: 24808359
autoimmunity; MDSC; CD24; gp96; HMGB1
8.  Intracellular CD24 disrupts the ARF–NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation 
Nature Communications  2015;6:5909.
CD24 is overexpressed in nearly 70% human cancers, whereas TP53 is the most frequently mutated tumour-suppressor gene that functions in a context-dependent manner. Here we show that both targeted mutation and short hairpin RNA (shRNA) silencing of CD24 retard the growth, progression and metastasis of prostate cancer. CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2 and decrease levels of p53 and the p53 target p21/CDKN1A. CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen. In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels. These data provide a general mechanism for functional inactivation of ARF and reveal an important cellular context for genetic and viral inactivation of TP53.
P53 is a tumour suppressor that is frequently mutated or downregulated in cancer. Here, Wang et al. show that CD24, a molecule frequently overexpressed in cancer, promotes p53 degradation by disrupting a regulatory ARF–MDM2 interaction, and silencing CD24 prevents the downregulation of p53.
PMCID: PMC4300525  PMID: 25600590
9.  The injury progression of T lymphocytes in a mouse model with subcutaneous injection of a high dose of sulfur mustard 
In clinical studies, the findings on sulfur mustard (SM) toxicity for CD3+CD4+ and CD3+CD8+ T lymphocyte subsets are contradictory. In animal experiments, the effect of SM on the T cell number and proliferation is incompatible and is even the opposite of the results in human studies. In this study, we observed the dynamic changes of T lymphocytes in the first week in a high-dose SM-induced model.
Mice were exposed to SM by subcutaneous injection (20 mg/kg) and were sacrificed 4 h, 24 h, 72 h and 168 h later. Spleen T lymphocyte proliferation was evaluated by 3H-TdR. Flow cytometric analysis was used to observe the percentage of CD3+CD4+ and CD3+CD8+ T lymphocyte subsets. The IL-1β, IL-6, IL-10 and TNF-α levels in plasma were assayed using the Luminex method. DNA damage in bone marrow cells was observed with the single cell gel electrophoresis technique (SCGE).
SM continuously inhibited the proliferation of lymphocytes for 7 days, and there was a significant rebound of Con A-induced T lymphocyte proliferation only at 24 h. The percentage of CD3+CD4+ and CD3+CD8+ lymphocytes was upregulated, which was accompanied by increased IL-1β and TNF-α and decreased IL-10. The IL-6 level was gradually decreased in the PG group at 4 h. The peak of lymphocytic apoptosis and DNA damage occurred at 24 h and 72 h, respectively.
Our results show that SM significantly inhibited T lymphocyte proliferation as well as induced CD3+CD4+ and CD3+CD8+ upregulation. SM intoxication also significantly increased the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and inhibited the level of anti-inflammatory cytokine IL-10. Our results may partly be due to the significant SM induced significant apoptosis and necrosis of lymphocytes as well as DNA damage of bone marrow cells. The results provided a favorable evaluation of SM immune toxicity in an animal model.
PMCID: PMC4341234  PMID: 25722879
Sulfur mustard; T lymphocyte; Apoptosis; Cytokine; DNA damage
10.  Broad and direct interaction between TLR and Siglec families of pattern recognition receptors and its regulation by Neu1 
eLife  2014;3:e04066.
Both pathogen- and tissue damage-associated molecular patterns induce inflammation through toll-like receptors (TLRs), while sialic acid-binding immunoglobulin superfamily lectin receptors (Siglecs) provide negative regulation. Here we report extensive and direct interactions between these pattern recognition receptors. The promiscuous TLR binders were human SIGLEC-5/9 and mouse Siglec-3/E/F. Mouse Siglec-G did not show appreciable binding to any TLRs tested. Correspondingly, Siglece deletion enhanced dendritic cell responses to all microbial TLR ligands tested, while Siglecg deletion did not affect the responses to these ligands. TLR4 activation triggers Neu1 translocation to cell surface to disrupt TLR4:Siglec-E interaction. Conversely, sialidase inhibitor Neu5Gc2en prevented TLR4 ligand-induced disruption of TLR4:Siglec E/F interactions. Absence of Neu1 in hematopoietic cells or systematic treatment with sialidase inhibitor Neu5Gc2en protected mice against endotoxemia. Our data raised an intriguing possibility of a broad repression of TLR function by Siglecs and a sialidase-mediated de-repression that allows positive feedback of TLR activation during infection.
eLife digest
Many living things have an immune system that is able to detect invading bacteria, viruses and other pathogens and trigger a response targeted against the threat before it causes lasting damage. Cells employ a number of different receptors that can detect these pathogens or the molecules that they produce.
In animals, toll-like receptors (or TLRs) are a type of protein that recognizes patterns or structures that are found in many different types of pathogen, known as pathogen-associated molecular patterns (or PAMPs). Injured cells release proteins that are also recognized by toll-like receptors and are called danger associated molecular patterns (or DAMPs). An immune response is triggered when PAMPs and DAMPs are recognized, but the response must be properly controlled. If it goes awry, it can result in an over-activation of the immune cells that can lead to life-threatening conditions, one of which is called sepsis.
Siglecs are proteins that bind to a sugar molecule, which is found attached to many other proteins, and are known to inhibit the immune response. However, it remained unclear how Siglecs do this and if they can interact directly with toll-like receptors. Chen et al. now show that most (although not all) Siglecs bind to TLRs, and that deleting the gene for a Siglec protein that can bind to multiple TLRs boosted the response of the immune cells to a range of microbial PAMPs. Deleting the gene for another Siglec that did not bind to any TLRs had no effect on the immune response.
Chen et al. suggest that the Siglec proteins that interact with toll-like receptors act a bit like a brake that slows down the activation of the receptors. However, when an immune cell detects a foreign molecule through a TLR, an enzyme called Neu1 is relocated from the inside of the cell to the cell's surface, where it removes the sugar molecules from the TLRs. This disrupts the interaction between the TLRs and the Siglecs, thus activating the receptors and triggering an immune response against the invading pathogen or damaged cells. This represents a newly discovered mechanism that can regulate the signaling of TLRs.
Chen et al. also show that a chemical compound that stops the function of the Neu1 enzyme prevents the toll-like receptors—and hence the immune cells—from becoming overly activated. Mice treated with this compound are protected against sepsis triggered by the presence of a bacterial PAMP. These results suggest that the Neu1 enzyme may be a promising new target for treating sepsis; further work will now be required to assess the potential side effects caused by inhibiting this enzyme.
PMCID: PMC4168287  PMID: 25187624
Siglec; toll-like receptor; sialidase; E. coli; mouse
11.  Ribosomal protein S27-like is a physiological regulator of p53 that suppresses genomic instability and tumorigenesis 
eLife  2014;3:e02236.
Cell-based studies showed that several Mdm2-binding ribosomal proteins, upon overexpression, stabilize and activate p53. In contrast, here we show in a mouse knockout study that Mdm2-binding ribosomal protein S27-like (Rps27l), upon disruption, activates p53. Germline inactivation of Rps27l triggers ribosomal stress to stabilize Mdm2, which degrades Mdm4 to reduce Mdm2-Mdm4 E3 ligase towards p53, leading to p53-dependent apoptotic depletion of hematopoietic stem cells and postnatal death, which is rescued by Trp53 deletion. Paradoxically, while increased p53 is expected to inhibit tumorigenesis, Rps27l−/−;Trp53+/− mice develop lymphomas at higher incidence with p53 loss-of-heterozygosity and severe genome aneuploidy, suggesting that Rps27l disruption impose a selection pressure against p53. Thus, Rps27l has dual functions in p53 regulation: under Trp53+/+ background, Rps27l disruption triggers ribosomal stress to induce p53 and apoptosis, whereas under Trp53+/− background, Rps27l disruption triggers genomic instability and Trp53 deletion to promote tumorigenesis. Our study provides a new paradigm of p53 regulation.
eLife digest
There are over a hundred different types of cancer that can affect humans; but, in general, all cancers are caused by mutations that cause cells to grow and divide abnormally. ‘Tumor suppressor genes’ are genes that normally protect a cell from genetic changes that can lead a cell towards becoming cancerous.
About half of all cancers in humans have a mutation in one of the two copies of a tumor suppressor gene that encodes a protein called p53, which helps to control how and when cells grow and divide. In normal cells, the p53 protein can be activated in various ways. Damage to a cell's DNA triggers p53 to stop the cell growing, which gives the cell time to repair the DNA damage. However, if the damage is too severe and cannot be repaired, p53 essentially causes the cell to kill itself, via a process called apoptosis. Furthermore, if a cell has problems building new copies of its protein-making machinery, some of the parts (called ribosomal proteins) that make up these molecular machines can also lead to p53 being activated.
By deleting the gene for a protein called Rps27l that is a newly characterized ribosomal protein, Xiong et al. have discovered that, in mice, Rps27I regulates the p53 protein in two different ways. In normal cells, Rps27l appears to inhibit p53, which is likely to encourage cancer to develop. But, if a cell has already lost a copy of the p53 gene—a situation that would normally encourage the cells to accrue further mutations and become cancerous—Rps27l acts as a tumor suppressor. In these mutated cells, the Rps27l protein helps to maintain the stability of the genome and prevent the loss of the second copy of gene for p53, and so protects the cell from becoming cancerous.
Thus Rps27l can either activate or inactivate p53 activity depending on how many copies of the gene for p53 remain intact. The next challenge is to investigate if Rps27l levels determine the early-onset of tumor development in cancer-prone cells seen in patients with Li-Fraumeni syndrome, who are born with a mutated copy of the p53 gene.
PMCID: PMC4163686  PMID: 25144937
p53; Mdm2-Mdm4; ribosomal proteins; mouse knockout; aneuploidy; tumorigenesis; human; mouse
12.  Laforin Prevents Stress-Induced Polyglucosan Body Formation and Lafora Disease Progression in Neurons 
Molecular neurobiology  2013;48(1):49-61.
Glycogen, the largest cytosolic macromolecule, is soluble because of intricate construction generating perfect hydrophilic-surfaced spheres. Little is known about neuronal glycogen function and metabolism, though progress is accruing through the neurodegenerative epilepsy Lafora disease (LD) proteins laforin and malin. Neurons in LD exhibit Lafora bodies (LBs), large accumulations of malconstructed insoluble glycogen (polyglucosans). We demonstrated that the laforin-malin complex reduces LBs and protects neuronal cells against endoplasmic reticulum stress-induced apoptosis. We now show that stress induces polyglucosan formation in normal neurons in culture and in brain. This is mediated by increased glucose-6-phosphate allosterically hyperactivating muscle glycogen synthase (GS1), and is followed by activation of the glycogen digesting enzyme glycogen phosphorylase. In the absence of laforin, stress-induced polyglucosans are undigested and accumulate into massive LBs, and in laforin-deficient mice stress drastically accelerates LB accumulation and LD. The mechanism through which laforin-malin mediates polyglucosan degradation remains unclear but involves GS1 dephosphorylation by laforin. Our work uncovers the presence of rapid polyglucosan metabolism as part of the normal physiology of neuroprotection. We propose that deficiency in the degradative phase of this metabolism, leading to LB accumulation and resultant seizure predisposition and neurodegeneration, underlies LD.
PMCID: PMC3722335  PMID: 23546741
Laforin; muscle glycogen synthase; polyglucosan; ER stress; neuron; Lafora body; Lafora disease; neurodegeneration
13.  Preserving Sialic Acid-dependent Pattern Recognition by CD24-Siglec G Interaction for Therapy of Polybacterial Sepsis 
Nature biotechnology  2011;29(5):428-435.
Control of inflammation is critical for therapy of infectious diseases. Pathogen-associated and/or danger-associated molecular patterns (PAMPs and DAMPs, respectively) are the two major inducers of inflammation. Because the CD24-Siglec G/10 interactions selectively repress inflammatory response to DAMPs, microbial disruption of the negative regulation would provide a general mechanism to exacerbate inflammation. Here we show that the sialic acid-based pattern recognitions of CD24 by Siglec G/10 are targeted by sialidases in polybacterial sepsis. Sialidase inhibitors protect mice against sepsis by a CD24-Siglecg-dependent mechanism, whereas a targeted mutation of either CD24 or Siglecg exacerbates sepsis. Bacterial sialidase and host CD24 and Siglecg genes interact to determine pathogen virulence. Our data demonstrate a critical role for disrupting sialic acid-based pattern recognitions in microbial virulence and suggest a therapeutic approach to dampen harmful inflammatory response during infection.
PMCID: PMC4090080  PMID: 21478876
14.  mTOR Regulation and Therapeutic Rejuvenation of Aging Hematopoietic Stem Cells 
Science signaling  2009;2(98):ra75.
Age-related declines in hematopoietic stem cell (HSC) function may contribute to anemia, poor response to vaccination, and tumorigenesis. Here, we show that mammalian target of rapamycin (mTOR) activity is increased in HSCs from old mice compared to those from young mice. mTOR activation through conditional deletion of Tsc1 in the HSC of young mice mimicked the phenotype of HSC from aged mice in various ways. These included increased abundance of the mRNA encoding the CDK inhibitors p16Ink4a, p19Arf, and p21Cip1, a relative decrease in lymphopoiesis, and impaired capacity to reconstitute the hematopoietic system. In the old mice, rapamycin increased the life span, restored the self renewal and hematopoiesis of HSC, and enabled effective vaccination against a lethal challenge with influenza virus. Together, our data implicate mTOR signaling in HSC aging and demonstrate the potential of mTOR inhibitors for restoring hematopoiesis in the elderly.
PMCID: PMC4020596  PMID: 19934433
15.  FOXP3 Regulates Sensitivity of Cancer Cells to Irradiation by Transcriptional Repression of BRCA1 
Cancer research  2013;73(7):10.1158/0008-5472.CAN-12-2481.
FOXP3 is an X-linked tumor suppressor gene and a master regulator in T regulatory cell function. This gene has been found to be mutated frequently in breast and prostate cancers and to inhibit tumor cell growth, but its functional significance in DNA repair has not been studied. We found that FOXP3 silencing stimulates homologous recombination-mediated DNA repair and also repair of γ-irradiation-induced DNA damage. Expression profiling and chromatin-immunoprecipitation analyses revealed that FOXP3 regulated the BRCA1-mediated DNA repair program. Among 48 FOXP3-regulated DNA repair genes, BRCA1 and 12 others were direct targets of FOXP3 transcriptional control. Site-specific interaction of FOXP3 with the BRCA1 promoter repressed its transcription. Somatic FOXP3 mutants identified in breast cancer samples had reduced BRCA1 repressor activity, while FOXP3 silencing and knock-in of a prostate cancer-derived somatic FOXP3 mutant increased the radioresistance of cancer cells. Together our findings provide a missing link between FOXP3 function and DNA repair programs.
PMCID: PMC3815443  PMID: 23319807
16.  FOXP3: Genetic and Epigenetic Implications for Autoimmunity 
Journal of autoimmunity  2013;41:72-78.
FOXP3 plays an essential role in the maintenance of self-tolerance and, thus, in preventing autoimmune diseases. Inactivating mutations of FOXP3 cause immunodysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome. FOXP3-expressing regulatory T cells attenuate autoimmunity as well as immunity against cancer and infection. More recent studies demonstrated that FOXP3 is an epithelial cell-intrinsic tumor suppressor for breast, prostate, ovary and other cancers. Corresponding to its broad function, FOXP3 regulates a broad spectrum of target genes. While it is now well established that FOXP3 binds to and regulates thousands of target genes in mouse and human genomes, the fundamental mechanisms of its broad impact on gene expression remain to be established. FOXP3 is known to both activate and repress target genes by epigenetically regulating histone modifications of target promoters. In this review, we first focus on germline mutations found in the FOXP3 gene among IPEX patients, then outline possible molecular mechanisms by which FOXP3 epigenetically regulates its targets. Finally, we discuss clinical implications of the function of FOXP3 as an epigenetic modifier. Accumulating results reveal an intriguing functional convergence between FOXP3 and inhibitors of histone deacetylases. The essential epigenetic function of FOXP3 provides a foundation for experimental therapies against autoimmune diseases.
PMCID: PMC3622774  PMID: 23313429
17.  Cytopenia and Autoimmune Diseases: a Vicious Cycle fueled by mTOR Dysregulation in Hematopoietic Stem Cells 
Journal of autoimmunity  2013;41:182-187.
A long-standing but poorly understood defect in autoimmune diseases is dysfunction of the hematopoietic cells. Leukopenia is often associated with systemic lupus erythematous (SLE) and other autoimmune diseases. In addition, homeostatic proliferation of T cells, which is a host response to T cell lymphopenia, has been implicated as potential cause of rheumatoid arthritis (RA) in human and experimental models of autoimmune diabetes in the NOD mice and the BB rats. Conversely, successful treatments of aplastic anemia by immune suppression suggest that the hematologic abnormality may have a root in autoimmune diseases. Traditionally, the link between autoimmune diseases and defects in hematopoietic cells has been viewed from the prism of antibody-mediated hemolytic cytopenia. While autoimmune destruction may well be part of pathogenesis of defects in hematopoietic system, it is worth considering the hypothesis that either leukopenia or pancytopenia may also result directly from defective hematopoietic stem cells (HSC). We have recently tested this hypothesis in the autoimmune Scurfy mice which has mutation Foxp3, the master regulator of regulatory T cells. Our data demonstrated that due to hyperactivation of mTOR, the HSC in the Scurfy mice are extremely poor in hematopoiesis. Moreover, rapamycin, an mTOR inhibitor rescued HSC defects and prolonged survival of the Scurfy mice. Our data raised the intriguing possibility that targeting mTOR dysregulation in the HSC may help to break the vicious cycle between cytopenia and autoimmune diseases.
PMCID: PMC3622805  PMID: 23375848
Cytopenia; homeostatic proliferation; CD24; mTOR; hematopoietic stem cells; autoimmune diseases; inflammatory cytokines; rapamycin
18.  A Critical Role for Rictor in T-lymphopoiesis1 
Apart from a critical role for Notch and pre-TCR, the signaling pathway required for T-lymphopoiesis is largely unknown. Given the potential link between Notch and mTOR signaling in cancer cells, we used mice with conditional deletion of either Raptor or Rictor genes to determine potential contribution of the mTOR complex I and II in T-lymphopoiesis. Our data demonstrated that targeted mutation of Rictor in the thymocytes drastically reduced the thymic cellularity, primarily by reducing proliferation of the immature thymocytes. Rictor-deficiency caused a partial block of thymocyte development at the double negative 3 stage. The effect of Rictor deficiency is selective for the T cell lineage, as the development of B cells, erythorocytes and myeloid cells are largely unaffected. Analysis of bone marrow chimera generated from a mixture of WT and Rictor-deficient hematopoietic stem cells demonstrated that the function of Rictor is cell-intrinsic. These data revealed a critical function of TORC2 in T-lymphopoiesis.
PMCID: PMC3412163  PMID: 22815285
19.  Laforin is Required for the Functional Activation of Malin in Endoplasmic Reticulum Stress Resistance in Neuronal Cells 
The FEBS journal  2012;279(14):2467-2478.
Mutations in either EPM2A, the gene encoding a dual-specificity phosphatase named laforin, or NHLRC1, the gene encoding an E3 ubiquitin ligase named malin, cause Lafora disease (LD) in humans. LD is a fatal neurological disorder characterized by progressive myoclonus epilepsy, severe neurological deterioration, and accumulation of poorly branched glycogen inclusions, called Lafora bodies (LBs) or polyglucosan bodies (PGBs), within the cell cytoplasm. The molecular mechanism underlying the neuropathogenesis of LD remains unknown. Here we present data demonstrating that in the cells expressing low levels of laforin protein, overexpressed malin and its LD-causing missense mutants are stably polyubiquitinated. Malin and malin mutants form ubiquitin-positive aggregates in or around the nuclei of the cells in which they are expressed. Neither wild type (WT) malin nor its mutants elicit endoplasmic reticulum (ER) stress, although the mutants exaggerate the response to ER stress. Overexpressed laforin impairs the polyubiquitination of malin and recruits malin to PGBs. The recruitment and activities of laforin and malin are both required for the PGB disruption. Consistently, targeted deletion of laforin in brain cells from Epm2a knockout (KO) mice increases polyubiquitinated proteins. Knockdown of Epm2a or Nhlrc1 in neuronal Neuro2a cells shows that they cooperate to allow cells to resist ER stress and apoptosis. These results reveal that a functional laforin-malin complex plays a critical role in destroying LB and relieving ER stress, implying that a causative pathogenic mechanism underlies their deficiency in LD.
PMCID: PMC3407668  PMID: 22578008
Laforin; Malin; Endoplasmic Reticulum Stress; Neuronal Cells and Polyglucosan
20.  CD24 on thymic antigen presenting cells regulates negative selection of myelin antigen specific T lymphocytes 
European Journal of Immunology  2012;42(4):924-935.
Negative selection plays a key role in the clonal deletion of autoreactive T cells in the thymus. However, negative selection is incomplete; as high numbers of autoreactive T cells can be detected in normal individuals, mechanisms that regulate negative selection must exist. In this regard, we previously reported that CD24, a GPI-anchored glycoprotein, is required for thymic generation of autoreactive T lymphocytes. The CD24-deficient 2D2 TCR transgenic mice (2D2+CD24-/-), whose TCR recognizes myelin oligodendrocyte glycoprotein (MOG), fail to generate functional 2D2 T cells. However, it was unclear if the CD24 function involved regulation of negative selection, and if so, what cellular mechanisms were involved. Here we show that elimination of MOG or Aire gene expression in 2D2+CD24-/- mice - through the creation of 2D2+CD24-/-MOG-/- or 2D2+CD24-/-Aire-/-mice - completely restores thymic cellularity and function of 2D2 T cells. Restoration of CD24 expression on dendritic cells (DCs), but not on thymocytes also partially restores 2D2 T-cell generation in 2D2+CD24-/- mice. Taken together, we propose that CD24 expression on thymic antigen presenting cells (mTECs, DCs) down-regulates autoantigen-mediated clonal deletion of autoreactive thymocytes.
PMCID: PMC3359065  PMID: 22213356
21.  FOXP3 Orchestrates H4K16 Acetylation and H3K4 Tri-Methylation for Activation of Multiple Genes through Recruiting MOF and Causing Displacement of PLU-1 
Molecular cell  2011;44(5):770-784.
Both H4K16 acetylation and H3K4 tri-methylation are required for gene activation. However, it is still largely unclear how these modifications are orchestrated by transcriptional factors. Here we analyzed the mechanism of the transcriptional activation by FOXP3, an X-linked suppressor of autoimmune diseases and cancers. FOXP3 binds near transcriptional start sites of its target genes. By recruiting MOF and displacing histone H3K4 demethylase PLU-1, FOXP3 increases both H4K16 acetylation and H3K4 tri-methylation at the FOXP3-associated chromatins of multiple FOXP3-activated genes. RNAi-mediated silencing of MOF reduced both gene activation and tumor suppression by FOXP3, while both somatic mutations in clinical cancer samples and targeted mutation of FOXP3 in mouse prostate epithelial disrupted nuclear localization of MOF. Our data demonstrate a pull-push model in which a single transcription factor orchestrates two epigenetic alterations necessary for gene activation and provide a mechanism for somatic inactivation of the FOXP3 protein function in cancer cells.
PMCID: PMC3243051  PMID: 22152480
22.  The Tuberous Sclerosis Complex -mTOR Pathway Maintains the Quiescence and Survival of Naïve T Cells 
Naïve T cells receive stimulation from the positive selecting ligand in the periphery for their survival. This stimulation does not normally lead to overt activation of T cells, as the T cells remain largely quiescent until they receive either antigenic or lymphopenic stimuli. The underlying mechanism responsible for survival and quiescence of the naïve T cells remain largely unknown. Here we report that T cell-specific deletion of Tsc1, a negative regulator of mTOR, resulted in both spontaneous losses of quiescence and cellularity, especially within the CD8 subset. The Tsc1-deficient T cells have increased cell proliferation and apoptosis. Tsc1 deletion affects the survival and quiescence of T cells in the absence of antigenic stimulation. Loss of quiescence but not cellularity was inhibited by rapamycin. Our data demonstrate that TSC-mTOR maintains quiescence and survival of T cells.
PMCID: PMC3151493  PMID: 21709159
23.  A hypermorphic SP1-binding CD24 variant associates with risk and progression of multiple sclerosis 
A large number of risk alleles have been identified for multiple sclerosis (MS). However, how genetic variations may affect pathogenesis remains largely unknown for most risk alleles. Through direct sequencing of CD24 promoter region, we identified a cluster of 7 new single nucleotide polymorphisms in the CD24 promoter. A hypermorphic haplotype consisting of 3 SNPs was identified through association studies consisting of 935 control and 764 MS patients (P=0.001, odds ratio 1.3). The variant is also associated with more rapid progression of MS (P=0.016, log rank test). In cells that are heterozygous for the risk allele, chromatin immunoprecipitation revealed that risk allele specifically bind to a transcription factor SP1, which is selectively required for the hypermorphic promoter activity of the variant. In MS patients, the CD24 transcript levels associate with the SP1-binding variant in a dose-dependent manner (P=7x10-4). Our data revealed a potential role for SP1-mediated transcriptional regulation in MS pathogenesis.
PMCID: PMC3426393  PMID: 22937211
Multiple sclerosis (MS); SP-binding CD24; promoter; risk alleles; single nucleotide polymorphisms (SNP)
24.  α-Lactosylceramide as a novel “sugar-capped” CD1d ligand for Natural Killer T-cells: biased cytokine profile and therapeutic activities 
Chembiochem  2008;9(9):1423-1430.
The iNKT cells have emerged as an important regulator for immunity to infection, cancer as well as autoimmune diseases. The iNKT cells can be activated by glycolipids binding CD1d. The most effective iNKT ligand reported to date is α-galactosylceramide (α-GalCer) which stimulates iNKT cells to secrete both Th-1 and Th-2 cytokines. Indiscriminative induction of both types of cytokines may limit the therapeutic potential of iNKT ligands, as Th-1 and Th-2 cytokines play different roles under physiological and pathological conditions. Therefore a ligand with a biased cytokine release profile is highly desirable. Here we report the synthesis and biological activity of α-lactosylceramide (α-LacCer). Our data demonstrates that the α-LacCer can stimulate the iNKT cells to proliferate and release cytokines, both in vitro and in vivo. Interestingly, while α-LacCer is approximately 1,000-fold less efficient in inducing Th-1 cytokine, it is as potent as α-GalCer in the induction of Th-2 cytokine. Thus, α-LacCer is a novel compound that induces a biased cytokine release. The processing by β-glycosidase was critical for α-LacCer activity. Moreover, in experimental therapies, α-LacCer is at least as potent as α-GalCer in the treatment of tumors and experimental autoimmune encephalomyelitis.
PMCID: PMC3398384  PMID: 18478523
α-GalCer; α-LacCer; iNKT cell; glycolipids; CD1d
25.  Targeting HIF1α eliminates cancer stem cells in hematological malignancies 
Cell stem cell  2011;8(4):399-411.
Molecular targeting of cancer stem cells has therapeutic potential for efficient treatment of cancer although relatively few specific targets have so far been identified. Hypoxia-inducible factor was recently shown to regulate tumorigenic capacity of glioma stem cells under hypoxic condition. Surprisingly, we found that, under normoxia, HIF1α signaling was selectively activated in the stem cells of mouse lymphoma and human acute myeloid leukemia (AML). HIF1a ShRNA and HIF inhibitors abrogated the colony forming unit activity of mouse lymphoma and human AML CSCs. Importantly, the HIF inhibitor echinomycin efficiently eradicated mouse lymphoma and serially transplantable human AML in xenogeneic model by preferential elimination of CSCs. HIF1α maintains mouse lymphoma CSCs by repressing a negative feedback loop in the Notch pathway. Taken together, our results demonstrate an essential function of HIF1α-Notch interaction in maintaining CSCs and provide an effective approach to target CSCs for therapy of hematological malignancies.
PMCID: PMC3084595  PMID: 21474104

Results 1-25 (64)