SATB1 Defines the Developmental Context for Gene Silencing by Xist in Lymphoma and Embryonic Cells

SummaryThe noncoding Xist RNA triggers silencing of one of the two female X chromosomes during X inactivation in mammals. Gene silencing by Xist is restricted to a special developmental context in early embryos and specific hematopoietic precursors. Here, we show that Xist can initiate silencing in a lymphoma model. We identify the special AT-rich binding protein SATB1 as an essential silencing factor. Loss of SATB1 in tumor cells abrogates the silencing function of Xist. In lymphocytes Xist localizes along SATB1-organized chromatin and SATB1 and Xist influence each other's pattern of localization. SATB1 and its homolog SATB2 are expressed during the initiation window for X inactivation in ES cells. Importantly, viral expression of SATB1 or SATB2 enables gene silencing by Xist in embryonic fibroblasts, which normally do not provide an initiation context. Thus, our data establish SATB1 as a crucial silencing factor contributing to the initiation of X inactivation

T-bet negatively regulates autoimmune myocarditis by suppressing local production of interleukin 17

Experimental autoimmune myocarditis (EAM) appears after infectious heart disease, the most common cause of dilated cardiomyopathy in humans. Here we report that mice lacking T-bet, a T-box transcription factor required for T helper (Th)1 cell differentiation and interferon (IFN)-γ production, develop severe autoimmune heart disease compared to T-bet−/− control mice. Experiments in T-bet−/− IL-4−/− and T-bet−/− IL-4Rα−/− mice, as well as transfer of heart-specific Th1 and Th2 cell lines, showed that autoimmune heart disease develops independently of Th1 or Th2 polarization. Analysis of T-bet−/− IL-12Rβ1−/− and T-bet−/− IL-12p35−/− mice then identified interleukin (IL)-23 as critical for EAM pathogenesis. In addition, T-bet−/− mice showed a marked increase in production of the IL-23–dependent cytokine IL-17 by heart-infiltrating lymphocytes, and in vivo IL-17 depletion markedly reduced EAM severity in T-bet−/− mice. Heart-infiltrating T-bet−/− CD8+ but not CD8− T cells secrete IFN-γ, which inhibits IL-17 production and protects against severe EAM. In contrast, T-bet−/− CD8+ lymphocytes completely lost their capacity to release IFN-γ within the heart. Collectively, these data show that severe IL-17–mediated EAM can develop in the absence of T-bet, and that T-bet can regulate autoimmunity via the control of nonspecific CD8+ T cell bystander functions in the inflamed target organ

PI3Kγ Protects from Myocardial Ischemia and Reperfusion Injury through a Kinase-Independent Pathway

BACKGROUND: PI3Kgamma functions in the immune compartment to promote inflammation in response to G-protein-coupled receptor (GPCR) agonists and PI3Kgamma also acts within the heart itself both as a negative regulator of cardiac contractility and as a pro-survival factor. Thus, PI3Kgamma has the potential to both promote and limit M I/R injury. METHODOLOGY/PRINCIPAL FINDINGS: Complete PI3Kgamma-/- mutant mice, catalytically inactive PI3KgammaKD/KD (KD) knock-in mice, and control wild type (WT) mice were subjected to in vivo myocardial ischemia and reperfusion (M I/R) injury. Additionally, bone-marrow chimeric mice were constructed to elucidate the contribution of the inflammatory response to cardiac damage. PI3Kgamma-/- mice exhibited a significantly increased infarction size following reperfusion. Mechanistically, PI3Kgamma is required for activation of the Reperfusion Injury Salvage Kinase (RISK) pathway (AKT/ERK1/2) and regulates phospholamban phosphorylation in the acute injury response. Using bone marrow chimeras, the cardioprotective role of PI3Kgamma was mapped to non-haematopoietic cells. Importantly, this massive increase in M I/R injury in PI3Kgamma-/- mice was rescued in PI3Kgamma kinase-dead (PI3KgammaKD/KD) knock-in mice. However, PI3KgammaKD/KD mice exhibited a cardiac injury similar to wild type animals, suggesting that specific blockade of PI3Kgamma catalytic activity has no beneficial effects. CONCLUSIONS/SIGNIFICANCE: Our data show that PI3Kgamma is cardioprotective during M I/R injury independent of its catalytic kinase activity and that loss of PI3Kgamma function in the hematopoietic compartment does not affect disease outcome. Thus, clinical development of specific PI3Kgamma blockers should proceed with caution

The Set3/Hos2 Histone Deacetylase Complex Attenuates cAMP/PKA Signaling to Regulate Morphogenesis and Virulence of Candida albicans

Candida albicans, like other pleiomorphic fungal pathogens, is able to undergo a reversible transition between single yeast-like cells and multicellular filaments. This morphogenetic process has long been considered as a key fungal virulence factor. Here, we identify the evolutionarily conserved Set3/Hos2 histone deacetylase complex (Set3C) as a crucial repressor of the yeast-to-filament transition. Cells lacking core components of the Set3C are able to maintain all developmental phases, but are hypersusceptible to filamentation-inducing signals, because of a hyperactive cAMP/Protein Kinase A signaling pathway. Strikingly, Set3C-mediated control of filamentation is required for virulence in vivo, since set3Δ/Δ cells display strongly attenuated virulence in a mouse model of systemic infection. Importantly, the inhibition of histone deacetylase activity by trichostatin A exclusively phenocopies the absence of a functional Set3C, but not of any other histone deacetylase gene. Hence, our work supports a paradigm for manipulating morphogenesis in C. albicans through alternative antifungal therapeutic strategies

Induction of RNA-directed DNA methylation upon decondensation of constitutive heterochromatin

Centromeric constitutive heterochromatin is marked by DNA methylation and dimethylated histone H3 Lys 9 (H3K9me2) in Arabidopsis. RNA-directed DNA methylation (RdDM) is a process that uses 24-nucleotide (nt) small interfering RNAs (siRNAs) to induce de novo methylation to its homologous DNA sequences. Despite the presence of centromeric 24-nt siRNAs, mutations in genes required for RdDM do not appreciably influence the methylation of centromeric repeats. The mechanism by which constitutive heterochromatin is protected from RdDM remains puzzling. Here, we report that the vegetative cell nuclei (VN) of the male gametophyte (pollen) invariably undergo extensive decondensation of centromeric heterochromatin and lose centromere identity. VN show greatly reduced H3K9me2, phenocopying nuclei carrying a mutation in the chromatin remodeller DECREASE IN DNA METHYLATION 1 (DDM1). However, unlike the situation in ddm1 nuclei, the decondensed heterochromatin retains dense CG methylation and transcriptional silencing, and, unexpectedly, is subjected to RdDM-dependent hypermethylation in non-CG contexts. These findings reveal two assembly orders of silent heterochromatin and implicate the condensed form in blocking the RdDM machinery

Loss of PI3Kγ results in massive myocardial ischemia/reperfusion induced heart damage.

<p>(<b>A</b>) Following M I/R injury, serum TroponinT is significantly increased in PI3Kγ^−/− (KO) mice compared to wild type (WT) controls. Data are from 3 hours after reperfusion. n = 13 per group. (<b>B</b>) No significant difference in the numbers of inflammatory cells in the injured hearts of PI3Kγ^−/− and WT mice. Inflammatory cell infiltrates were assessed 3 hours after reperfusion. n = 7 per group. (<b>C</b>) No significant difference in the area at risk (AAR)/left ventricle (LV) in PI3Kγ^−/− and WT mice. (<b>D</b>) PI3Kγ KO mice display a markedly increased area of infarction/area at risk (AAR) 24 hours after reperfusion. n = 6 per group for (<b>C</b>) and (<b>D</b>). (<b>E</b>) Representative left ventricular TTC-stained sections from the basis (left) to the apex (right) of wild type (WT; upper panels) and PI3Kγ^−/− (KO; lower panels) hearts after 24 hours of reperfusion. Pale areas represent necrotic regions (arrows). Bars indicate 1 mm. In all bar graphs mean values +/− SEM are shown. *p<0.05. **p<0.01. NS = not significant.</p

PI3Kγ^−/− lipid kinase activity is not required for cardioprotection.

<p>(<b>A</b>) TroponinT levels measured after 30 minutes of ischemia and 3 hours of reperfusion are comparable between PI3Kγ^KD/KD (KD) and control wild type (WT) mice. In total PI3Kγ knock-out (KO) mice TroponinT levels are significantly increased (*p<0.05). n = 13 for WT and KO, n = 11 for KD mice. (<b>B</b>) No significant difference in the area at risk (AAR)/left ventricle (LV) in PI3Kγ^−/− (KO), PI3Kγ^KD/KD (KD), and control WT mice. Data are at 24 hours after M/I injury. (<b>C</b>) PI3Kγ KO, but not PI3Kγ^KD/KD (KD) mice display a markedly increased area of infarction/area at risk (AAR) 24 hours after reperfusion evaluated by TTCstaining. n = 6 per group. *p<0.05. NS = not significant. (<b>D</b>) Representative left ventricular Trichrome-stained sections from the basis (left) to the apex (right) of hearts from wild type (WT), PI3Kγ^KD/KD knock-in (KD), and PI3Kγ^−/− (KO) mice challenged with 30 minutes of ischemia plus 1 week of reperfusion. Arrows represent infarcted regions. Bars indicate 1 mm. (<b>E</b>) PI3Kγ^KD/KD (KD) mice display similar infarctions/left ventricle (LV) compared to wild type controls after 30 minutes of ischemia and 1 week of reperfusion. Data from PI3Kγ^−/− (KO) are also shown. n = 6 per group. (<b>F</b>) PI3Kγ^−/− (KO), but not PI3Kγ^KD/KD knock-in (KD) mice exhibit a significant greater impairment in percentage fractional shortening (FS) relative to WT controls after 30 minutes of ischemia and 1 week of reperfusion. n = 6 per group. Note that the data are calculated to baseline FS of the respective genotype. All bar graphs show mean values +/− SEM. *p<0.05. (<b>G</b>) Representative M-mode echocardiography of wild type (WT), PI3Kγ^KD/KD knock-in (KD), and PI3Kγ^−/− (KO) mice one week after M I/R injury. Note, that baseline contractility is enhanced in PI3Kγ^−/− (KO), but not PI3Kγ^KD/KD knock-in (KD) mice as reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009350#pone.0009350-Crackower1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009350#pone.0009350-Patrucco1" target="_blank">[12]</a>. Bars indicate 50 ms (horizontal) and 1 mm (vertical).</p

Loss of PI3Kγ results in long term myocardial ischemia/reperfusion induced heart damage.

<p>(<b>A</b>) Areas of infarction (arrows) in representative left mid-ventricular sections 1 week after M I/R injury. (H&E staining). Bars indicate 1 mm. (<b>B</b>) PI3Kγ^−/− (KO) hearts show a markedly greater infarction size in the left ventricle (LV) at 1 week after M I/R. n = 7 per group. (<b>C</b>) No significant difference between PI3Kγ^−/− and WT mice in the numbers of infiltrating inflammatory cells 1 week after M I/R. n = 7 per group. (<b>D</b>) Representative left mid-ventricular sections stained with Trichrome and Aniline blue to visualize collagen deposits (arrows) in hearts of PI3Kγ^−/− (KO) and wild type (WT) mice 3 weeks after M I/R. Magnifications ×20. (<b>E</b>) Markedly increased scar tissue in PI3Kγ^−/− 3 weeks after M I/R injury. n = 7 per group. (<b>F</b>) PI3Kγ^−/− mice exhibit a significant greater loss in percentage fractional shortening (FS) relative to their respective baseline FS after 3 weeks of M I/R compared to WT controls. n = 9 per group. In all bar graphs mean values +/− SEM are shown. *p<0.05. NS = not significant.</p