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NLRs (nucleotide-binding domain [NBD] leucine-rich repeat [LRR]-containing proteins) exhibit diverse functions in innate and adaptive immunity. NAIPs (NLR family, apoptosis inhibitory proteins) are NLRs that appear to function as cytosolic immunoreceptors for specific bacterial proteins, including flagellin and the inner rod and needle proteins of bacterial type III secretion systems (T3SSs). Despite strong biochemical evidence implicating NAIPs in specific detection of bacterial ligands, genetic evidence has been lacking. Here we report the use of CRISPR/Cas9 to generate Naip1(-/-) and Naip2(-/-) mice, as well as Naip1-6(Δ/Δ) mice lacking all functional Naip genes. By challenging Naip1(-/-) or Naip2(-/-) mice with specific bacterial ligands in vivo, we demonstrate that Naip1 is uniquely required to detect T3SS needle protein and Naip2 is uniquely required to detect T3SS inner rod protein, but neither Naip1 nor Naip2 is required for detection of flagellin. Previously generated Naip5(-/-) mice retain some residual responsiveness to flagellin in vivo, whereas Naip1-6(Δ/Δ) mice fail to respond to cytosolic flagellin, consistent with previous biochemical data implicating NAIP6 in flagellin detection. Our results provide genetic evidence that specific NAIP proteins function to detect specific bacterial proteins in vivo.

Preadipocyte factor-1 (Pref-1) is made as a transmembrane protein containing EGF-repeats at the extracellular domain that can be cleaved to generate a biologically active soluble form. Pref-1 is found in islet β-cells and its level has been reported to increase in neonatal rat islets upon growth hormone treatment. We found here that Pref-1 can promote growth of pancreatic tumor derived AR42J cells. To examine Pref-1 function in pancreatic islets in vivo, we generated transgenic mouse lines overexpressing the Pref-1/hFc in islet β-cells using rat insulin II promoter (RIP). These transgenic mice exhibit an increase in islet mass with higher proportion of larger islets in pancreas compared to wild-type littermates. This is in contrast to pancreas from Pref-1 null mice that show higher proportion of smaller islets. Insulin expression and insulin secretion from pancreatic islets from RIP-Pref-1/hFc transgenic mice are increased also. Thus, RIP-Pref-1/hFc transgenic mice show normal glucose levels but with higher plasma insulin levels in both fasting and fed conditions. These mice show improved glucose tolerance. Taken together, we conclude Pref-1 as a positive regulator of islet β-cells and insulin production.

Uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis and, upon cold exposure, is induced in brown adipose tissue (BAT) and subcutaneous white adipose tissue (iWAT). Here, by high-throughput screening using the UCP1 promoter, we identify Zfp516 as a transcriptional activator of UCP1 as well as PGC1α, thereby promoting a BAT program. Zfp516 itself is induced by cold and sympathetic stimulation through the cAMP-CREB/ATF2 pathway. Zfp516 directly binds to the proximal region of the UCP1 promoter, not to the enhancer region where other transcription factors bind, and interacts with PRDM16 to activate the UCP1 promoter. Although ablation of Zfp516 causes embryonic lethality, knockout embryos still show drastically reduced BAT mass. Overexpression of Zfp516 in adipose tissue promotes browning of iWAT even at room temperature, increasing body temperature and energy expenditure and preventing diet-induced obesity. Zfp516 may represent a future target for obesity therapeutics.

Pref-1 is an EGF-repeat-containing protein that inhibits adipocyte differentiation. To better understand the origin and development of white adipose tissue (WAT), we generated transgenic mouse models for transient or permanent fluorescent labeling of cells using the Pref-1 promoter, facilitating inducible ablation. We show that Pref-1-marked cells retain proliferative capacity and are very early adipose precursors, prior to expression of Zfp423 or PPARγ. In addition, the Pref-1-marked cells establish that adipose precursors are mesenchymal, but not endothelial or pericytal, in origin. During embryogenesis, Pref-1-marked cells first appear in the dorsal mesenteric region as early as embryonic day 10.5 (E10.5). These cells become lipid-laden adipocytes at E17.5 in the subcutaneous region, whereas visceral WAT develops after birth. Finally, ablation of Pref-1-marked cells prevents not only embryonic WAT development but also later adult adipose expansion upon high-fat feeding, demonstrating the requirement of Pref-1 cells for adipogenesis.

The production of distinct sets of T cell receptor (TCR) gammadelta(+) T cells occurs in an ordered fashion in thymic development. The Vgamma3 and Vgamma4 genes, located downstream in the TCRgamma Cgamma1 gene cluster, are expressed by the earliest waves of developing TCRgammadelta(+) T cells in the fetal thymus, destined for intraepithelial locations. Upstream Vgamma2 and Vgamma5 genes are expressed in later waves in the adult and constitute most TCRgammadelta(+) T cells in secondary lymphoid tissue. This developmental pattern is caused in part by a preference for rearrangements of the downstream Vgamma3 and Vgamma4 genes in the early fetal stage, which switches to a preference for rearrangements of the upstream Vgamma2 and Vgamma5 gene rearrangements in the adult. Our gene targeting studies show that the downstream Vgamma genes rearrange preferentially in the early fetal thymus because of their downstream location, independent of promoter or recombination signal sequences and unrelated to the extent of germline transcription. Remarkably, gene deletion studies show that the downstream Vgamma genes competitively inhibit upstream Vgamma rearrangements at the fetal stage. These data provide a mechanism for specialization of the fetal thymus for the production of T cells expressing specific Vgamma genes.

The production of distinct sets of T cell receptor (TCR) gammadelta(+) T cells occurs in an ordered fashion in thymic development. The Vgamma3 and Vgamma4 genes, located downstream in the TCRgamma Cgamma1 gene cluster, are expressed by the earliest waves of developing TCRgammadelta(+) T cells in the fetal thymus, destined for intraepithelial locations. Upstream Vgamma2 and Vgamma5 genes are expressed in later waves in the adult and constitute most TCRgammadelta(+) T cells in secondary lymphoid tissue. This developmental pattern is caused in part by a preference for rearrangements of the downstream Vgamma3 and Vgamma4 genes in the early fetal stage, which switches to a preference for rearrangements of the upstream Vgamma2 and Vgamma5 gene rearrangements in the adult. Our gene targeting studies show that the downstream Vgamma genes rearrange preferentially in the early fetal thymus because of their downstream location, independent of promoter or recombination signal sequences and unrelated to the extent of germline transcription. Remarkably, gene deletion studies show that the downstream Vgamma genes competitively inhibit upstream Vgamma rearrangements at the fetal stage. These data provide a mechanism for specialization of the fetal thymus for the production of T cells expressing specific Vgamma genes.

A main function of white adipose tissue is to release fatty acids from stored triacylglycerol for other tissues to use as an energy source. Whereas endocrine regulation of lipolysis has been extensively studied, autocrine and paracrine regulation is not well understood. Here we describe the role of the newly identified major adipocyte phospholipase [A.sub.2], AdPLA (encoded by Pla2g16, also called HREV107), in the regulation of lipolysis and adiposity. AdPLA-null mice have a markedly higher rate of lipolysis owing to increased cyclic AMP levels arising from the marked reduction in the amount of adipose prostaglandin [E.sub.2] that binds the G[[alpha].sub.i];-coupled receptor, EP3. AdPLA-null mice have markedly reduced adipose tissue mass and triglyceride content but normal adipogenesis. They also have higher energy expenditure with increased fatty acid oxidation within adipocytes. AdPLA- deficient ob/ob mice remain hyperphagic but lean, with increased energy expenditure, yet have ectopic triglyceride storage and insulin resistance. AdPLA is a major regulator of adipocyte lipolysis and is crucial for the development of obesity., Triacylglycerol in adipose tissue is the major energy storage form in mammals. An imbalance between energy intake and expenditure can result in excess triacylglycerol accumulation in this tissue, resulting in [...]

Major histocompatibility complex (MHC) class I molecules display tens of thousands of peptides on the cell surface, derived from virtually all endogenous proteins, for inspection by cytotoxic T cells (CTLs). We show that, in normal mouse cells, MHC I molecules present a peptide encoded in the 3' 'untranslated' region. Despite its rarity, the peptide elicits CTL responses and induces self-tolerance, establishing that immune surveillance extends well beyond conventional polypeptides. Furthermore, translation of this cryptic peptide occurs by a previously unknown mechanism that decodes the CUG initiation codon as leucine rather than the canonical methionine., The bicistronic transgene we used encoded two peptides. The first peptide, WI9, derived from the Y-chromosome Uty gene (8), was in a conventional translational context with an upstream inflame AUG [...]

The PI3K-AKT pathway can mediate diverse biological responses and is crucial for optimal immune responses and lymphocyte development. Deletion of PI3K subunits or AKT leads to blockage of T-cell development at the TCR-beta checkpoint. Studies with over-expression of constitutively activated AKT have implicated this pathway in anti-apoptosis of developing thymocytes and in development of regulatory T cells. However, the role of endogenous PI3K-AKT in T-cell development beyond the TCR-beta checkpoint remains unclear. Here, we inhibited the endogenous PI3K-AKT pathway in thymocytes after double negative stages by expressing the negative regulator, PTEN. These mice exhibit normal early T-cell development, but the transition from intermediate single positive to double positive (DP) thymocytes is inhibited, leading to a significantly decreased number of DP, single positive thymocytes and peripheral T cells. Proliferation of peripheral T cells is reduced but apoptosis of DP cells and subsequent T-cell maturation, including regulatory T cells, are normal. AKT phosphorylation can be readily observed in most WT T-cell compartments but not DP thymocytes in response to TCR activation. Thus, the PI3K-AKT pathway is crucial for the transition of intermediate single positive to DP thymocytes but is dispensable for apoptosis and maturation of developing thymocytes.

Nur77 is a nuclear orphan steroid receptor that has been implicated in negative selection. Expression of Nur77 in thymocytes and cell lines leads to apoptosis through a mechanism that remains unclear. In some cell lines, Nur77 was reported to act through a transcription-independent mechanism involving translocation to mitochondria, leading to cytochrome c release. However, we show here that Nur77-mediated apoptosis in thymocytes does not involve cytoplasmic cytochrome c release and cannot be rescued by Bcl-2. Microarray analysis shows that Nur77 induces many genes, including two novel genes (NDG1, NDG2) and known apoptotic genes FasL and TRAIL. Characterization of NDG1 and NDG2 indicates that NDG1 initiates a novel apoptotic pathway in a Bcl-2-independent manner. Thus Nur77-mediated apoptosis in T cells involves Bcl-2 independent transcriptional activation of several known and novel apoptotic pathways.

Upstream regulatory factor (USF) and sterol regulatory element binding protein (SREBP) play key roles in the transcriptional regulation of the fatty acid synthase (FAS) gene by feeding and insulin. Due to the dual binding specificity of SREBP, as well as the presence of multiple consensus sites for these transcription factors in the FAS promoter, their physiologically relevant functional binding sites have been controversial. Here, in order to determine the occupancy of the putative USF and SREBP binding sites, we examined their protein-DNA interactions in living animals by using formaldehyde cross-linking and immunoprecipitation of chromatin and tested the function of these elements by employing mice transgenic for a reporter gene driven by various 5' deletions as well as site-specific mutations of the FAS promoter. We show that the -332 and -65 E-boxes are bound by USF in both fasted and refed mice, while the -150 SRE is bound by SREBP-1 only in refed mice. We also found that mutation of either the -150 SRE or the -65 E-box abolishes the feeding-induced activation of the FAS promoter in transgenic mice. Furthermore, in vivo occupancy of the FAS promoter by SREBP in the fed state can be prevented by mutation not only of the -150 SRE but, unexpectedly, of the -65 E-box as well. We conclude that the FAS promoter is activated during refeeding via the induced binding of SREBP to the -150 SRE and that USF binding to the -65 E-box is also required for SREBP binding and activation of the FAS promoter.

Many mammalian genes, including those encoding antigen receptors, contain more than one enhancer element. Deleting one element often does not prevent expression, but functional redundancy has never been directly demonstrated by gene targeting of multiple elements. We demonstrate that simultaneous deletion of two enhancer/LCR-like elements in the TCR Cgamma1 cluster, HsA and 3'E(Cgamma1), severely diminishes TCRgamma transcription, selectively impairs development of gammadelta thymocyte subsets, but only modestly reduces TCRgamma gene rearrangement, while deletion of each element separately has little effect. In contrast to these results in thymocytes, deletion of HsA alone reduces transcription of one Vgamma gene specifically in peripheral gammadelta T cells. Thus, the two elements exhibit functional redundancy in thymocytes but also have unique functions in other settings.