The Polycomb-Dependent Epigenome Controls ß Cell Dysfunction, Dedifferentiation, and Diabetes.

To date, it remains largely unclear to what extent chromatin machinery contributes to the susceptibility and progression of complex diseases. Here, we combine deep epigenome mapping with single-cell transcriptomics to mine for evidence of chromatin dysregulation in type 2 diabetes. We find two chromatin-state signatures that track ß cell dysfunction in mice and humans: ectopic activation of bivalent Polycomb-silenced domains and loss of expression at an epigenomically unique class of lineage-defining genes. ß cell-specific Polycomb (Eed/PRC2) loss of function in mice triggers diabetes-mimicking transcriptional signatures and highly penetrant, hyperglycemia-independent dedifferentiation, indicating that PRC2 dysregulation contributes to disease. The work provides novel resources for exploring ß cell transcriptional regulation and identifies PRC2 as necessary for long-term maintenance of ß cell identity. Importantly, the data suggest a two-hit (chromatin and hyperglycemia) model for loss of ß cell identity in diabetes.

Trim28 Haploinsufficiency Triggers Bi-stable Epigenetic Obesity.

More than one-half billion people are obese, and despite progress in genetic research, much of the heritability of obesity remains enigmatic. Here, we identify a Trim28-dependent network capable of triggering obesity in a non-Mendelian, "on/off" manner. Trim28(+/D9) mutant mice exhibit a bi-modal body-weight distribution, with isogenic animals randomly emerging as either normal or obese and few intermediates. We find that the obese-"on" state is characterized by reduced expression of an imprinted gene network including Nnat, Peg3, Cdkn1c, and Plagl1 and that independent targeting of these alleles recapitulates the stochastic bi-stable disease phenotype. Adipose tissue transcriptome analyses in children indicate that humans too cluster into distinct sub-populations, stratifying according to Trim28 expression, transcriptome organization, and obesity-associated imprinted gene dysregulation. These data provide evidence of discrete polyphenism in mouse and man and thus carry important implications for complex trait genetics, evolution, and medicine.

Hedgehog partial agonism drives Warburg-like metabolism in muscle and brown fat.

Diabetes, obesity, and cancer affect upward of 15% of the world's population. Interestingly, all three diseases juxtapose dysregulated intracellular signaling with altered metabolic state. Exactly which genetic factors define stable metabolic set points in vivo remains poorly understood. Here, we show that hedgehog signaling rewires cellular metabolism. We identify a cilium-dependent Smo-Ca(2+)-Ampk axis that triggers rapid Warburg-like metabolic reprogramming within minutes of activation and is required for proper metabolic selectivity and flexibility. We show that Smo modulators can uncouple the Smo-Ampk axis from canonical signaling and identify cyclopamine as one of a new class of "selective partial agonists," capable of concomitant inhibition of canonical and activation of noncanonical hedgehog signaling. Intriguingly, activation of the Smo-Ampk axis in vivo drives robust insulin-independent glucose uptake in muscle and brown adipose tissue. These data identify multiple noncanonical endpoints that are pivotal for rational design of hedgehog modulators and provide a new therapeutic avenue for obesity and diabetes.

Deletion of the aceE gene (encoding a component of pyruvate dehydrogenase) attenuates Salmonella enterica serovar Enteritidis.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major food-borne pathogen. From a transposon insertion mutant library created previously using S. Enteritidis 10/02, one of the mutants was identified to have a 50% lethal dose (LD(50) ) at least 100 times that of the parental strain in young chicks, with an attenuation in a poorly studied gene encoding a component of pyruvate dehydrogenase, namely the aceE gene. Evaluation of the in vitro virulence characteristics of the ΔaceE∷kan mutant revealed that it was less able to invade epithelial cells, less resistant to reactive oxygen intermediate, less able to survive within a chicken macrophage cell line and had a retarded growth rate compared with the parental strain. Young chicks vaccinated with 2 × 10(9) CFU of the ΔaceE∷kan mutant were protected from the subsequent challenge of the parental strain, with the mutant colonized in the liver and spleen in a shorter time than the group infected with the parental strain. In addition, compared with the parental strain, the ΔaceE∷kan mutant did not cause persistent eggshell contamination of vaccinated hens.

Porcine circovirus type 2 ORF3 protein competes with p53 in binding to Pirh2 and mediates the deregulation of p53 homeostasis.

The ORF3 protein of porcine circovirus type 2 (PCV2) causes apoptosis in virus-infected cells and is not essential for virus replication. The ORF3 protein plays an important role in the pathogenesis of the PCV2 infection in mouse models and SPF piglets. The ORF3 protein interacts with the porcine homologue of Pirh2 (pPirh2), a p53-induced ubiquitin-protein E3 ligase, which regulates p53 ubiquitination. Here, we present our study analyzing the details of the molecular interaction between these three factors. Our experiments, in vitro and in vivo, show that ORF3 protein competes with p53 in binding to pPirh2. The amino acid residues 20 to 65 of the ORF3 protein are essential in this competitive interaction of ORF3 protein with pPirh2 over p53. The interaction of ORF3 protein with pPirh2 also leads to an alteration in the physiological cellular localization of pPirh2 and a significant reduction in the stability of pPirh2. These events contribute to the deregulation of p53 by pPirh2, leading to increased p53 levels and apoptosis of the infected cells.

Attenuation of porcine circovirus 2 in SPF piglets by abrogation of ORF3 function.

Porcine circovirus 2 (PCV2), open reading frame 3 (ORF3) codes a 105 amino acid protein that causes apoptosis of PCV2 infected cells. In infected cells, the ORF3 causes the accumulation of p53 by interacting with pPirh2 and possibly by disrupting the association of p53 and pPirh2 (J.Virol.81(2007)9560). Mutant PCV2 lacking the expression of ORF3 are infectious and replicate in cells in vitro, but do not cause apoptosis of the infected cells. The ORF3 of PCV2 has been shown to be involved in pathogenesis of the virus in mice model (J. Virol. 80(2006)5065). Here we report the experimental inoculation of ORF3 deficient PCV2 in its natural host, the piglets. The pathogenicity of the ORF3 deficient virus is attenuated in the piglets. The mutant virus did not cause any observable disease or perturbation of the lymphocyte count in the inoculated piglets and elicited an efficient immune response. When compared with the wildtype virus infection, the mutant virus infection was characterized by mild viremia and absence of pathological lesions. The findings highlight the role of ORF3 in the pathogenesis of PCV2 infection in its host.