Precisely defining the roles of specific cell types is an intriguing and challenging frontier in the study of intact biological systems, and has stimulated the rapid development of genetically-encoded observation and control tools. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location, and connectivity. Here we have combined recombinase tools with engineered introns to achieve expression of genetically-encoded payloads conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We use this approach to target intersectionally-specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically-encoded interventional and observational tools for intact-systems biology.

BackgroundObesity, an inflammatory condition linked to cardiovascular disease, is associated with expansion of adipose tissue. Highly prevalent coplanar polychlorinated biphenyls (PCBs) such as 3,3′,4,4′-tetrachlorobiphenyl (PCB-77) accumulate in adipose tissue because of their lipophilicity and increase with obesity. However, the effects of PCBs on adipocytes, obesity, and obesity-associated cardiovascular disease are unknown.ObjectivesIn this study we examined in vitro and in vivo effects of PCB-77 on adipocyte differentiation, proinflammatory adipokines, adipocyte morphology, body weight, serum lipids, and atherosclerosis.MethodsPCB-77 or 2,2′,4,4,5,5′-hexachlorobiphenyl (PCB-153) was incubated with 3T3-L1 adipocytes either during differentiation or in mature adipocytes. Concentration-dependent effects of PCB-77 were contrasted with those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For in vivo studies, we treated C57BL/6 wild-type (WT) or aryl hydrocarbon receptor (AhR)−/− mice with vehicle or PCB-77 (49 mg/kg, by intraperitoneal injection) and examined body weight gain. In separate studies, we injected ApoE−/− mice with vehicle or PCB-77 over a 6-week period and examined body weight, adipocyte size, serum lipids, and atherosclerosis.ResultsLow concentrations of PCB-77 or TCDD increased adipocyte differentiation, glycerol–3-phosphate dehydrogenase activity, and expression of peroxisome proliferator–activated receptor γ, whereas higher concentrations inhibited adipocyte differentiation. Effects of PCB-77 were abolished by the AhR antagonist α-naphthoflavone. PCB-77 promoted the expression and release of various proinflammatory cytokines from 3T3-L1 adipocytes. Administration of PCB-77 increased body weight gain in WT but not AhR−/− mice. ApoE−/− mice injected with PCB-77 exhibited greater body weight, adipocyte hypertrophy, serum dyslipidemia, and augmented atherosclerosis.ConclusionsOur findings suggest that PCB-77 may contribute to the development of obesity and obesity-associated atherosclerosis.

The Ah receptor (AHR), the Ah receptor nuclear translocator protein (ARNT), and single-minded protein (SIM) are members of the basic helix-loop-helix-PAS (bHLH-PAS) family of regulatory proteins. In this study, we examine the DNA half-site recognition and pairing rules for these proteins using oligonucleotide selectionamplification and coprecipitation protocols. Oligonucleotide selection-amplification revealed that a variety of bHLH-PAS protein combinations could interact, with each generating a unique DNA binding specificity. To validate the selection-amplification protocol, we demonstrated the preference of the AHR⅐ARNT complex for the sequence commonly found in dioxin-responsive enhancers in vivo (TNGCGTG). We then demonstrated that the ARNT protein is capable of forming a homodimer with a binding preference for the palindromic E-box sequence, CACGTG. Further examination indicated that ARNT may have a relaxed partner specificity, since it was also capable of forming a heterodimer with SIM and recognizing the sequence GT(G/A)CGTG. Coprecipitation experiments using various PAS proteins and ARNT were consistent with the idea that the ARNT protein has a broad range of interactions among the bHLH-PAS proteins, while the other members appear more restricted in their interactions. Comparison of this in vitro data with sites known to be bound in vivo suggests that the high affinity half-site recognition sequences for the AHR, SIM, and ARNT are T(C/T)GC, GT(G/A)C (5-halfsites), and GTG (3-half-sites), respectively. The AHR1 is a bHLH protein that mediates the metabolic, carcinogenic, and teratogenic effects of compounds such as TCDD (1). In response to agonists, the AHR interacts with a related protein known as ARNT to form a dimeric 2 complex that is capable of binding genomic enhancer elements, known as DREs, and activating transcription at adjacent promoters (2-5). The AHR and ARNT have sequence similarities to two regulatory proteins found in Drosophila, SIM, and PER (6 -10). SIM is a developmentally regulated bHLH protein involved in controlling central nervous system midline gene expression (11). PER lacks a bHLH domain and thus may be an inhibitor of a related signaling pathway involved in the maintenance of circadian rhythms (12). The hallmark of this family of proteins is that they all possess homology in a sequence of 200 -300 amino acids termed a PAS domain (13). In the AHR, the PAS domain has been shown to be involved in ligand binding, interaction with Hsp90, and may serve as a secondary surface to support ARNT dimerization (2, 14 -16). Basic/helix-loop-helix proteins are involved in a variety of tightly regulated biological processes, such as the regulation of myogenesis (MyoD/E47) (17), neurogenesis (Achaete-scute/ Daughterless) (18), regulation of immunoglobulin genes (TFEC/TFE3) (19), cellular proliferation (Myc/Max) (20, 21), and xenobiotic metabolism (AHR/ARNT) (10). Biochemical and crystallographic data suggest that the HLH domains often act in concert with secondary dimerization surfaces (e.g. "le...

No abstract

The Ah receptor (AHR) is a basic helix-oophelix protein that mediates the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin. In this report, we describe a rabbit reticulocyte system that allows functional expression ofboth the AHR and its dimeric partner, the AHR nuclear translocator protein (ARNT). By using this in vitro system, we were able to reconstitute agonist binding to the AHR and agonist-induced AHR-ARNT recognition ofa cognate DNA enhancer sequence. Expression of AHR deletion mutants revealed the location of N-terminal domains responsible for ligand and DNA recognition and C-terminal domains that play roles in agonist-induced DNA recognition.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) serves as the prototype for a number of highly toxic environmental contaminants (1). Genetic studies in TCDDsensitive and -resistant murine strains and structure-activity analysis of congener potency indicate that the effects of this compound are mediated through its binding to a soluble protein known as the Ah receptor (AHR) (2-4). Although the exact mechanisms underlying many ofthe receptor-mediated toxic effects are unclear, it has been demonstrated that ligand-activated AHR interacts with dioxin-responsive enhancers (DREs) lying upstream of target promoters to increase the expression of a number of genes involved in xenobiotic metabolism (5-7).Recent results indicate that at least two other proteins play a role in receptor signaling. The 90-kDa heat shock protein (Hsp9O) appears to associate with the AHR, holding it in a conformation able to bind ligand and also repressing the receptor's intrinsic DNA binding properties (8,9). A second protein, the AHR nuclear translocator (ARNT), was identified and cloned by virtue of its ability to rescue a Hepa lclc7 mutant cell line deficient in transducing the signal of receptor