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Supplementary Figures 1-4 from PDK1 Attenuation Fails to Prevent Tumor Formation in PTEN-Deficient Transgenic Mouse Models

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

Objective: Reported associations between kidney risk variants (G1 and G2) in APOL1 (apolipoprotein L1), encoding APOL1, and cardiovascular disease have been conflicting. We sought to explore associations of APOL1 risk variants with cause of sudden death using the CVPath Sudden Death Autopsy Registry. Approach and Results: APOL1 haplotypes and causes of sudden death, as determined through autopsy and histopathology, were obtained for 764 Black subjects. Genotyping revealed APOL1 risk alleles in 452 of 764 (59%) subjects with 347 (77%) subjects carrying one risk allele and 105 (23%) subjects harboring 2 risk alleles. APOL1 risk allele carrier status was associated with a significantly increased risk of coronary thrombosis due to plaque rupture, versus noncarriers (odds ratio for rupture, 1.655 [95% CI, 1.079–2.539]; P =0.021). Histological examinations showed coronary plaques in carriers of 2 APOL1 risk alleles had larger necrotic cores compared with noncarriers (necrotic core area/total plaque area: 46.79%±6.47% versus 20.57%±5.11%; P =0.0343 in ruptured plaques, and 41.48%±7.49% versus 18.93%±3.97%; P =0.0342 in nonruptured plaques), and immunohistochemical and immunofluorescent staining revealed APOL1-positive areas localized primarily to the necrotic core. Conclusions: APOL1 risk alleles were independently associated with an increased risk of thrombotic coronary death due to plaque rupture. Our results suggest that carriers of both 1 and 2 APOL1 risk alleles have greater accumulation of APOL1 protein within culprit plaques and greater necrotic core sizes than noncarriers. These findings suggest that APOL1 plays a role in determining plaque stability.

Background Apolipoprotein L1, APOL1, is a trypanosome lytic factor present in human and certain other primates. APOL1 gene variants, present in individuals of recent sub-Saharan African descent, increase risk for glomerular disease and associate with the disease progression, but the molecular mechanisms have not been defined. Objectives We focus on the mechanism how APOL1 variant proteins enhance podocyte injury in the stressed kidney. Methods First, we investigated the expression of APOL1 protein isoform and the localization of APOL1 protein in the kidney. Next, we examined the role of APOL1 in the podocyte stress and the inflammatory signaling in the kidney after hemi-nephrectomy. Results We identified a novel RNA variant that lacks a secretory pathway signal sequence and we found that the predicted APOL1-B3 protein isoform was expressed in human podocytes in vivo and by BAC-APOL1 transgenic mice. APOL1-B3-G2 transgenic mice, carrying a renal risk variant, manifested podocyte injury and increased pro-IL-1β mRNA in isolated glomeruli and increased IL-1β production in the remnant kidney after uninephrectomy. APOL1-B3 interacted with NLRP12, a key regulator of Toll-like receptor signaling. Conclusions These results suggest a possible mechanism for podocyte injury by which one of the APOL1 protein isoforms, APOL1-B3 and its renal risk variants, enhances inflammatory signaling.

The functional interpretation of genome-wide association studies (GWAS) is challenging due to the cell-type-dependent influences of genetic variants. Here, we generated comprehensive maps of expression quantitative trait loci (eQTLs) for 659 microdissected human kidney samples and identified cell-type-eQTLs by mapping interactions between cell type abundances and genotypes. By partitioning heritability using stratified linkage disequilibrium score regression to integrate GWAS with single-cell RNA sequencing and single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing data, we prioritized proximal tubules for kidney function and endothelial cells and distal tubule segments for blood pressure pathogenesis. Bayesian colocalization analysis nominated more than 200 genes for kidney function and hypertension. Our study clarifies the mechanism of commonly used antihypertensive and renal-protective drugs and identifies drug repurposing opportunities for kidney disease.

The functional interpretation of GWAS remains challenging due to cell-type dependent influences of genetic variants.Here, we generated comprehensive maps of expression quantitative trait loci (eQTL) for 659 microdissected human kidney samples and identified cell-type eQTLs by mapping interactions between cell type abundance and genotype. Separately, we generated single cell open chromatin maps (by snATAC-seq) for human kidney samples. We highlight critical enrichment of proximal tubules in kidney function and endothelial cells and distal tubule segments in blood pressure by partitioning heritability using stratified LD-score regression to integrate GWAS with scRNA-seq and snATAC-seq data. Bayesian colocalization analysis nominated more than 200 genes for kidney function and hypertension. Our study clarifies the mechanism of the most commonly used antihypertensive and renal protective drugs and identifies drug repurposing opportunities for kidney disease.One Sentence SummaryWe define causal cell types, genes and mechanism for kidney dysfunction.

Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by the modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.

Genetic variation in the FAM13A (Family with Sequence Similarity 13 Member A) locus has been associated with several glycemic and metabolic traits in genome-wide association studies (GWAS). Here, we demonstrate that in humans, FAM13A alleles are associated with increased FAM13A expression in subcutaneous adipose tissue (SAT) and an insulin resistance-related phenotype (e.g. higher waist-to-hip ratio and fasting insulin levels, but lower body fat). In human adipocyte models, knockdown of FAM13A in preadipocytes accelerates adipocyte differentiation. In mice, Fam13a knockout (KO) have a lower visceral to subcutaneous fat (VAT/SAT) ratio after high-fat diet challenge, in comparison to their wild-type counterparts. Subcutaneous adipocytes in KO mice show a size distribution shift toward an increased number of smaller adipocytes, along with an improved adipogenic potential. Our results indicate that GWAS-associated variants within the FAM13A locus alter adipose FAM13A expression, which in turn, regulates adipocyte differentiation and contribute to changes in body fat distribution., Genetic variants in the FAM13A locus have been associated with anthropometric and glycemic traits. Here, using fine-mapping, in vitro knockdown studies in pre-adipocytes and in vivo knockout in mice, the authors show that FAM13A is involved in regulating fat distribution and metabolic traits.

The kidney expression pattern of APOL1 was examined using both protein and mRNAin situmethods onAPOL1bacterial artificial chromosome transgenic mice, with and without proteinuria. APOL1 was detected in podocytes and endothelial cells of the kidney, but was not expressed in tubular epithelia, nor was plasma APOL1 protein filtered and reabsorbed by the proximal tubule. APOL1 expression in podocytes and endothelia should remain the focus for mechanistic studies of APOL1-mediated pathogenesis.

The mechanism of pathogenesis associated with APOL1 polymorphisms and risk for non-diabetic chronic kidney disease (CKD) is not fully understood. Prior studies have minimized a causal role for the circulating APOL1 protein, thus efforts to understand kidney pathogenesis have focused on APOL1 expressed in renal cells. Of the kidney cells reported to express APOL1, the proximal tubule expression patterns are inconsistent in published reports, and whether APOL1 is synthesized by the proximal tubule or possibly APOL1 protein in the blood is filtered and reabsorbed by the proximal tubule remains unclear. Using both protein and mRNA in situ methods, the kidney expression pattern of APOL1 was examined in normal human and APOL1 bacterial artificial chromosome transgenic mice with and without proteinuria. APOL1 protein and mRNA was detected in podocytes and endothelial cells, but not in tubular epithelia. In the setting of proteinuria, plasma APOL1 protein did not appear to be filtered or reabsorbed by the proximal tubule. A side-by-side examination of commercial antibodies used in prior studies suggest the original reports of APOL1 in proximal tubules likely reflects antibody non-specificity. As such, APOL1 expression in podocytes and endothelia should remain the focus for mechanistic studies in the APOL1-mediated kidney diseases.

APOL1 risk alleles associate with chronic kidney disease in African Americans, but the mechanisms remain to be fully understood. We show that APOL1 risk alleles activate protein kinase R (PKR) in cultured cells and transgenic mice. This effect is preserved when a premature stop codon is introduced to APOL1 risk alleles, suggesting that APOL1 RNA but not protein is required for the effect. Podocyte expression of APOL1 risk allele RNA, but not protein, in transgenic mice induces glomerular injury and proteinuria. Structural analysis of the APOL1 RNA shows that the risk variants possess secondary structure serving as a scaffold for tandem PKR binding and activation. These findings provide a mechanism by which APOL1 variants damage podocytes and suggest novel therapeutic strategies., Okamoto et al. report that APOL1 RNA, but not protein, expressed from chronic kidney disease (CKD)-associated alleles activates protein kinase R, inducing glomerular injury in a mouse model. The secondary structure of APOL1 risk variant RNA acts as a scaffold for protein kinase R, suggesting potential therapeutic strategies for CKD.

Summary Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters. In contrast, experiments in primary human, rhesus, and cynomolgus hepatocytes demonstrated that selective inhibition of DGAT2 has only a modest effect. Acute and chronic inhibition of DGAT2 in rhesus primates recapitulated the in vitro data yielding no significant effects on production of plasma TG or VLDL apolipoprotein B. These results call into question whether selective inhibition of DGAT2 is sufficient for remediation of dyslipidemia.

Apolipoprotein L1 (APOL1) genetic variants G1 and G2, compared to the common allele G0, are major risk factors for non-diabetic kidney disease in African descent populations. APOL1 is a minor protein component of HDL, as well as being expressed in podocytes and vascular cells. Reverse cholesterol transport involves the transport of cholesterol to HDL by cellular ATP-binding cassette; ABCA1 and ABCG1 with subsequent delivery from peripheral tissues to the liver. With impaired reverse cholesterol transport, lipid accumulation occurs and macrophages morphologically transform into foam cells, releasing inflammatory factors. We asked whether the APOL1 risk variants alter peripheral cholesterol metabolism and specifically affect macrophage cholesterol efflux. Tissues and bone marrow (BM)-derived monocytes were isolated from wild-type mice (WT) and from BAC/APOL1 transgenic (APOL1-G0, APOL1-G1, and APOL1-G2) mice, which carry a bacterial artificial chromosome that contains the human APOL1 genomic region. Monocytes were differentiated into macrophages using M-CSF, and then polarized into M1 and M2 macrophages. Cholesterol content, cholesterol efflux, and ABCA1 and ABCG1 mRNA expression were measured. Kidney, spleen, and bone marrow-derived macrophages from APOL1-G1 and -G2 mice showed increased cholesterol accumulation and decreased ABCA1 and ABCG1 mRNA levels. BM-derived macrophages from APOL1-G1 and -G2 mice showed significantly reduced cholesterol efflux compared to WT or APOL1-G0 macrophages. Taken together, the evidence suggests that APOL1-G1 and -G2 risk variants impaired reverse cholesterol transport through decreased expression of cholesterol efflux transporters suggesting a possible mechanism to promote macrophage foam cell formation, driving inflammation in the glomerulus and renal interstitium.

The enteric nervous system (ENS) arises from the coordinated migration, expansion and differentiation of vagal and sacral neural crest progenitor cells. During development, vagal neural crest cells enter the foregut and migrate in a rostro-to-caudal direction, colonizing the entire gastrointestinal tract and generating the majority of the ENS. Sacral neural crest contributes to a subset of enteric ganglia in the hindgut, colonizing the colon in a caudal-to-rostral wave. During this process, enteric neural crest-derived progenitors (ENPs) self-renew and begin expressing markers of neural and glial lineages as they populate the intestine. Our earlier work demonstrated that the transcription factor Foxd3 is required early in neural crest-derived progenitors for self-renewal, multipotency and establishment of multiple neural crest-derived cells and structures including the ENS. Here, we describe Foxd3 expression within the fetal and postnatal intestine: Foxd3 was strongly expressed in ENPs as they colonize the gastrointestinal tract and was progressively restricted to enteric glial cells. Using a novel Ednrb-iCre transgene to delete Foxd3 after vagal neural crest cells migrate into the midgut, we demonstrated a late temporal requirement for Foxd3 during ENS development. Lineage labeling of Ednrb-iCre expressing cells in Foxd3 mutant embryos revealed a reduction of ENPs throughout the gut and loss of Ednrb-iCre lineage cells in the distal colon. Although mutant mice were viable, defects in patterning and distribution of ENPs were associated with reduced proliferation and severe reduction of glial cells derived from the Ednrb-iCre lineage. Analyses of ENS-lineage and differentiation in mutant embryos suggested activation of a compensatory population of Foxd3-positive ENPs that did not express the Ednrb-iCre transgene. Our findings highlight the crucial roles played by Foxd3 during ENS development including progenitor proliferation, neural patterning, and glial differentiation and may help delineate distinct molecular programs controlling vagal versus sacral neural crest development.

The development of zinc finger nuclease (ZFN) technology has enabled the genetic engineering of the rat genome. The ability to manipulate the rat genome has great promise to augment the utility of rats for biological and pharmacological studies. A Wistar Hannover rat model lacking the multidrug resistance protein Mdr1a P-glycoprotein (P-gp) was generated using a rat Mdr1a-specific ZFN. Mdr1a was completely absent in tissues, including brain and small intestine, of the knockout rat. Pharmacokinetic studies with the Mdr1a P-gp substrates loperamide, indinavir, and talinolol indicated that Mdr1a was functionally inactive in the blood-brain barrier and intestine in Mdr1a(-/-) rats. To identify possible compensatory mechanisms in Mdr1a(-/-) rats, the expression levels of drug-metabolizing enzyme and transporter-related genes were compared in brain, liver, kidney, and intestine of male and female Mdr1a(-/-) and control rats. In general, alterations in gene expression of these genes in Mdr1a(-/-) rats seemed to be modest, with more changes in female than in male rats. Taken together, our studies demonstrate that the ZFN-generated Mdr1a(-/-) rat will be a valuable tool for central nervous system drug target validation and determining the role of P-gp in drug absorption and disposition.

RAD18 is an ubiquitin ligase that is involved in replication damage bypass and DNA double-strand break (DSB) repair processes in mitotic cells. Here, we investigated the testicular phenotype of Rad18-knockdown mice to determine the function of RAD18 in meiosis, and in particular, in the repair of meiotic DSBs induced by the meiosis-specific topoisomerase-like enzyme SPO11. We found that RAD18 is recruited to a specific subfraction of persistent meiotic DSBs. In addition, RAD18 is recruited to the chromatin of the XY chromosome pair, which forms the transcriptionally silent XY body. At the XY body, RAD18 mediates the chromatin association of its interaction partners, the ubiquitin-conjugating enzymes HR6A and HR6B. Moreover, RAD18 was found to regulate the level of dimethylation of histone H3 at Lys4 and maintain meiotic sex chromosome inactivation, in a manner similar to that previously observed for HR6B. Finally, we show that RAD18 and HR6B have a role in the efficient repair of a small subset of meiotic DSBs.

This report aims at exploring quantitatively the relationship between FXII inhibition and thromboprotection. FXII full and partial null in rats were established via zinc finger nuclease-mediated knockout and siRNA-mediated knockdown, respectively. The rats were subsequently characterized in thrombosis and hemostasis models. Knockout rats exhibited complete thromboprotection in both the arteriovenous shunt model (∼100% clot weight reduction) and the FeCl3-induced arterial thrombosis model (no reduction in blood flow), without any increase in cuticle bleeding time compared with wild-type control rats. Ex-vivo aPTT and the ellagic acid-triggered thrombin generation assay (TGA) exhibited anticoagulant changes. In contrast, ex-vivo PT or high tissue factor-triggered TGA was indistinguishable from control. Rats receiving single doses (0, 0.01, 0.03, 0.1, 0.3, 1 mg/kg) of FXII siRNA exhibited dose-dependent knockdown in liver FXII mRNA and plasma FXII protein (95 and 99%, respectively, at 1 mg/kg) at day 7 post dosing. FXII knockdown was associated with dose-dependent thromboprotection (maximal efficacy achieved with 1 mg/kg in both models) and negligible change in cuticle bleeding times. Ex-vivo TGA triggered with low-level (0.5 μmol/l) ellagic acid tracked best with the knockdown levels and efficacy. Our findings confirm and extend literature reports of an attractive benefit-to-risk profile of targeting FXII for antithrombotic therapies. Titrating of FXII is instructive for its pharmacological inhibition. The knockout rat is valuable for evaluating both mechanism-based safety concerns and off-target effects of FXII(a) inhibitors. Detailed TGA analyses will inform on optimal trigger conditions in studying pharmacodynamic effects of FXII(a) inhibition.

SummaryHaemophilia A and B are characterised by a life-long bleeding predisposition, and several lines of evidence suggest that risks of atherothrombotic events may also be reduced. Establishing a direct correlation between coagulation factor levels, thrombotic risks and bleeding propensity has long been hampered by an inability to selectively and specifically inhibit coagulation factor levels. Here, the exquisite selectivity of gene silencing combined with a gene knockout (KO) approach was used to define the relative contribution of factor IX (fIX) to thrombosis and primary haemostasis in the rat. Using a lipid nanoparticle (LNP) formulation, we successfully delivered fIX siRNAs to the liver by intravenous administration. The knockdown (KD) of target gene mRNA was achieved rapidly (within 24 hour post-siRNA dosing), sustained (maintained for at least 7 days post dosing) and not associated with changes in mRNA expression levels of other coagulation factors. We found that intermediate levels of liver fIX mRNA silencing (60–95 %) translating into a 50–99 % reduction of plasma fIX activity provided protection from thrombosis without prolonging the cuticle bleeding time. Over 99 % inhibition of fIX activity was required to observe increase in bleeding, a phenotype confirmed in fIX KO rats. These data provide substantial evidence of a participation of fIX in the mechanisms regulating thrombosis prior to those regulating primary haemostasis, therefore highlighting the potential of fIX as a therapeutic target. In addition, hepatic mRNA silencing using LNP-encapsulated siRNAs may represent a promising novel approach for the chronic treatment and prevention of coagulation-dependent thrombotic disorders in humans.

Genetic manipulation techniques are widely used in mice to study the functions of genes. The most common strategy for assessing in vivo function involves making irreversible changes in the genome by homologous recombination. To complement this approach, a number of systems have been developed that allow specific and controlled expression of a gene. One of the more versatile and promising systems is based on the tetracycline (tet) responsive bacterial tetracycline repressor (TetR). In recent years, the tet system has proven to be a valuable method for understanding the function of genes involved in a number of physiological processes, including mouse models for human diseases such as cancer and neurological and pigment disorders. This review will highlight the power and elegance of the tet system by focusing on its utility in the study of two pigment cell-related biological problems, the pathogenesis of melanomas and melanocyte development in the embryo.

The renal outer medullary potassium channel (ROMK, KCNJ1 ) mediates potassium recycling and facilitates sodium reabsorption through the Na + /K + /2Cl − cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Human genetic studies indicate that ROMK homozygous loss-of-function mutations cause type II Bartter syndrome, featuring polyuria, renal salt wasting, and hypotension; humans heterozygous for ROMK mutations identified in the Framingham Heart Study have reduced blood pressure. ROMK null mice recapitulate many of the features of type II Bartter syndrome. We have generated an ROMK knockout rat model in Dahl salt-sensitive background by using zinc finger nuclease technology and investigated the effects of knocking out ROMK on systemic and renal hemodynamics and kidney histology in the Dahl salt-sensitive rats. The ROMK −/− pups recapitulated features identified in the ROMK null mice. The ROMK +/− rats, when challenged with a 4% salt diet, exhibited a reduced blood pressure compared with their ROMK +/+ littermates. More importantly, when challenged with an 8% salt diet, the Dahl salt-sensitive rats with 50% less ROMK expression showed increased protection from salt-induced blood pressure elevation and signs of protection from renal injury. Our findings in ROMK knockout Dahl salt-sensitive rats, together with the previous reports in humans and mice, underscore a critical role of ROMK in blood pressure regulation.

Diacylglycerol acyltransferase-1 (DGAT1) is a potential therapeutic target for treatment of obesity and related metabolic diseases. However, the degree of DGAT1 inhibition required for metabolic benefits is unclear. Here we show that partial DGAT1 deficiency in mice suppressed postprandial triglyceridemia, led to elevations in glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) only following meals with very high lipid content, and did not protect from diet-induced obesity. Maximal DGAT1 inhibition led to enhanced GLP-1 and PYY secretion following meals with physiologically relevant lipid content. Finally, combination of DGAT1 inhibition with dipeptidyl-peptidase-4 (DPP-4) inhibition led to further enhancements in active GLP-1 in mice and dogs. The current study suggests that targeting DGAT1 to enhance postprandial gut hormone secretion requires maximal inhibition, and suggests combination with DPP-4i as a potential strategy to develop DGAT1 inhibitors for treatment of metabolic diseases.

The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received2H2O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings,2H2O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered.

PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, long-term PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTEN-deficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target. Cancer Res; 71(8); 3052–65. ©2011 AACR.

Expression of small hairpin RNA (shRNA) in mammalian cells can trigger potent RNAi-mediated gene silencing. The dominant-acting RNAi can often result in phenotypes similar to that of a null allele. Moreover, the generation of shRNA knockdown mice and subsequent phenotypic analysis can be achieved in a condensed timeline compared to that of conventional gene-targeting knockout strategies. Here, we discuss methods for the in vivo analysis of gene function in adult mouse tissues following tetracycline-induced RNA knockdown in a single-copy inducible polymerase III promoter-driven shRNA system.

Expression of small hairpin RNA (shRNA) in mammalian cells can trigger potent RNAi-mediated gene silencing. The dominant-acting RNAi can often result in phenotypes similar to that of a null allele. Moreover, the generation of shRNA knockdown mice and subsequent phenotypic analysis can be achieved in a condensed timeline compared to that of conventional gene-targeting knockout strategies. Here, we discuss methods for the in vivo analysis of gene function in adult mouse tissues following tetracycline-induced RNA knockdown in a single-copy inducible polymerase III promoter-driven shRNA system.

The endothelin receptor B gene (Ednrb) encodes a G-protein-coupled receptor that is expressed in a variety of cell types and is specifically required for the development of neural crest-derived melanocytes and enteric ganglia. In humans, mutations in this gene are associated with Waardenburg-Shah syndrome, a disorder characterized by pigmentation defects,deafness and megacolon. To address the question of whether melanocyte development depends entirely on a cell-autonomous action of Ednrb, we performed a series of tissue recombination experiments in vitro, using neural crest cell cultures from mouse embryos carrying a novel Ednrb-null allele characterized by the insertion of a lacZ marker gene. The results show that Ednrb is not required for the generation of early neural crest-derived melanoblasts but is required for the expression of the differentiation marker tyrosinase. Tyrosinase expression can be rescued,however, by the addition of Ednrb wild-type neural tubes. These Ednrb wild-type neural tubes need not be capable of generating melanocytes themselves, but must be capable of providing KIT ligand, the cognate ligand for the tyrosine kinase receptor KIT. In fact, soluble KIT ligand is sufficient to induce tyrosinase expression in Ednrb-deficient cultures. Nevertheless, these tyrosinase-expressing, Ednrb-deficient cells do not develop to terminally differentiated,pigmented melanocytes. Pigmentation can be induced, however, by treatment with tetradecanoyl phorbol acetate, which mimics EDNRB signaling, but not by treatment with endothelin 1, which stimulates the paralogous receptor EDNRA. The results suggest that Ednrb plays a significant role during melanocyte differentiation and effects melanocyte development by both cell non-autonomous and cell-autonomous signaling mechanisms.

Hirschsprung disease (HSCR) is a multigenic, congenital disorder that affects 1 in 5,000 newborns and is characterized by the absence of neural crest-derived enteric ganglia in the colon. One of the primary genes affected in HSCR encodes the G protein-coupled endothelin receptor-B (EDNRB). The expression of Ednrb is required at a defined time period during the migration of the precursors of the enteric nervous system (ENS) into the colon. In this study, we describe a conserved spatiotemporal ENS enhancer of Ednrb. This 1-kb enhancer is activated as the ENS precursors approach the colon, and partial deletion of this enhancer at the endogenous Ednrb locus results in pigmented mice that die postnatally from megacolon. We identified binding sites for SOX10, an SRY-related transcription factor associated with HSCR, in the Ednrb ENS enhancer, and mutational analyses of these sites suggested that SOX10 may have multiple roles in regulating Ednrb in the ENS.

Endothelin receptor B (EDNRB) is a G-protein-coupled receptor with seven transmembrane domains which is required for the development of melanocytes and enteric neurons. Mice that are homozygous for a null mutation in the Ednrb gene are almost completely white and die as juveniles from megacolon. To determine when EDNRB signalling is required during embryogenesis, we have exploited the tetracycline-inducible system to generate strains of mice in which the endogenous Ednrb locus is under the control of the tetracycline-dependant transactivators tTa or rtTA. By using this system to express Ednrb at different stages of embryogenesis, we have determined that EDNRB is required during a restricted period of neural crest development between embryonic days 10 and 12.5. Moreover, our results imply that EDNRB is required for the migration of both melanoblasts and enteric neuroblasts.

The piebald locus on mouse chromosome 14 encodes the endothelin-B receptor (EDNRB), a G protein-coupled, seven-transmembrane domain protein, which is required for neural crest-derived melanocyte and enteric neuron development. A spontaneous null allele of Ednrb results in homozygous mice that are predominantly white and die as juveniles from megacolon. To identify the important domains for EDNRB function, four recessive juvenile lethal alleles created by either radiation or chemical mutagens ( Ednrb 27Pub , Ednrb 17FrS , Ednrb 1Chlc , and Ednrb 3Chlo ) were examined at the molecular level. Ednrb 27Pub mice harbor a mutation at a critical proline residue in the fifth transmembrane domain of the EDNRB protein. A gross genomic alteration within the Ednrb gene in Ednrb 3Chlo results in the production of aberrantly sized transcripts and no authentic Ednrb mRNA. Ednrb 17FrS mice exhibited a decreased level of Ednrb mRNA, supporting previous observations that the degree of spotting in piebald mice is dependent on the amount of EDNRB expressed. Finally, no molecular defect was detected in Ednrb 1Chlc mice, which produce normal levels of Ednrb mRNA in adult brain, suggesting that the mutation affects important regulatory elements that mediate the expression of the gene during development.

Mutations in the genes encoding endothelin receptor-B (Ednrb) and its ligand endothelin-3 (Edn3) affect the development of two neural crest-derived cell types, melanocytes and enteric neurons. EDNRB signaling is exclusively required between E10.5 and E12.5 during the migratory phase of melanoblast and enteric neuroblast development. To determine the fate of Ednrb-expressing cells during this critical period, we generated a strain of mice with the bacterial beta-galactosidase (lacZ) gene inserted downstream of the endogenous Ednrb promoter. The expression of the lacZ gene was detected in melanoblasts and precursors of the enteric neuron system (ENS), as well as other neural crest cells and nonneural crest-derived lineages. By comparing Ednrb(lacZ)/+ and Ednrb(lacZ)/Ednrb(lacZ) embryos, we determined that the Ednrb pathway is not required for the initial specification and dispersal of melanoblasts and ENS precursors from the neural crest progenitors. Rather, the EDNRB-mediated signaling is required for the terminal migration of melanoblasts and ENS precursors, and this pathway is not required for the survival of the migratory cells.

Several physical and chemical signals from the extracellular environment are known to be transduced into changes in gene expression through multiple step pathways; however, mechanisms for triggering cellular responses to heavy metal stress have yet to be elucidated. We demonstrate here one such mechanism that employs a single heavy metal receptor protein, MerR, to directly activate transcription of the bacterial mercuric ion resistance operon. The mercuric ion-MerR complex and E. coli RNA polymerase holoenzyme synergistically bind to the metal responsive promoter in an unprecedented spatial relationship to form transcriptionally competent complexes. The activator binds adjacent to and overlaps with the polymerase molecule between the consensus −35 and −10 promoter regions. Our results support a model for transcriptional activation that includes both effector-induced protein-protein interactions and activator-induced alteration in DNA structure.

The s15DttMb, s36Pub, s1Acrg and s24Pub piebald deletion alleles belong to a set of overlapping deficiencies on the distal portion of chromosome 14. Molecular analysis was used to define the extent of the deletions. Mice homozygous for the smallest deletion, s15DttMb, die shortly after delivery and display alterations in the central nervous system, including hydrocephalus and a dorsally restricted malformation of the spinal cord. These mice also display homeotic transformations of vertebrae in the midthoracic and lumbar regions. Homozygous s27Pub mice contain a point mutation in the piebald gene, survive to weaning, and display no central nervous system or skeletal defects, arguing that the s15DttMb phenotype results from the loss of genes in addition to piebald. A larger deletion, s36Pub, exhibits additional cartilage malformations and defects in the anterior axial and cranial skeleton. The skeletal defects in both s15DttMb and s36Pub mice resemble transformations associated with the targeted disruption of Hox genes and genes encoding the retinoic acid receptors, which play a role in the specification of segmental identity along the anteroposterior axis. Complementation analysis of the s15DuMb and s36Pub phenotypes, using two additional deletions, localized the gene(s) associated with each phenotype to a defined chromosomal region.