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A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.

Targeted gene ablation studies of the endocrine pancreas have long suffered from suboptimal Cre deleter strains. In many cases, Cre lines purportedly specific for beta cells also displayed expression in other islet endocrine cells or in a subset of neurons in the brain. Several pancreas and endocrine Cre lines have experienced silencing or mosaicism over time. In addition, many Cre transgenic constructs were designed to include the hGH mini-gene, which by itself increases beta-cell replication and decreases beta-cell function. More recently, driver lines with Cre or CreER inserted into the Ins1 locus were generated, with the intent of producing β cell-specific Cre lines with faithful recapitulation of insulin expression. These lines were bred in multiple labs to several different mouse lines harboring various lox alleles. In our hands, the ability of the Ins1-Cre and Ins1-CreER lines to delete target genes varied from that originally reported, with both alleles displaying low levels of expression, increased levels of methylation compared to the wild-type allele, and ultimately inefficient or absent target deletion. Thus, caution is warranted in the interpretation of results obtained with these genetic tools, and Cre expression and activity should be monitored regularly when using these lines.

Objective Both Type I and Type II diabetes mellitus result from insufficient functional β-cell mass. Efforts to increase β-cell proliferation as a means to restore β-cell mass have been met with limited success. Suppression of Tumorigenicity 5 (ST5) activates Ras/Erk signaling in the presence of Epidermal Growth Factor (EGF). In the pancreatic islet, Ras/Erk signaling is required for augmented β-cell proliferation during pregnancy, suggesting that ST5 is an appealing candidate to enhance adult β-cell proliferation. We aimed to test the hypothesis that overexpression of ST5 drives adult β-cell proliferation. Methods We utilized a doxycycline-inducible bitransgenic mouse model to activate β-cell-specific expression of human ST5 in adult mice at will. Islet morphology, β-cell proliferation, and β-cell mass in control and ST5-overexpressing (ST5 OE) animals were analyzed by immunofluorescent staining, under basal and two stimulated metabolic states: pregnancy and streptozotocin (STZ)-induced β-cell loss. Results Doxycycline treatment resulted in robust ST5 overexpression in islets from 12-16 week-old ST5 OE animals compared to controls, without affecting the islet morphology and identity of the β-cells. Under both basal and metabolically stimulated pregnancy states, β-cell proliferation and mass were comparable in ST5 OE and control animals. Furthermore, there was no detectable difference in β-cell proliferation between ST5 OE and control animals in response to STZ-induced β-cell loss. Conclusions We successfully derived an inducible bitransgenic mouse model to overexpress ST5 specifically in β-cells. However, our findings demonstrate that ST5 overexpression by itself has no mitogenic effect on the adult β-cell under basal and metabolically challenged states., Highlights • Hypothesized that overexpression of ST5 would drive adult β-cell proliferation due to its role in activating MAPK/ERK. • Generated a doxycycline-inducible bitransgenic mouse model to activate β-cell-specific expression of ST5. • ST5 overexpression has no mitogenic effect on adult β-cellsunder basal and metabolically challenged states.

Most platelet agonists activate platelets by binding to G-protein-coupled receptors. We have shown previously that a critical node in the G-protein signaling network in platelets is formed by a scaffold protein, spinophilin (SPL), the tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1 (SHP-1), and the regulator of G-protein signaling family member, RGS18. Here, we asked whether SPL and other RGS18 binding proteins such as 14-3-3γ regulate platelet reactivity by sequestering RGS18 and, if so, how this is accomplished. The results show that, in resting platelets, free RGS18 levels are relatively low, increasing when platelets are activated by thrombin. Free RGS18 levels also rise when platelets are rendered resistant to activation by exposure to prostaglandin I2 (PGI2) or forskolin, both of which increase platelet cyclic adenosine monophosphate (cAMP) levels. However, the mechanism for raising free RGS18 is different in these 2 settings. Whereas thrombin activates SHP-1 and causes dephosphorylation of SPL tyrosine residues, PGI2 and forskolin cause phosphorylation of SPL Ser94 without reducing tyrosine phosphorylation. Substituting alanine for Ser94 blocks cAMP-induced dissociation of the SPL/RGS/SHP-1 complex. Replacing Ser94 with aspartate prevents formation of the complex and produces a loss-of-function phenotype when expressed in mouse platelets. Together with the defect in platelet function we previously observed in SPL(-/-) mice, these data show that (1) regulated sequestration and release of RGS18 by intracellular binding proteins provides a mechanism for coordinating activating and inhibitory signaling networks in platelets, and (2) differential phosphorylation of SPL tyrosine and serine residues provides a key to understanding both.

The loss of insulin-secreting β cells is characteristic among type I and type II diabetes. Stimulating proliferation to expand sources of β cells for transplantation remains a challenge because adult β cells do not proliferate readily. The cell cycle inhibitor p57 has been shown to control cell division in human β cells. Expression of p57 is regulated by the DNA methylation status of the imprinting control region 2 (ICR2), which is commonly hypomethylated in Beckwith-Wiedemann syndrome patients who exhibit massive β cell proliferation. We hypothesized that targeted demethylation of the ICR2 using a transcription activator-like effector protein fused to the catalytic domain of TET1 (ICR2-TET1) would repress p57 expression and promote cell proliferation. We report here that overexpression of ICR2-TET1 in human fibroblasts reduces p57 expression levels and increases proliferation. Furthermore, human islets overexpressing ICR2-TET1 exhibit repression of p57 with concomitant upregulation of Ki-67 while maintaining glucose-sensing functionality. When transplanted into diabetic, immunodeficient mice, the epigenetically edited islets show increased β cell replication compared with control islets. These findings demonstrate that epigenetic editing is a promising tool for inducing β cell proliferation, which may one day alleviate the scarcity of transplantable β cells for the treatment of diabetes.

Background— Genome-wide association studies have established ADAMTS7 as a locus for coronary artery disease in humans. However, these studies fail to provide directionality for the association between ADAMTS7 and coronary artery disease. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell migration, but a role for and the direction of impact of this gene in atherogenesis have not been shown in relevant model systems. Methods and Results— We bred an Adamts7 whole-body knockout mouse onto both the Ldlr and Apoe knockout hyperlipidemic mouse models. Adamts7 −/− / Ldlr −/− and Adamts7 −/− / Apoe −/− mice displayed significant reductions in lesion formation in aortas and aortic roots compared with controls. Adamts7 knockout mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later time points, and primary Adamts7 knockout vascular smooth muscle cells showed reduced migration in the setting of tumor necrosis factor-α stimulation. ADAMTS7 localized to cells positive for smooth muscle cell markers in human coronary artery disease lesions, and subcellular localization studies in cultured vascular smooth muscle cells placed ADAMTS7 at the cytoplasm and cell membrane, where it colocalized with markers of podosomes. Conclusions— These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.

Background: Common diseases such as coronary heart disease (CHD) are complex in etiology. The interaction of genetic susceptibility with lifestyle factors may play a prominent role. However, gene-lifestyle interactions for CHD have been difficult to identify. Here, we investigate interaction of smoking behavior, a potent lifestyle factor, with genotypes that have been shown to associate with CHD risk. Methods: We analyzed data on 60 919 CHD cases and 80 243 controls from 29 studies for gene-smoking interactions for genetic variants at 45 loci previously reported to be associated with CHD risk. We also studied 5 loci associated with smoking behavior. Study-specific gene-smoking interaction effects were calculated and pooled using fixed-effects meta-analyses. Interaction analyses were declared to be significant at a P value of –3 (Bonferroni correction for 50 tests). Results: We identified novel gene-smoking interaction for a variant upstream of the ADAMTS7 gene. Every T allele of rs7178051 was associated with lower CHD risk by 12% in never-smokers ( P =1.3×10 –16 ) in comparison with 5% in ever-smokers ( P =2.5×10 –4 ), translating to a 60% loss of CHD protection conferred by this allelic variation in people who smoked tobacco (interaction P value=8.7×10 –5 ). The protective T allele at rs7178051 was also associated with reduced ADAMTS7 expression in human aortic endothelial cells and lymphoblastoid cell lines. Exposure of human coronary artery smooth muscle cells to cigarette smoke extract led to induction of ADAMTS7. Conclusions: Allelic variation at rs7178051 that associates with reduced ADAMTS7 expression confers stronger CHD protection in never-smokers than in ever-smokers. Increased vascular ADAMTS7 expression may contribute to the loss of CHD protection in smokers.

Rationale : Hemodynamic disturbed flow (DF) is associated with susceptibility to atherosclerosis. Endothelial Kruppel-Like Factor 4 (KLF4) is an important anti-inflammatory atheroprotective transcription factor that is suppressed in regions of DF. Objective : The plasticity of epigenomic KLF4 transcriptional regulation by flow-mediated DNA methylation was investigated in vitro and in arterial tissue. Methods and Results : To recapitulate dominant flow characteristics of atheroprotected and atherosusceptible arteries, human aortic endothelial cells were subjected to pulsatile undisturbed flow or oscillatory DF containing a flow-reversing phase. Differential CpG site methylation was measured by methylation-specific polymerase chain reaction, bisulfite pyrosequencing, and restriction enzyme-polymerase chain reaction. The methylation profiles of endothelium from disturbed and undisturbed flow sites of adult swine aortas were also investigated. In vitro, DF increased DNA methylation of CpG islands within the KLF4 promoter that significantly contributed to suppression of KLF4 transcription; the effects were mitigated by DNA methyltransferase (DNMT) inhibitors and knockdown of DNMT3A. Contributory mechanisms included DF-induced increase of DNMT3A protein (1.7-fold), DNMT3A enrichment (11-fold) on the KLF4 promoter, and competitive blocking of a myocyte enhancer factor-2 binding site in the KLF4 promoter near the transcription start site. DF also induced DNMT-sensitive propathological expression of downstream KLF4 transcription targets nitric oxide synthase 3, thrombomodulin, and monocyte chemoattractant protein-1. In support of the in vitro findings, swine aortic endothelium isolated from DF regions expressed significantly lower KLF4 and nitric oxide synthase 3, and bisulfite sequencing of KLF4 promoter identified a hypermethylated myocyte enhancer factor-2 binding site. Conclusions : Hemodynamics influence endothelial KLF4 expression through DNMT enrichment/myocyte enhancer factor-2 inhibition mechanisms of KLF4 promoter CpG methylation with regional consequences for atherosusceptibility.

Common diseases such as coronary heart disease (CHD) are complex in etiology. The interaction of genetic susceptibility with lifestyle factors may play a prominent role. However, gene-lifestyle interactions for CHD have been difficult to identify. Here, we investigate interaction of smoking behavior, a potent lifestyle factor, with genotypes that have been shown to associate with CHD risk.We analyzed data on 60 919 CHD cases and 80 243 controls from 29 studies for gene-smoking interactions for genetic variants at 45 loci previously reported to be associated with CHD risk. We also studied 5 loci associated with smoking behavior. Study-specific gene-smoking interaction effects were calculated and pooled using fixed-effects meta-analyses. Interaction analyses were declared to be significant at aWe identified novel gene-smoking interaction for a variant upstream of the

ADAMTS7 , a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, has been recently identified as a novel genetic locus for coronary artery disease. Although a handful of ADAMTS7 substrates have been identified through traditional yeast two-hybrid screening and standard proteomic profiling, the molecular mechanisms of ADAMTS7 contribution to atherosclerosis have not been fully explored in the context of its proteolytic action. Here we utilized terminal amine isotope labeling of substrates (TAILS), a cutting-edge proteomic approach to enrich N-terminal peptides, enable systemic characterization of proteolytic events and substrate identification for ADAMTS7. Briefly, primary vascular smooth muscle cells (SMC) from ADAMTS7 WT and KO mice were treated with TNFα to stimulated endogenous ADAMTS7 expression, followed by TAILS using dimethyl labeling in cellular proteome and secretome. In 3 biological replicates, we identified 1,720 and 1,616 quantified N-terminal peptides in SMC proteome and secretome, which composed of 415 and 122 unique N-termini of mature protein as well as 1,185 and 1,161 cleaved neo-N-termini driven by proteolytic processing. As expected, most of acetylated peptides in SMC proteome and secretome are natural N-termini of mature proteins (82% and 77%, respectively) while free N-terminal peptides are remarkably enriched with cleaved neo-N termini (85% and 96%, respectively). Importantly, 106 and 86 neo-N-termini (corresponding to 65 and 54 unique proteins) were enriched in ADAMTS7 WT SMC proteome and secretome respectively (WT/KO isotope ratio >2), suggesting that these proteins underwent elevated proteolysis in ADAMTS WT SMCs and thus are proteolytic substrate candidates for ADAMTS7. Interestingly, gene pathway analysis indicates that these proteins are enriched with genes involved in proteinaceous extracellular matrix and extracellular region, such as vimentin, fibulin and MMPs. Biochemistry studies are ongoing to validate these ADAMTS7 substrate candidates. In summary, our study presents a systemic profiling on proteolytic events in SMC cellular proteome and secretome and identifies candidate ADAMTS7 substrates relevant to smooth muscle function and vascular biology.

SNPs in the genomic region containing the gene ADAMTS7, a gene that codes for a metalloproteinase involved in vascular smooth muscle cell migration, are significantly associated with CAD in humans. Recently, our lab demonstrated that knock out of Adamts7 in mice is protective against atherosclerosis, suggesting that ADAMTS7 is involved in the pathogenesis of cardiovascular disease. Several of the top CAD-associated SNPs (pADAMTS7 .. We investigated the functionality of CAD associated SNPs that overlap 5 active regulatory elements as predicted by vascular smooth muscle cell (VSMC) ChIP-seq signals, ENCODE histone methylation peaks, and aortic smooth muscle cell DNase hypersensitivity peaks. Several regions containing association SNPs showed enhancer activity in minimal promoter luciferase reporter assays. Region 2 showed an 8.2 (pADAMTS7 , but our current data suggest that top CAD-associated SNPs alter regulatory activity at the ADAMTS7 locus.

ADAMTS7 has recently been identified as a novel genomic locus associated with coronary artery disease (CAD) in humans. We have recently shown that deficiency of Adamts7 in hyperlipidemic mouse models reduces atherosclerosis following prolonged western diet feeding, however the mechanism of this reduction remains unknown. Using a β-galactosidase (β-gal) reporter, we observed that vascular Adamts7 expression is induced in response to both mechanical injury and western diet feeding in a transient manner, and this expression predominantly colocalizes to vascular smooth muscle cells (VSMCs). We investigated the ability of multiple cytokines and growth factors (PDGF-BB, TNFα, IFNγ, and Angiotensin II) to stimulate Adamts7 expression in primary mouse VSMCs, and found that of these only TNFα induced Adamts7 expression (5.5-fold, p=0.0007). We determined the expression of Adamts7 and TNFα in whole aortas and aortic roots of Apoe-/- mice at various timepoints of western diet feeding, and found trends towards increased Adamts7 expression while TNFα expression consistently increased over the course of the experiment (4.4-fold increase, p=0.002). We next examined proliferation and migration of primary VSMCs from Adamts7 WT and KO animals. Unstimulated Adamts7 KO VSMCs had no observable difference in migration as compared to WT controls. However, in the setting of TNFα stimulation we observed a decrease in migration of Adamts7 KO VSMCs plated on collagen I (50% reduction, p=0.02) and laminin (60% reduction, p=0.0002). Finally, using both public and private data sets, we assessed if associations exist between SNPs in and around the human ADAMTS7 locus and the expression of ADAMTS7 in relevant tissues. In small data sets we find that SNPs with genome-wide association with CAD also associate with ADAMTS7 expression, and the direction of this association is consistent with our in vivo reduction of atherosclerosis in Adamts7 KO animals. In summary, Adamts7 expression in mice is induced in response to vascular injury, perhaps mediated by TNFα expression, and this induction can modulate VSMC migration. These observations present a novel mechanistic paradigm for ADAMTS7 modulation of atherogenesis and suggest its inhibition might reduce CHD in clinic.

Recent genome-wide association studies (GWAS) have identified an association between the ADAMTS7 locus and coronary artery disease (CAD) in humans. While ADAMTS7 is proposed to play a role in vascular smooth muscle cell (VSMC) migration and neointimal formation, the molecular regulation of human ADAMTS7 gene expression has not yet been explored. We assessed ADAMTS7 expression levels in primary mouse aortic smooth muscle cells (mAoSMC) as well as primary human coronary and human pulmonary artery smooth muscle cells (hCASMC, hPASMC) in response to treatment with various stimulatory agents. No differences in ADAMTS7 expression were observed upon treatment with PDGF, angiotensin II, and nicotine in any of the cell lines tested. However, TNFα upregulated ADAMTS7 by 4-fold in mAoSMC but not in human VSMCs while H 2 O 2 upregulated ADAMTS7 by 4-fold in hCASMC but not mAoSMCs. No agents modulated expression in hPASMC. Basal levels of ADAMTS7 varied among different VSMC lines; hCASMC had 2-fold greater levels of ADAMTS7 when compared to hPASMC and hAoSMC. These data demonstrate that ADAMTS7 is differentially regulated not only across different species, but also among different VSMC types, underscoring the complex genetic regulation of ADAMTS7 . In an attempt to elucidate important regulatory genomic regions controlling ADAMTS7 expression, we utilized data from the ENCODE project to identify regions surrounding ADAMTS7 that are enriched for regulatory domains, e.g. DNase hypsersensitivity (DHS) and H3K27 acetylation, in relevant cell types. We then overlayed data from CARDIoGRAM and C4D CAD GWAS to look for CAD-associated SNPs in these potential enhancers. Ultimately we selected 5 regions of interest (e.g., rs5029904 lies in region with increased H3K27 acetylation in ENCODE layered 7 cell lines, DHS peaks in serum fed and starved hAoSMCs, binding peaks for transcription factors via ENCODE ChIP-Seq experiments) for further studies in luciferase reporter assays multiple cell types to look for tissue specific expression mediated by these genomic regions. GWAS variants in these domains may affect the transcriptional regulation of ADAMTS7 , thus providing insight into the role for ADAMTS7 in human atherosclerosis.

Multiple genome-wide association studies demonstrate that the genomic locus containing the gene ADAMTS7 is associated with coronary artery disease in humans. ADAMTS7 was not linked to cardiovascular disease prior to those genetic studies, and to date this association has not been validated in experimental models of atherosclerosis. An Adamts7 ( Ats7 ) knockout mouse, with knock-in of a β-galactosidase reporter gene, was bred onto both the LDLR and APOE KO hyperlipidemic mouse models. Atherosclerosis studies were performed on the resultant Ats7/LDLR and Ats7 / APOE double KO (dKO) mice and control littermates. Mice were fed western diet for 16 weeks ( LDLR ) or 10 weeks ( APOE ), and aortas and aortic roots were harvested for lesion area quantification. In addition, brachiocephalic arteries were dissected for immunohistochemical analysis of lesion composition. No differences in plasma lipids were observed between experimental groups in either study. Ats7 / LDLR dKO males (N=7) and females (N=13) displayed significant reductions in lesion formation in aortas by en face (58%, p p p =0.04 and 22%, p =0.002 respectively) compared to control LDLR -/- male (N=6) and female (N=10) littermates. Additionally, Ats7 / APOE dKO males (N=16) and females (N=13) displayed significantly reduced lesion formation by en face (62%, p p p =0.15 and 22%, p =0.10 respectively) compared to control APOE -/- males (N=9) and females (N=10). Femoral wire injury experiments suggested reduced neointimal formation in Ats7 KO mice at 28 days post-injury, and X-gal staining at multiple timepoints showed adventitial Ats7 expression peaking at 7 days post-injury. Preliminary studies suggest that aortic rings from Ats7 / LDLR dKO mice embedded in collagen have reduced cell migration compared to control LDLR aortic rings. In summary, these data represent the first in vivo experimental validation of Adamts7 as a gene involved in atherogenesis, likely through modulation of vascular cell migration into atherosclerotic lesions. These data support the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans.