Many of the type 2 diabetes loci identified through genome-wide association studies localize to non-protein-coding intronic and intergenic regions and likely contain variants that regulate gene ...transcription. The CDC123/CAMK1D type 2 diabetes association signal on chromosome 10 spans an intergenic region between CDC123 and CAMK1D and also overlaps the CDC123 3'UTR. To gain insight into the molecular mechanisms underlying the association signal, we used open chromatin, histone modifications and transcription factor ChIP-seq data sets from type 2 diabetes-relevant cell types to identify SNPs overlapping predicted regulatory regions. Two regions containing type 2 diabetes-associated variants were tested for enhancer activity using luciferase reporter assays. One SNP, rs11257655, displayed allelic differences in transcriptional enhancer activity in 832/13 and MIN6 insulinoma cells as well as in human HepG2 hepatocellular carcinoma cells. The rs11257655 risk allele T showed greater transcriptional activity than the non-risk allele C in all cell types tested. Using electromobility shift and supershift assays we demonstrated that the rs11257655 risk allele showed allele-specific binding to FOXA1 and FOXA2. We validated FOXA1 and FOXA2 enrichment at the rs11257655 risk allele using allele-specific ChIP in human islets. These results suggest that rs11257655 affects transcriptional activity through altered binding of a protein complex that includes FOXA1 and FOXA2, providing a potential molecular mechanism at this GWAS locus.
The growth of many cancers depends on self-renewing cells called cancer stem cells or tumor-propagating cells (TPCs). In human brain tumors, cells expressing the stem cell marker CD133 have been ...implicated as TPCs. Here we show that tumors from a model of medulloblastoma, the
Patched mutant mouse, are propagated not by CD133
+ cells but by cells expressing the progenitor markers Math1 and CD15/SSEA-1. These cells have a distinct expression profile that suggests increased proliferative capacity and decreased tendency to undergo apoptosis and differentiation. CD15 is also found in a subset of human medulloblastomas, and tumors expressing genes similar to those found in murine CD15
+ cells have a poorer prognosis. Thus, CD15 may represent an important marker for TPCs in medulloblastoma.
Tissue-specific transcriptional regulation is central to human disease. To identify regulatory DNA active in human pancreatic islets, we profiled chromatin by formaldehyde-assisted isolation of ...regulatory elements coupled with high-throughput sequencing (FAIRE-seq). We identified ∼80,000 open chromatin sites. Comparison of FAIRE-seq data from islets to that from five non-islet cell lines revealed ∼3,300 physically linked clusters of islet-selective open chromatin sites, which typically encompassed single genes that have islet-specific expression. We mapped sequence variants to open chromatin sites and found that rs7903146, a TCF7L2 intronic variant strongly associated with type 2 diabetes, is located in islet-selective open chromatin. We found that human islet samples heterozygous for rs7903146 showed allelic imbalance in islet FAIRE signals and that the variant alters enhancer activity, indicating that genetic variation at this locus acts in cis with local chromatin and regulatory changes. These findings illuminate the tissue-specific organization of cis-regulatory elements and show that FAIRE-seq can guide the identification of regulatory variants underlying disease susceptibility.
Genome-wide association studies (GWASs) have identified more than 150 loci associated with blood lipid and cholesterol levels; however, the functional and molecular mechanisms for many associations ...are unknown. We examined the functional regulatory effects of candidate variants at the GALNT2 locus associated with high-density lipoprotein cholesterol (HDL-C). Fine-mapping and conditional analyses in the METSIM study identified a single locus harboring 25 noncoding variants (r2 > 0.7 with the lead GWAS variants) strongly associated with total cholesterol in medium-sized HDL (e.g., rs17315646, p = 3.5 × 10−12). We used luciferase reporter assays in HepG2 cells to test all 25 variants for allelic differences in regulatory enhancer activity. rs2281721 showed allelic differences in transcriptional activity (75-fold T versus 27-fold C more than the empty-vector control), as did a separate 780-bp segment containing rs4846913, rs2144300, and rs6143660 (49-fold AT– haplotype versus 16-fold CC+ haplotype more). Using electrophoretic mobility shift assays, we observed differential CEBPB binding to rs4846913, and we confirmed this binding in a native chromatin context by performing chromatin-immunoprecipitation (ChIP) assays in HepG2 and Huh-7 cell lines of differing genotypes. Additionally, sequence reads in HepG2 DNase-I-hypersensitivity and CEBPB ChIP-seq signals spanning rs4846913 showed significant allelic imbalance. Allelic-expression-imbalance assays performed with RNA from primary human hepatocyte samples and expression-quantitative-trait-locus (eQTL) data in human subcutaneous adipose tissue samples confirmed that alleles associated with increased HDL-C are associated with a modest increase in GALNT2 expression. Together, these data suggest that at least rs4846913 and rs2281721 play key roles in influencing GALNT2 expression at this HDL-C locus.
Insulin secretion has a crucial role in glucose homeostasis, and failure to secrete sufficient insulin is a hallmark of type 2 diabetes. Genome-wide association studies (GWAS) have identified loci ...contributing to insulin processing and secretion; however, a substantial fraction of the genetic contribution remains undefined. To examine low-frequency (minor allele frequency (MAF) 0.5-5%) and rare (MAF < 0.5%) nonsynonymous variants, we analyzed exome array data in 8,229 nondiabetic Finnish males using the Illumina HumanExome Beadchip. We identified low-frequency coding variants associated with fasting proinsulin concentrations at the SGSM2 and MADD GWAS loci and three new genes with low-frequency variants associated with fasting proinsulin or insulinogenic index: TBC1D30, KANK1 and PAM. We also show that the interpretation of single-variant and gene-based tests needs to consider the effects of noncoding SNPs both nearby and megabases away. This study demonstrates that exome array genotyping is a valuable approach to identify low-frequency variants that contribute to complex traits.
Translation of noncoding common variant association signals into meaningful molecular and biological mechanisms explaining disease susceptibility remains challenging. For the type 2 diabetes ...association signal in JAZF1 intron 1, we hypothesized that the underlying risk variants have cis-regulatory effects in islets or other type 2 diabetes-relevant cell types. We used maps of experimentally predicted open chromatin regions to prioritize variants for functional follow-up studies of transcriptional activity. Twelve regions containing type 2 diabetes-associated variants were tested for enhancer activity in 832/13 and MIN6 insulinoma cells. Three regions exhibited enhancer activity and only rs1635852 displayed allelic differences in enhancer activity; the type 2 diabetes risk allele T showed lower transcriptional activity than the nonrisk allele C. This risk allele showed increased binding to protein complexes, suggesting that it functions as part of a transcriptional repressor complex. We applied DNA affinity capture to identify factors in the complex and determined that the risk allele preferentially binds the pancreatic master regulator PDX1. These data suggest that the rs1635852 region in JAZF1 intron 1 is part of a cis-regulatory complex and that maps of open chromatin are useful to guide identification of variants with allelic differences in regulatory activity at type 2 diabetes loci.
The Sonic hedgehog (Shh) and FGF signaling pathways regulate growth and differentiation in many regions of the nervous system, but interactions between these pathways have not been studied ...extensively. Here, we examine the relationship between Shh and FGF signaling in granule cell precursors (GCPs), which are the most abundant neural progenitors in the cerebellum and the putative cell of origin for the childhood brain tumor medulloblastoma. In these cells, Shh induces a potent proliferative response that is abolished by coincubation with basic FGF. FGF also inhibits transcription of Shh target genes and prevents activation of a Gli-responsive promoter in fibroblasts, which suggests that it blocks Shh signaling upstream of Gli-mediated transcription. FGF-mediated inhibition of Shh responses requires activation of FGF receptors and of ERK and JNK kinases, because it can be blocked by inhibitors of these enzymes. Finally, FGF promotes differentiation of GCPs in vitro and in vivo and halts proliferation of tumor cells from patched (ptc) mutant mice, a model for medulloblastoma. These findings suggest that FGF is a potent inhibitor of Shh signaling and may be a useful therapy for tumors involving activation of the hedgehog pathway.
Δ
9
‐Tetrahydrocannabinol (THC), the main psychoactive ingredient of marijuana, induces apoptosis in cultured cortical neurons. THC exerts its apoptotic effects in cortical neurons by binding to the ...CB
1
cannabinoid receptor. The CB
1
receptor has been shown to couple to the stress‐activated protein kinase, c‐Jun N‐terminal kinase (JNK). However, the involvement of specific JNK isoforms in the neurotoxic properties of THC remains to be established.
The present study involved treatment of rat cultured cortical neurons with THC (0.005–50
μ
M
), and combinations of THC with the CB
1
receptor antagonist, AM 251 (10
μ
M
) and pertussis toxin (PTX; 200 ng ml
−1
). Antisense oligonucleotides (AS) were used to deplete neurons of JNK1 and JNK2 in order to elucidate their respective roles in THC signalling.
Here we report that THC induces the activation of JNK
via
the CB
1
receptor and its associated G‐protein, G
i/o
. Treatment of cultured cortical neurons with THC resulted in a differential timeframe of activation of the JNK1 and JNK2 isoforms.
Use of specific JNK1 and JNK2 AS identified activation of caspase‐3 and DNA fragmentation as downstream consequences of JNK1 and JNK2 activation.
The results from this study demonstrate that activation of the CB
1
receptor induces JNK and caspase‐3 activation, an increase in Bax expression and DNA fragmentation. The data demonstrate that the activation of both JNK1 and JNK2 isoforms is central to the THC‐induced activation of the apoptotic pathway in cortical neurons.
British Journal of Pharmacology
(2003)
140
, 547–557. doi:
10.1038/sj.bjp.0705464
Pherobrine and Burley are siphoviruses infecting Gordonia rubripertincta. Pherobrine has a 60,305-bp genome with 89 predicted protein-coding genes, and Burley has a 60,111-bp genome with 90 predicted ...protein-coding genes. Both phages are assigned to cluster DJ, where they share 78% gene content similarity with each other.
Characterization of the epigenome promises to yield the functional elements buried in the human genome sequence, thus helping to annotate non-coding DNA polymorphisms with regulatory functions. Here, ...we develop two novel strategies to combine epigenomic data with transcriptomic profiles in humans or mice to prioritize potential candidate SNPs associated with lipid levels by genome-wide association study (GWAS). First, after confirming that lipid-associated loci that are also expression quantitative trait loci (eQTL) in human livers are enriched for ENCODE regulatory marks in the human hepatocellular HepG2 cell line, we prioritize candidate SNPs based on the number of these marks that overlap the variant position. This method recognized the known SORT1 rs12740374 regulatory SNP associated with LDL-cholesterol, and highlighted candidate functional SNPs at 15 additional lipid loci. In the second strategy, we combine ENCODE chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) data and liver expression datasets from knockout mice lacking specific transcription factors. This approach identified SNPs in specific transcription factor binding sites that are located near target genes of these transcription factors. We show that FOXA2 transcription factor binding sites are enriched at lipid-associated loci and experimentally validate that alleles of one such proxy SNP located near the FOXA2 target gene BIRC5 show allelic differences in FOXA2-DNA binding and enhancer activity. These methods can be used to generate testable hypotheses for many non-coding SNPs associated with complex diseases or traits.
•We combined eQTL and ENCODE data to prioritize functional variants at lipid loci.•We integrated mouse transcriptomic and ENCODE data to fine-map GWAS loci.•We validated in silico predictions using functional experiments for a lipid locus.
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