Genomic Characterization of Metformin Hepatic Response.

Metformin is used as a first-line therapy for type 2 diabetes (T2D) and prescribed for numerous other diseases. However, its mechanism of action in the liver has yet to be characterized in a systematic manner. To comprehensively identify genes and regulatory elements associated with metformin treatment, we carried out RNA-seq and ChIP-seq (H3K27ac, H3K27me3) on primary human hepatocytes from the same donor treated with vehicle control, metformin or metformin and compound C, an AMP-activated protein kinase (AMPK) inhibitor (allowing to identify AMPK-independent pathways). We identified thousands of metformin responsive AMPK-dependent and AMPK-independent differentially expressed genes and regulatory elements. We functionally validated several elements for metformin-induced promoter and enhancer activity. These include an enhancer in an ataxia telangiectasia mutated (ATM) intron that has SNPs in linkage disequilibrium with a metformin treatment response GWAS lead SNP (rs11212617) that showed increased enhancer activity for the associated haplotype. Expression quantitative trait locus (eQTL) liver analysis and CRISPR activation suggest that this enhancer could be regulating ATM, which has a known role in AMPK activation, and potentially also EXPH5 and DDX10, its neighboring genes. Using ChIP-seq and siRNA knockdown, we further show that activating transcription factor 3 (ATF3), our top metformin upregulated AMPK-dependent gene, could have an important role in gluconeogenesis repression. Our findings provide a genome-wide representation of metformin hepatic response, highlight important sequences that could be associated with interindividual variability in glycemic response to metformin and identify novel T2D treatment candidates.

Genome-wide discovery of drug-dependent human liver regulatory elements.

Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks (p300, H3K4me1, H3K27ac) on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements.

Genome-wide identification of signaling center enhancers in the developing limb.

The limb is widely used as a model developmental system and changes to gene expression patterns in its signaling centers, notably the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER), are known to cause limb malformations and evolutionary differences in limb morphology. Although several genes that define these limb signaling centers have been described, the identification of regulatory elements that are active within these centers has been limited. By dissecting mouse E11.5 limbs that fluorescently mark the ZPA or AER, followed by fluorescence-activated cell sorting and low-cell H3K27ac ChIP-seq, we identified thousands of specific signaling-center enhancers. Our ChIP-seq datasets show strong correlation with ZPA- and AER-expressed genes, previously characterized functional ZPA and AER enhancers and enrichment for relevant biological terms related to limb development and malformation for the neighboring genes. Using transgenic assays, we show that several of these sequences function as ZPA and AER enhancers. Our results identify novel ZPA and AER enhancers that could be important regulators of genes involved in the establishment of these specialized regions and the patterning of tetrapod limbs.

Comparative genomic analysis of Atlantic salmon, Salmo salar, from Europe and North America.

BACKGROUND: Several lines of evidence including allozyme analysis, restriction digest patterns and sequencing of mtDNA as well as mini- and micro-satellite allele frequencies indicate that Atlantic salmon (Salmo salar) from North America and Europe are genetically distinct. These observations are supported by karyotype analysis, which revealed that North American Atlantic salmon have 27 pairs of chromosomes whereas European salmon have 29 pairs. We set out to construct a linkage map for a North American Atlantic salmon family and to compare this map with the well developed map for European Atlantic salmon. RESULTS: We used microsatellite markers, which had previously been mapped in the two Atlantic salmon SALMAP mapping families from the River Tay, Scotland, to carry out linkage analysis in an Atlantic salmon family (NB1) whose parents were derived from the Saint John River stock in New Brunswick, Canada. As large differences in recombination rates between female and male Atlantic salmon have been noted, separate genetic maps were constructed for each sex. The female linkage map comprises 218 markers in 37 linkage groups while the male map has 226 markers in 28 linkage groups. We combined 280 markers from the female and male maps into 27 composite linkage groups, which correspond to the haploid number of chromosomes in Atlantic salmon from the Western Atlantic. CONCLUSIONS: A comparison of the composite NB1 and SALMAP linkage maps revealed the reason for the difference in the chromosome numbers between European and North American Atlantic salmon: Linkage groups AS-4 and AS-32 in the Scottish salmon, which correspond to chromosomes Ssa-6 and Ssa-22, are combined into a single NB1 linkage group as are linkage groups AS-21 and AS-33 (corresponding to chromosomes Ssa-26 and Ssa-28). The comparison of the linkage maps also suggested some additional chromosomal rearrangements, but it will require finer mapping, potentially using SNPs, to test these predictions. Our results provide the first comparison of the genomic architecture of Atlantic salmon from North America and Europe with respect to chromosome organization.