Phosphatidylinositol-(4,5)-Bisphosphate Regulates Plasma Cholesterol Through LDL (Low-Density Lipoprotein) Receptor Lysosomal Degradation.

OBJECTIVE: TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of Tmem55b knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P2) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against Tmem55b or a nontargeting control for 3 to 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ≈70%, and plasma non-HDL (high-density lipoprotein) cholesterol was increased ≈1.8-fold (P<0.0001). Immunoblot analysis of fast protein liquid chromatography (FPLC) fractions revealed enrichment of ApoE-containing particles in the LDL size range. In contrast, Tmem55b knockdown had no effect on plasma cholesterol in Ldlr-/- mice. In primary hepatocytes and liver tissues from Tmem55b knockdown mice, there was decreased LDLR protein. In the hepatocytes, there was increased lysosome staining and increased LDLR-lysosome colocalization. Impairment of lysosome function (incubation with NH4Cl or knockdown of the lysosomal proteins LAMP1 or RAB7) abolished the effect of TMEM55B knockdown on LDLR in HepG2 (human hepatoma) cells. Colocalization of the recycling endosome marker RAB11 (Ras-related protein 11) with LDLR in HepG2 cells was reduced by 50% upon TMEM55B knockdown. Finally, knockdown increased hepatic PI(4,5)P2 levels in vivo and in HepG2 cells, while TMEM55B overexpression in vitro decreased PI(4,5)P2. TMEM55B knockdown decreased, whereas overexpression increased, LDL uptake in HepG2 cells. Notably, the TMEM55B overexpression effect was reversed by incubation with PI(4,5)P2. Conclusions: These findings indicate a role for TMEM55B in regulating plasma cholesterol levels by affecting PI(4,5)P2-mediated LDLR lysosomal degradation.

Rare Variants in the ABCG2 Promoter Modulate In Vivo Activity.

ABCG2 encodes the breast cancer resistance protein (BCRP), an efflux membrane transporter important in the detoxification of xenobiotics. In the present study, the basal activity of the ABCG2 promoter in liver, kidney, intestine, and breast cell lines was examined using luciferase reporter assays. The promoter activities of reference and variant ABCG2 sequences were compared in human hepatocellular carcinoma cell (HepG2), human embryonic kidney cell (HEK293T), human colorectal carcinoma cell (HCT116), and human breast adenocarcinoma cell (MCF-7) lines. The ABCG2 promoter activity was strongest in the kidney and intestine cell lines. Four variants in the basal ABCG2 promoter (rs76656413, rs66664036, rs139256004, and rs59370292) decreased the promoter activity by 25%-50% in at least three of the four cell lines. The activity of these four variants was also examined in vivo using the hydrodynamic tail vein assay, and two single nucleotide polymorphisms (rs76656413 and rs59370292) significantly decreased in vivo liver promoter activity by 50%-80%. Electrophoretic mobility shift assays confirmed a reduction in nuclear protein binding to the rs59370292 variant probe, whereas the rs76656413 probe had a shift in transcription factor binding specificity. Although both rs59370292 and rs76656413 are rare variants in all populations, they could contribute to patient-level variation in ABCG2 expression in the kidney, liver, and intestine.

ABCG2 regulatory single-nucleotide polymorphisms alter in vivo enhancer activity and expression.

OBJECTIVES: The expression and activity of the breast cancer resistance protein (ABCG2) contributes toward the pharmacokinetics of endogenous and xenobiotic substrates. The effect of genetic variation on the activity of cis-regulatory elements and nuclear response elements in the ABCG2 locus and their contribution toward ABCG2 expression have not been investigated systematically. In this study, the effect of genetic variation on the in vitro and in vivo enhancer activity of six previously identified liver enhancers in the ABCG2 locus was examined. METHODS: Reference and variant liver enhancers were tested for their ability to alter luciferase activity in vitro in HepG2 and HEK293T cell lines and in vivo using a hydrodynamic tail vein assay. Positive in vivo single-nucleotide polymorphisms (SNPs) were tested for association with gene expression and for altered protein binding in electrophoretic mobility shift assays. RESULTS: Multiple SNPs were found to alter enhancer activity in vitro. Four of these variants (rs9999111, rs12508471, ABCG2RE1*2, and rs149713212) decreased and one (rs2725263) increased enhancer activity in vivo. In addition, rs9999111 and rs12508471 were associated with ABCG2 expression in lymphoblastoid cell lines, lymphocytes, and T cells, and showed increased HepG2 nuclear protein binding. CONCLUSION: This study identifies SNPs within regulatory regions of the ABCG2 locus that alter enhancer activity in vitro and in vivo. Several of these SNPs correlate with tissue-specific ABCG2 expression and alter DNA/protein binding. These SNPs could contribute toward reported tissue-specific variability in ABCG2 expression and may influence the correlation between ABCG2 expression and disease risk or the pharmacokinetics and pharmacodynamics of breast cancer resistance protein substrates.

In Vivo Hepatic Enhancer Elements in the Human ABCG2 Locus.

ABCG2 encodes the mitoxantrone resistance protein (MXR; breast cancer resistance protein), an ATP-binding cassette (ABC) efflux membrane transporter. Computational analysis of the ∼300 kb region of DNA surrounding ABCG2 (chr4:88911376-89220011, hg19) identified 30 regions with potential cis-regulatory capabilities. These putative regulatory regions were tested for their enhancer and suppressor activity in a human liver cell line using luciferase reporter assays. The in vitro enhancer and suppressor assays identified four regions that decreased gene expression and five regions that increased expression >1.6-fold. Four of five human hepatic in vitro enhancers were confirmed as in vivo liver enhancers using the mouse hydrodynamic tail vein injection assay. Two of the in vivo liver enhancers (ABCG2RE1 and ABCG2RE9) responded to 17ß-estradiol or rifampin in human cell lines, and ABCG2RE9 had ChIP-seq evidence to support the binding of several transcription factors and the transcriptional coactivator p300 in human hepatocytes. This study identified genomic regions surrounding human ABCG2 that can function as regulatory elements, some with the capacity to alter gene expression upon environmental stimulus. The results from this research will drive future investigations of interindividual variation in ABCG2 expression and function that contribute to differences in drug response.

SUGP1 is a novel regulator of cholesterol metabolism.

A large haplotype on chromosome 19p13.11 tagged by rs10401969 in intron 8 of SURP and G patch domain containing 1 (SUGP1) is associated with coronary artery disease (CAD), plasma LDL cholesterol levels, and other energy metabolism phenotypes. Recent studies have suggested that TM6SF2 is the causal gene within the locus, but we postulated that this locus could harbor additional CAD risk genes, including the putative splicing factor SUGP1 Indeed, we found that rs10401969 regulates SUGP1 exon 8 skipping, causing non-sense-mediated mRNA decay. Hepatic Sugp1 overexpression in CD1 male mice increased plasma cholesterol levels 20-50%. In human hepatoma cell lines, SUGP1 knockdown stimulated 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) alternative splicing and decreased HMGCR transcript stability, thus reducing cholesterol synthesis and increasing LDL uptake. Our results strongly support a role for SUGP1 as a novel regulator of cholesterol metabolism and suggest that it contributes to the relationship between rs10401969 and plasma cholesterol.

Systematic dissection of coding exons at single nucleotide resolution supports an additional role in cell-specific transcriptional regulation.

In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons) could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6) at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types.

Genetic association study of adiposity and melanocortin-4 receptor (MC4R) common variants: replication and functional characterization of non-coding regions.

Common genetic variants 3' of MC4R within two large linkage disequilibrium (LD) blocks spanning 288 kb have been associated with common and rare forms of obesity. This large association region has not been refined and the relevant DNA segments within the association region have not been identified. In this study, we investigated whether common variants in the MC4R gene region were associated with adiposity-related traits in a biracial population-based study. Single nucleotide polymorphisms (SNPs) in the MC4R region were genotyped with a custom array and a genome-wide array and associations between SNPs and five adiposity-related traits were determined using race-stratified linear regression. Previously reported associations between lower BMI and the minor alleles of rs2229616/Val103Ile and rs52820871/Ile251Leu were replicated in white female participants. Among white participants, rs11152221 in a proximal 3' LD block (closer to MC4R) was significantly associated with multiple adiposity traits, but SNPs in a distal 3' LD block (farther from MC4R) were not. In a case-control study of severe obesity, rs11152221 was significantly associated. The association results directed our follow-up studies to the proximal LD block downstream of MC4R. By considering nucleotide conservation, the significance of association, and proximity to the MC4R gene, we identified a candidate MC4R regulatory region. This candidate region was sequenced in 20 individuals from a study of severe obesity in an attempt to identify additional variants, and the candidate region was tested for enhancer activity using in vivo enhancer assays in zebrafish and mice. Novel variants were not identified by sequencing and the candidate region did not drive reporter gene expression in zebrafish or mice. The identification of a putative insulator in this region could help to explain the challenges faced in this study and others to link SNPs associated with adiposity to altered MC4R expression.

Functional characterization of SIM1-associated enhancers.

Haploinsufficiency of the single-minded homology 1 (SIM1) gene in humans and mice leads to severe obesity, suggesting that altered expression of SIM1, by way of regulatory elements such as enhancers, could predispose individuals to obesity. Here, we identified transcriptional enhancers that could regulate SIM1, using comparative genomics coupled with zebrafish and mouse transgenic enhancer assays. Owing to the dual role of Sim1 in hypothalamic development and in adult energy homeostasis, the enhancer activity of these sequences was annotated from embryonic to adult age. Of the seventeen tested sequences, two SIM1 candidate enhancers (SCE2 and SCE8) were found to have brain-enhancer activity in zebrafish. Both SCE2 and SCE8 also exhibited embryonic brain-enhancer expression in mice, and time course analysis of SCE2 activity showed overlapping expression with Sim1 from embryonic to adult age, notably in the hypothalamus in adult mice. Using a deletion series, we identified the critical region in SCE2 that is needed for enhancer activity in the developing brain. Sequencing this region in obese and lean cohorts revealed a higher prevalence of single nucleotide polymorphisms (SNPs) that were unique to obese individuals, with one variant reducing developmental-enhancer activity in zebrafish. In summary, we have characterized two brain enhancers in the SIM1 locus and identified a set of obesity-specific SNPs within one of them, which may predispose individuals to obesity.

Sequence signatures extracted from proximal promoters can be used to predict distal enhancers.

BACKGROUND: Gene expression is controlled by proximal promoters and distal regulatory elements such as enhancers. While the activity of some promoters can be invariant across tissues, enhancers tend to be highly tissue-specific. RESULTS: We compiled sets of tissue-specific promoters based on gene expression profiles of 79 human tissues and cell types. Putative transcription factor binding sites within each set of sequences were used to train a support vector machine classifier capable of distinguishing tissue-specific promoters from control sequences. We obtained reliable classifiers for 92% of the tissues, with an area under the receiver operating characteristic curve between 60% (for subthalamic nucleus promoters) and 98% (for heart promoters). We next used these classifiers to identify tissue-specific enhancers, scanning distal non-coding sequences in the loci of the 200 most highly and lowly expressed genes. Thirty percent of reliable classifiers produced consistent enhancer predictions, with significantly higher densities in the loci of the most highly expressed compared to lowly expressed genes. Liver enhancer predictions were assessed in vivo using the hydrodynamic tail vein injection assay. Fifty-eight percent of the predictions yielded significant enhancer activity in the mouse liver, whereas a control set of five sequences was completely negative. CONCLUSIONS: We conclude that promoters of tissue-specific genes often contain unambiguous tissue-specific signatures that can be learned and used for the de novo prediction of enhancers.

The hydrodynamic tail vein assay as a tool for the study of liver promoters and enhancers.

The hydrodynamic tail vein injection is a technique that is used to deliver nucleic acids into live mice. Delivery through this method results in the in vivo transfection of foreign DNA primarily in the liver. Here, we describe the use of this technique to test for regulatory activity of liver promoters and enhancers, using a dual luciferase reporter system as the readable/measureable output and how this application can be used for pharmacogenomic studies.

Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model.

Despite continual progress in the cataloging of vertebrate regulatory elements, little is known about their organization and regulatory architecture. Here we describe a massively parallel experiment to systematically test the impact of copy number, spacing, combination and order of transcription factor binding sites on gene expression. A complex library of ∼5,000 synthetic regulatory elements containing patterns from 12 liver-specific transcription factor binding sites was assayed in mice and in HepG2 cells. We find that certain transcription factors act as direct drivers of gene expression in homotypic clusters of binding sites, independent of spacing between sites, whereas others function only synergistically. Heterotypic enhancers are stronger than their homotypic analogs and favor specific transcription factor binding site combinations, mimicking putative native enhancers. Exhaustive testing of binding site permutations suggests that there is flexibility in binding site order. Our findings provide quantitative support for a flexible model of regulatory element activity and suggest a framework for the design of synthetic tissue-specific enhancers.

Coding exons function as tissue-specific enhancers of nearby genes.

Enhancers are essential gene regulatory elements whose alteration can lead to morphological differences between species, developmental abnormalities, and human disease. Current strategies to identify enhancers focus primarily on noncoding sequences and tend to exclude protein coding sequences. Here, we analyzed 25 available ChIP-seq data sets that identify enhancers in an unbiased manner (H3K4me1, H3K27ac, and EP300) for peaks that overlap exons. We find that, on average, 7% of all ChIP-seq peaks overlap coding exons (after excluding for peaks that overlap with first exons). By using mouse and zebrafish enhancer assays, we demonstrate that several of these exonic enhancer (eExons) candidates can function as enhancers of their neighboring genes and that the exonic sequence is necessary for enhancer activity. Using ChIP, 3C, and DNA FISH, we further show that one of these exonic limb enhancers, Dync1i1 exon 15, has active enhancer marks and physically interacts with Dlx5/6 promoter regions 900 kb away. In addition, its removal by chromosomal abnormalities in humans could cause split hand and foot malformation 1 (SHFM1), a disorder associated with DLX5/6. These results demonstrate that DNA sequences can have a dual function, operating as coding exons in one tissue and enhancers of nearby gene(s) in another tissue, suggesting that phenotypes resulting from coding mutations could be caused not only by protein alteration but also by disrupting the regulation of another gene.

Massively parallel functional dissection of mammalian enhancers in vivo.

The functional consequences of genetic variation in mammalian regulatory elements are poorly understood. We report the in vivo dissection of three mammalian enhancers at single-nucleotide resolution through a massively parallel reporter assay. For each enhancer, we synthesized a library of >100,000 mutant haplotypes with 2-3% divergence from the wild-type sequence. Each haplotype was linked to a unique sequence tag embedded within a transcriptional cassette. We introduced each enhancer library into mouse liver and measured the relative activities of individual haplotypes en masse by sequencing the transcribed tags. Linear regression analysis yielded highly reproducible estimates of the effect of every possible single-nucleotide change on enhancer activity. The functional consequence of most mutations was modest, with ∼22% affecting activity by >1.2-fold and ∼3% by >2-fold. Several, but not all, positions with higher effects showed evidence for purifying selection, or co-localized with known liver-associated transcription factor binding sites, demonstrating the value of empirical high-resolution functional analysis.

Identification and characterization of novel polymorphisms in the basal promoter of the human transporter, MATE1.

OBJECTIVES: Human multidrug and toxin extrusion member 1, MATE1 (SLC47A1), plays an important role in the renal and biliary excretion of endogenous and exogenous organic cations including many therapeutic drugs. In this study, we characterized the transcriptional effects of five polymorphic variants and six common haplotypes in the basal promoter region of MATE1 that were identified in 272 DNA samples from ethnically diverse US populations. METHODS: We measured luciferase activities of the six common promoter haplotypes of MATE1 using in-vitro and in-vivo reporter assays. RESULTS: Haplotypes that contain the most common variant (mean allele frequency in four ethnic groups: 0.322), g.-66T>C, showed a significant decrease in reporter activities compared to the reference. Two transcription factors, activating protein-1 (AP-1) and activating protein-2 repressor (AP-2rep), were predicted to bind to the promoter in the region of g.-66T>C. Results from electrophoretic mobility shift assays showed that the g.-66T allele, exhibited greater binding to AP-1 than the g.-66C allele. AP-2rep inhibited the binding of AP-1 to the MATE1 basal promoter region, and the effect was considerably greater for the g.-66T>C. These data suggest that the reduced transcriptional activity of g.-66T>C results from a reduction in the binding potency of the transcriptional activator, AP-1, and an enhanced binding potency of the repressor, AP-2rep to the MATE1 basal promoter region. Consistent with the reporter assays, MATE1 mRNA expression levels were significantly lower in kidney samples from individuals who were homozygous or heterozygous for g.-66T>C in comparison with samples from individuals who were homozygous for the g.-66T allele. CONCLUSION: Our study suggests that the rate of transcription of MATE1 is regulated by AP-1 and AP-2rep and that a common promoter variant, g.-66T>C may affect the expression level of MATE1 in human kidney, and ultimately result in variation in drug disposition and response.