Association of genetic variation in COL11A1 with adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a common and progressive spinal deformity in children that exhibits striking sexual dimorphism, with girls at more than fivefold greater risk of severe disease compared to boys. Despite its medical impact, the molecular mechanisms that drive AIS are largely unknown. We previously defined a female-specific AIS genetic risk locus in an enhancer near the PAX1 gene. Here, we sought to define the roles of PAX1 and newly identified AIS-associated genes in the developmental mechanism of AIS. In a genetic study of 10,519 individuals with AIS and 93,238 unaffected controls, significant association was identified with a variant in COL11A1 encoding collagen (α1) XI (rs3753841; NM_080629.2_c.4004C>T; p.(Pro1335Leu); p=7.07E-11, OR = 1.118). Using CRISPR mutagenesis we generated Pax1 knockout mice (Pax1-/-). In postnatal spines we found that PAX1 and collagen (α1) XI protein both localize within the intervertebral disc-vertebral junction region encompassing the growth plate, with less collagen (α1) XI detected in Pax1-/- spines compared to wild-type. By genetic targeting we found that wild-type Col11a1 expression in costal chondrocytes suppresses expression of Pax1 and of Mmp3, encoding the matrix metalloproteinase 3 enzyme implicated in matrix remodeling. However, the latter suppression was abrogated in the presence of the AIS-associated COL11A1P1335L mutant. Further, we found that either knockdown of the estrogen receptor gene Esr2 or tamoxifen treatment significantly altered Col11a1 and Mmp3 expression in chondrocytes. We propose a new molecular model of AIS pathogenesis wherein genetic variation and estrogen signaling increase disease susceptibility by altering a PAX1-COL11a1-MMP3 signaling axis in spinal chondrocytes.

Adolescent idiopathic scoliosis (AIS) is a twisting deformity of the spine that occurs during periods of rapid growth in children worldwide. Children with severe cases of AIS require surgery to stop it from getting worse, presenting a significant financial burden to health systems and families. Although AIS is known to cluster in families, its genetic causes and its inheritance pattern have remained elusive. Additionally, AIS is known to be more prevalent in females, a bias that has not been explained. Advances in techniques to study the genetics underlying diseases have revealed that certain variations that increase the risk of AIS affect cartilage and connective tissue. In humans, one such variation is near a gene called Pax1, and it is female-specific. The extracellular matrix is a network of proteins and other molecules in the space between cells that help connect tissues together, and it is particularly important in cartilage and other connective tissues. One of the main components of the extracellular matrix is collagen. Yu, Kanshour, Ushiki et al. hypothesized that changes in the extracellular matrix could affect the cartilage and connective tissues of the spine, leading to AIS. To show this, the scientists screened over 100,000 individuals and found that AIS is associated with variants in two genes coding for extracellular matrix proteins. One of these variants was found in a gene called Col11a1, which codes for one of the proteins that makes up collagen. To understand the relationship between Pax1 and Col11a1, Yu, Kanshour, Ushiki et al. genetically modified mice so that they would lack the Pax1 gene. In these mice, the activation of Col11a1 was reduced in the mouse spine. They also found that the form of Col11a1 associated with AIS could not suppress the activation of a gene called Mmp3 in mouse cartilage cells as effectively as unmutated Col11a1. Going one step further, the researchers found that lowering the levels of an estrogen receptor altered the activation patterns of Pax1, Col11a1, and Mmp3 in mouse cartilage cells. These findings suggest a possible mechanism for AIS, particularly in females. The findings of Yu, Kanshour, Ushiki et al. highlight that cartilage cells in the spine are particularly relevant in AIS. The results also point to specific molecules within the extracellular matrix as important for maintaining proper alignment in the spine when children are growing rapidly. This information may guide future therapies aimed at maintaining healthy spinal cells in adolescent children, particularly girls.

Integrative single-cell characterization of a frugivorous and an insectivorous bat kidney and pancreas.

Frugivory evolved multiple times in mammals, including bats. However, the cellular and molecular components driving it remain largely unknown. Here, we use integrative single-cell sequencing (scRNA-seq and scATAC-seq) on insectivorous (Eptesicus fuscus; big brown bat) and frugivorous (Artibeus jamaicensis; Jamaican fruit bat) bat kidneys and pancreases and identify key cell population, gene expression and regulatory differences associated with the Jamaican fruit bat that also relate to human disease, particularly diabetes. We find a decrease in loop of Henle and an increase in collecting duct cells, and differentially active genes and regulatory elements involved in fluid and electrolyte balance in the Jamaican fruit bat kidney. The Jamaican fruit bat pancreas shows an increase in endocrine and a decrease in exocrine cells, and differences in genes and regulatory elements involved in insulin regulation. We also find that these frugivorous bats share several molecular characteristics with human diabetes. Combined, our work provides insights from a frugivorous mammal that could be leveraged for therapeutic purposes.

Chromatin compartmentalization regulates the response to DNA damage.

The DNA damage response is essential to safeguard genome integrity. Although the contribution of chromatin in DNA repair has been investigated1,2, the contribution of chromosome folding to these processes remains unclear3. Here we report that, after the production of double-stranded breaks (DSBs) in mammalian cells, ATM drives the formation of a new chromatin compartment (D compartment) through the clustering of damaged topologically associating domains, decorated with γH2AX and 53BP1. This compartment forms by a mechanism that is consistent with polymer-polymer phase separation rather than liquid-liquid phase separation. The D compartment arises mostly in G1 phase, is independent of cohesin and is enhanced after pharmacological inhibition of DNA-dependent protein kinase (DNA-PK) or R-loop accumulation. Importantly, R-loop-enriched DNA-damage-responsive genes physically localize to the D compartment, and this contributes to their optimal activation, providing a function for DSB clustering in the DNA damage response. However, DSB-induced chromosome reorganization comes at the expense of an increased rate of translocations, also observed in cancer genomes. Overall, we characterize how DSB-induced compartmentalization orchestrates the DNA damage response and highlight the critical impact of chromosome architecture in genomic instability.

Association of genetic variation in COL11A1 with adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a common and progressive spinal deformity in children that exhibits striking sexual dimorphism, with girls at more than five-fold greater risk of severe disease compared to boys. Despite its medical impact, the molecular mechanisms that drive AIS are largely unknown. We previously defined a female-specific AIS genetic risk locus in an enhancer near the PAX1 gene. Here we sought to define the roles of PAX1 and newly-identified AIS-associated genes in the developmental mechanism of AIS. In a genetic study of 10,519 individuals with AIS and 93,238 unaffected controls, significant association was identified with a variant in COL11A1 encoding collagen (α1) XI (rs3753841; NM_080629.2_c.4004C>T; p.(Pro1335Leu); P=7.07e-11, OR=1.118). Using CRISPR mutagenesis we generated Pax1 knockout mice (Pax1-/-). In postnatal spines we found that PAX1 and collagen (α1) XI protein both localize within the intervertebral disc (IVD)-vertebral junction region encompassing the growth plate, with less collagen (α1) XI detected in Pax1-/- spines compared to wildtype. By genetic targeting we found that wildtype Col11a1 expression in costal chondrocytes suppresses expression of Pax1 and of Mmp3, encoding the matrix metalloproteinase 3 enzyme implicated in matrix remodeling. However, this suppression was abrogated in the presence of the AIS-associated COL11A1P1335L mutant. Further, we found that either knockdown of the estrogen receptor gene Esr2, or tamoxifen treatment, significantly altered Col11a1 and Mmp3 expression in chondrocytes. We propose a new molecular model of AIS pathogenesis wherein genetic variation and estrogen signaling increase disease susceptibility by altering a Pax1-Col11a1-Mmp3 signaling axis in spinal chondrocytes.

Implantation of engineered adipocytes that outcompete tumors for resources suppresses cancer progression.

Tumors acquire an increased ability to obtain and metabolize nutrients. Here, we engineered and implanted adipocytes to outcompete tumors for nutrients and show that they can substantially reduce cancer progression. Growing cells or xenografts from several cancers (breast, colon, pancreas, prostate) alongside engineered human adipocytes or adipose organoids significantly suppresses cancer progression and reduces hypoxia and angiogenesis. Transplanting modulated adipocyte organoids in pancreatic or breast cancer mouse models nearby or distal from the tumor significantly suppresses its growth. To further showcase therapeutic potential, we demonstrate that co-culturing tumor organoids derived from human breast cancers with engineered patient-derived adipocytes significantly reduces cancer growth. Combined, our results introduce a novel cancer therapeutic approach, termed adipose modulation transplantation (AMT), that can be utilized for a broad range of cancers.

Risk scoring based on DNA methylation-driven related DEGs for colorectal cancer prognosis with systematic insights.

Colorectal cancer is a common malignant tumor of the digestive tract. Despite advances in diagnostic techniques and medications. Its prognosis remains challenging. DNA methylation-driven related circulating tumor cells have attracted enormous interest in diagnosing owing to their non-invasive nature and early recognition properties. However, the mechanism through which risk biomarkers act remains elusive. Here, we designed a risk model based on differentially expressed genes, DNA methylation, robust, and survival-related factors in the framework of Cox regression. The model has satisfactory performance and is independently verified by an external and isolated dataset in terms of C-index value, ROC, and tROC. The model was applied to Colorectal cancer patients who were subsequently divided into high- and low-risk groups. Functional annotations, genomic alterations, tumor immune environment, and drug sensitivity were analyzed. We observed that up-regulated genes are associated with epithelial cell differentiation and MAPK signaling pathways. The down-regulated genes are related to IL-7 signaling and apoptosis-induced DNA fragmentation. Interestingly, the immune system was inhibited in high-risk groups. High-frequency mutation genes tend to co-occur. High-risk score patients are related to copy number amplification events. To address the challenges, we suggested eleven and twenty-one drugs that are sensitive to low- and high-risk patients. Finally, an artificial neural network was provided to evaluate the immunotherapeutic efficiency. Taken together, the findings demonstrated that our risk score model is robust and reliable for evaluating the prognosis with novel diagnostic and treatment targets. It also yields benefits for the treatment and provides unique insights into developing therapeutic strategies.

Oestrogen engages brain MC4R signalling to drive physical activity in female mice.

Oestrogen depletion in rodents and humans leads to inactivity, fat accumulation and diabetes1,2, underscoring the conserved metabolic benefits of oestrogen that inevitably decrease with age. In rodents, the preovulatory surge in 17ß-oestradiol (E2) temporarily increases energy expenditure to coordinate increased physical activity with peak sexual receptivity. Here we report that a subset of oestrogen-sensitive neurons in the ventrolateral ventromedial hypothalamic nucleus (VMHvl)3-7 projects to arousal centres in the hippocampus and hindbrain, and enables oestrogen to rebalance energy allocation in female mice. Surges in E2 increase melanocortin-4 receptor (MC4R) signalling in these VMHvl neurons by directly recruiting oestrogen receptor-α (ERα) to the Mc4r gene. Sedentary behaviour and obesity in oestrogen-depleted female mice were reversed after chemogenetic stimulation of VMHvl neurons expressing both MC4R and ERα. Similarly, a long-term increase in physical activity is observed after CRISPR-mediated activation of this node. These data extend the effect of MC4R signalling - the most common cause of monogenic human obesity8 - beyond the regulation of food intake and rationalize reported sex differences in melanocortin signalling, including greater disease severity of MC4R insufficiency in women9. This hormone-dependent node illuminates the power of oestrogen during the reproductive cycle in motivating behaviour and maintaining an active lifestyle in women.

Absent from DNA and protein: genomic characterization of nullomers and nullpeptides across functional categories and evolution.

Nullomers and nullpeptides are short DNA or amino acid sequences that are absent from a genome or proteome, respectively. One potential cause for their absence could be their having a detrimental impact on an organism. RESULTS: Here, we identify all possible nullomers and nullpeptides in the genomes and proteomes of thirty eukaryotes and demonstrate that a significant proportion of these sequences are under negative selection. We also identify nullomers that are unique to specific functional categories: coding sequences, exons, introns, 5'UTR, 3'UTR, promoters, and show that coding sequence and promoter nullomers are most likely to be selected against. By analyzing all protein sequences across the tree of life, we further identify 36,081 peptides up to six amino acids in length that do not exist in any known organism, termed primes. We next characterize all possible single base pair mutations that can lead to the appearance of a nullomer in the human genome, observing a significantly higher number of mutations than expected by chance for specific nullomer sequences in transposable elements, likely due to their suppression. We also annotate nullomers that appear due to naturally occurring variants and show that a subset of them can be used to distinguish between different human populations. Analysis of nullomers and nullpeptides across vertebrate evolution shows they can also be used as phylogenetic classifiers. CONCLUSIONS: We provide a catalog of nullomers and nullpeptides in distinct functional categories, develop methods to systematically study them, and highlight the use of variability in these sequences in other analyses.

Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution.

Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.

Asymmetron: a toolkit for the identification of strand asymmetry patterns in biological sequences.

DNA strand asymmetries can have a major effect on several biological functions, including replication, transcription and transcription factor binding. As such, DNA strand asymmetries and mutational strand bias can provide information about biological function. However, a versatile tool to explore this does not exist. Here, we present Asymmetron, a user-friendly computational tool that performs statistical analysis and visualizations for the evaluation of strand asymmetries. Asymmetron takes as input DNA features provided with strand annotation and outputs strand asymmetries for consecutive occurrences of a single DNA feature or between pairs of features. We illustrate the use of Asymmetron by identifying transcriptional and replicative strand asymmetries of germline structural variant breakpoints. We also show that the orientation of the binding sites of 45% of human transcription factors analyzed have a significant DNA strand bias in transcribed regions, that is also corroborated in ChIP-seq analyses, and is likely associated with transcription. In summary, we provide a novel tool to assess DNA strand asymmetries and show how it can be used to derive new insights across a variety of biological disciplines.

A systematic evaluation of the design and context dependencies of massively parallel reporter assays.

Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. To date, there are limited studies that systematically compare differences in MPRA design. Here, we screen a library of 2,440 candidate liver enhancers and controls for regulatory activity in HepG2 cells using nine different MPRA designs. We identify subtle but significant differences that correlate with epigenetic and sequence-level features, as well as differences in dynamic range and reproducibility. We also validate that enhancer activity is largely independent of orientation, at least for our library and designs. Finally, we assemble and test the same enhancers as 192-mers, 354-mers and 678-mers and observe sizable differences. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements and to a lesser degree the precise assay, influence MPRA results.

Characterization of functional transposable element enhancers in acute myeloid leukemia.

Transposable elements (TEs) have been shown to have important gene regulatory functions and their alteration could lead to disease phenotypes. Acute myeloid leukemia (AML) develops as a consequence of a series of genetic changes in hematopoietic precursor cells, including mutations in epigenetic factors. Here, we set out to study the gene regulatory role of TEs in AML. We first explored the epigenetic landscape of TEs in AML patients using ATAC-seq data. We show that a large number of TEs in general, and more specifically mammalian-wide interspersed repeats (MIRs), are more enriched in AML cells than in normal blood cells. We obtained a similar finding when analyzing histone modification data in AML patients. Gene Ontology enrichment analysis showed that genes near MIRs in open chromatin regions are involved in leukemogenesis. To functionally validate their regulatory role, we selected 19 MIR regions in AML cells, and tested them for enhancer activity in an AML cell line (Kasumi-1) and a chronic myeloid leukemia (CML) cell line (K562); the results revealed several MIRs to be functional enhancers. Taken together, our results suggest that TEs are potentially involved in myeloid leukemogenesis and highlight these sequences as potential candidates harboring AML-associated variation.

MPRAnalyze: statistical framework for massively parallel reporter assays.

Massively parallel reporter assays (MPRAs) can measure the regulatory function of thousands of DNA sequences in a single experiment. Despite growing popularity, MPRA studies are limited by a lack of a unified framework for analyzing the resulting data. Here we present MPRAnalyze: a statistical framework for analyzing MPRA count data. Our model leverages the unique structure of MPRA data to quantify the function of regulatory sequences, compare sequences' activity across different conditions, and provide necessary flexibility in an evolving field. We demonstrate the accuracy and applicability of MPRAnalyze on simulated and published data and compare it with existing methods.

A multidisciplinary review of triphalangeal thumb.

Despite being a rare congenital limb anomaly, triphalangeal thumb is a subject of research in various scientific fields, providing new insights in clinical research and evolutionary biology. The findings of triphalangeal thumb can be predictive for other congenital anomalies as part of an underlying syndrome. Furthermore, triphalangeal thumb is still being used as a model in molecular genetics to study gene regulation by long-range regulatory elements. We present a review that summarizes a number of scientifically relevant topics that involve the triphalangeal thumb phenotype. Future initiatives involving multidisciplinary teams collaborating in the field of triphalangeal thumb research can lead to a better understanding of the pathogenesis and molecular mechanisms of this condition as well as other congenital upper limb anomalies.

CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency.

A wide range of human diseases result from haploinsufficiency, where the function of one of the two gene copies is lost. Here, we targeted the remaining functional copy of a haploinsufficient gene using CRISPR-mediated activation (CRISPRa) in Sim1 and Mc4r heterozygous mouse models to rescue their obesity phenotype. Transgenic-based CRISPRa targeting of the Sim1 promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in Sim1 heterozygous mice. To evaluate the therapeutic potential of CRISPRa, we injected CRISPRa-recombinant adeno-associated virus into the hypothalamus, which led to reversal of the obesity phenotype in Sim1 and Mc4r haploinsufficient mice. Our results suggest that endogenous gene up-regulation could be a potential strategy to treat altered gene dosage diseases.

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.

CRISPR-Cas9-mediated functional dissection of 3'-UTRs.

Many studies using reporter assays have demonstrated that 3' untranslated regions (3'-UTRs) regulate gene expression by controlling mRNA stability and translation. Due to intrinsic limitations of heterologous reporter assays, we sought to develop a gene editing approach to investigate the regulatory activity of 3'-UTRs in their native context. We initially used dual-CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 targeting to delete DNA regions corresponding to nine chemokine 3'-UTRs that destabilized mRNA in a reporter assay. Targeting six chemokine 3'-UTRs increased chemokine mRNA levels as expected. However, targeting CXCL1, CXCL6 and CXCL8 3'-UTRs unexpectedly led to substantial mRNA decreases. Metabolic labeling assays showed that targeting these three 3'-UTRs increased mRNA stability, as predicted by the reporter assay, while also markedly decreasing transcription, demonstrating an unexpected role for 3'-UTR sequences in transcriptional regulation. We further show that CRISPR-Cas9 targeting of specific 3'-UTR elements can be used for modulating gene expression and for highly parallel localization of active 3'-UTR elements in the native context. Our work demonstrates the duality and complexity of 3'-UTR sequences in regulation of gene expression and provides a useful approach for modulating gene expression and for functional annotation of 3'-UTRs in the native context.

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.

Use antibiotics in cell culture with caution: genome-wide identification of antibiotic-induced changes in gene expression and regulation.

Standard cell culture guidelines often use media supplemented with antibiotics to prevent cell contamination. However, relatively little is known about the effect of antibiotic use in cell culture on gene expression and the extent to which this treatment could confound results. To comprehensively characterize the effect of antibiotic treatment on gene expression, we performed RNA-seq and ChIP-seq for H3K27ac on HepG2 cells, a human liver cell line commonly used for pharmacokinetic, metabolism and genomic studies, cultured in media supplemented with penicillin-streptomycin (PenStrep) or vehicle control. We identified 209 PenStrep-responsive genes, including transcription factors such as ATF3 that are likely to alter the regulation of other genes. Pathway analyses found a significant enrichment for "xenobiotic metabolism signaling" and "PXR/RXR activation" pathways. Our H3K27ac ChIP-seq identified 9,514 peaks that are PenStrep responsive. These peaks were enriched near genes that function in cell differentiation, tRNA modification, nuclease activity and protein dephosphorylation. Our results suggest that PenStrep treatment can significantly alter gene expression and regulation in a common liver cell type such as HepG2, advocating that antibiotic treatment should be taken into account when carrying out genetic, genomic or other biological assays in cultured cells.

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.