Bioinformatics and Analytics Research Collaborative https://www.med.unc.edu/barc/ We analyze, visualize, interpret, and manage big data Tue, 04 Nov 2025 16:38:38 +0000 en-US hourly 1 https://www.med.unc.edu/barc/wp-content/uploads/sites/1132/2020/07/cropped-MwQYH5Rx_400x400-32x32.jpg Bioinformatics and Analytics Research Collaborative https://www.med.unc.edu/barc/ 32 32 Interindividual Variability in Imatinib Metabolism in Human Liver Microsomes and Primary Human Hepatocytes: Impact of CYP2C8 and CYP3A Phenotypes https://doi.org/10.1016/j.dmd.2025.100196 Tue, 04 Nov 2025 16:38:38 +0000 https://www.med.unc.edu/barc/?p=2862 Abstract Imatinib is a kinase inhibitor used in the treatment of chronic myeloid leukemia (CML) and other cancers. Although its pharmacokinetics are generally predictable, substantial interindividual variability in clearance and exposure remains. In this study, we investigated the impact of cytochrome P450 (CYP) enzymes: CYP2C8, CYP3A4, and CYP3A5 phenotypes and genotypes on imatinib metabolism using … Read more

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Imatinib is a kinase inhibitor used in the treatment of chronic myeloid leukemia (CML) and other cancers. Although its pharmacokinetics are generally predictable, substantial interindividual variability in clearance and exposure remains. In this study, we investigated the impact of cytochrome P450 (CYP) enzymes: CYP2C8, CYP3A4, and CYP3A5 phenotypes and genotypes on imatinib metabolism using both human liver microsomes (HLM) and primary human hepatocytes (PHH). Imatinib clearance and N-desmethyl imatinib formation were quantified by LC-MS/MS and correlated with enzyme activity, protein concentration, and compared by genotype groups. In single-donor HLM (n = 21), imatinib clearance varied 45-fold between donors and was strongly associated with CYP2C8 (r = 0.91) and CYP3A (r = 0.90) protein concentrations and CYP2C8 (r = 0.66) and CYP3A (r = 0.86) enzyme activities. A multiple linear regression model identified CYP3A activity and biological sex as significant predictors of imatinib clearance (adjusted R2 = 0.90, p < 0.0001). In PHH (n = 14), imatinib clearance varied 10-fold and was significantly correlated with CYP2C8 activity and protein concentration, but not with CYP3A activity. CYP2C8*3 carriers had significantly lower clearance than *1/*1 donors (p = 0.012). N-desmethyl imatinib formation was consistently associated with both CYP2C8 and CYP3A protein concentration and enzyme activity in HLM and PHH. These findings provide further insight into the enzymes driving imatinib metabolism and highlight the relevance of CYP2C8 and CYP3A variability to interindividual differences in drug disposition. These results support the integration of enzyme phenotyping into predictive models to inform imatinib precision dosing approaches.

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]]> NAT1 and NAT2 Enzyme Activity Drive Interindividual Variability in Sulfamethoxazole N-Acetylation https://doi.org/10.1016/j.dmd.2025.100195 Tue, 04 Nov 2025 16:37:25 +0000 https://www.med.unc.edu/barc/?p=2860 Abstract Sulfamethoxazole (SMX) is associated with idiosyncratic drug induced liver injury (DILI), which remains difficult to predict. SMX is metabolized by N-acetyltransferases (NAT1/NAT2) to form N4-acetyl sulfamethoxazole (NA-SMX), and by cytochrome P450-mediated oxidation to form SMX-hydroxylamine (SMX-HA). This study aimed to characterize SMX metabolism in vitro and investigate how NAT1 and NAT2 variation influences NA-SMX formation, including the relationship between NAT2 protein levels … Read more

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Sulfamethoxazole (SMX) is associated with idiosyncratic drug induced liver injury (DILI), which remains difficult to predict. SMX is metabolized by N-acetyltransferases (NAT1/NAT2) to form N4-acetyl sulfamethoxazole (NA-SMX), and by cytochrome P450-mediated oxidation to form SMX-hydroxylamine (SMX-HA). This study aimed to characterize SMX metabolism in vitro and investigate how NAT1 and NAT2 variation influences NA-SMX formation, including the relationship between NAT2 protein levels and metabolite formation. Human liver microsomes (HLM), S9 fractions, and primary human hepatocytes (PHH), were used to generate SMX metabolites. NA-SMX was the most abundant metabolite in PHH, showing 4.2-fold variability across n = 26 donors. Interestingly, NAT2 genotype-inferred acetylator phenotype did not reliably predict NA-SMX formation in six of nine slow acetylators, whose formation exceeded the mean of intermediate acetylators. However, N-acetyl sulfamethazine (NA-SMZ) formation was accurately predicted using the NAT2 probe substrate, sulfamethazine (SMZ), revealing significant differences between NAT2 phenotype groups (p < 0.05). Activities of NAT1 and NAT2, as measured by p-aminobenzoic acid (PABA) and sulfamethazine (SMZ) N-acetylation, respectively, significantly correlated with NA-SMX formation (r = 0.576, p = 0.006; r = 0.459, p = 0.036). The stronger correlation with NAT1 activity supports the relationship of NAT1 to SMX metabolism. Novel targeted proteomic quantification of NAT2 showed significant correlations between NAT2 protein concentration and NAT2 activity (r = 0.823; p < 0.0001 and r = 0.734, p = 0.0002; for two peptides). This work demonstrates interindividual variability in SMX metabolism and highlights the importance of considering genetic and non-genetic factors in SMX-induced DILI risk.

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]]> Dynamic activation of rAAV transgene expression by a small molecule that recruits endogenous transcriptional machinery https://doi.org/10.1093/nar/gkaf345 Wed, 07 May 2025 21:47:28 +0000 https://www.med.unc.edu/barc/?p=2813 Abstract Adeno-associated virus (AAV) gene therapies typically use constitutive transgene expression vectors that cannot be altered after vector administration. Here, we describe a bioorthogonal platform for tuning AAV expression which enables the controlled activation of viral transgenes after transduction. This platform uses a small, synthetic DNA-binding protein embedded in the AAV genome coupled with a … Read more

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Adeno-associated virus (AAV) gene therapies typically use constitutive transgene expression vectors that cannot be altered after vector administration. Here, we describe a bioorthogonal platform for tuning AAV expression which enables the controlled activation of viral transgenes after transduction. This platform uses a small, synthetic DNA-binding protein embedded in the AAV genome coupled with a heterobifunctional small molecule that recruits endogenous transcriptional machinery to chemically induce transgene expression in a dose-dependent and reversible manner. In human cells, this strategy successfully activates AAV expression across different viral serotypes, cassette configurations, and transgene payloads. Epigenomic analysis reveals that this technology facilitates direct and specific recruitment of the transcriptional regulator BRD4 to AAV genomes. Our results demonstrate that the expression of native AAV genomes can be tuned through chemically induced proximity, opening the possibility of a new class of AAV vectors that can be dynamically potentiated.

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]]> Spt6-Spn1 interaction is required for RNA Polymerase II association and precise nucleosome positioning along transcribed genes https://www.jbc.org/article/S0021-9258(25)00285-6/fulltext Mon, 24 Mar 2025 13:59:36 +0000 https://www.med.unc.edu/barc/?p=2785 Abstract Spt6-Spn1 is an essential histone chaperone complex that associates with RNA Polymerase II (RNAPII) and reassembles nucleosomes during gene transcription. While the interaction between Spt6 and Spn1 is important for its histone deposition and transcription functions, a precise mechanistic understanding is still limited. Here, using temperature sensitive alleles of spt6 and spn1 that disrupt … Read more

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Spt6-Spn1 is an essential histone chaperone complex that associates with RNA Polymerase II (RNAPII) and reassembles nucleosomes during gene transcription. While the interaction between Spt6 and Spn1 is important for its histone deposition and transcription functions, a precise mechanistic understanding is still limited. Here, using temperature sensitive alleles of spt6 and spn1 that disrupt their interaction in yeast, we show that Spt6-Spn1 association is important for its stable interaction with the elongating RNAPII complex and nucleosomes. Using micrococcal nuclease (MNase)-based chromatin occupancy profiling, we further find that Spt6-Spn1 interaction is required to maintain a preferred nucleosome positioning at actively transcribed genes; in the absence of Spt6-Spn1 interaction, we observe a return to replication-dependent phasing. In addition to positioning defects, Spt6-Spn1 disrupting mutants also resulted in an overall shift of nucleosomes towards the 5’ end of genes that was correlated with decreased RNAPII levels. As loss of Spt6-Spn1 association results in cryptic transcription at a subset of genes, we examined these genes for their nucleosome profiles. These findings revealed that the chromatin organization at these loci is similar to other active genes, thus underscoring the critical role of DNA sequence in mediating cryptic transcription when nucleosome positioning is altered. Taken together, these findings reveal Spt6-Spn1 interaction is key to its association with elongating RNAPII and for its ability to precisely organize nucleosomes across transcription units.

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]]> Early life exposure to vitamin D deficiency impairs molecular mechanisms that regulate liver cholesterol biosynthesis, energy metabolism, inflammation, and detoxification https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2024.1335855/full Fri, 10 May 2024 17:59:17 +0000 https://www.med.unc.edu/barc/?p=2657 Introduction: Emerging data suggests liver disease may be initiated during development when there is high genome plasticity and the molecular pathways supporting liver function are being developed.

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]]> Introduction: Emerging data suggests liver disease may be initiated during development when there is high genome plasticity and the molecular pathways supporting liver function are being developed.

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]]> CHD4 and SMYD1 repress common transcriptional programs in the developing heart https://pubmed.ncbi.nlm.nih.gov/38619323/ Fri, 10 May 2024 17:57:40 +0000 https://www.med.unc.edu/barc/?p=2655 Abstract Regulation of chromatin states is essential for proper temporal and spatial gene expression. Chromatin states are modulated by remodeling complexes composed of components that have enzymatic activities. CHD4 is the catalytic core of the nucleosome remodeling and deacetylase (NuRD) complex, which represses gene transcription. However, it remains to be determined how CHD4, a ubiquitous … Read more

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Regulation of chromatin states is essential for proper temporal and spatial gene expression. Chromatin states are modulated by remodeling complexes composed of components that have enzymatic activities. CHD4 is the catalytic core of the nucleosome remodeling and deacetylase (NuRD) complex, which represses gene transcription. However, it remains to be determined how CHD4, a ubiquitous enzyme that remodels chromatin structure, functions in cardiomyocytes to maintain heart development. In particular, whether other proteins besides the NuRD components interact with CHD4 in the heart is controversial. Using quantitative proteomics, we identified that CHD4 interacts with SMYD1, a striated muscle-restricted histone methyltransferase that is essential for cardiomyocyte differentiation and cardiac morphogenesis. Comprehensive transcriptomic and chromatin accessibility studies of Smyd1 and Chd4 null embryonic mouse hearts revealed that SMYD1 and CHD4 repress a group of common genes and pathways involved in glycolysis, response to hypoxia, and angiogenesis. Our study reveals a mechanism by which CHD4 functions during heart development, and a previously uncharacterized mechanism regarding how SMYD1 represses cardiac transcription in the developing heart.

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]]> The tardigrade Hypsibius exemplaris dramatically upregulates DNA repair pathway genes in response to ionizing radiation https://www.sciencedirect.com/science/article/pii/S0960982224003166?dgcid=author Mon, 15 Apr 2024 16:22:25 +0000 https://www.med.unc.edu/barc/?p=2653 Summary   Tardigrades can survive remarkable doses of ionizing radiation, up to about 1,000 times the lethal dose for humans. How they do so is incompletely understood. We found that the tardigrade Hypsibius exemplaris suffers DNA damage upon gamma irradiation, but the damage is repaired. We show that this species has a specific and robust … Read more

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Tardigrades can survive remarkable doses of ionizing radiation, up to about 1,000 times the lethal dose for humans. How they do so is incompletely understood. We found that the tardigrade Hypsibius exemplaris suffers DNA damage upon gamma irradiation, but the damage is repaired. We show that this species has a specific and robust response to ionizing radiation: irradiation induces a rapid upregulation of many DNA repair genes. This upregulation is unexpectedly extreme—making some DNA repair transcripts among the most abundant transcripts in the animal. By expressing tardigrade genes in bacteria, we validate that increased expression of some repair genes can suffice to increase radiation tolerance. We show that at least one such gene is important in vivo for tardigrade radiation tolerance. We hypothesize that the tardigrades’ ability to sense ionizing radiation and massively upregulate specific DNA repair pathway genes may represent an evolved solution for maintaining DNA integrity.

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]]> Von Hippel Lindau tumor suppressor controls m6A-dependent gene expression in renal tumorigenesis https://www.jci.org/articles/view/175703 Mon, 15 Apr 2024 16:20:43 +0000 https://www.med.unc.edu/barc/?p=2651 Abstract N6-Methyladenosine (m6A) is the most abundant posttranscriptional modification, and its contribution to cancer evolution has recently been appreciated. Renal cancer is the most common adult genitourinary cancer, approximately 85% of which is accounted for by the clear cell renal cell carcinoma (ccRCC) subtype characterized by VHL loss. However, it is unclear whether VHL loss … Read more

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N6-Methyladenosine (m6A) is the most abundant posttranscriptional modification, and its contribution to cancer evolution has recently been appreciated. Renal cancer is the most common adult genitourinary cancer, approximately 85% of which is accounted for by the clear cell renal cell carcinoma (ccRCC) subtype characterized by VHL loss. However, it is unclear whether VHL loss in ccRCC affects m6A patterns. In this study, we demonstrate that VHL binds and promotes METTL3/METTL14 complex formation while VHL depletion suppresses m6A modification, which is distinctive from its canonical E3 ligase role. m6A RNA immunoprecipitation sequencing (RIP-Seq) coupled with RNA-Seq allows us to identify a selection of genes whose expression may be regulated by VHL-m6A signaling. Specifically, PIK3R3 is identified to be a critical gene whose mRNA stability is regulated by VHL in a m6A-dependent but HIF-independent manner. Functionally, PIK3R3 depletion promotes renal cancer cell growth and orthotopic tumor growth while its overexpression leads to decreased tumorigenesis. Mechanistically, the VHL-m6A–regulated PIK3R3 suppresses tumor growth by restraining PI3K/AKT activity. Taken together, we propose a mechanism by which VHL regulates m6A through modulation of METTL3/METTL14 complex formation, thereby promoting PIK3R3 mRNA stability and protein levels that are critical for regulating ccRCC tumorigenesis.

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]]> Gastrulation-stage alcohol exposure induces similar rates of craniofacial malformations in male and female C57BL/6J mice https://doi.org/10.1002/bdr2.2292 Wed, 20 Dec 2023 15:22:32 +0000 https://www.med.unc.edu/barc/?p=2643 Background Prenatal alcohol exposure during gastrulation (embryonic day [E] 7 in mice, ~3rd week of human pregnancy) impairs eye, facial, and cortical development, recapitulating birth defects characteristic of Fetal Alcohol Syndrome (FAS). However, it is not known whether the prevalence or severity of craniofacial features associated with FAS is affected by biological sex. Methods The … Read more

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Background

Prenatal alcohol exposure during gastrulation (embryonic day [E] 7 in mice, ~3rd week of human pregnancy) impairs eye, facial, and cortical development, recapitulating birth defects characteristic of Fetal Alcohol Syndrome (FAS). However, it is not known whether the prevalence or severity of craniofacial features associated with FAS is affected by biological sex.

Methods

The current study administered either alcohol (2.9 g/kg, two i.p. doses, 4 hr apart) or vehicle to pregnant C57BL/6J females on E7, prior to gonadal sex differentiation, and assessed fetal morphology at E17.

Results

Whereas sex did not affect fetal size in controls, alcohol-exposed females were smaller than both control females and alcohol-treated males. Alcohol exposure increased the incidence of eye defects to a similar degree in males and females. Together, these data suggest that females might be more sensitive to the general developmental effects of alcohol, but not effects specific to the craniofacies. Whole transcriptomic analysis of untreated E7 embryos found 214 differentially expressed genes in females vs. males, including those in pathways related to cilia and mitochondria, histone demethylase activity, and pluripotency.

Conclusion

Gastrulation-stage alcohol induces craniofacial malformations in male and female mouse fetuses at similar rates and severity, though growth deficits are more prevalent females. These findings support the investigation of biological sex as a contributing factor in prenatal alcohol studies.

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]]> New BARC article: Gestational diabetes mellitus placentas exhibit epimutations at placental development genes https://pubmed.ncbi.nlm.nih.gov/35993304/ Thu, 20 Oct 2022 18:57:15 +0000 https://www.med.unc.edu/barc/?p=2582 Gestational diabetes mellitus (GDM) is a maternal metabolic disorder that perturbs placental development and increases the risk of offspring short- and long-term metabolic disorders. The mechanisms by which GDM impairs placental development remain poorly understood. Here, we defined the DNA methylome of GDM placentas and determined whether GDM perturbs methylation at genes important for placental … Read more

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Gestational diabetes mellitus (GDM) is a maternal metabolic disorder that perturbs placental development and increases the risk of offspring short- and long-term metabolic disorders. The mechanisms by which GDM impairs placental development remain poorly understood. Here, we defined the DNA methylome of GDM placentas and determined whether GDM perturbs methylation at genes important for placental development. We conducted an epigenome-wide association study of 42 placentas from pregnancies in the South African Soweto First 1000 days cohort (S1000). Using genome-wide bisulfite sequencing, we compared non-GDM placentas to GDM placentas with similar proportions from obese and non-obese mothers. Compared to non-GDM, GDM placentas exhibited a distinct methylation profile consisting of 12,210 differentially methylated CpGs (DMCs) that mapped to 3,875 genes. Epigenetically altered genes were enriched in Wnt and cadherin signalling pathways, both critical in placentation and embryogenesis. We also defined regional DNA methylation perturbation in GDM placentas at 11 placental development genes. These findings reveal extensive changes to the placental epigenome of GDM pregnancies and highlight perturbation enriched at important placental development genes. These molecular changes represent potential mechanisms for GDM-induced placental effects that may serve as candidate biomarkers for placental, maternal, and foetal health. Using a study design that used similar proportions of obese and non-obese mothers in our case and control pregnancies, we minimized the detection of changes due to obesity alone. Further work will be necessary to investigate the extent of the influence of obesity on these GDM-related placental epigenetic changes.

Keywords: DNA methylation; Epigenetics; development; developmental programming; gestational diabetes mellitus; placenta.

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