ERα targetome in mammary epithelial cells.

(A) Proportional Venn diagram representing unique estradiol-induced genes at 2h has identified by RNA-seq (orange), unique genes with at least one ERα -binding region as detected by ChIP-seq (purple), and overlap indicates-induced genes at 2 h with at least one ER binding site. (B) ERα binding distribution at genes induced by estradiol at 2 h. (C) SeqPos Motif enrichment in direct ER target genes was induced at 2 h after estradiol exposure. (D) Proportional Venn diagram representing unique estradiol-repressed genes at 2 h as detected by RNA-seq (orange), unique genes with at least one ERα -binding region as detected by ChIP-seq (purple), and estradiol-repressed genes at 2 h with at least one ERα binding site (overlap). (E) ERα binding distribution at genes was repressed by estradiol treatment. (F) SeqPos Motif enrichment in the direct ER target genes repressed at 2 h after estradiol exposure. (G) Enriched GO terms of ER targetome by using the DAVID functional annotation tool.</p

Validation of estrogen regulated genes by qPCR.

Quantitative Real-Time PCR validation of estradiol-induced (A) and repressed (B) genes. Expression of selected genes was normalized using Ppid as the internal control. A total of 4–5 mice per treatment replicate, tested in triplicate per treatment group. Results are means ± SEM of three independent experimental replicates. *, p p p < 0.001.</p

Global gene expression analysis in mouse mammary gland.

(A) Heatmap of 493 genes (FDR<0.05) that are differentially expressed between mammary gland samples from vehicle (2 h) and estradiol (2 h) treatment. Duplicate pools of mammary glands from mice (5 mice per pool) were used for RNA-seq analyses under each treatment condition. (B) Pearson correlation of RNA-seq replicates (Control r = 0.96 and estrogen treatment = 0.96). (C) Ingenuity Pathway Analysis of RNA-seq. Interactions of estrogen regulated genes was analyzed by Ingenuity Pathway Analysis. A top enriched molecular network revolves around FOS. Red color indicates upregulated genes and green color indicates downregulated genes after acute treatment with estradiol. (D) Summary of enriched GO terms (FDR< 0.05) of ER target genes induced and repressed at 2 h after estradiol treatment using the DAVID Functional Annotation Tool.</p

Validation of ERα ChIP-seq accuracy, binding sites distribution and motif enrichment.

(A) Proportional Venn diagram representing the intersection of ERα binding sites identified in two ChIP-seq replicates; Six mice per replicate. (B) Heatmap showing ERα binding events found in the mammary gland after 2h of treatment with estradiol (ERα ChIP-seq replicate 1 and 2 [left] and input [right]). The window shows ±5kb regions from the center of the binding sites. (C) Pearson correlation of the ERα binding sites of two ChIP-seq replicates (r = 0.95). (D) Conservation plot of mouse ERα binding sites with high conservation around peak centers compared to flanking regions. (E) Distribution of ERα binding sites in the overlap of ERα binding events were identified in two ChIP-seq replicates. (F) SeqPos motif enrichment in distal (up and down) and proximal regions of ERα binding sites.</p

The genomic landscape of estrogen receptor α binding sites in mouse mammary gland - Fig 2

Representative screen shots of ChIP-seq data showing gene ERα recruitment at 2 hours after exposure to estradiol IGV screen shots showing ERα binding sites in relation to the TSS of (A) Greb1, (B) Pgr, (C) Fos (D) Gata3 and (E) Areg. Peak locations relative to the TSS are listed below each screen shot and the numbers indicate peak value of each gene. Red boxes represent peaks that were validated by ChIP-qPCR. Graphs representing validation of ERα occupancy using ChIP followed by qPCR for Greb1, Pgr, Fos, Gata3 and Areg. A total of six mice per replicate; Results are means ± SEM of three independent experimental replicates. **, p p < 0.001.</p

Table_8_A human stem cell-derived neuronal model of morphine exposure reflects brain dysregulation in opioid use disorder: Transcriptomic and epigenetic characterization of postmortem-derived iPSC neurons.XLSX

IntroductionHuman-derived induced pluripotent stem cell (iPSC) models of brain promise to advance our understanding of neurotoxic consequences of drug use. However, how well these models recapitulate the actual genomic landscape and cell function, as well as the drug-induced alterations, remains to be established. New in vitro models of drug exposure are needed to advance our understanding of how to protect or reverse molecular changes related to substance use disorders.MethodsWe engineered a novel induced pluripotent stem cell-derived model of neural progenitor cells and neurons from cultured postmortem human skin fibroblasts, and directly compared these to isogenic brain tissue from the donor source. We assessed the maturity of the cell models across differentiation from stem cells to neurons using RNA cell type and maturity deconvolution analyses as well as DNA methylation epigenetic clocks trained on adult and fetal human tissue. As proof-of-concept of this model’s utility for substance use disorder studies, we compared morphine- and cocaine-treated neurons to gene expression signatures in postmortem Opioid Use Disorder (OUD) and Cocaine Use Disorder (CUD) brains, respectively.ResultsWithin each human subject (N = 2, 2 clones each), brain frontal cortex epigenetic age parallels that of skin fibroblasts and closely approximates the donor’s chronological age; stem cell induction from fibroblast cells effectively sets the epigenetic clock to an embryonic age; and differentiation of stem cells to neural progenitor cells and then to neurons progressively matures the cells via DNA methylation and RNA gene expression readouts. In neurons derived from an individual who died of opioid overdose, morphine treatment induced alterations in gene expression similar to those previously observed in OUD ex-vivo brain tissue, including differential expression of the immediate early gene EGR1, which is known to be dysregulated by opioid use.DiscussionIn summary, we introduce an iPSC model generated from human postmortem fibroblasts that can be directly compared to corresponding isogenic brain tissue and can be used to model perturbagen exposure such as that seen in opioid use disorder. Future studies with this and other postmortem-derived brain cellular models, including cerebral organoids, can be an invaluable tool for understanding mechanisms of drug-induced brain alterations.</p

Table_2_A human stem cell-derived neuronal model of morphine exposure reflects brain dysregulation in opioid use disorder: Transcriptomic and epigenetic characterization of postmortem-derived iPSC neurons.XLSX

IntroductionHuman-derived induced pluripotent stem cell (iPSC) models of brain promise to advance our understanding of neurotoxic consequences of drug use. However, how well these models recapitulate the actual genomic landscape and cell function, as well as the drug-induced alterations, remains to be established. New in vitro models of drug exposure are needed to advance our understanding of how to protect or reverse molecular changes related to substance use disorders.MethodsWe engineered a novel induced pluripotent stem cell-derived model of neural progenitor cells and neurons from cultured postmortem human skin fibroblasts, and directly compared these to isogenic brain tissue from the donor source. We assessed the maturity of the cell models across differentiation from stem cells to neurons using RNA cell type and maturity deconvolution analyses as well as DNA methylation epigenetic clocks trained on adult and fetal human tissue. As proof-of-concept of this model’s utility for substance use disorder studies, we compared morphine- and cocaine-treated neurons to gene expression signatures in postmortem Opioid Use Disorder (OUD) and Cocaine Use Disorder (CUD) brains, respectively.ResultsWithin each human subject (N = 2, 2 clones each), brain frontal cortex epigenetic age parallels that of skin fibroblasts and closely approximates the donor’s chronological age; stem cell induction from fibroblast cells effectively sets the epigenetic clock to an embryonic age; and differentiation of stem cells to neural progenitor cells and then to neurons progressively matures the cells via DNA methylation and RNA gene expression readouts. In neurons derived from an individual who died of opioid overdose, morphine treatment induced alterations in gene expression similar to those previously observed in OUD ex-vivo brain tissue, including differential expression of the immediate early gene EGR1, which is known to be dysregulated by opioid use.DiscussionIn summary, we introduce an iPSC model generated from human postmortem fibroblasts that can be directly compared to corresponding isogenic brain tissue and can be used to model perturbagen exposure such as that seen in opioid use disorder. Future studies with this and other postmortem-derived brain cellular models, including cerebral organoids, can be an invaluable tool for understanding mechanisms of drug-induced brain alterations.</p

Table_9_A human stem cell-derived neuronal model of morphine exposure reflects brain dysregulation in opioid use disorder: Transcriptomic and epigenetic characterization of postmortem-derived iPSC neurons.XLSX

IntroductionHuman-derived induced pluripotent stem cell (iPSC) models of brain promise to advance our understanding of neurotoxic consequences of drug use. However, how well these models recapitulate the actual genomic landscape and cell function, as well as the drug-induced alterations, remains to be established. New in vitro models of drug exposure are needed to advance our understanding of how to protect or reverse molecular changes related to substance use disorders.MethodsWe engineered a novel induced pluripotent stem cell-derived model of neural progenitor cells and neurons from cultured postmortem human skin fibroblasts, and directly compared these to isogenic brain tissue from the donor source. We assessed the maturity of the cell models across differentiation from stem cells to neurons using RNA cell type and maturity deconvolution analyses as well as DNA methylation epigenetic clocks trained on adult and fetal human tissue. As proof-of-concept of this model’s utility for substance use disorder studies, we compared morphine- and cocaine-treated neurons to gene expression signatures in postmortem Opioid Use Disorder (OUD) and Cocaine Use Disorder (CUD) brains, respectively.ResultsWithin each human subject (N = 2, 2 clones each), brain frontal cortex epigenetic age parallels that of skin fibroblasts and closely approximates the donor’s chronological age; stem cell induction from fibroblast cells effectively sets the epigenetic clock to an embryonic age; and differentiation of stem cells to neural progenitor cells and then to neurons progressively matures the cells via DNA methylation and RNA gene expression readouts. In neurons derived from an individual who died of opioid overdose, morphine treatment induced alterations in gene expression similar to those previously observed in OUD ex-vivo brain tissue, including differential expression of the immediate early gene EGR1, which is known to be dysregulated by opioid use.DiscussionIn summary, we introduce an iPSC model generated from human postmortem fibroblasts that can be directly compared to corresponding isogenic brain tissue and can be used to model perturbagen exposure such as that seen in opioid use disorder. Future studies with this and other postmortem-derived brain cellular models, including cerebral organoids, can be an invaluable tool for understanding mechanisms of drug-induced brain alterations.</p

Image_4_A human stem cell-derived neuronal model of morphine exposure reflects brain dysregulation in opioid use disorder: Transcriptomic and epigenetic characterization of postmortem-derived iPSC neurons.JPEG

IntroductionHuman-derived induced pluripotent stem cell (iPSC) models of brain promise to advance our understanding of neurotoxic consequences of drug use. However, how well these models recapitulate the actual genomic landscape and cell function, as well as the drug-induced alterations, remains to be established. New in vitro models of drug exposure are needed to advance our understanding of how to protect or reverse molecular changes related to substance use disorders.MethodsWe engineered a novel induced pluripotent stem cell-derived model of neural progenitor cells and neurons from cultured postmortem human skin fibroblasts, and directly compared these to isogenic brain tissue from the donor source. We assessed the maturity of the cell models across differentiation from stem cells to neurons using RNA cell type and maturity deconvolution analyses as well as DNA methylation epigenetic clocks trained on adult and fetal human tissue. As proof-of-concept of this model’s utility for substance use disorder studies, we compared morphine- and cocaine-treated neurons to gene expression signatures in postmortem Opioid Use Disorder (OUD) and Cocaine Use Disorder (CUD) brains, respectively.ResultsWithin each human subject (N = 2, 2 clones each), brain frontal cortex epigenetic age parallels that of skin fibroblasts and closely approximates the donor’s chronological age; stem cell induction from fibroblast cells effectively sets the epigenetic clock to an embryonic age; and differentiation of stem cells to neural progenitor cells and then to neurons progressively matures the cells via DNA methylation and RNA gene expression readouts. In neurons derived from an individual who died of opioid overdose, morphine treatment induced alterations in gene expression similar to those previously observed in OUD ex-vivo brain tissue, including differential expression of the immediate early gene EGR1, which is known to be dysregulated by opioid use.DiscussionIn summary, we introduce an iPSC model generated from human postmortem fibroblasts that can be directly compared to corresponding isogenic brain tissue and can be used to model perturbagen exposure such as that seen in opioid use disorder. Future studies with this and other postmortem-derived brain cellular models, including cerebral organoids, can be an invaluable tool for understanding mechanisms of drug-induced brain alterations.</p

Additional file 1 of Systemic interindividual epigenetic variation in humans is associated with transposable elements and under strong genetic control

Additional file 1: Supplementary Materials and Methods, Supplementary Figures