Single sample sequencing (S3EQ) of epigenome and transcriptome in nucleus accumbens

Xu, Song-Jun; Heller, Elizabeth A.

doi:10.1016/j.jneumeth.2018.07.006

Cited by 8 publications

(9 citation statements)

References 62 publications

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“…S3EQ (33) was conducted as previously published with modified buffer volume. Tissue samples were homogenized in 350 μL cell lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 3 mM MgCl2 and 0.5% NP-40 in H2O) and spun for five minutes (1,500 x g, 4°C).…”

Section: Methodsmentioning

confidence: 99%

“…Prior to initiating the protocol, the following solutions were prepared as previously described (33,34): BSA Blocking Buffer (0.5% Bovine serum albumin in 1x PBS), Dilution Buffer (16.7 mM Tris-HCl (pH 8.0), 1.1% Triton-X 100, 0.01% SDS, 167 mM NaCl, 1.2mM EDTA in H2O), Nuclear Lysis Buffer (50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1% SDS in H2O), Wash Buffer 1 (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS in H2O), Wash Buffer 2 (20 mM Tris-Cl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS in H2O), Wash Buffer 3 (250 mM LiCl, 10 mM Tris-HCl pH 8.0, 1% sodium deoxycholate, 1 mM EDTA, 1% IGEPAL CA-630 in H2O), TE Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA in H2O), Elution Buffer (0.1 M NaHCO3, 1% SDS in H2O). M280 Sheep anti-Rabbit Dynabeads (Invitrogen, 11204D) were washed three times in BSA blocking buffer.…”

Section: Single and Sequential Chromatin Immunoprecipitation (Chip) A...mentioning

confidence: 99%

“…In this study, we utilized single sample sequencing (S3EQ) (33) and sequential ChIP (23,34) to investigate the sex-specific transcriptomic and epigenomic profiles of Nr4a1 from three different brain regions. We also utilized Pearson’s correlation matrices to enable us to capture the individual variability in biological changes induced by cocaine exposure in males and females.…”

Section: Introductionmentioning

confidence: 99%

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Cocaine regulation of Nr4a1 chromatin bivalency and mRNA in male and female mice

Fischer

Krick

Han

et al. 2022

Preprint

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BACKGROUNDCocaine epigenetically regulates gene expression via changes in histone post-translational modifications (HPTMs). We previously found that the immediate early gene Nr4a1 is epigenetically activated by cocaine in mouse brain reward regions. HPTMs act combinatorically, yet few studies examine multiple HPTMs at a single gene. Bivalent gene promoters are simultaneously enriched in both activating (H3K4me3 (K4)) and repressive (H3K27me3 (K27)) HPTMs. As such, bivalent genes are lowly expressed but poised for activity-dependent gene regulation. In the current study, we defined regulation of K4&K27 bivalency at Nr4a1 following cocaine treatment in male and female mice. The inclusion of female mice can shed light on the epidemiological relevance of sex to cocaine use disorder.METHODSMale and female mice were injected with saline or cocaine (i.p. 20mg/kg). We applied sequential chromatin immunoprecipitation and qPCR to define Nr4a1 bivalency and expression in striatum (STR), prefrontal cortex (PFC), and hippocampus (HPC). Pearson’s correlation matrices quantified relationships within each brain region across treatment conditions for each sex.RESULTSWe defined K4&K27 bivalency at the Nr4a1 promoter in all three brain regions, in both sexes. In female STR, cocaine increased Nr4a1 mRNA, coupled to maintenance of Nr4a1 K4&K27 bivalency. In male STR, cocaine enriched repressive H3K27me3 and K4&K27 bivalency at Nr4a1 and failed to increase Nr4a1 mRNA. Furthermore, cocaine epigenetically regulated a putative NR4A1 target, Cartpt, in male PFC.CONCLUSIONThis study defined the epigenetic regulation of Nr4a1 in reward brain regions in male and female mice. Cocaine treatment in female mice increased Nr4a1 mRNA in STR, but there was no change in Nr4a1 H3K27me3 or K4&K27 promoter bivalency. Following cocaine treatment in male mice, Nr4a1 mRNA did not change in STR, HPC, or PFC, and Nr4a1 H3K27me3 and K4&K27 promoter bivalency increased in the STR.

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Section: Methodsmentioning

confidence: 99%

Section: Single and Sequential Chromatin Immunoprecipitation (Chip) A...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cocaine regulation of Nr4a1 chromatin bivalency and mRNA in male and female mice

Fischer

Krick

Han

et al. 2022

Preprint

View full text Add to dashboard Cite

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“…Given the evidence that H3K36me3 positioning strongly predicts alternative exon usage across various tissues and development, Xu and Heller (2018) next analyzed the role of H3K36me3 in influencing alternative splicing in adult mouse nucleus accumbens. Analysis was performed on ChIP/RNA-Seq datasets from nucleus accumbens following either cocaine (or saline) selfadministration or overexpression of SET2, the histone methyltransferase that catalyzes H3K36me3.…”

Section: Chromatin-mediated Alternative Splicing Is Linked To Reward mentioning

confidence: 99%

Mechanisms of Neuronal Alternative Splicing and Strategies for Therapeutic Interventions

Soto

Gandal

Gonatopoulos-Pournatzis

et al. 2019

J. Neurosci.

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Many cellular and physiological processes are coordinated by regulatory networks that produce a remarkable complexity of transcript isoforms. In the mammalian nervous system, alternative pre-mRNA splicing generates functionally distinct isoforms that play key roles in normal physiology, supporting development, plasticity, complex behaviors, and cognition. Neuronal splicing programs controlled by RNA-binding proteins, are influenced by chromatin modifications and can exhibit neuronal subtype specificity. As highlighted in recent publications, aberrant alternative splicing is a major contributor to disease phenotypes. Therefore, understanding the underlying mechanisms of alternative splicing regulation and identifying functional splicing isoforms with critical phenotypic roles are expected to provide a comprehensive resource for therapeutic development, as illuminated by recent successful interventions of spinal muscular atrophy. Here, we discuss the latest progress in the study of the emerging complexity of alternative splicing mechanisms in neurons, and how these findings inform new therapies to correct and control splicing defects.

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“…In this study, we utilized single sample sequencing (S3EQ) 45 and sequential chromatin immunoprecipitation (ChIP) 35 , 46 to independently investigate the transcriptomic and epigenomic profiles of Nr4a1 from three different brain regions in males and females. We also utilized Pearson’s correlation matrices to capture the individual variability in biological changes induced by cocaine exposure in males and females.…”

Section: Introductionmentioning

confidence: 99%

Cocaine regulation of Nr4a1 chromatin bivalency and mRNA in male and female mice

Fischer

Krick

Han

et al. 2022

Sci Rep

View full text Add to dashboard Cite

Cocaine epigenetically regulates gene expression via changes in histone post-translational modifications (HPTMs). We previously found that the immediate early gene Nr4a1 is epigenetically activated by cocaine in mouse brain reward regions. However, few studies have examined multiple HPTMs at a single gene. Bivalent gene promoters are simultaneously enriched in both activating (H3K4me3 (K4)) and repressive (H3K27me3 (K27)) HPTMs. As such, bivalent genes are lowly expressed but poised for activity-dependent gene regulation. In this study, we identified K4&K27 bivalency at Nr4a1 following investigator-administered cocaine in male and female mice. We applied sequential chromatin immunoprecipitation and qPCR to define Nr4a1 bivalency and expression in striatum (STR), prefrontal cortex (PFC), and hippocampus (HPC). We used Pearson’s correlation to quantify relationships within each brain region across treatment conditions for each sex. In female STR, cocaine increased Nr4a1 mRNA while maintaining Nr4a1 K4&K27 bivalency. In male STR, cocaine enriched repressive H3K27me3 and K4&K27 bivalency at Nr4a1 and maintained Nr4a1 mRNA. Furthermore, cocaine epigenetically regulated a putative NR4A1 target, Cartpt, in male PFC. This study defined the epigenetic regulation of Nr4a1 in reward brain regions in male and female mice following cocaine, and, thus, shed light on the biological relevance of sex to cocaine use disorder.

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Single sample sequencing (S3EQ) of epigenome and transcriptome in nucleus accumbens

Abstract: The S3EQ method can be applied to improve the correlative power of transcriptomic and epigenomic studies in neuronal tissue.

Cited by 8 publications

References 62 publications

Cocaine regulation of Nr4a1 chromatin bivalency and mRNA in male and female mice

Cocaine regulation of Nr4a1 chromatin bivalency and mRNA in male and female mice

Mechanisms of Neuronal Alternative Splicing and Strategies for Therapeutic Interventions

Cocaine regulation of Nr4a1 chromatin bivalency and mRNA in male and female mice

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