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Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for Zmiz1 in Neuro-2a (N2a) cells

ABSTRACT: Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Here, we use Neuro-2a cell line to perform ChIP sequencing on Zmiz1 bound, direct or indirect to profile Zmiz1 associated genomic regulation. Methods: Neuro-2a (N2a) (ATCC, CCL-131) were cultured in DMEM-high glucose (Cytiva, SH30022) supplemented with 5 % FBS (Cytiva, SH3008803) and 50 U/ml Penicillin-Streptomycin (Thermo Fisher Scientific, 15070063) at 37 °C and 5% CO2. Zmiz1-HA was overexpressed in N2a cells and subsequently, ChIP DNA samples were obtained by using ChIP-IT Express Enzymatic Kit (Active Motif, 53009) and processed according to manufacturer specifications. Anti-HA-Tag Antibody (Cell Signaling, 3724S) was used for immunoprecipitation. Pull down DNA quantity was measured using Qubit DNA HS kit (Thermo Fisher Scientific, Q32851). The sequencing library was prepared using TruSeq ChIP Library Preparation Kit (Illumina, IP-202–1012). Ten nanograms of starting material were used for library preparation. Library quality and quantity were assessed using Agilent DNA 1000 chip (Agilent 5067-1504) and Qubit DNA HS kit respectively. Libraries were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 (Illumina, 20046812) on a Nextseq1000/2000 system. Sequencing analysis was done using the ChIPSeq App from BaseSpace Labs (Illumina), which uses MACS2 for region enrichment and HOMER for motif analysis. Results: ChIP-seq peaks analysis identified thousands of peaks which are mostly located in intergenic regions followed by introns. Conclusions: We identify Zmiz1 bound DNA (direclty or indireclty) in Neuro-2a cells thus providing genomic regulation in neurodevelopmental events.

ORGANISM(S): Mus musculus

PROVIDER: GSE241255 | GEO | 2026/02/20

REPOSITORIES: GEO

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Project description:Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 critical role in angiogenesis, its role in lymphatic vasculature is unknown. Here, we use HDLECs cell line to profile transcriptional changes upon Zmiz1 knockdown using siRNA. Methods: Total RNA was extracted from control and Zmiz1 siRNA transfected HDLECs using GeneJET RNA Purification Kit (Thermo Fisher Scientific, K0732) following the manufacturer instructions. RNA concentration and RNA integrity (RIN) number were determined using Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32852) and Bioanalyzer RNA 6000 Nano assay kit (Agilent, 5067-1511) respectively. RNA library was prepared using TruSeq RNA Library Prep Kit v2 (Illumina, RS-122-2001) according to the manufacturer instructions. mRNA library was quantified using Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32851) and verified using the Bioanalyzer DNA1000 assay kit (Agilent, 5067-1505). Verified samples were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 (Illumina, 20046812) on a Nextseq1000/2000. Sequenced reads were aligned to the human (hg19) reference genome with RNA-Seq alignment tool (STAR aligner). The aligned reads were used to quantify mRNA expression and determine differentially expressed genes using the RNA-Seq Differential Expression tool (version 1.0.1). Both alignment and differential expression analysis were performed using the tools in the illumina BaseSpace Sequence Hub. Results: We found 2,228 differentially expressed genes of which 1,115 genes were upregulated while 1,113 genes were downregulated. Downregulated genes were enriched in biological processes such as lymph vessel development, cell migration and heart valve development. Conclusions: We identify Zmiz1 regulates Prox1 in lymphatic endothelial cells

Project description:Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 mutations association with neurodevelopmental disorders such as ASD, ADHD, and intellectual disability, its role in physiological and pathological neurodevelopment is significantly unknown. Here, we use murine model to knockout Zmiz1 using Emx1Cre and performed RNA sequencing on P7 cortex to profile transcriptional changes upon Zmiz1 deletion. Timed pregnancy was carried out. Mice was euthanized using CO2 and embryos brain were extracted at E15.5. Cortex was dissected and dissociated into single cell suspension using Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, 130-092-628) following manufacture instruction. Intermediate progenitors were magnetically selected and isolated using Anti-Prominin-1 MicroBeads, mouse (Miltenyi Biotec, 130-092-333). Total RNA was extracted from wildtype and Zmiz1-KO samples. RNA concentration and RNA integrity number were determined. RNA library was prepared, quantified, and verified using TruSeq RNA Library Prep Kit v2, Qubit dsDNA High Sensitivity Assay kit and Bioanalyzer DNA1000 assay kit respectively. Verified samples were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 on a Nextseq1000/2000. RNA-seq data analysis was performed using illumina BaseSpace Sequence Hub. Briefly, sequenced reads were aligned to mouse (mm10) reference genome with RNA-Seq alignment tool (STAR aligner) and differentially expressed genes (DEG) were determined using the RNA-Seq Differential Expression tool (version 1.0.1). Results: We found 114 differentially expressed genes of which 35 genes were upregulated while 69 genes were downregulated. Downregulated genes were enriched in biological processes such as forebrain deveopment, axon development, neuron differentiation etc. Conclusions: We assessed P7 cortex specific transcriptional profile changes which are potentially regulated by Zmiz1.

Project description:Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 mutations association with neurodevelopmental disorders such as ASD, ADHD, and intellectual disability, its role in physiological and pathological neurodevelopment is significantly unknown. Here, we use murine model to knockout Zmiz1 using Emx1Cre and performed basal progenitors isolation at E15.5 and RNA sequencing to profile transcriptional changes upon Zmiz1 deletion. Timed pregnancy was carried out. Mice was euthanized using CO2 and embryos brain were extracted at E15.5. Cortex was dissected and dissociated into single cell suspension using Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, 130-092-628) following manufacture instruction. Intermediate progenitors were magnetically selected and isolated using Anti-Prominin-1 MicroBeads, mouse (Miltenyi Biotec, 130-092-333). Total RNA was extracted from wildtype and Zmiz1-KO samples. RNA concentration and RNA integrity number were determined. RNA library was prepared, quantified, and verified using TruSeq RNA Library Prep Kit v2, Qubit dsDNA High Sensitivity Assay kit and Bioanalyzer DNA1000 assay kit respectively. Verified samples were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 on a Nextseq1000/2000. RNA-seq data analysis was performed using illumina BaseSpace Sequence Hub. Briefly, sequenced reads were aligned to mouse (mm10) reference genome with RNA-Seq alignment tool (STAR aligner) and differentially expressed genes (DEG) were determined using the RNA-Seq Differential Expression tool (version 1.0.1). Results: We found 261 differentially expressed genes of which 67 genes were upregulated while 194 genes were downregulated. Downregulated genes were enriched in biological processes such as central nervous system differentiation, axon development, neuron migration, progenitors proliferation, etc. Conclusions: We assessed cortical intermediate progenitors transcriptional profile potentially regulated by Zmiz1.

Project description:Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 mutations association with neurodevelopmental disorders such as ASD, ADHD, and intellectual disability, its role in physiological and pathological neurodevelopment is significantly unknown. Here, we use murine model to knockout Zmiz1 using Emx1Cre and performed neuron enrichment on P1 and P14 cortex followed by RNA sequencing to profile transcriptional changes upon Zmiz1 deletion. Mice was euthanized using ice (P1) or CO2 (P14) and brain were extracted at P1 or P14. Cortex was dissected and dissociated into single cell suspension using Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, 130-092-628 (P1) or 130-107-677 (P14)) following manufacture instruction. Neurons were magnetically isolated/enriched by negative selection through depletion of non-neuronal cell types using cocktail of antibodies for non-neuronal cell types (Miltenyi Biotec, 130-115-390). Total RNA was extracted from wildtype and Zmiz1-KO samples. RNA concentration and RNA integrity number were determined. RNA library was prepared, quantified, and verified using TruSeq RNA Library Prep Kit v2, Qubit dsDNA High Sensitivity Assay kit and Bioanalyzer DNA1000 assay kit respectively. Verified samples were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 on a Nextseq1000/2000. RNA-seq data analysis was performed using illumina BaseSpace Sequence Hub. Briefly, sequenced reads were aligned to mouse (mm10) reference genome with RNA-Seq alignment tool (STAR aligner) and differentially expressed genes (DEG) were determined using the RNA-Seq Differential Expression tool (version 1.0.1). Results: For P1 enriched neurons, we found 291 differentially expressed genes of which 124 genes were upregulated while 167 genes were downregulated. Downregulated genes were enriched in biological processes such as neuron development, central nervous system differentiation, and axon development. For P14 enriched neurons, we found 2,116 differentially expressed genes of which 749 genes were upregulated while 1,367 genes were downregulated. Downregulated genes were enriched in biological processes such as synaptic signalling and organization and neuron development and differentitation. Conclusions: We assessed P1 and P14 cortical neurons transcriptional landscape following Zmiz1 deletion in the cortex.

Project description:Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 association with vascular system, its role in physiological and pathological angiogenesis is significantly unknown. Here, we use (1) MS1 cell line treated with control shRNA and Zmiz1 shRNA and (2) murine model to knockout Zmiz1 in endothelial cells using Cdh5CreERT2 and isolate retinal endothelial cells (iRECs) then performed RNA sequencing to profile transcriptional changes upon Zmiz1 deletion. Retinal ECs were isolated from P7 pups as previously described (Patel et al., 2022. JCI Insight). Briefly, isolation of ECs from the retinas was performed using the Miltenyi Neural tissue Dissociation Kit (P) (Miltenyi, 130-092-628). For each sample, 8-10 retinas were pooled and digested to obtain a single cell suspension. CD31+ cells were separated from the cell mixture via magnetic activated cell sorting. MS1 cells were transfected with 20μM of either control-shRNA or Zmiz1-shRNA using Lipofectamine3000 (Thermofisher, L3000015). Next, RNA was isolated from the cells and used for downstream RNA-sequencing. RNA concentration and RNA integrity number were determined. RNA library was prepared, quantified, and verified using TruSeq RNA Library Prep Kit v2, Qubit dsDNA High Sensitivity Assay kit and Bioanalyzer DNA1000 assay kit respectively. Verified samples were sequenced on a Nextseq 500/550 platform. RNA-seq data analysis was performed using illumina BaseSpace Sequence Hub. Results: For iRECs , we found 2,364 differentially expressed genes of which 834 genes were upregulated while 1530 genes were downregulated. Downregulated genes were enriched in biological processes such as angiogenesis, vascular development, and cell growth. For MS1 cells, we found 1,711 differentially expressed genes of which 718 genes were upregulated while 993 genes were downregulated. Downregulated genes were enriched in biological processes such as regulation of angiogenesis. Conclusions: We assessed changes in transcriptional landscape in MS1 and iRECs following Zmiz1 deletion.

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Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for Zmiz1 in Neuro-2a (N2a) cells

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