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RNA sequencing of (1) isolated retinal endothelial cells from wildtype and Zmiz1-KO endothelial cells and (2) control and Zmiz1 shRNA treated MS1 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 . 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE242406 | GEO | 2025/05/28

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 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 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

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RNA sequencing of (1) isolated retinal endothelial cells from wildtype and Zmiz1-KO endothelial cells and (2) control and Zmiz1 shRNA treated MS1 cells

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