Transcription profiling by array of VAMP7 knockdown using siRNA in human embryonal carcinoma NTERA2/D1 cells
ABSTRACT: To unravel the function of VAMP7 in the male sexual differentiation, we carried out in vitro studies of VAMP7 knockdown using siRNA, in the human embryonal carcinoma NTERA2/D1 cells. We knocked down VAMP7 expression in the testicular teratocarcinoma cell line, NTERA2/D1. By comparing the knockdown conditions with the control scrambled samples, we are able to dissect the cellular pathways affected by alteration of VAMP7 gene expression.
Project description:To unravel the function of VAMP7 in the male sexual differentiation, we carried out in vitro studies of VAMP7 knockdown using siRNA, in the human embryonal carcinoma NTERA2/D1 cells. We knocked down VAMP7 expression in the testicular teratocarcinoma cell line, NTERA2/D1. By comparing the knockdown conditions with the control scrambled samples, we are able to dissect the cellular pathways affected by alteration of VAMP7 gene expression.
Project description:This SuperSeries is composed of the following subset Series: GSE16858: Human NTERA2 (NT2/D1) cells lines: shZRF1 vs. shRandom during Retinoic Acid (RA) administration GSE16878: Human NTERA2 (NT2/D1) cells lines: Genomewide distribution of ZRF1 at gene promoters during RA administration Refer to individual Series
Project description:Although liganded nuclear receptors have been established to regulate RNA polymerase II (Pol II)-dependent transcription units, their role in regulating Pol III-transcribed DNA repeats remains largely unknown. Here we report that ~2-3% of the ~100,000-200,000 total human DR2 Alu repeats located in proximity to activated Pol II transcription units are activated by the retinoic acid receptor (RAR) in human embryonic stem cells to generate Pol III-dependent RNAs. These transcripts are processed, initially in a DICER-dependent fashion, into small RNAs (~28-65 nt) referred to as repeat-induced RNAs that cause the degradation of a subset of crucial stem-cell mRNAs, including Nanog mRNA, which modulate exit from the proliferative stem-cell state. This regulation requires AGO3-dependent accumulation of processed DR2 Alu transcripts and the subsequent recruitment of AGO3-associated decapping complexes to the target mRNA. In this way, the RAR-dependent and Pol III-dependent DR2 Alu transcriptional events in stem cells functionally complement the Pol II-dependent neuronal transcriptional program. RNA-sequencing of polyA selected RNA molecules in NTera2/D1 cells and Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq).
Project description:Transcriptional profiling of human NT2 cell lines comparing control cells (shRandom) with ZRF1 knockdown cells (shZRF1) both either untreated (0 h) or treated with Retinoic Acid (3 h) at 0.01 µM. The cell lines were established by retroviral infection allowing for the transcription of either a random shRNA (shRandom) or a shRNA specific for ZRF1 (shZRF1). The goal was to determine the effect of ZRF1 on transcriptional activation at the onset of differentiation. Four-condition experiment, shRandom vs. shZRF1 cells either uninduced (0 h) or RA induced (3 h). Biological replicates: 6 replicates (shRandom) taken on 3 different days without or with RA administration. 6 shZRF1 replicates taken on three different days with or without RA administration.
Project description:Embryonic stem cells (ESCs) maintain high genomic plasticity, essential for their capacity to enter diverse differentiation pathways. Post-transcriptional modifications of chromatin histones play a pivotal role in maintaining this plasticity. We now report that one such modification, monoubiquitylation of histone H2B on lysine 120 (H2BK120ub1), catalyzed by the E3 ligase RNF20, increases during ESC differentiation and is required for efficient execution of this process. This increase is particularly important for the transcriptional induction of long genes during ESC differentiation. Furthermore, we identify USP44 as a deubiquitinase whose downregulation by differentiation signals contributes to the increase in H2BK120ub1. Our findings suggest that optimal ESC differentiation requires dynamic changes in H2B ubiquitylation patterns, which must occur in a timely and well-coordinated manner. RNF20 depleted or control NTera2 cells stimulated with RA for 72 hours
Project description:We performed Chromatin Isolation by RNA Purification (ChIRP) of SRA and ChIP of p68 following by high-throughput sequencing in NTERA2 cell line. We find that SRA localizes with p68 genome-wide at genes whose function is involved in embryonic development. SRA ChIRP and p68 ChIP of triplicate samples.
Project description:Chip-on-chip experiment with NT2 wildtype cells untreated (0 h) or treated with Retinoic Acid (1h and 3 h) at 0.01 µM. The goal was to determine the genome-wide occupancy of ZRF1 at gene promoters in undifferentiated cells and at the onset of differentiation. Three-condition experiment. Biological replicates: NT2 cells were treated with or without RA on three different days from cycling NT2 cells (0h, 1h and 3h of RA). Chromatin was prepared and a triplicate of IPs was performed with specific ZRF1 antibodies for every condition. A triplicate of control IPs was performed with IgGs.
Project description:Human embryonal carcinoma (EC) cells are the stem cells of nonseminoma testicular germ cells tumors (TGCTs) and share remarkable similarities to human embryonic stem (ES) cells. In prior work we found that EC cells are hypersensitive to low nanomolar doses of 5-aza deoxycytidine (5-aza) and that this hypersensitivity partially depended on unusually high levels of the DNA methyltransferase, DNMT3B. We show here that low-dose 5-aza treatment results in DNA damage and induction of p53 in NT2/D1 cells. In addition, low-dose 5-aza results in global and gene specific promoter DNA hypomethylation. Low-dose 5-aza induces a p53 transcriptional signature distinct from that induced with cisplatin in NT2/D1 cells and also uniquely downregulates genes associated with pluripotency including NANOG, SOX2, GDF3 and Myc target genes. Changes in the p53 and pluripotency signatures with 5-aza were to a large extent dependent on high levels of DNMT3B. In contrast to the majority of p53 target genes upregulated by 5-aza that did not show DNA hypomethylation, several other genes induced with 5-aza had corresponding decreases in promoter methylation. These genes include RIN1, SOX15, GPER, and TLR4 and are novel candidate tumors suppressors in TGCTs. Our studies suggest that the hypersensitivity of NT2/D1 cells to low-dose 5-aza is multifactorial and involves the combined activation of p53 targets, repression of pluripotency genes, and activation of genes repressed by DNA methylation. Low-dose 5-aza therapy may be a general strategy to treat those tumors that are sustained by cells with embryonic stem-like properties. Genomic DNA obtained from NT2/D1-R1 sh control or NT2/D1-R1 sh DNMT3B knockdown cells treated with vehicle or 10 nM 5-aza deoxycytidine for 3 days. Four groups in biological triplicate for total of 12 hybridizations on Illumina HumanMethylation27 BeadChip.