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: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:Human tissue based proteomics projects are challenging due to low abundance of proteins and tissue specificity of protein expression. In this study, we aimed to develop a cell-based approach to profile the male specific region of the Y chromosome (MSY) proteins. First, we profiled the expression of 23 Y chromosome genes and 15 of their X-linked homologues during neural cell differentiation from NT2 cells at three different developmental stages using qRT-PCR, western blotting and immunofluorescent (IF) techniques. The expression level of 12 Y-linked genes significantly increased over neural differentiation. Including RBMY1, EIF1AY, DDX3Y1, HSFY1, BPY2, PCDH11Y, UTY, RPS4Y1, USP9Y, SRY, PRY, and ZFY. Subsequently, DDX3Y was selected as a candidate for knockdown as it was significantly expressed in neural progenitor cells and it is known to be expressed in a gender specific manner and play a role in spermatogenesis. A siRNA-mediated DDX3Y knockdown in neural progenitor cells impaired cell cycle progression and increased apoptosis, consequently interrupting differentiation. Label-free quantitative shotgun proteomics based on a spectral counting approach was then used to characterize the proteomic profile of the cells after DDX3Y knockdown. Among 920 reproducibly identified proteins detected, 74 proteins were differentially expressed following DDX3Y siRNA treatment compared to mock treated cells. Functional grouping indicated these proteins were associated with cell cycle, cell-to-cell signaling, apoptosis and other important networks such as RNA processing and transcription regulation. Disease-based analysis confirmed DDX3Y involvement primarily in neurological and RNA metabolism disorders. Our results confirm that MSY genes are expressed in male neuronal cells, and demonstrate that Y linked DDX3 (DDX3Y) could play a multifunctional role in neural cell development in a sexually dimorphic manner.
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: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 aM-BM- 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, includingM-BM- NanogM-BM- mRNA, which modulate exit from the proliferative stem-cell state. This regulation requiresM-BM- AGO3-dependent accumulation of processed DR2 Alu transcripts and the subsequent recruitment ofM-BM- 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: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: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 M-BM-5M. 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:Background: MicroRNAs (miRNAs) are short non-coding RNAs predicted to regulate one third of protein-coding genes via mRNA targeting. In conjunction with key transcription factors, such as the repressor REST (RE1 silencing transcription factor), miRNAs play crucial roles in neurogenesis, which requires a highly orchestrated program of gene expression to ensure the appropriate development and function of diverse neural cell types. Whilst previous studies have highlighted select groups of miRNAs during neural development, there remains a need for amenable models in which miRNA expression and function can be analyzed over the duration of neurogenesis. Principal Findings: We performed large-scale expression profiling of miRNAs in human NTera2/D1 (NT2) cells during retinoic acid (RA)-induced transition from progenitors to fully differentiated neural phenotypes. Our results revealed dynamic changes of miRNA patterns, resulting in distinct miRNA subsets that could be linked to specific neurodevelopmental stages. Moreover, the cell-type specific miRNA subsets were very similar in NT2-derived differentiated cells and human primary neurons and astrocytes. Further analysis identified miRNAs as putative regulators of REST, as well as candidate miRNAs targeted by REST. Finally, we confirmed the existence of two predicted miRNAs; pred-MIR191 and pred-MIR222 associated with SLAIN1 and FOXP2, respectively, and provided some evidence of their potential co-regulation. Conclusions: In the present study, we demonstrate that regulation of miRNAs occurs in precise patterns indicative of their roles in cell fate commitment, progenitor expansion and differentiation into neurons and glia. Furthermore, the similarity between our NT2 system and primary human cells suggests their roles in molecular pathways critical for human in vivo neurogenesis. The experiment consists of a total of 51 arrays: 29 retinoic acid time series arrays (0,2,4,6,8,12,14,21 and 28 days), 2 each of NT2-derived neurons and astrocytes, 12 primary human fetal astrocytes, 3 primary human embryonic astrocytes and 3 primary human neurons. Each condition has a minimum of 2 biological replicates. The samples were compared as single channel experiments. NOTE: The raw data files were submitted as generated in Quantarray with 2 channels, but due to issues with the control sample dye (Cy5 on Channel 1), only the Channel 2 (Cy3) data was analysed.
Project description:The study focuses on an extensive biochemical fractionation with in-depth quantitative mass spectrometric profiling in the mitochondrial (mt) extracts of cultured human NTera2 embryonal carcinoma stem cells (i.e. ECSCs or undifferentiated state) and upon exposure to retinoic acid-induced differentiated neurons (DNs) to establish a network of high-quality mt protein-protein interactions. The resulting network showed that most of the native mt protein complexes with predicted subunits are previously unreported and endured extensive changes during neuronal differentiation and influence neuronal function and neurodegenerative disorder attributes.