RNA-seq of human MCF10A EditR breast epithelial cells expressing Cas9, with PTBP1 mutation as compared to control
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ABSTRACT:
INSTRUMENT(S): Illumina NovaSeq 6000
ORGANISM(S): Homo sapiens
SUBMITTER: Declan Bennett
PROVIDER: E-MTAB-12578 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Background: Genome-wide transcriptome profiling generated by microarray and RNA-Seq often provides deregulated genes or pathways applicable only to larger cohort. On the other hand, individualized interpretation of transcriptomes is increasely pursued to improve diagnosis, prognosis, and patient treatment processes. Yet, robust and accurate methods based on a single paired-sample remain an unmet challenge. Methods: M-bM-^@M-^\N-of-1-pathwaysM-bM-^@M-^] translates gene expression data profiles into mechanism-level profiles on single pairs of samples (one p-value per geneset). It relies on three principles: i) statistical universe is a single paired sample, which serves as its own control; ii) statistics can be derived from multiple gene expression measures that share common biological mechanisms assimilated to genesets; iii) semantic similarity metric takes into account inter-mechanismsM-bM-^@M-^Y relationships to better assess commonality and differences, within and cross study-samples (e.g. patients, cell-lines, tissues, etc.), which helps the interpretation of the underpinning biology. Results: In the context of underpowered experiments, N-of-1-pathways predictions perform better or comparable to those of GSEA and Differentially Expressed Genes enrichment (DEG enrichment), within- and cross-datasets. N-of-1-pathways uncovered concordant PTBP1-dependent mechanisms across datasets (Odds-RatiosM-bM-^IM-%13, p-valuesM-bM-^IM-$1M-CM-^W10-5), such as RNA splicing and cell cycle. In addition, it unveils tissue-specific mechanisms of alternatively transcribed PTBP1-dependent genesets. Furthermore, we demonstrate that GSEA and DEG Enrichment preclude accurate analysis on single paired samples. Conclusions: N-of-1-pathways enables robust and biologically relevant mechanism-level classifiers with small cohorts and one single paired samples that surpasses conventional methods. Further, it identifies unique sample/ patient mechanisms, a requirement for precision medicine. Cell lines, culture conditions: The epithelial human breast cancer cell line MDA-MB231 (ER-/ PR-/ HER2-) were obtained from the American Type Culture Collection (Manassas, VA). The epithelial human ovarian tumor cell line A2780 was received as a generous gift from Dr. Thomas C. Hamilton (Fox Chase Cancer Center, Philadelphia, PA) Cancer Center, Philadelphia, PA). Cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine in a humid environment at 37M-BM-0C, with 5% CO2. Both cell lines were free of Mycoplasma species and were maintained for no longer than 10 weeks in culture after recovery from frozen stocks. Mycoplasma levels were checked periodically using the MycoAlertM-BM-. Mycoplasma Detection Kit (Lonza Inc., Allendale, NJ). The authenticity of cell lines was assessed by the ATCC carrying out short tandem repeat (STR) analysis (Verified STR Profiling Service, ATCCM-BM-. 135-XV). Additionally, we compared A2780 to the original STR profile collected by the European Collection of Cell Culture (Catalogue number 93112519). Doxycycline-inducible knockdown of PTBP1 regulated by small hairpin RNA (shRNA): In order to analyze the effect of PTBP1 depletion, two consecutive viral transductions were performed in both MDA-MB231 and A2780 cell lines. Cells were plated on 24-well plate (10-20M-CM-^W104 cells/well), maintained in culture for 16 hours, and then medium containing LV-tTR/KRAB-Red lentiviral particles was added. Following 16 h of incubation, cells were transduced a second time by LV-THM/PTBshRNA or LV-THM/LUCshRNA lentiviral particles. Clones expressing both red and green fluorescent protein (dsRED and GFP respectively) were selected and expanded. Following 16 h of incubation, cells were washed and split in two subcultures, one without doxycycline (PTBP1/-DOX; Control in Figure 1) and the other with Doxycycline (DOX) at a final concentration of 1 M-NM-<g/ml (PTBP1/+DOX; PTBP1-KD in Figure 1). Doxycycline was prepared according to the manufacturerM-bM-^@M-^Ys recommendations (Sigma-Aldrich, St. Louis, MO). Five days later, cells were analyzed by fluorescence microscopy, and PTBP1 gene expression was assessed using PCR and Western Blotting (data not shown). The cells that were transduced by LV-LUCshRNA express PTBP1 regardless of the presence of DOX (LUCshRNA/+DOX). Constructs and lentivirus preparation were performed as previously described in literature [He X et al., Knockdown of polypyrimidine tract-binding protein suppresses ovarian tumor cell growth and invasiveness in vitro, Oncogene 2007, 26(34):4961-4968]. Microarray Analysis: For each of the cell lines, MDA-MB231 and A2780, total RNAs were extracted from four biological replicates of PTBP1-depleted cells, PTBP1-KD (4M-CM-^W PTBP1/+DOX) and eight biological replicates control cells (4M-CM-^W PTBP1/-DOX and 4M-CM-^W LUCshRNA/+DOX) by Direct-zol RNA kit (Zymo Research, Irvine, CA). All paired samples consist of PTBP1-depleted cells (PTBP1-KD) and matched control cells. Qualities of RNA were assessed based on the RNA quality indicator (RQI M-bM-^IM-% 8) using Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA). Gene expression microarray measurements were performed using the GeneChip PrimeView Human Gene Expression Array that contains 49,395 probes and measures 36,000 transcripts and variants per sample. Labeling and hybridization were performed following Affymetrix protocols. The raw data were normalized according to the Robust Multiple-array Average (RMA) technique, using Affymetrix Power Tools (APT). biological replicate: Ovarian_LucsH_DOX_1, Ovarian_LucsH_DOX_2, Ovarian_LucsH_DOX_3, Ovarian_LucsH_DOX_4 biological replicate: Ovarian_PTBsh1_NODOX_1, Ovarian_PTBsh1_NODOX_2, Ovarian_PTBsh1_NODOX_3, Ovarian_PTBsh1_NODOX_4 biological replicate: Ovarian_PTBsh1_DOX_1, Ovarian_PTBsh1_DOX_2, Ovarian_PTBsh1_DOX_3, Ovarian_PTBsh1_DOX_4 biological replicate: Breast_LucsH_DOX_1, Breast_LucsH_DOX_2, Breast_LucsH_DOX_3, Breast_LucsH_DOX_4 biological replicate: Breast_PTBsh1_NODOX_1, Breast_PTBsh1_NODOX_2, Breast_PTBsh1_NODOX_3, Breast_PTBsh1_NODOX_4 biological replicate: Breast_PTBsh1_DOX_1, Breast_PTBsh1_DOX_2, Breast_PTBsh1_DOX_3, Breast_PTBsh1_DOX_4
2013-11-20 | E-GEOD-52493 | biostudies-arrayexpress
Project description:We describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions with 1 million HEK293 cells, and with public eCLIP and iCLIP PTBP1 data. There are 3 PTBP1 iiCLIP libraries, 1 input iiCLIP library and 1 PTBP1 iCLIP2 library produced in this study.
2022-08-01 | E-MTAB-10881 | biostudies-arrayexpress
Project description:We examined the role of PTBP1 in regulation of co-transcriptional splicing process by depleting this RNA-binding protein from embryonic stem cells using the auxin-inducible degron technology and analysing the total and chromatin-associated RNA fractions by RNA-seq. We also performed mNET-seq and ChIP-seq analyses using RNA polymerase II- and PTBP1-specific antibodies, respectively. Our data suggest that PTBP1 activates co-transcriptional splicing of hundreds of introns, a surprising effect given that PTBP1 is better known as a splicing repressor. Importantly, some co-transcriptionally activated introns fail to be spliced post-transcriptionally without PTBP1. In a striking example of this regulation, lasting retention of a PTBP1-dependent intron triggers nonsense-mediated decay of mRNAs encoding DNA methyltransferase DNMT3B, explaining their natural expression dynamics in development. Our further analyses suggest that this mechanism may protect differentiation-specific genes from aberrant methylation. We conclude that PTBP1-activated co-transcriptional splicing underlies biologically important decisions.
2023-01-08 | E-MTAB-11544 | biostudies-arrayexpress
Project description:CDC7 is a serine-threonine kinase, which has an important role in the activation of replication origins in the S-phase. CDC7 is also a potential molecular target for the development of anticancer drugs. Multiple studies have shown that different cell lines have a wide range of sensitivity to CDC7 inhibition. However, how the manner in which cells respond to prolonged CDC7 inhibition has yet to be investigated. Hence, we performed an RNA-sequencing experiment to investigate variation in gene expression in cells undergoing prolonged CDC7 inhibition.
2024-05-14 | E-MTAB-13179 | biostudies-arrayexpress
Project description:RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Polypyrimidine tract binding proteins, PTBP1 and PTBP2, are paralogous RNA binding proteins that play a critical role in the process of neuronal differentiation and maturation; changes in the concentration of PTBP proteins during neuronal development direct splicing changes in many transcripts that code for proteins critical for neuronal differentiation. How the two related proteins regulate different sets of neuronal exons is unclear. The distinct splicing activities of PTBP1 and PTBP2 can be recapitulated in an in vitro splicing system with the differentially regulated N1 exon of the c-src pre-mRNA. Here, we conducted experiments under these in vitro splicing conditions to identify PTBP1 and PTBP2 interacting partner proteins.
2022-01-20 | PXD029899 | Pride
Project description:PTBP1 and PTBP2 control alternative splicing programs during neuronal development, but the cellular functions of most PTBP1/2-regulated isoforms remain unknown. We show that PTBP1 guides developmental gene expression by regulating the transcription factor Pbx1. We identify exons that are differentially spliced when mouse embryonic stem cells (ESCs) differentiate into neuronal progenitor cells (NPCs) and neurons, and transition from PTBP1 to PTBP2 expression. We define those exons controlled by PTBP1 in ESCs and NPCs by RNA-seq analysis after PTBP1 depletion and PTBP1 crosslinking-immunoprecipitation. We find that PTBP1 represses Pbx1 exon 7 and the expression of its neuronal isoform Pbx1a in ESC. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of specific neuronal genes including known Pbx1 targets. Thus PTBP1 controls the activity of Pbx1 and suppresses its neuronal transcriptional program prior to differentiation. 46C mESCs were treated with 20 nM control, Ptbp1, Ptbp2, or Ptbp1 and Ptbp2 siRNAs for 72 hours. The knockdowns were performed using 2 independent sets of siRNAs, including one biological replicate. Poly-A RNA was isolated for RNA-sequencing and splicing analyses.
2016-07-03 | E-GEOD-71075 | biostudies-arrayexpress
Project description:PTBP1 and PTBP2 control alternative splicing programs during neuronal development, but the cellular functions of most PTBP1/2-regulated isoforms remain unknown. We show that PTBP1 guides developmental gene expression by regulating the transcription factor Pbx1. We identify exons that are differentially spliced when mouse embryonic stem cells (ESCs) differentiate into neuronal progenitor cells (NPCs) and neurons, and transition from PTBP1 to PTBP2 expression. We define those exons controlled by PTBP1 in ESCs and NPCs by RNA-seq analysis after PTBP1 depletion and PTBP1 crosslinking-immunoprecipitation. We find that PTBP1 represses Pbx1 exon 7 and the expression of its neuronal isoform Pbx1a in ESC. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of specific neuronal genes including known Pbx1 targets. Thus PTBP1 controls the activity of Pbx1 and suppresses its neuronal transcriptional program prior to differentiation. 46C mESCs were differentiated in mNPCs. The mNPCs were treated with 10 nM control, Ptbp1, Ptbp2, or Ptbp1 and Ptbp2 siRNAs for 48 hours. The knockdowns were performed using 2 independent sets of siRNAs. Poly-A RNA was isolated for RNA-sequencing and splicing analyses.
2016-07-03 | E-GEOD-70985 | biostudies-arrayexpress
Project description:The aim of the experiment was to compare binding of PTBP1 in presence and absence of MATR3. HEK293T cells were transfected with siRNA targeting MATR3 or control sequences and labelled with 4thio-uridine for 8 hours (100µM, crosslinking 2x400mJ/cm2 365nm UV light).
2018-05-01 | E-MTAB-6286 | biostudies-arrayexpress
Project description:Using basal‐like untransformed cells, MCF10A, as a model system, data from our laboratory showed that both mRNAs and microRNAs exhibit dynamic changes in expression following EGF stimulation (Amit et al, 2007; Avraham et al, 2010; Kostler et al, 2013). We further demonstrated that the inducible mRNAs and microRNAs are embedded into regulatory subnetworks, which are deregulated in diverse tumor types. Considering the emerging roles for long noncoding RNAs (lncRNAs) in metastasis of breast cancer (Serviss et al, 2014), we raised the possibility that some EGF‐inducible lncRNAs might play a role in basal‐like breast cancer. Thus, MCF10A cells were stimulated with EGF (10 ng/ml) for 0, 20, 40, 60, 120, 240 and 480 minutes. RNA was then extracted from cells and expression of lncRNAs was measured using Agilent SurePrint microarrays.
2017-07-27 | E-MTAB-4822 | biostudies-arrayexpress
Project description:Chromatin immuno-precipitation using SUMO2 (Life Technologies) antibody in MCF10A cell line, treated with epidermal growth factor for 30 minutes.
2019-07-16 | E-MTAB-7759 | biostudies-arrayexpress