Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse fibroblast cells transfected with zinc-inducible C/EBP expression vectors identifies Per2 as a gene implicated in myeloid leukemia

ABSTRACT: The zinc-inducible C/EBP expression vectors pMTa, pMTb, pMTd and pMTe were constructed by cloning the human C/EBPa, C/EBPb, C/EBPd and C/EBPe cDNAs, respectively, into the pMTCB6+ vector. NIH 3T3 cells were transfected with zinc-inducible C/EBP vectors as well as control empty vector using the GenePORTERâ¢ transfection Reagent (GTS Inc.). Multiple polyclonal clones were obtained by selection with G418 (700 mg/ml). Clones were screened by Western blot analysis for C/EBP protein expression following induction for 16 h with ZnSO4 (100 mM). Triplicate clones of NIH 3T3 cells were induced by addition of ZnSO4 (100 mM) for 16 h to the medium. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). Biotinylated cRNAs were prepared and hybridized to murine MG_74Av2 microarrays (Affymetrix, Santa Clara, CA, USA), which contains >12 000 genes. The probed arrays were scanned with a Hewlett Packard Gene Array scanner. The scanned output image files were analyzed using Affymetrix Microarray Suite version 5.0. To identify genes that were differently expressed between the five-sample sets (empty vector, C/EBPa, C/EBPb, C/EBPd and C/EBPe) class compression analysis were performed using BRBArray Tools (developed by Richard Simon and Amy Peng Lam; http://linus.nci.nih.gov/BRB-ArrayTools.html). Medium normalization was applied to the arrays and the percent absent filter was set at 80% to exclude probe sets that were unreliable. A list of 158 genes with probability of 95% that contain no more then 10% false discoveries was generated. Of these genes, 117 were significant at the 0.001 level of univariate F-test used and the remaining 41 were significant at the 0.027 level.

ORGANISM(S): Mus musculus

SUBMITTER: sigal gery

PROVIDER: E-GEOD-2188 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transcription profiling of C/EBP targets identifies Per2 as a gene implicated in myeloid leukemia.

Gery Sigal S Gombart Adrian F AF Yi William S WS Koeffler Chloe C Hofmann Wolf-K WK Koeffler H Phillip HP

Blood 20050628 8

CCAAT/enhancer-binding proteins (C/EBPs) are a family of transcription factors that regulate cell growth and differentiation in numerous cell types. To identify novel C/EBP-target genes, we performed transcriptional profiling using inducible NIH 3T3 cell lines expressing 1 of 4 members of the C/EBP family. Functional analysis revealed a previously unknown link between C/EBP proteins and circadian clock genes. Our microarray data showed that the expression levels of 2 core components of the circa ...[more]

PMID: 15985538

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Project description:Background: LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the aggressive cancer state that stems from Lkb1 deficiency can be reverted remains unknown. Purpose: To ascertain the tumor-suppressive capacity C/EBP family members, including Cebpa, Cebpb, and Cebpd, through CRISPR/Cas9-mediated targeting in a genetically engineered mouse model of oncogenic KRAS-driven lung adenocarcinoma. Approach: Measurements of tumor size across genetic perturbations were obtained by tumor barcode sequencing (Tuba-seq; Rogers et al., 2017 Nature Methods). Briefly, lung tumors were initiated in KrasLSL-G12D/+;R26LSL-tdTomato (KT) and KT;H11LSL-Cas9 mice using a pool of Lenti-sgRNA/Cre vectors that are each modified with two-component barcodes composed of sgRNA-specific and clonal identifiers. This particular pool of Lenti-sgRNA/Cre vectors was composed of vectors targeting each of the three C/ebp paralogs individually, in pairwise combinations, and all three simultaneously. Following tumor development, the two-component lentiviral barcodes integrated within transduced populations were amplified from genomic DNA of whole-lung homogenates. The resulting amplicons were then subjected to next-generation sequencing. From the resulting reads, barcode pileups were generated and filtered prior to conversion to absolute numbers of neoplastic cells via normalization to spiked-in samples of known quantities of barcoded neoplastic cells. The resulting tumor size distributions were then analyzed by multiple statistical measures as described previously (Rogers et al., 2017 Nature Methods). Results: The targeting of Cebpa significantly increased tumor size relative to tumors initiated with vectors encoding inert sgRNAs (oncogenic KRAS only tumors). The targeting of Cebpb and Cebpd had no significant impact on tumor growth. There were no additive effects of knocking out additional paralogs on top of Cebpa. Conclusions: C/EBPa is the dominant tumor-suppressive paralog and there was no evidence of functional redundancy among C/EBPa, C/EBPb, and C/EBPd.

Dataset Information

Transcription profiling of mouse fibroblast cells transfected with zinc-inducible C/EBP expression vectors identifies Per2 as a gene implicated in myeloid leukemia

Publications

Transcription profiling of C/EBP targets identifies Per2 as a gene implicated in myeloid leukemia.

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