Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Human NTERA2 (NT2/D1) cells lines: shZRF1 vs. shRandom during Retinoic Acid (RA) administration

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Luciano Di Croce

PROVIDER: E-GEOD-16858 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcriptional activation of polycomb-repressed genes by ZRF1.

Richly Holger H Rocha-Viegas Luciana L Ribeiro Joana Domingues JD Demajo Santiago S Gundem Gunes G Lopez-Bigas Nuria N Nakagawa Tekeya T Rospert Sabine S Ito Takashi T Di Croce Luciano L

Nature 20101201 7327

Covalent modification of histones is fundamental in orchestrating chromatin dynamics and transcription. One example of such an epigenetic mark is the mono-ubiquitination of histones, which mainly occurs at histone H2A and H2B. Ubiquitination of histone H2A has been implicated in polycomb-mediated transcriptional silencing. However, the precise role of the ubiquitin mark during silencing is still elusive. Here we show in human cell lines that ZRF1 (zuotin-related factor 1) is specifically recruit ...[more]

PMID: 21179169

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Project description:Our goal is to find new genes regulated by p21 in human primary cells . To get it we carried out a gene expression profiling in two different models, human myeloid leukemia K562 cells and human keratinocytes both of them with conditional expression of p21. In order to find genes regulated by p21 in human primary cells we carried out a gene expression profiling in human myeloid leukemia K562 cells with conditional expression of p21. We previously described a K562 derivative, termed Kp21-4, that carries a zinc-inducible p21 gene (Munoz-Alonso MJ et at., 2005). We performed a kinetic study to identify the expression peak of p21 in this system. This transient induction of p21 was accompanied by proliferation arrest and an increase in polyploid cells after 48-72 h (Munoz-Alonso MJ et at., 2005). Actually, 6-12 h of p21 induction with ZnSO4 is sufficient to irreversibly trigger proliferation arrest. Therefore, we chose 12 h as the induction time to analyse p21 effects on the transcriptome of these cells, as gene expression changes later on may be indirect due to other phenotypic effects. We next carried out the gene expression profiling of Kp21-4 cells upon p21 induction by ZnSO4. In order to identify genes specifically modulated by p21 we compared with the cell line Kp27-5, which carries a Zn2+-inducible p27 allele (Munoz-Alonso MJ et at., 2005). p27 is a close relative to p21 that also inhibits CDKs and induce cell cycle arrest . Thus, the comparison serves to identify genes specifically regulated by p21 in our analysis. We subtracted the gene expression changes occurring at 72 h in Kp21-4 cells those genes regulated by p27 in the Kp27-5 cells and genes changed by ZnSO4 treatment in parental K562 cells. So, our intention is to identify only genes regulated at short time of induction by p21 and not by p27. In order to confirm these results we studied the p21-dependent repression of mitotic genes in a different cellular system. A dramatic increase in p21 and p27 in Kp21 and Kp27 were demonstrated by RT-qPCR and immunoblot (as we show in the manuscript). We prepared RNA 12 h and 72h after induction with ZnSO4 and performed large-scale expression assay using the Afftymetrix platform. The clustering analysis revealed that p21 provoked the down-regulation of a number genes involved in cell cycle control not shared by cells expressing p27 (as we show in the manuscript). Our goal, has been getting genes regulated more strongly by p21 and not by p27, in cell cycle and itosis. Our result are supported because we have found the same genes in two different models and also we have validated (by RT-qPCR) more than 20 cell cycle and mitotic genes, found in our affymetrix arrays. Also we have found the region of p21 that is sufficient for gene regulation and for one gene we have described as p21 bind to the promoter. Finally, we have discussed in our manuscript how p21 can do this regulation by bioinformatic analysis of p21-target genes. The sucess of this study is describe a new role of p21 as a transcriptional co-repressor in some systems.

Project description:Our goal is to find new genes regulated by p21 in human primary cells . To get it we carried out a gene expression profiling in two different models, human myeloid leukemia K562 cells and human keratinocytes both of them with conditional expression of p21. In order to identify genes specifically modulated by p21 we compared with the cell line with overexpression of p27, because p21 and p27 belong to the same gene family and regulated the same genes specially in cell cycle. So, our intention is to identify only genes regulated by p21 and not p27. In order to confirm these results we studied the p21-dependent repression of mitotic genes in a different cellular system. We chose human primary keratinocytes because they are non-tumorigenic, non-immortalized and epithelial cells, in contrast to human myeloid leukemia K562 cells. Human primary keratinocytes were infected with recombinant adenoviruses expressing the full-length p21 protein. A dramatic increase in p21 in infected keratinocytes was demonstrated by RT-qPCR (as we show in the manuscript). As controls, we also infected the keratinocytes with adenovirus carrying the genes for p27 which overexpression was also confirmed by RT-qPCR (as we show in the manuscript). We prepared RNA 24 h after infection and performed large-scale expression assay using the Afftymetrix platform. The clustering analysis revealed that p21 provoked the down-regulation of a number genes involved in cell cycle control not shared by cells expressing p27 (as we show in the manuscript). Our goal, has been getting genes regulated more strongly by p21 and not by p27 in cell cycle and mitosis. Our result are supported because we have found the same genes in two different models and also we have validated (by RT-qPCR) more than 20 cell cycle and mitotic genes, found in our affymetrix arrays. Also we have found the region of p21 that is sufficient for gene regulation and for one gene we have described as p21 bind to the promoter. Finally, we have discussed in our manuscript how p21 can do this regulation by bioinformatic analysis of p21-target genes. The success of this study is to describe a new role of p21 as a transcriptional co-repressor in some systems.

Dataset Information

Human NTERA2 (NT2/D1) cells lines: shZRF1 vs. shRandom during Retinoic Acid (RA) administration

Publications

Transcriptional activation of polycomb-repressed genes by ZRF1.

Similar Datasets

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