Transcriptomics

Dataset Information

3

Transcription profiling by array of human primary keratinocytes to study the role of p21 as a co-represor of S-phase and mitotic control gene


ABSTRACT: 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.

INSTRUMENT(S): 418 [Affymetrix]

ORGANISM(S): Homo sapiens  

SUBMITTER: NURIA FERRANDIZ  

PROVIDER: E-MEXP-3431 | ArrayExpress | 2012-05-30

REPOSITORIES: ArrayExpress

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It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and st  ...[more]

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