Abstract
The protein p27Kip1 (p27), a member of the Cip-Kip family of cyclin-dependent kinase inhibitors, is involved in tumorigenesis and a correlation between reduced levels of this protein in human tumours and a worse prognosis has been established. Recent reports revealed that p27 also behaves as a transcriptional regulator. Thus, it has been postulated that the development of tumours with low amounts of p27 could be propitiated by deregulation of transcriptional programs under the control of p27. However, these programs still remain mostly unknown. The aim of this study has been to define the transcriptional programs regulated by p27 by first identifying the p27-binding sites (p27-BSs) on the whole chromatin of quiescent mouse embryonic fibroblasts. The chromatin regions associated to p27 have been annotated to the most proximal genes and it has been considered that the expression of these genes could by regulated by p27. The identification of the chromatin p27-BSs has been performed by Chromatin Immunoprecipitation Sequencing (ChIP-seq). Results revealed that p27 associated with 1839 sites that were annotated to 1417 different genes being 852 of them protein coding genes. Interestingly, most of the p27-BSs were in distal intergenic regions and introns whereas, in contrast, its association with promoter regions was very low. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by p27 as cell adhesion, intracellular signalling and neuron differentiation among others. We validated the interaction of p27 with different chromatin regions by ChIP followed by qPCR and demonstrated that the expressions of several genes belonging to these programs are actually regulated by p27. Finally, cell adhesion assays revealed that the adhesion of p27-/- cells to the plates was much higher that controls, revealing a role of p27 in the regulation of a transcriptional program involved in cell adhesion.
Highlights
Cell cycle progression is driven by different members of a family of serine/threonine kinases named cyclin dependent kinases (Cdks), characterized by their need for associating with a regulatory subunit, the cyclin, that potentiates Cdk catalytic activity [1]
As p27 has been identified as a transcriptional regulator, identifying the transcriptional programs regulated by p27 will help deciphering the role of this protein in tumor development
ChIP-seq analysis performed in quiescent mouse embryonic fibroblasts (MEFs) allowed the identification of 1417 genes displaying p27-binding sites (p27-BSs) in their vicinity and annotated as putative p27-target genes (p27-TGs)
Summary
Cell cycle progression is driven by different members of a family of serine/threonine kinases named cyclin dependent kinases (Cdks), characterized by their need for associating with a regulatory subunit, the cyclin, that potentiates Cdk catalytic activity [1]. In addition to the regulation by cyclins, Cdk activity is regulated by other mechanisms including phosphorylation, acetylation and binding to Cdk-inhibitors (CKIs)[1,4,5,6]. These mechanisms allow modulating, Cdk activity, and its intracellular location and degradation. The Cip/Kip family that includes p21Cip (p21), p27Kip (p27) and p57Kip that associate with most cyclin-cdk complexes involved in cell cycle regulation [7] and the INK4 family that includes p15INK4B, p16INK4A, p18INK4C and p19 INK4D and that acts on Cdk and Cdk6 [8]
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