ChIP-Seq analysis identifies p27(Kip1)-target genes involved in cell adhesion and cell signalling in mouse embryonic fibroblasts

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.

Role of p27Kip1 as a transcriptional regulator

“…This analysis confirms that cell adhesion is a major biological process regulated by p27 and this merits to be analyzed in detail in the next future. A recent report showing that cells lacking p27 have a much higher adherence to the culture plates than control cells, strongly support this concept [ 77 ].…”

“…Two different articles have reported ChIP-seq analyses that have been performed to identify the p27-BSs in the chromatin of two different cellular types. One has been performed in quiescent HCT116 colorectal cancer cells [ 76 ] and the other one in quiescent MEFs [ 77 ]. In HCT116 cells, 1981 specific p27-BSs that were annotated to 1012 genes were identified (Table 1 ).…”

“…These studies also revealed that in both cellular types only a small number of p27-BSs were on gene promoters while most of them were in distal intergenic (74% and 64%) or intronic regions (24% and 31%). Another interesting observation was that a significant number of p27-BSs (~40% in both cases) were close to pseudogenes or sequences of non-coding RNAs suggesting a role of p27, not only in the transcriptional regulation of protein encoding genes, but also of pseudogenes and non-coding RNAs [ 76 , 77 ]. Specifically, a significant number of microRNAs (Mirs) were annotated to p27-BSs in both cellular types.…”

“…Finally, sequence analysis of the p27-BSs identified by ChIP-seq in MEFs revealed enrichment of several TFs as AHR, Ap2α and Ap2γ, Pax5, Pax4, and MyoD, among others. The association of p27 with some of these TFs was subsequently validated by IP [ 77 ]. Thus, p27 plays a role in the regulation of transcription by associating with a significant number of different TFs (Figure 4 ).…”

“…Alteration of TF expression in p27KO brings up the possibility that the around of 4000 genes whose expression is altered in these cells could be a consequence of different transcription waves. First, a limited number of genes directly regulated by p27 would be initially expressed in p27KO cells, and among them, some TFs, as for instance Mef2c, Sox6, Zfpm2 and Shox2 that have p27-BSs in their vicinity [ 77 ]. As a consequence, a second transcriptional wave can de generated by the action of these TFs.…”