A proteomic analysis of p27kip1-binding proteins reveals a putative role in transcription regulation through RNA polymerase II interaction
Introduction and objectives: The protein p27(kip1) is a member of the Cip-Kip family of cyclin-dependent kinase (CDK) inhibitors. In addition to this role, p27 also behaves as a transcriptional regulator. It directly interacts with the p130/E2F4 subunit of the DREAM transcriptional repressor complex through its carboxyl moiety. To further identify new mechanism of transcriptional regulation mediated by p27 we aimed to identify nuclear proteins that associates with its carboxyl terminal domain.
Methods: Affinity chromatography columns of p27 peptide (including a.a. 161-198) bound to sepharose-beads was used to purify p27-binding proteins (p27-BPs). These proteins were subsequently identified by mass spectrometry (MS).
Results and Discussion: MS analysis of the purified p27-BPs revealed that 8 subunits of RNA polymerase II (RNA pol II) associate with p27.
The interaction of p27 with RNA pol II was validated by immunoprecipitation (IP) analysis using anti-p27 antibodies.
A subsequent Proto-Array analysis identified the RNA pol II-K subunit as the direct target of p27. This direct interaction was validated by affinity chromatography using purified proteins.
Finally, by Chromatin-IP (ChIP) we could establish a correlation between the loading of RNA pol II on the promoters of specific target genes and the presence of p27 on these promoters.
Conclusions: The protein p27Kip1 might regulate transcription by loading RNA polymerase II on the promoters of specific target genes.
Methods: Affinity chromatography columns of p27 peptide (including a.a. 161-198) bound to sepharose-beads was used to purify p27-binding proteins (p27-BPs). These proteins were subsequently identified by mass spectrometry (MS).
Results and Discussion: MS analysis of the purified p27-BPs revealed that 8 subunits of RNA polymerase II (RNA pol II) associate with p27.
The interaction of p27 with RNA pol II was validated by immunoprecipitation (IP) analysis using anti-p27 antibodies.
A subsequent Proto-Array analysis identified the RNA pol II-K subunit as the direct target of p27. This direct interaction was validated by affinity chromatography using purified proteins.
Finally, by Chromatin-IP (ChIP) we could establish a correlation between the loading of RNA pol II on the promoters of specific target genes and the presence of p27 on these promoters.
Conclusions: The protein p27Kip1 might regulate transcription by loading RNA polymerase II on the promoters of specific target genes.
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