CDKN1C Negatively Regulates RNA Polymerase II C-terminal Domain Phosphorylation in an E2F1-dependent Manner

Abstract

CDKN1C is a cyclin-dependent kinase inhibitor and is a candidate tumor suppressor gene. We previously found that the CDKN1C protein represses E2F1-driven transcription in an apparent negative feedback loop. Herein, we explore the mechanism by which CDKN1C represses transcription. We find that adenoviral-mediated overexpression of CDKN1C leads to a dramatic reduction in phosphorylation of the RNA polymerase II (pol II) C-terminal domain (CTD). RNA interference studies demonstrate that this activity is not an artifact of CDKN1C overexpression, because endogenous CDKN1C mediates an inhibition of RNA pol II CTD phosphorylation in HeLa cells upon treatment with dexamethasone. Surprisingly, we find that CDKN1C-mediated repression of RNA pol II phosphorylation is E2F1-dependent, suggesting that E2F1 may direct CDKN1C to chromatin. Chromatin immunoprecipitation assays demonstrate that CDKN1C is associated with E2F1-regulated promoters in vivo and that this association can dramatically reduce the level of RNA pol II CTD phosphorylation at both Ser-2 and Ser-5 of the C-terminal domain repeat. In addition, we show that CDKN1C interacts with both CDK7 and CDK9 (putative RNA pol II CTD kinases) and that CDKN1C blocks their ability to phosphorylate a glutathione S-transferase-CTD fusion protein in vitro. E2F1 and CDKN1C are found to form stable complexes both in vivo and in vitro. Molecular studies demonstrate that the E2F1-CDKN1C interaction is mediated by two E2F domains. A central E2F1 domain interacts directly with CDKN1C, whereas a C-terminal E2F1 domain interacts with CDKN1C via interaction with Rb. The results presented in this report highlight a novel mechanism of tumor suppression by CDKN1C.

p57Kip2 and Cancer: Time for a Critical Appraisal

“…E2F1 then directs p57 Kip2 to chromatin. Finally, p57 Kip2 inhibits CDK7 and CDK9 (putative RNA pol II CTD kinases), affecting CTD phosphorylation and in turn RNA synthesis (105). The authors proposed that these results highlight a novel mechanism of tumor suppression by p57 Kip2 ; however, further confirmation is needed to substantiate this promising conclusion.…”

“…Treatment with demethylating agents, such as 5-azacytidine or 5-aza-2'-deoxycytidine, resulted in CDKN1C reexpression or overexpression in several experimental cancer models (105,(147)(148)(149). This finding suggests that compounds that reduce DNA methylation might be employed, possibly in association with other drugs, for the therapy of cancers with low p57 Kip2 content.…”

Tripartite Motif Containing 28 (Trim28) Can Regulate Cell Proliferation by Bridging HDAC1/E2F Interactions

“…Chromatin Immunoprecipitation (ChIP) Assay and ChIPReChIP-ChIP assays were performed as previously described (30). Briefly, H1299 cells were treated with formaldehyde to create protein-DNA cross-links, and the cross-linked chromatin extracted and sheared by sonication.…”

“…Analytical Methods-Immunoprecipitation and immunoblot experiments were performed as previously described (30). E2F1 (sc-193), E2F3 (sc-878), E2F4 (sc-866), p107 (sc-318), and GFP (sc-9996) antibodies were purchased from Santa Cruz Biotechnology.…”

CD4+ T cells from elite controllers resist HIV-1 infection by selective upregulation of p21

“…The beads were sequentially washed with lysis buffer, lysis buffer plus 0.5 M LiCl, and kinase buffer (20 mM HEPES, pH 7.5; 50 mM NaCl; 10 mM MgCl2; and 1 mM dithiothreitol). The immunoprecipitate was resuspended in 25 μl kinase buffer containing 5 mM ATP and 0.5 μg purified GST-CTD polymerase II substrate (37). The kinase reaction was conducted at 30°C for 20 minutes and stopped by addition of SDS-PAGE loading buffer.…”