The Promoter of the Human Proliferating Cell Nuclear Antigen Gene Is Not Sufficient for Cell Cycle-dependent Regulation in Organotypic Cultures of Keratinocytes

Francisco Noya,Wei-Ming Chien,Xiaoyun Wu,Nilam S Banerjee,John C Kappes,Thomas R Broker,Louise T Chow

doi:10.1074/jbc.m112441200

Abstract

The proliferating cell nuclear antigen (PCNA) is essential for DNA replication of mammalian cells and their small DNA tumor viruses. The mechanism of the cell cycle-dependent regulation of the human PCNA promoter is not clear despite extensive investigations. In this report, we employed organotypic cultures of primary human keratinocytes, which closely resemble native skin comprising both proliferating and postmitotic, differentiated cells, to examine the cell cycle-dependent regulation of the human PCNA gene (hPCNA) in the absence or presence of the human papillomavirus type 18 (HPV-18) E7 protein. HPV-18 E7 promotes S phase re-entry in post-mitotic differentiated keratinocytes by abrogating the transcription repression of E2F transcription factors by the retinoblastoma susceptibility protein, pRb. We demonstrated that E7 reactivated the transcription of the endogenous hPCNA in differentiated keratinocytes. In contrast, with or without E7, the expression of a transduced hPCNA promoter-driven reporter did not correlate with that of the endogenous hPCNA gene in either proliferating or differentiated cells. Moreover, in Chinese hamster ovary and L-cells, HPV E7 and the adenovirus E1A protein repressed the transduced hPCNA promoter, but both activated an extended promoter construct spanning the first intron. Mutations of two E2F sites in the intron reduced the basal activity and abolished the response to E7 or E1A. Promoter repression or activation required the CR2 domain of E7 and, to a lesser extent, CR1 as well. However, in organotypic cultures, this extended promoter construct failed to recapitulate the cell cycle-dependent regulation of the endogenous hPCNA gene. Only when a full-length Myc-tagged hPCNA spanning the 5' promoter and all exons and introns was used was the native pattern of expression largely restored, indicative of the complexity of its regulation.

Highlights

The proliferating cell nuclear antigen (PCNA)1 is a conserved highly acidic 29-kDa nuclear protein that functions as a sliding hPCNAp activation by E1A is mediated through an element termed PERE that contains binding sites for transcription factors ATF/CRE and RFX1 [13, 17,18,19]. p300/CBP and p107, a pRb-related protein, interact indirectly with these sequences
human papillomavirus type 18 (HPV-18) E7 Reactivates the Transcription of human PCNA gene (hPCNA) in Differentiated Keratinocytes—E7 is known to induce the accumulation of proteins, such as p21cip1, via a post-transcriptional mechanism [40, 43]
We performed in situ hybridization with 35S-labeled, strand-specific RNA probes to detect hPCNA message and immunohistochemical staining for the PCNA protein in adjacent sections in raft cultures transduced with either the vector-only retrovirus or the retrovirus containing human papillomaviruses (HPVs)-18 URR-E7

Summary

Introduction

The proliferating cell nuclear antigen (PCNA) is a conserved highly acidic 29-kDa nuclear protein that functions as a sliding hPCNAp activation by E1A is mediated through an element termed PERE that contains binding sites for transcription factors ATF/CRE and RFX1 [13, 17,18,19]. p300/CBP and p107, a pRb-related protein, interact indirectly with these sequences. Conserved region 2 (CR2), which spans residues 16 –36, contains the pRb-binding domain [32], as well as a casein kinase II phosphorylation site [33] The integrity of both CR1 and CR2 is required for the transformation and transactivation activities of E7 (34 –37). When acutely transduced via a recombinant retrovirus into PHKs, the HPV-18 URR-driven E7 induces PCNA protein in the differentiated cells of raft cultures This activity requires the presence of both the pRb-binding motif and the casein kinase II phosphorylation site in CR2 [39]. These cells no longer replicate their DNA and are negative for PCNA [44] (see Fig. 1D, left panels) This culture system, in conjunction with the differentiation-dependent expression of HPV-18 E7 from the URR promoter, provides an exceptional opportunity to examine the mechanisms by which the cell controls the expression of genes necessary for S phase entry

Methods

Results

Conclusion