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. 2011 Oct 1;10(19):3317-26.
doi: 10.4161/cc.10.19.17618. Epub 2011 Oct 1.

Jab1/CSN5 mediates E2F dependent expression of mitotic and apoptotic but not DNA replication targets

Huarui Lu  1 , Xudong Liang, Olga A Issaenko, Timothy C Hallstrom
Affiliations

Jab1/CSN5 mediates E2F dependent expression of mitotic and apoptotic but not DNA replication targets

Huarui Lu et al. Cell Cycle. .

Abstract

The E2F transcription factors are critical regulators of cell cycle and cell fate control. Several classes of E2F target genes have been categorized based on their roles in DNA replication, mitosis, apoptosis, DNA repair, etc. How E2Fs coordinate the appropriate and timely expression of these functionally disparate gene products is poorly understood at a molecular level. We previously showed that the E2F1 binding partner Jab1/CSN5 promotes E2F1-dependent induction of apoptosis but not proliferation. To better understand how Jab1 regulates E2F1 dependent transcription, we performed gene expression analysis to identify E2F target genes most and least affected by shRNA depletion of Jab1. We find that a significant number of apoptotic and mitotic E2F target genes are poorly expressed in cells lacking Jab1/CSN5, whereas DNA replication genes are generally still highly expressed. Chromatin immunoprecipitation analysis indicates that both Jab1 and E2F1 co-occupy apoptotic and mitotic, but not DNA replication target genes. We explored a potential connection between PI3K activity and Jab1/E2F1 target gene induction, and found that E2F1/Jab1 co-induction of apoptotic target genes can be inhibited by activated PI3K. Furthermore, PI3K activity interferes with formation of the E2F1/Jab1 complex by co-immunoprecipitation. Jab1/CSN5 is upregulated in a variety of human tumors, but it's unclear how its pro-proliferatory and apoptotic functions are regulated in this context. We explored the link between increased Jab1 levels and PI3K function in tumors and detected a highly significant correlation between elevated Jab1/CSN5 levels and PI3K activity in breast, ovarian, lung and prostate cancers.

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Figures

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Figure 1. Identification of E2F1 target genes requiring Jab1/CSN5 for expression. Control and shJab1 REF52 cells were infected with equal multiplicities of infection (25 min.o.i.) of control or E2F1-expressing adenovirus. RNA was harvested from infected cells and analyzed by rat Affymetrix arrays (RAE230A). E2F1 target genes induced at least 2-fold were sorted based on the effect that loss of Jab1/CSN5 had on E2F1-dependent expression. FI refers to “fold-induction” by E2F1 compared with CMV infection in control REF52 cells. shJAB1 is the percent expression of the gene in shJAB1 REF52 cells compared with E2F1-induction of the gene in control cells.
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Figure 2. Depletion of Jab1/CSN5 impairs E2F1 dependent expression of mitotic and apoptotic but not replication targets. mRNA expression levels of PRKAA2 (AMPK>2), p19ARF, Cdc2, Cyclin B2 (CCNB2), Cyp26b1, RFC4, Dut and PCNA were measured using quantitative real-time PCR in control vs. shJAB1 REF52 cells. Expression levels were normalized to GAPDH levels and displayed as % expression in control REF52 cells compared with E2F1-dependent expression in shJAB1 cells.
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Figure 3. E2F1 and Jab1/CSN5 co-occupy mitotic and apoptotic target but not DNA replication gene promoters. E2F1 and Jab1 binding to E2F1 target gene promoters was assessed using chromatin IP (ChIP). REF52 cells were cross-linked with formaldehyde, chromatin was isolated and sonicated and immunoprecipitated with anti-E2F1 (A) or anti-Jab1/CSN5 (B) antisera. Precipitated DNA was analyzed by SYBR green real-time PCR using primers and results are displayed as a comparative fold-induced binding from precipitate using no antibody vs. experimental antibody. Specific promoters analyzed are listed below each graph. ChIP results are separated into two parts in section A because of the very different scales for the two classes of genes. (C) Serum deprived quiescent REF52 cells were transfected with E2F1 or vector control. Chromatin was isolated and immunoprecipitated with anti-E2F1 or anti-Jab1 antisera.
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Figure 4. PI3K counter-regulates Jab1 induction of E2F1 apoptotic target genes. (A) Microarray data sets from this study (shJab1) and a previous study looking at Serum/PI3K repression of E2F1 target genes were combined, normalized using distance weighted discrimination (DWD), and subjected to unsupervised hierarchical clustering. Clusters were visualized using Java TreeView. Red shading represents relatively higher gene expression, and green shading corresponds to relatively lower gene expression. Only expression arrays with E2F1 added are included, to better display reductions in gene expression caused by the shJAB1 or Serum/PI3K lanes. (B) PI3K activity blocks Jab1/E2F1 target gene induction. Quiescent REF52 cells were infected with control, E2F1, Jab1 and PI3K expressing adenovirus, mRNA was isolated 24 h post-infection and used for real-time PCR analysis looking at p19ARF and Cyp26b1 gene induction. Results are reported as fold-inductions compared with gene expression in control (CMV) infected cells.
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Figure 5. PI3K inhibition promotes Jab1 binding to E2F1. PI3K inhibition promotes Jab1/E2F1 complex formation. REF52 cells were infected with Ad-Jab1 (100 min.o.i.) and treated with vehicle or the PI3K inhibitor LY294002. Protein lysates were isolated and endogenous E2F1 was immunoprecipitated with anti-E2F1 antisera and immunoblotted with anti-Jab1 antisera. Anti-Jab1 and -Actin were used to confirm equal protein input levels.
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Figure 6. Increased Jab1/CSN5 levels in various tumor types correlates with elevated PI3K activity. Publically available data sets for different tumor types were downloaded from the NCBI website. Tumor data sets were sorted based on increasing Jab1/CSN5 mas5 levels. The x-axis are individual tumors, and the y-axis is relative Jab1/CSN5 mas5 levels in each tumor. A “score” for putative PI3K activity based on gene expression patterns was derived from binary regression analysis in MATLAB. PI3K scores are displayed as a color bar with blue representing low activity and red, high activity. Correlations between PI3K score and Jab1/CSN5 levels were determined by linear regression analysis using GraphPad software.
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References

    1. Müller H, Bracken AP, Vernell R, Moroni MC, Christians F, Grassilli E, Prosperini E, Vigo E, Oliner JD, Helin K. E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes Dev. 2001;15:267–85. doi: 10.1101/gad.864201. - DOI - PMC - PubMed
    1. Ishida S, Huang E, Zuzan H, Spang R, Leone G, West M, Nevins JR. Role for E2F in control of both DNA replication and mitotic functions as revealed from DNA microarray analysis. Mol Cell Biol. 2001;21:4684–99. doi: 10.1128/MCB.21.14.4684-4699.2001. - DOI - PMC - PubMed
    1. van den Heuvel S, Dyson NJ. Conserved functions of the pRB and E2F families. Nat Rev Mol Cell Biol. 2008;9:713–24. doi: 10.1038/nrm2469. - DOI - PubMed
    1. Zhang HS, Gavin M, Dahiya A, Postigo AA, Ma D, Luo RX, Harbour JW, Dean DC. Exit from G1 and S phase of the cell cycle is regulated by repressor complexes containing HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF. Cell. 2000;101:79–89. doi: 10.1016/S0092-8674(00)80625-X. - DOI - PubMed
    1. Wu X, Levine AJ. p53 and E2F-1 cooperate to mediate apoptosis. Proc Natl Acad Sci U S A. 1994;91:3602–6. doi: 10.1073/pnas.91.9.3602. - DOI - PMC - PubMed

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