Abstract
NF-kappaB family members play a pivotal role in many cellular and organismal functions, including the cell cycle. As an activator of cyclin D1 and p21(Waf1) genes, NF-kappaB has been regarded as a critical modulator of cell cycle. To study the involvement of NF-kappaB in G(1)/S phase regulation, the levels of selected transcriptional regulators were monitored following overexpression of NF-kappaB or its physiological induction by tumor necrosis factor-alpha. Cyclin E gene was identified as a major transcriptional target of NF-kappaB. Recruitment of NF-kappaB to the cyclin E promoter was correlated with the transrepression of cyclin E gene. Ligation-mediated PCR and micrococcal nuclease-Southern assays suggested the nucleosomal nature of this region while chromatin immunoprecipitation analysis confirmed the exchange of cofactors following tumor necrosis factor-alpha treatment or release from serum starvation. There was a progressive reduction in cyclin E transcription along with the accumulation of catalytically inactive cyclin E-cdk2 complexes and arrest of cells in G(1)/S-phase. Thus, our study clearly establishes NF-kappaB as a negative regulator of cell cycle through transcriptional repression of cyclin E.
Highlights
Genetic Engineering and Biotechnology, New Delhi. □S The on-line version of this article contains supplemental Figs
The expression of cyclin E gene is under the control of an autonomous mechanism that peaks around the G1/S phase boundary [10, 11]
We report that multiple NF-B binding sites present in the promoter region of cyclin E gene are associated with transcriptional repression without involving a chromatin remodeling
Summary
Expression Vectors and Reporter DNA Constructs—Description of different expression vectors can be found elsewhere: pCMV-E2F1 and pCMV-E2F1⌬C [1–374] [28]; HA-p65 and HA-p50 [29]; p65K218A, p65K222A, and p65K310A [30]; IB␣ [31]; and human retinoblastoma, pRB [32]. Nuclear extracts (10 g of protein) from HEK293 cells were incubated on ice for 60 min with 50 fmol of 32Plabeled EMSA probes (Table 2). Samples were pre-cleared for 2 h with protein A-Sepharose beads (Amersham Biosciences) and incubated overnight with 2 g/ml specific antibodies (p50, p52, p65, p300, E2F1, HDAC1, c-Rel, RelB, Ac-H3-K9, and Ac-H4-K12). Nuclei were isolated from HEK293 cells (108 cells) and incubated in 100-l aliquots at 25 °C for 5 min with MNase Nuclei were prepared as described in MNase-Southern assay and incubated with the restriction enzymes (BstEII, EcoNI, and NdeI) at 37 °C for different time periods. DNA was extracted by phenol-chloroform method, and the cyclin E promoter region was amplified using cyclin E-NF-B ChIP-PCR primer (Table 1). Bioinformatic Analysis—The TFSEARCH program owed to the TRANSFAC data base was used to predict transcription factor binding sites on human cyclin E promoter sequence (TFSEARCH is available on-line) [43]
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