The retinoblastoma gene and its product are targeted by ICBP90: a key mechanism in the G1/S transition during the cell cycle.
The retinoblastoma protein (pRB) is encoded by the RB1 gene whose promoter contains several putative binding sites for ICBP90 (Inverted CCAAT box Binding Protein of 90 kDa), a transcriptional regulator of the topoisomerase IIalpha gene. ICBP90 has two consensus binding sites for pRB in its primary sequence. Here, we show that pRB and ICBP90 co-immunoprecipitate in cell extracts of proliferating human lung fibroblasts and of proliferating or confluent Jurkat cells. GST pull-down assays and immunocytochemistry, after cell synchronization in late G1 phase, confirmed this interaction. Overexpression of ICBP90 induces downregulation of pRB expression in lung fibroblasts as a result of mRNA decrease. DNA chromatin immunoprecipitation experiment shows that ICBP90 binds to the RB1 gene promoter under its methylated status. Overexpression of ICBP90 increases the S and G2/M phase cell fractions of serum-starved lung fibroblasts as assessed by flow cytometry analysis and increases topoisomerase IIalpha expression. Together, these results show that ICBP90 regulates pRB at the protein and gene transcription levels, thus favoring the entry into the S phase of the cells. We propose that ICBP90 overexpression, found in cancer cells, is involved in the altered checkpoint controls occurring in cancerogenesis.
Jeanblanc
Michaël
M
Inserm UMR S392, Faculté de Pharmacie, 74 route du Rhin, BP 60024, 67401 Illkirch Cedex, France.
Mousli
Marc
M
Hopfner
Raphaël
R
Bathami
Kawtar
K
Martinet
Nadine
N
Abbady
Abdul-Qader
AQ
Siffert
Jean-Claude
JC
Mathieu
Eric
E
Muller
Christian D
CD
Bronner
Christian
C
D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D004250:DNA Topoisomerases, Type II / Q000502:physiology; D015536:Down-Regulation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D016193:G1 Phase; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells / Q000166:cytology / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016160:Retinoblastoma Protein / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase; D044767:Ubiquitin-Protein Ligases
D022762:CCAAT-Enhancer-Binding Proteins; D016160:Retinoblastoma Protein; C406364:UHRF1 protein, human; D044767:Ubiquitin-Protein Ligases; D004250:DNA Topoisomerases, Type II