p16, also known as INK4a, CDKN2, or MTS1, plays an important role in the co
ntrol of the cell cycle progression, and retinoblastoma protein (pRb) is su
ggested to be involved in the transcriptional regulation of p16. However, i
t is not fully understood how pRb regulates transcription of the p16 gene.
Nuclear proteins prepared only from the pRb-nonfunctional human tumor cells
were found to bind to the 5'-flanking sequence of the p16 gene in the pres
ence of Zn2+ by electrophoretic mobility shift assay (EMSA). EMSA using mut
agenized 5'-flanking sequences as competitors suggested that the sequence a
t position -97 to -87 relative to first ATG of the p16 gene was critical fo
r protein binding. Transient reporter assay indicated that the sequence ide
ntified by EMSA acted as a silencer element in the pRb-nonfunctional tumor
cells, showing the presence of a transcriptional repressor associated with
functional pRb (RBAR1).