This dissertation, "Transcriptional Regulation of P16INK4a Expression by the Forkhead Box Transcription Factor FOXM1" by Chi-yun, Johannes, Ching, 程子忻, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled INK4a "Transcriptional Regulation of p16 Expression by the Forkhead Box Transcription Factor FOXM1" Submitted by CHING Chi Yun Johannes for the degree of Master of Philosophy at The University of Hong Kong in August 2003 FOXM1 is a forkhead box transcription factor ubiquitously expressed in proliferating cells. It has been suggested to facilitate G2/M transition by up-regulating the expressions of mitotic genes. The effect of FOXM1 expression on G1/S transition is, however, uncertain. One model proposes that FOXM1 facilitates both G1/S and G2/M transitions. On the contrary, the other model suggests that FOXM1 suppresses G1/S transition and facilitates G2/M transition in order to ensure the coupling of S phase and M phase (S-M coupling). Recently, FOXM1 is shown to increase the protein INK4a INK4a and RNA levels of p16 . Whether or not the up-regulation of p16 expression is a direct transactivation effect of FOXM1 at the transcriptional level has not yet been INK4a known. As p16 is a cyclin-dependent kinase inhibitor that suppresses G1/S transition, FOXM1 may suppress G1/S transition through the modulation of p16 transcription in order to regulate S-M coupling. The objectives of this study are to INK4a study the transcriptional regulation of p16 by FOXM1 and to study the effect of FOXM1 over-expression on the cell cycle. By learning more about the effect of INK4a FOXM1 on p16 expression, the exact role FOXM1 plays in the regulation of cell cycle and S-M coupling will be better understood. In this research, reporter assay was used to study the effect of co-transfection INK4a of FOXM1 expression plasmids on the promoter activity of p16 . It was found that INK4a Cip1/Waf1 FOXM1 activated p16 promoter but not p21 promoter. Deletion analysis INK4a showed that the 5'-untranslated region of p16 was essential for the FOXM1-dependent promoter activation. A putative FOXM1 binding sequence was INK4a identified in the 5'-untranslated region of p16 . Site-directed mutagenesis and subsequent reporter assay showed that the mutation of the putative FOXM1 binding INK4a sequence in p16 promoter abolished the FOXM1-dependent promoter activation. INK4a Binding of FOXM1 to the p16 promoter was shown by chromatin immunoprecipitation assay. To study the cell cycle effect of FOXM1, especially on G1/S transition, hTERT-BJ1-derived cell lines that are 4-hydroxytamoxifen (4OHT)-inducible to express ectopic FOXM1 in nuclei were used. hTERT-BJ1-derived cell lines have intact cell cycle checkpoints, making them suitable for studying cell cycle and G1/S INK4a transition. Increase in p16 protein level was observed in the 4OHT-treated cells. Cell counting of actively dividing 4OHT-inducible cells revealed that the number of 4OHT-treated cells increased at a rate slower than that of untreated cells. Flow cytometric analysis was used to study the effect of nuclear expression of FOXM1 on the cell cycle distribution of synchronized 4OHT-inducible cells. It was found that G1/S transition was slowed down in 4OHT-treated cells. The decreased rate of cell proliferation and retarded G1/S transition observed in 4OHT-treated cells, which over-express FOXM1 in nuclei, is consistent with the proposed G1/S transition-suppressing role of FOXM1. This study, therefore, supports the model proposing that FOXM1 regulates S-M coupling by facilitating G2/M transition, an