The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts
Fig 4
Conserved regions in the Pak1 promoter contain E2F binding sites.
(A) Alignment of a region of 1,000 bp (from -700 to +300 bp) of the mouse and human Pak1 promoters generated by the Data Base of Transcriptional Start Sites (DBTSS), showing 7 conserved regions labeled 0–6. The longest conserved stretch is region 0, which spans a 406-bp region from -186 to +220 with 66% identity. The table below the promoter diagram shows the start and stop positions for each conserved region in mouse (m) and human (h). (B) Schematic of the human and mouse Pak1 promoters showing 5 E2F1 binding sites (labeled as 1–3 in the human promoter and 4 and 5 in the mouse promoter), as identified by Genomatrix analysis. These E2F1 binding sites are in the conserved region (labeled as 0 in A). The table shows the positions, strand, and sequences of each E2F1 binding site, with the core nucleotides in each binding site indicated capitalized in bold. (C) A Pak1 mouse promoter-Firefly luciferase construct containing the 2 E2F binding sites was transfected into Rb+/+ and Rb-/- MC3T3 cells, and promoter activation was measured by its luciferase activity and normalized against a co-transfected Renilla luciferase construct. The Pak1 promoter region containing the E2F binding sites was amplified using the forward (F) and reverse (R) primers illustrated in (B) and in bold in S1 Fig. Transcriptional activity in Rb-/- cells was stronger than in Rb+/+ cells by a factor of 2.1 after normalization with Renilla luciferase, a value that is close to the 2.7-fold transcriptional induction that we observed in our nuclear run-on assays. *P < 0.05.