Abstract
To understand the role of the CCAAT binding factor (CBF) in transcription during the cell cycle, we studied the mouse topoisomerase II alpha (topo II alpha) promoter, which is activated during the late S and G(2)/M phases of the cell cycle and contains multiple CBF binding sites. Mutational analysis of the promoter shows that CBF binding to an inverted orientation of the CCAAT motif in the topo II alpha promoter, but not to a direct orientation, is required for transcription activation during the cell cycle. In contrast, analysis of the promoter in an in vitro reconstituted transcription system shows that CBF activates transcription of the topo II alpha promoter irrespective of the orientation of the CBF binding sites. This analysis demonstrates that only one of the three transcription start sites of the topo II alpha promoter is stimulated by CBF, indicating that transcription activation by CBF is dependent on basal promoter structure. Interestingly, mutations of the start site that abolish CBF-dependent transcription activation in vitro do not inhibit activation of the promoter during the cell cycle. Consistent with this observation, expression of a truncated CBF-B subunit lacking a transcription activation domain, which inhibits activity of a collagen promoter, does not affect activity of the topo II alpha promoter in fibroblast cells. In contrast, expression of an allele-specific CBF-B mutant that binds high affinity to a mutant CBF binding site containing a CCAAC motif revives transcription activation of an inactive mutant topo II alpha promoter containing CCAAC during the cell cycle. Altogether, this study indicates that CBF binding, but not conventional CBF activation domains, are required for activation of the topo II alpha promoter during the cell cycle. Considering these results together with results of another recent study, we hypothesize that binding of CBF that disrupts the nucleosomal structure in the topo II alpha promoter is a major function of CBF by which it regulates the cell cycle-dependent transcription of the topo II alpha promoter and possibly many other cell cycle-regulated promoters containing CBF binding sites.
Highlights
From the Department of Molecular Genetics, The University of Texas, M
The addition of the third form of CCAAT binding factor (CBF) containing both truncated CBF-B and CBF-C resulted in no activation from start site II. These results indicate that the two glutamine-rich activation domains of CBF-B and CBF-C subunits mediate transcription activation of the topo
The fact that a collagen promoter containing multiple CBF binding sites in the inverted orientation is not activated during the cell cycle is strongly indicative that the role of CBF in transcription activation during the cell cycle is specific to the topoisomerase II (topo II)␣ promoter
Summary
In a recent study [7], we showed that the mammalian heterotrimeric CCAAT binding factor (CBF) regulates expression of the cellular topo II␣ gene as well as transcription activity of the topo II␣ promoter. Because the promoters of various growth-regulated genes, such as topo II␣, cyclin B1, CDC25C, E2F1, and thymidine kinase, are activated at different stages of the cell cycle and contain multiple CBF binding sites (8 –12), we hypothesized that these promoters are highly dependent on CBF activity in vivo. One paradox of this hypothesis is that the DNA binding activity of CBF is unchanged during the cell cycle; it remains unclear how constitutively expressed CBF is involved in regulating transcription of various promoters in specific cell-cycle stages
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