The BMAL1 C terminus regulates the circadian transcription feedback loop

The circadian clock is driven by cell-autonomous transcription/translation feedback loops. The BMAL1 transcription factor is an indispensable component of the positive arm of this molecular oscillator in mammals. Here, we present a molecular genetic screening assay for mutant circadian clock proteins that is based on real-time circadian rhythm monitoring in cultured fibroblasts. By using this assay, we identified a domain in the extreme C terminus of BMAL1 that plays an essential role in the rhythmic control of E-box-mediated circadian transcription. Remarkably, the last 43 aa of BMAL1 are required for transcriptional activation, as well as for association with the circadian transcriptional repressor CRYPTOCHROME 1 (CRY1), depending on the coexistence of CLOCK protein. C-terminally truncated BMAL1 mutant proteins still associate with mPER2 (another protein of the negative feedback loop), suggesting that an additional repression mechanism may converge on the N terminus. Taken together, these results suggest that the C-terminal region of BMAL1 is involved in determining the balance between circadian transcriptional activation and suppression

The cis-Acting Family of Repeats Can Inhibit as well as Stimulate Establishment of an oriP Replicon

Previously we have shown that the establishment of an oriP replicon is dependent on its epigenetic modification, which occurs in only 1 to 10% of proliferating cells (E. R. Leight and B. Sugden, Mol. Cell. Biol. 21:4149–4161, 2001). To gain insights into the cis-acting requirements for the establishment of oriP replicons, we monitored the replication of oriP plasmid derivatives for several weeks following their introduction into cells. In EBNA-1-positive 143B and H1299 cells, plasmids containing only the region of dyad symmetry (DS) of oriP replicated but were lost more rapidly from cells than were oriP plasmids, demonstrating that the family of repeats (FR) of oriP acts in cis to stimulate replication in these cells. Unexpectedly, we found that the DS plasmid was established efficiently in 293/EBNA-1 cells, being lost at a rate of only 8% per cell generation over 24 days posttransfection. However, plasmids containing the FR in addition to the DS of oriP replicated but were lost at a rate of approximately 30% per cell generation in 293/EBNA-1 cells, indicating that the FR inhibits oriP's establishment in this cell line. FR's enhancement of transcription of a promoter in cis and FR's ability to inhibit replication fork movement do not account solely for oriP's inefficient establishment. In addition, DNA looping between FR and DS neither stimulates nor inhibits replication. Deletion of 11 EBNA-1 binding sites in the FR or replacement of the FR with DS sequences, however, does overcome the inhibitory activity of the FR, thereby allowing efficient establishment of the oriP derivative in 293/EBNA-1 cells