Abstract
The BMAL1 and CLOCK heterodimer in the mammalian circadian transcriptional complex is thought to be repressed by PER2 and CRY1 via direct interactions. We recently reported that PER2 is largely cytosolic in Pml−/− cells and did not co-immunoprecipitate (co-IP) with BMAL1 or CLOCK. Here, using multi-color immunofluorescence (IF) staining and co-IP, we observed a nuclear distribution of BMAL1 and a predominately cytosolic distribution of CLOCK in Pml−/− MEF. In the presence of WT PML, PER2 co-localized with BMAL1 in the nucleus. In Pml−/− MEF transfected with mutant K487R PML, we observed that BMAL1 and PER2 co-localized with K487R PML in the cytosol. Furthermore, cytosolic CLOCK and PER2 displayed a significant non-overlapping IF staining pattern. In Bmal1−/− MEF, CLOCK was primarily cytosolic while PML and PER2 were nuclear. Together, our studies suggest that PML mediates the binding of PER2 to BMAL1 in the BMAL1/CLOCK heterodimer and is an important component in the organization of a functional clock complex in the nucleus. Our studies also support that BMAL1 is important for CLOCK nuclear localization.
Highlights
Nuclear localization presented an opportunity to determine whether PER2 interaction with the BMAL1/CLOCK heterodimer complex is direct or occurs via an intermediary partner(s)
It is widely believed that PER2 regulates its own transcription via direct interaction with CRY1 to bind the BMAL1/CLOCK transcriptional heterodimer leading to repression of its promoter activity
Compelling evidence supporting this model has come from studies showing PER2 co-IPs with BMAL1 or CLOCK indicating that these proteins are in a complex
Summary
Nuclear localization presented an opportunity to determine whether PER2 interaction with the BMAL1/CLOCK heterodimer complex is direct or occurs via an intermediary partner(s). We observed that PML enhances BMAL1/CLOCK transcriptional activity only in the presence of PER28, suggesting that PML may be involved in PER2/BMAL1/ CLOCK complex formation. We investigate how PML organizes PER2/BMAL1/CLOCK complex formation. We undertook immunofluorescence (IF) and co-immunoprecipitation (co-IP) studies using Pml+/+ and Pml−/− cells to investigate the cellular distribution and protein-protein interactions of PER2 with BMAL1 and CLOCK. Our studies revealed that PML mediates the binding of PER2 to BMAL1 in the BMAL1/CLOCK heterodimer. Our studies revealed that BMAL1 but not CLOCK is pivotal for BMAL1/CLOCK heterodimer interaction with PML. We observed that the cellular distribution of PER2 and CLOCK but not BMAL1 is influenced by PML. Using Bmal1−/− MEF, we observed that nuclear distribution of CLOCK but not PML is influenced by BMAL1
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