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Magel2, a Prader-Willi syndrome candidate gene, modulates the activities of circadian rhythm proteins in cultured cells.

Devos J, Weselake SV, Wevrick R - J Circadian Rhythms (2011)

Bottom Line: Magel2 interacts with Bmal1 and with Per2 as measured by co-immunoprecipitation in co-transfected cells, and exhibits a subcellular distribution consistent with these interactions when visualized by immunofluorescence.As well, Magel2 induces the redistribution of the subcellular localization of Clock towards the cytoplasm, in contrast to the nucleus-directed effect of Bmal1 on Clock subcellular localization.Consistent with the blunted circadian rhythm observed in Magel2- mice, these data suggest that Magel2 normally promotes negative feedback regulation of the cellular circadian cycle, through interactions with key core circadian rhythm proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, University of Alberta, Edmonton, AB Canada T6G 2H7. rachel.wevrick@ualberta.ca.

ABSTRACT

Background: The Magel2 gene is most highly expressed in the suprachiasmatic nucleus of the hypothalamus, where its expression cycles in a circadian pattern comparable to that of clock-controlled genes. Mice lacking the Magel2 gene have hypothalamic dysfunction, including circadian defects that include reduced and fragmented total activity, excessive activity during the subjective day, but they have a normal circadian period. Magel2 is a member of the MAGE family of proteins that have various roles in cellular function, but the specific function of Magel2 is unknown.

Methods: We used a variety of cell-based assays to determine whether Magel2 modifies the properties of core circadian rhythm proteins.

Results: Magel2 represses the activity of the Clock:Bmal1 heterodimer in a Per2-luciferase assay. Magel2 interacts with Bmal1 and with Per2 as measured by co-immunoprecipitation in co-transfected cells, and exhibits a subcellular distribution consistent with these interactions when visualized by immunofluorescence. As well, Magel2 induces the redistribution of the subcellular localization of Clock towards the cytoplasm, in contrast to the nucleus-directed effect of Bmal1 on Clock subcellular localization.

Conclusion: Consistent with the blunted circadian rhythm observed in Magel2- mice, these data suggest that Magel2 normally promotes negative feedback regulation of the cellular circadian cycle, through interactions with key core circadian rhythm proteins.

No MeSH data available.


Related in: MedlinePlus

Subcellular localization of circadian proteins in the presence of Magel2. pXpressMagel2 and plasmids encoding other epitope-tagged circadian proteins (A, Clock, B, Bmal1, C, Per2, and D, Cry1) were detected in co-transfected NIH3T3 cells by immunofluorescence. Plots of fluorescence intensity were generated to assess overlap between the immunofluorescence signals from each protein within transfected cells, which were also stained with Hoechst 33342 to label the nucleus.
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Figure 3: Subcellular localization of circadian proteins in the presence of Magel2. pXpressMagel2 and plasmids encoding other epitope-tagged circadian proteins (A, Clock, B, Bmal1, C, Per2, and D, Cry1) were detected in co-transfected NIH3T3 cells by immunofluorescence. Plots of fluorescence intensity were generated to assess overlap between the immunofluorescence signals from each protein within transfected cells, which were also stained with Hoechst 33342 to label the nucleus.

Mentions: The co-immunoprecipitation experiments show that Magel2 can physically interact with Bmal1, and likely also with Per2. We next examined whether Magel2 co-localized with either protein in transfected cells, by immunofluorescence (Figure 3). We acquired fluorescence images of co-transfected individual cells, using Hoechst staining to visualize the nucleus. We then converted the images to RGB stacks and plotted the fluorescence intensity in lines drawn across individual cells. Fluorescence intensities were acquired in three channels, plotted, and visually examined for overlap in the profiles. The profile of Magel2 fluorescence most resembled that of Clock, with overlapping signals in the cytoplasm in most cells, but also in the nucleus in some cells (e.g. right hand profile in Figure 3A, Magel2/Clock). In about half the cells in which Magel2 and Bmal1 were co-expressed, the fluorescence overlapped in the cytoplasm but overlap was not seen in the nucleus (Figure 3B). We also detected overlapping profiles for Magel2 and Per2, in both the nucleus and the cytoplasm, in some but not all cells (Figure 3C). Lastly, there was no evidence of overlapping profiles in cells co-transfected with Magel2 and Cry1 (Figure 3D).


Magel2, a Prader-Willi syndrome candidate gene, modulates the activities of circadian rhythm proteins in cultured cells.

Devos J, Weselake SV, Wevrick R - J Circadian Rhythms (2011)

Subcellular localization of circadian proteins in the presence of Magel2. pXpressMagel2 and plasmids encoding other epitope-tagged circadian proteins (A, Clock, B, Bmal1, C, Per2, and D, Cry1) were detected in co-transfected NIH3T3 cells by immunofluorescence. Plots of fluorescence intensity were generated to assess overlap between the immunofluorescence signals from each protein within transfected cells, which were also stained with Hoechst 33342 to label the nucleus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3278377&req=5

Figure 3: Subcellular localization of circadian proteins in the presence of Magel2. pXpressMagel2 and plasmids encoding other epitope-tagged circadian proteins (A, Clock, B, Bmal1, C, Per2, and D, Cry1) were detected in co-transfected NIH3T3 cells by immunofluorescence. Plots of fluorescence intensity were generated to assess overlap between the immunofluorescence signals from each protein within transfected cells, which were also stained with Hoechst 33342 to label the nucleus.
Mentions: The co-immunoprecipitation experiments show that Magel2 can physically interact with Bmal1, and likely also with Per2. We next examined whether Magel2 co-localized with either protein in transfected cells, by immunofluorescence (Figure 3). We acquired fluorescence images of co-transfected individual cells, using Hoechst staining to visualize the nucleus. We then converted the images to RGB stacks and plotted the fluorescence intensity in lines drawn across individual cells. Fluorescence intensities were acquired in three channels, plotted, and visually examined for overlap in the profiles. The profile of Magel2 fluorescence most resembled that of Clock, with overlapping signals in the cytoplasm in most cells, but also in the nucleus in some cells (e.g. right hand profile in Figure 3A, Magel2/Clock). In about half the cells in which Magel2 and Bmal1 were co-expressed, the fluorescence overlapped in the cytoplasm but overlap was not seen in the nucleus (Figure 3B). We also detected overlapping profiles for Magel2 and Per2, in both the nucleus and the cytoplasm, in some but not all cells (Figure 3C). Lastly, there was no evidence of overlapping profiles in cells co-transfected with Magel2 and Cry1 (Figure 3D).

Bottom Line: Magel2 interacts with Bmal1 and with Per2 as measured by co-immunoprecipitation in co-transfected cells, and exhibits a subcellular distribution consistent with these interactions when visualized by immunofluorescence.As well, Magel2 induces the redistribution of the subcellular localization of Clock towards the cytoplasm, in contrast to the nucleus-directed effect of Bmal1 on Clock subcellular localization.Consistent with the blunted circadian rhythm observed in Magel2- mice, these data suggest that Magel2 normally promotes negative feedback regulation of the cellular circadian cycle, through interactions with key core circadian rhythm proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, University of Alberta, Edmonton, AB Canada T6G 2H7. rachel.wevrick@ualberta.ca.

ABSTRACT

Background: The Magel2 gene is most highly expressed in the suprachiasmatic nucleus of the hypothalamus, where its expression cycles in a circadian pattern comparable to that of clock-controlled genes. Mice lacking the Magel2 gene have hypothalamic dysfunction, including circadian defects that include reduced and fragmented total activity, excessive activity during the subjective day, but they have a normal circadian period. Magel2 is a member of the MAGE family of proteins that have various roles in cellular function, but the specific function of Magel2 is unknown.

Methods: We used a variety of cell-based assays to determine whether Magel2 modifies the properties of core circadian rhythm proteins.

Results: Magel2 represses the activity of the Clock:Bmal1 heterodimer in a Per2-luciferase assay. Magel2 interacts with Bmal1 and with Per2 as measured by co-immunoprecipitation in co-transfected cells, and exhibits a subcellular distribution consistent with these interactions when visualized by immunofluorescence. As well, Magel2 induces the redistribution of the subcellular localization of Clock towards the cytoplasm, in contrast to the nucleus-directed effect of Bmal1 on Clock subcellular localization.

Conclusion: Consistent with the blunted circadian rhythm observed in Magel2- mice, these data suggest that Magel2 normally promotes negative feedback regulation of the cellular circadian cycle, through interactions with key core circadian rhythm proteins.

No MeSH data available.


Related in: MedlinePlus