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Pax6 localizes to chromatin-rich territories and displays a slow nuclear mobility altered by disease mutations

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Abstract

The transcription factor Pax6 is crucial for the embryogenesis of multiple organs, including the eyes, parts of the brain and the pancreas. Mutations in one allele of PAX6 lead to eye diseases including Peter's anomaly and aniridia. Here, we use fluorescence recovery after photobleaching to show that Pax6 and also other Pax family proteins display a strikingly low nuclear mobility compared to other transcriptional regulators. For Pax6, the slow mobility is largely due to the presence of two DNA-binding domains, but protein-protein interactions also contribute. Consistently, the subnuclear localization of Pax6 suggests that it interacts preferentially with chromatin-rich territories. Some aniridia-causing missense mutations in Pax6 have impaired DNA-binding affinity. Interestingly, when these mutants were analyzed by FRAP, they displayed a pronounced increased mobility compared to wild-type Pax6. Hence, our results support the conclusion that disease mutations result in proteins with impaired function because of altered DNA- and protein-interaction capabilities.

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Abbreviations

FRAP:

fluorescence recovery after photobleaching

PD:

paired domain

HD:

homeodomain

TAD:

transactivation domain

GFP:

green fluorescent protein

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Acknowledgments

We are grateful to Ernst Thomassen, Ingvild Mikkola, Endalkachew A. Alemu, J.Y. Thuret and J.A. Goldman for their generous gifts of the plasmid constructs used in this work. We thank the BioImaging FUGE core facility at IMB for use of instrumentation and expert assistance. We are indebted to Kenneth Bowitz Larsen for guidance on the use of confocal microscopy software. This study was supported by grants from the Norwegian Cancer Society and the Blix Foundation to TJ.

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  1. Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø, 9037, Tromso, Norway

    Julianne Elvenes, Eva Sjøttem, Turid Holm, Geir Bjørkøy & Terje Johansen

  2. University College of Sør-Trøndelag, 7006, Trondheim, Norway

    Geir Bjørkøy

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Supplementary material 1 (DOC 68.5 kb)

Supplementary figure S1

Confocal pictures of HeLa cells transiently expressing GFP, Cherry, GFP and Cherry, GFP and Cherry-Pax6, or GFP-Pax6 and Cherry. The pictures show that the punctuated nuclear pattern observed for Pax6 is not caused by the tags used. Subconfluent HeLa cells were transfected with the different constructs as described in the methods section. The following day, confocal images were taken with the cells kept in 1 × HBSS (Invitrogen) supplemented with 10% fetal calf serum. Scale bars: 10 μm. Supplementary material 2 (TIFF 2.18 mb)

Supplementary figure S2

a FRAP recovery curves for GFP-Pax6 in HeLa and U2OS cells. b FRAP recovery curves for zebrafish Pax6 with N-terminal GFP or C-terminal YFP tag. Changing the location of the tag does not alter the mobility significantly. c Transactivation assay with HeLa and U2OS cells transiently transfected with the Pax6 paired domain reporter P6xCON-luc and 50 to 150 ng of GFP-Pax6 and Pax6-GFP. pCMV-βgal was used to normalize the transfection efficiency, and pcDNA3 was used to equal the DNA concentration in each well. The fold activation is determined based on the background level obtained with pcDNA3 alone. The results are the average of three independent experiments, triplicates each time, with standard error bars included. Supplement to figure 2. Supplementary material 3 (TIFF 252 kb)

Supplementary figure S3

FRAP recovery curves for different N-terminal GFP-tagged transcriptional regulators compared with GFP-Pax6. Supplement to figure 3. Supplementary material 4 (TIFF 558 kb)

Supplementary figure S4

FRAP recovery curves for different N-terminal GFP-tagged Pax family members compared with GFP-Pax6. z, zebrafish; m, mouse; h, human. Supplement to figure 4. Supplementary material 5 (TIFF 551 kb)

Supplementary figure S5

FRAP recovery curves for different N-terminal GFP-tagged parts of Pax6 and the splice variant Pax6(5A) compared with full-length GFP-Pax6. PD, paired domain; HD, homeodomain; TAD, transactivation domain. Supplement to figure 5. Supplementary material 6 (TIFF 440 kb)

Supplementary figure S6

FRAP recovery curves for different N-terminal GFP-tagged point mutations in Pax6 compared with the wild-type protein. Supplement to figure 6. Supplementary material 7 (TIFF 725 kb)

Supplementary figure S7

Subnuclear localization of mutated versus wild-type GFP-tagged Pax6. Subconfluent HeLa cells were transfected with the different constructs as described in the methods section. The following day, confocal images were taken with the cells kept in 1 × HBSS (Invitrogen) supplemented with 10% fetal calf serum. Scale bars: 5 μm. Supplementary material 8 (TIFF 12.4 mb)

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Elvenes, J., Sjøttem, E., Holm, T. et al. Pax6 localizes to chromatin-rich territories and displays a slow nuclear mobility altered by disease mutations. Cell. Mol. Life Sci. 67, 4079–4094 (2010). https://doi.org/10.1007/s00018-010-0429-0

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