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Sumoylation of Hes6 Regulates Protein Degradation and Hes1-Mediated Transcription.

Lee J, Chun SK, Son GH, Kim K - Endocrinol Metab (Seoul) (2015)

Bottom Line: Protein stability of Hes6 was decreased by sumoylation.In addition, sumoylation was associated with both the rhythmic expression and transcriptional regulation of Hes6.These results suggest that sumoylation may be crucial for rhythmic expression of Hes6 and downstream target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Brain and Cognitive Sciences, Seoul National University School of Biological Sciences, Seoul, Korea.

ABSTRACT

Background: Hes6 is a transcriptional regulator that induces transcriptional activation by binding to transcription repressor Hes1 and suppressing its activity. Hes6 is controlled by the ubiquitin-proteosome-mediated degradation system. Here we investigated the sumoylation of Hes6 and its functional role in its rhythmic expression.

Methods: Hes6, SUMO, and ubiquitin were transfected into HeLa cells and the expression pattern was observed by Western blot and immunoprecipitation. To confirm the effect of sumoylation on the rhythmic expression of Hes6, we generated mouse Hes6 promoter-driven GFP-Hes6 fusion constructs and expressed these constructs in NIH 3T3 cells.

Results: Overexpression of SUMO led to sumoylation of Hes6 at both lysine 27 and 30. Protein stability of Hes6 was decreased by sumoylation. Moreover, expression of a Hes6 sumoylation-defective mutant, the 2KR (K27/30R) mutant, or co-expression of SUMO protease SUSP1 with native Hes6, strongly reduced ubiquitination. In addition, sumoylation was associated with both the rhythmic expression and transcriptional regulation of Hes6. Wild type Hes6 showed oscillatory expression with about 2-hour periodicity, whereas the 2KR mutant displayed a longer period. Furthermore, sumoylation of Hes6 derepressed Hes1-induced transcriptional repression.

Conclusion: Hes6 sumoylation plays an important role in the regulation of its stability and Hes1-mediated transcription. These results suggest that sumoylation may be crucial for rhythmic expression of Hes6 and downstream target genes.

No MeSH data available.


Hes6 is sumoylated at lysine residues 27 and 30. (A) Proteins were extracted from HeLa cells transfected with Hes6 constructs encoding wild type (WT) or its Lys-Arg mutants. Western blot was performed with the indicated antibodies. (B) Cells were transfected with Hes6 constructs encoding WT, the single mutants (K27R or K30R), or the 2KR (K27/30R) double mutant together with SUMO and Ubc9 and analyzed by Western blot using anti-Hes6 antibodies.
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Figure 2: Hes6 is sumoylated at lysine residues 27 and 30. (A) Proteins were extracted from HeLa cells transfected with Hes6 constructs encoding wild type (WT) or its Lys-Arg mutants. Western blot was performed with the indicated antibodies. (B) Cells were transfected with Hes6 constructs encoding WT, the single mutants (K27R or K30R), or the 2KR (K27/30R) double mutant together with SUMO and Ubc9 and analyzed by Western blot using anti-Hes6 antibodies.

Mentions: Next, to determine which lysine residues of Hes6 are targets of sumoylation, we next examined which lysine residues of Hes6 are targets for sumoylation. Within the bHLH domain of Hes6, there were five lysine residues positioned at K27, K30, K35, K36, and K60. We generated point mutants of Hes6 with arginine substituted for lysine residues. As shown in Fig. 2A, sumoylation of K27R or K30R single mutant was reduced in comparison with wild type Hes6, while the other mutants (K35R, K36R, or K60R mutants) did not show any decrease in sumoylation. Moreover, Hes6 mutations at both K27R and K30R (2KR mutant) completely abolished sumoylation (Fig. 2B). These data indicate that both lysine 27 and 30 of Hes6 are the target sites for sumoylation.


Sumoylation of Hes6 Regulates Protein Degradation and Hes1-Mediated Transcription.

Lee J, Chun SK, Son GH, Kim K - Endocrinol Metab (Seoul) (2015)

Hes6 is sumoylated at lysine residues 27 and 30. (A) Proteins were extracted from HeLa cells transfected with Hes6 constructs encoding wild type (WT) or its Lys-Arg mutants. Western blot was performed with the indicated antibodies. (B) Cells were transfected with Hes6 constructs encoding WT, the single mutants (K27R or K30R), or the 2KR (K27/30R) double mutant together with SUMO and Ubc9 and analyzed by Western blot using anti-Hes6 antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4595364&req=5

Figure 2: Hes6 is sumoylated at lysine residues 27 and 30. (A) Proteins were extracted from HeLa cells transfected with Hes6 constructs encoding wild type (WT) or its Lys-Arg mutants. Western blot was performed with the indicated antibodies. (B) Cells were transfected with Hes6 constructs encoding WT, the single mutants (K27R or K30R), or the 2KR (K27/30R) double mutant together with SUMO and Ubc9 and analyzed by Western blot using anti-Hes6 antibodies.
Mentions: Next, to determine which lysine residues of Hes6 are targets of sumoylation, we next examined which lysine residues of Hes6 are targets for sumoylation. Within the bHLH domain of Hes6, there were five lysine residues positioned at K27, K30, K35, K36, and K60. We generated point mutants of Hes6 with arginine substituted for lysine residues. As shown in Fig. 2A, sumoylation of K27R or K30R single mutant was reduced in comparison with wild type Hes6, while the other mutants (K35R, K36R, or K60R mutants) did not show any decrease in sumoylation. Moreover, Hes6 mutations at both K27R and K30R (2KR mutant) completely abolished sumoylation (Fig. 2B). These data indicate that both lysine 27 and 30 of Hes6 are the target sites for sumoylation.

Bottom Line: Protein stability of Hes6 was decreased by sumoylation.In addition, sumoylation was associated with both the rhythmic expression and transcriptional regulation of Hes6.These results suggest that sumoylation may be crucial for rhythmic expression of Hes6 and downstream target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Brain and Cognitive Sciences, Seoul National University School of Biological Sciences, Seoul, Korea.

ABSTRACT

Background: Hes6 is a transcriptional regulator that induces transcriptional activation by binding to transcription repressor Hes1 and suppressing its activity. Hes6 is controlled by the ubiquitin-proteosome-mediated degradation system. Here we investigated the sumoylation of Hes6 and its functional role in its rhythmic expression.

Methods: Hes6, SUMO, and ubiquitin were transfected into HeLa cells and the expression pattern was observed by Western blot and immunoprecipitation. To confirm the effect of sumoylation on the rhythmic expression of Hes6, we generated mouse Hes6 promoter-driven GFP-Hes6 fusion constructs and expressed these constructs in NIH 3T3 cells.

Results: Overexpression of SUMO led to sumoylation of Hes6 at both lysine 27 and 30. Protein stability of Hes6 was decreased by sumoylation. Moreover, expression of a Hes6 sumoylation-defective mutant, the 2KR (K27/30R) mutant, or co-expression of SUMO protease SUSP1 with native Hes6, strongly reduced ubiquitination. In addition, sumoylation was associated with both the rhythmic expression and transcriptional regulation of Hes6. Wild type Hes6 showed oscillatory expression with about 2-hour periodicity, whereas the 2KR mutant displayed a longer period. Furthermore, sumoylation of Hes6 derepressed Hes1-induced transcriptional repression.

Conclusion: Hes6 sumoylation plays an important role in the regulation of its stability and Hes1-mediated transcription. These results suggest that sumoylation may be crucial for rhythmic expression of Hes6 and downstream target genes.

No MeSH data available.