Endocrinol Metab.  2015 Sep;30(3):381-388. 10.3803/EnM.2015.30.3.381.

Sumoylation of Hes6 Regulates Protein Degradation and Hes1-Mediated Transcription

• Lee J
• Chun SK
• Son GH
• Kim K

• KMID: 2407090
• DOI: http://doi.org/10.3803/EnM.2015.30.3.381

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.

Keyword

Hes6; Sumoylation; Ubiquitination; Degradation; Rhythmic expression

MeSH Terms

Animals
Blotting, Western
HeLa Cells
Humans
Immunoprecipitation
Lysine
Mice
NIH 3T3 Cells
Periodicity
Protein Stability
Proteolysis*
Repression, Psychology
Sumoylation*
Transcriptional Activation
Ubiquitin
Ubiquitination
Lysine
Ubiquitin