Analysis of Wdr68 function in the differentiation of a mouse myoblast cell line
Authors:Estibaliz Alvarado, Tatiana Vela
Mentor:Robert Nissen, Professor of Biological Sciences, CSULA
Skeletal muscle development requires sequential activation of a group of myogenic regulatory factors (MRFs) including MEF2. In the C2C12 mouse myoblast cells, differentiation is facilitated by a dual-specificity tyrosine-regulated kinase, Dyrk1b, and a WD40-repeat protein, Wdr68. This complex inhibits an inhibitor of MEF2 to allow myogenin (myog) expression. In zebrafish, Dyrk1b and Wdr68 are important for jaw development and heart laterality. The Dyrk1b-Wdr68 complex is also important for myog expression in differentiating C2C12 rat myoblast cells cells. In a dual luciferase assay, myog-luc expression was enhanced by a Wdr68 construct and blocked by a Nuclear Export Sequence fusion, suggesting that nuclear localization is required for Wdr68 function. A transcriptional repression domain fusion, MadFlagWdr68, was able to repress myog-luc activity, while an activation domain fusion increased it. MEKK1 inhibits C2C12 cell differentiation via p38gamma, a mitogen activated kinase. Physical complexes of MEKK1-Wdr68-Dyrk1b have been previously detected, suggesting Wdr68 may bridge MEKK1 signals to Dyrk1b during differentiation. However, co-expression of a dnMEKK1 construct did not alter the Wdr68-mediated increase in myog-luc activity; Wdr68's effect on myog-luc activity does not appear to be dependent on upstream MEKK1 function. Transcriptional co-regulators modulate promoter activities via indirect associations with promoter regions, and recent findings in plants suggest Wdr68 orthologs may similarly do so. We hypothesized a Gal4Wdr68 fusion could modulate a synthetic GAL4/UAS reporter in C2C12 cells. The Gal4Wdr68 fusion increased reporter activity 3-fold above that of Gal4DBD alone during cellular differentiation. Together, these findings add additional support to a model in which Wdr68 functions in the cell nucleus to enhance transcription of downstream target genes independent of MEKK1 activity.