Background:
We previously demonstrated that Sox17, an Sry-box-containing transcription factor that interacts with the Wnt/ β-catenin pathway, is essential for cardiac myogenesis in differentiating embryonic stem (ES) cells. Sox17 shRNA blocks cardiac myogenesis and selectively impairs the induction of Hex, a member of the homeobox family of transcription factors, many of which are involved in developmental processes. However, whether Hex is a direct target of Sox17 or not and the function of Hex in mouse ES cell differentiation to cardiomyocytes have not been defined.
Hypothesis:
Hex is a direct target of Sox17, and
Hex is essential for cardiac myogenesis in ES cells.
Methods:
Cells were subjected to lentiviral transduction for a chimeric protein, contains Sox17 fused to protein A, using a protocol that reconstructs the native Sox17 expression pattern. Protein A-TEV-tagged Chromatin Immuno-precipitation (PAT-ChIP) technology was used to purify the DNA fragments bound to Sox17. Chromatin-immunoprecipitated DNA was subjected to PCR for promoter regions of Hex that contain putative Sox-binding motifs. Lentiviral vectors encoding shRNAs that knock down Hex were transduced into AB2.2 cells. Transduced cells, distinguished by expression of EGFP, were flow-sorted and subjected to embryonic body (EB) culture. Total RNA was extracted from EBs at 10 time points. Real-time QRT-PCR was carried out for representative genes related to mesoderm formation, mesoderm patterning, and cardiac myogenesis. Spontaneously beating EBs were scored.
Results:
Several Hex promoter regions containing predicted Sox17 binding sites were confirmed as potential direct targets of Sox17. The prevalence of beating EBs and the expression of cardiogenic transcription factors (Nkx2.5, Tbx5, Mef2c, Gata4 and myocardin) and cardiac structural genes (Ryr2 and α-MHC) both were suppressed by Hex shRNA. Hex shRNA did not impair the progressive down-regulation of Sox2 and Oct4 (master regulators of pluripotency) or the induction of Brachyury/T and Mesp1/2 (markers of primitive and precardiac mesoderm, respectively).
Conclusion:
Hex is potentially a direct target of Sox17, and is essential for cardiac myogenesis in differentiating ES cells at the stage of cardiac specification.
Early incorporation of obscurin into nascent sarcomeres: implication for myofibril assembly during cardiac myogenesis
