Transcription factor HESX1 enhances mesendodermal commitment of human embryonic stem cells by modulating ERK1/2 signaling

Transcription factors are key determinants of lineage commitment during mammalian development. However, the function and molecular mechanism for the transcription factors in the formation of three primary germ layers during human embryonic development are not fully elucidated. Here, we report that homeobox-containing transcription factor HESX1 plays a critical role in mesendodermal (ME) commitment of human embryonic stem cells (hESCs). Our results show that expression of HESX1 in hESCs is regulated by OCT4 and NANOG, and that its expression level changes with hESC differentiation. We find that knockdown of HESX1 does not disrupt the undifferentiated state of hESCs, in terms of cell morphology and expression levels of pluripotency- associated genes. However, HESX1 deficiency in hESCs impairs their ME commitment, whereas forced expression of HESX1 significantly enhances ME marker expression during ME commitment. Interestingly, HESX1 knockdown in hESCs represses ERK1/2 signaling activated by ME induction, while overexpression of HESX1 markedly enhances ERK1/2 activity during ME commitment of hESCs. Of note, MEK inhibitor PD0325901 weakens or even eliminates HESX1 overexpression- mediated promotive effects on ME induction in a dosage- dependent manner. Together, this study identifies a novel role of HESX1 in hESC commitment to ME cells and establishes the functional link between a transcription factor and lineage- associated signaling. These findings would help to better understand early human development and develop more efficient protocols to induce hESC differentiation to desired lineages. Overall design: To investigate the role of HESX1 in hESC self-renewal and mesendodermal(ME) differentiation, we depleted HESX1 in hESCs by two small interfering RNAs (siRNAs) targeting different coding regions of HESX1. A non-targeting (NT) siRNA was used as a negative control. To determine whether HESX1 deficiency would alter hESC gene expression program, the RNA-seq experiment was performed for siNT(NT_mTe), siHESX1 #1(si5_mTe) and siHESX1 #2(si8_mTe) hESC samples, with each having two biological replicates. To determine whether HESX1 would play a role in ME lineage commitment, we applied a previously published protocol for efficient ME induction from hESCs. There were 4 groups of cells: siNT mTeSR1 cells, siNT ME(NT_ME) cells, siHESX1 #1 ME(si5_ME) and siHESX1 #2 ME(si8_ME) cells. We performed RNA-seq experiments for the 4 groups of cells described above with 2 biological replicates for each group.