CRISPR/Cas9 genomic editing was utilized to delete the first intronic region containing six highly conserved GLl1 binding sites. The decreased GLl1 gene expression impacted GLl1 regulatory capacity within the Sonic Hedgehog (SHH) signaling pathway. The result was a reduced transcriptional activation of upstream and downstream GLl1 targets, which ultimately impacted endodermal, mesodermal, and ectodermal differentiation via down‐regulation of key genes.
Abstract
GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1‐edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells.
The roles of GLI1 in human ESC differentiation were investigated by comparing RNA‐Seq, q‐rtPCR, and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer‐related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT‐61 had similar in‐vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage‐specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.
© AlphaMed Press 2021
Significance Statement
Using the CRISPR/Cas9 strategy, we edited out a regulatory region of the first intron of the GLI1 gene in a human embryonic stem cell model. This region contains six highly conserved GLI1 binding sites (GBS). The editing significantly lowered GLI1 transcription in the heterozygous state and reduced GLI1 expression to barely detectable levels in the homozygous state. Such alteration in GLI1 expression significantly decreased ectodermal, mesodermal, and endodermal marker expression. This resulted in significant differentiation defects: a reduction in blast colonies and hematopoietic potential, a delay in the onset of early osteogenic markers, delayed neural differentiation, and a reduction in cancer‐related gene expression.