Runx1 shapes the chromatin landscape via a cascade of direct and indirect targets

Runt-related transcription factor 1 (Runx1) can act as both an activator and a repressor. Here we show that CRISPR-mediated deletion of Runx1 in mouse metanephric mesenchyme-derived mK4 cells results in large-scale genome-wide changes to chromatin accessibility and gene expression. Open chromatin regions near down-regulated loci enriched for Runx sites in mK4 cells lose chromatin accessibility in Runx1 knockout cells, despite remaining Runx2-bound. Unexpectedly, regions near upregulated genes are depleted of Runx sites and are instead enriched for Zeb transcription factor binding sites. Re-expressing Zeb2 in Runx1 knockout cells restores suppression, and CRISPR mediated deletion of Zeb1 and Zeb2 phenocopies the gained expression and chromatin accessibility changes seen in Runx1KO due in part to subsequent activation of factors like Grhl2. These data confirm that Runx1 activity is uniquely needed to maintain open chromatin at many loci, and demonstrate that Zeb proteins are required and sufficient to maintain Runx1-dependent genome-scale repression. Author summary Runt-related transcription factor (Runx) 1 & 2 impact development and disease by activating or repressing transcription. In this manuscript we used genome editing tools to remove Runx1, and as expected, observed widespread changes in chromatin accessibility. Newly closed areas contained Runx1 binding sites and were enriched near genes whose expression depended on Runx1. Interestingly, this occurred despite continued binding of Runx2 to the same regions of DNA, which suggests that Runx2 is insufficient to maintain open chromatin and expression of Runx1 target genes in this cellular context. By contrast, newly opened chromatin regions, many near genes that were upregulated in Runx1 knockout cells, did not enrich for Runx1 binding sites. Instead, these regions were enriched for sites for the repressor Zeb proteins. We found that the loss of Zeb 1 & 2 expression, direct transcriptional targets of Runx1, resulted in the opening of chromatin and upregulation of genes residing near the newly open sites in Runx1 knockout cells. The same sites were also open and nearby genes expressed in edited Zeb1 and Zeb2 knockout cells. Among them were transcription factors, such as the Grhl2 gene, which in turn bind to and upregulate their target genes. Thus, the loss of a single transcription factor initiates a cascade of direct and indirect ramifications with likely negative effects on development and health.

Automated summary

The study demonstrates that Runx1 plays a unique role in maintaining open chromatin at many loci, while Zeb proteins are necessary and sufficient for the maintenance of Runx1-dependent genome-scale repression. This cascade of effects initiated by the loss of a single transcription factor may have negative consequences on development and health.