Analyses of the Atoh1, Pou4f3 and Gfi1 genome binding profile during hair cell and neuronal differentiation

To gain new insights into the genetic networks regulating hair cell development, we developed a new transcriptional programming strategy to promote in vitro hair cell differentiation, starting from mouse embryonic stem cells (mESCs). In vivo, Atoh1 is the only transcription factor (TF) known to be necessary and sufficient for HC differentiation, but in vitro its overexpression induces neuronal rather than hair cell differentiation. We discovered that Atoh1 expression combined with two other TFs (Pou4f3, Gfi1) resulted in efficient hair cell generation. This work offers a new paradigm to understand the molecular mechanisms governing transcription factor specificity. For example, how do Pou4f3-Gfi1 modulate Atoh1 activity to orchestrate a HC differentiation program? To address this question, we have engineered several mESCs lines containing Atoh1, Gfi1 and/or Pou4f3 in a doxycycline-inducible locus to allow a controllable overexpression of every combination of these 3 transcriptional factors either using a polycistronic approach (mESCs lines: iG+A, iG+P, iP+A and iGPA) or by promoting their individual overexpression (mESCs lines: iAtoh1, iGfi1 and iPou4f3). These findings highlight the diversity of mechanisms by which one TF can redirect the activity of another to enable combinatorial control of cell identity. Overall design: 7 different doxycycline-inducible mESCs lines containing every combination of Atoh1, Gfi1 and Pou4f3 were analysed at day 6 of embryoid body differentiation. Doxycycline-inducible mESCs lines were labelled using the following criteria: 1) iAtoh1- Atoh1 overexpression only; 2) iGfi1- Gfi1 overexpression only; 3) iPou4f3- Pou4f3 overexpression only; 4) iG+A- Gfi1 and Atoh1 combined overexpression; 5) iG+P- Gfi1 and Pou4f3 combined overexpression; 6) iP+A- Pou4f3 and Atoh1 combined overexpression; 7) iGPA- Gfi1, Pou4f3 and Atoh1 combined overexpression. In order to promote overexpression of these TFs, 2 µg/ml of doxycycline was added to all doxycycline-inducible mESCs lines at day 4 of embryoid body differentiation and maintained until day 6. In parallel, a control group was generated by not adding doxycycline to the culture media but subjecting embryoid bodies to the same differentiation conditions. Please note that doxycycline treatment and control groups are indicated in Sample titles (e.g. *Dox* and *Ctrl*) For each doxycycline-inducible mESCs lines, 2 independent biological replicates were performed using different clones for the same mESCs line and these are indicated in Sample titles (e.g. *Rep1 and *Rep2). In summary, a total of 28 samples comprising of 7 different doxycycline-inducible mESCs lines, 2 experimental groups (control and doxycycline treatment) and 2 independent biological replicates were analysed. All samples are present in 4 lanes of sequencing, reads from which were demultiplexed and provided here.