Chromatin and Transcriptional Response to Loss of TBX1 in Early Differentiation of Mouse Cells.

Andrea Cirino,Monica Franzese,Rosa Ferrentino,Ilaria Aurigemma,Dario Righelli,Gabriella Lania,Elizabeth Illingworth,Claudia Angelini,Antonio Baldini

doi:10.3389/fcell.2020.571501

Abstract

The T-box transcription factor TBX1 has critical roles in the cardiopharyngeal lineage and the gene is haploinsufficient in DiGeorge syndrome, a typical developmental anomaly of the pharyngeal apparatus. Despite almost two decades of research, if and how TBX1 function triggers chromatin remodeling is not known. Here, we explored genome-wide gene expression and chromatin remodeling in two independent cellular models of Tbx1 loss of function, mouse embryonic carcinoma cells P19Cl6, and mouse embryonic stem cells (mESCs). The results of our study revealed that the loss or knockdown of TBX1 caused extensive transcriptional changes, some of which were cell type-specific, some were in common between the two models. However, unexpectedly we observed only limited chromatin changes in both systems. In P19Cl6 cells, differentially accessible regions (DARs) were not enriched in T-BOX binding motifs; in contrast, in mESCs, 34% (n = 47) of all DARs included a T-BOX binding motif and almost all of them gained accessibility in Tbx1–/– cells. In conclusion, despite a clear transcriptional response of our cell models to loss of TBX1 in early cell differentiation, chromatin changes were relatively modest.

Highlights

TBX1 is a transcription factor encoded by a gene that is haploinsufficient in DiGeorge/22q11.2 deletion syndromes and in the mouse (Greulich et al, 2011; McDonald-McGinn et al, 2015; Baldini et al, 2017)
We have used two cellular models that respond transcriptionally to Tbx1 gene dosage, mouse P19Cl6 and embryonic stem cells, but that are at an early stage of differentiation, and we tested the effects of Tbx1 inactivation on transcription and on chromatin remodeling. mESCs (Tbx1+/+ and Tbx1−/−) were subjected to a widely used cardiac mesoderm differentiation protocol (Kattman et al, 2011) and selected using a fluorescence activated cell sorting (FACS) approach
Data analysis of two cell culture models provided a snapshot of the chromatin accessibility, as measured by ATAC-seq, with and without TBX1 function, or dosage reduction

Summary

Introduction

TBX1 is a transcription factor encoded by a gene that is haploinsufficient in DiGeorge/22q11.2 deletion syndromes and in the mouse (Greulich et al, 2011; McDonald-McGinn et al, 2015; Baldini et al, 2017). Timed-deletion of the gene has revealed a requirement as early as E7.5–E8.0 (Xu et al, 2005) for the development of the 4th pharyngeal arch artery that will form much later While this phenomenon could be explained by a number of mechanisms, one possibility is that TBX1 primes enhancers for downstream activation or repression, thereby creating asynchrony between the time of requirement and the phenotypic consequences. To address this issue, we have used two cellular models that respond transcriptionally to Tbx gene dosage, mouse P19Cl6 and embryonic stem cells (mESCs), but that are at an early stage of differentiation, and we tested the effects of Tbx inactivation on transcription and on chromatin remodeling.

Methods

Results

Conclusion