Inactivation of the SRF cofactors Mrtfa and Mrtfb in MEFs results in proliferation defects and reduced expression of cytoskeletal and cell cycle progression genes

The MRTFs (Myocardin-related transcription factors) are cofactors of the SRF transcription factor, and together they control dozens of growth factor-responsive, cytoskeletal and muscle specific genes. We used a conditional knockout strategy to demonstrate that in fibroblasts, inactivation of both Mrtfa and Mrtfb, or Srf, induces a severe defect in proliferation. This state exhibits features of both quiescence and senescence, with MRTF-null cells upregulating similar cell cycle regulators to serum-deprived wildtype cells, but is fully reversible by MRTF re-expression. We analysed gene expression in three MEF pools derived from Wildtype (Rosa26Tam-Cre) animals, and three MEF pools derived from Mrtfa-/-;Mrtfbfl/fl;Rosa26Tam-Cre mice. RNA was collected 0, 2, 4 or 12 days after 4-hydroxytamoxifen treatment, which inactivate Mrtfb in Mrtfa-/-;Mrtfbfl/fl;Rosa26Tam-Cre and generate MRTF-null cells. The data indicate that substantial numbers of genes exhibit altered expression in Mrtfa-/-;Mrtfb-/- cells, the difference being most pronouced 12 days following 4-OHT treatment. Genes downregulated at this point include many genes identified as serum-inducible MRTF-SRF target cells in NIH3T3 cells (Esnault et al, 2014, DOI: 10.1101/gad.239327.114) or as derepressed in MEFs following inactivation of all members of the TCF family of SRF cofactors (Gualdrini et al, 2016, DOI: 10.1016/j.molcel.2016.10.016). They are most enriched in GO categories related to actin cytoskeletal dynamics and cell signalling, and GSEA hallmarks connected with cell growth, division, and cell cycle. Overall design: mRNA library was prepared using polyA_KAPA_mRNA_Hyper_Prep of 24 samples consisting of three biological replicates of WT MEFs (Rosa26Tam-Cre) and three biological replicates of MRTF-floxed MEFs (Mrtfa-/-;Mrtfbfl/fl;Rosa26Tam-Cre) each treated with 1µM 4-hydroxytamoxifen for 2 days before switching back to regular medium. RNA was collected 0, 2, 4, and 12 days after tamoxifen treatment.