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. 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.