Fig. 2
From: MKL1-actin pathway restricts chromatin accessibility and prevents mature pluripotency activation
MKL1 drives actin cytoskeletal gene expression and blocks mature pluripotency. a Left: schematic diagram of constitutively active MKL1 (caMKL1), with the RPEL domain deleted and replaced with a nuclear localization signal (NLS). Right: experimental design illustrating a retroviral vector expressing caMKL1-IRES-mCherry to be transduced into reprogrammable MEFs, which express Col1a:OKSM, Rosa26:M2rtTA and Oct4:GFP. The co-expressed mCherry from the vector was used to isolate the transduced cells for all further experiments. b Realtime QPCR analysis of total MKL1 mRNA level in primary MEFs, as compared to the cells at reprogramming day 25 coexpressing either a control vector or caMKL1. n = 3 for MEF and control; n = 4 for caMKL1-overexpressing condition. Error bars denote standard deviation. c AP stained reprogramming cultures on day 10, coexpressing either control vector or caMKL1. The number of AP+ colonies derived from 4,000 reprogrammable MEFs are quantified. d Percentage of Oct4:GFP+ cells arising from reprogramming MEFs coexpressing control vector or caMKL1, as determined by FACS. Cells were trypsinized and analyzed at indicated time points starting from day 10. e AP stained reprogramming cultures from control- or caMKL1-expressing reprogramming MEFs on day25, when cells were passaged and replated into continued cultures with or without Dox. Bottom panel shows colonies at higher magnification. Note that no AP+ colonies could be detected in the caMKL1-expressing cultures without Dox. f, g MA plots of differentially expressed genes between control- and caMKL1-overexpressing cells at reprogramming day 6 (f) and day 25 (g), respectively. h Meta analysis of Oct4-bound regions in day 25 reprogramming cells expressing the control vector or caMKL1 (left). The Oct4-bound regions in control iPS cells show reduced Oct4 binding in caMKL1-expressing cells. Oct4 ChIP-qPCR confirmed reduced binding at the Oct4 enhancer region in caMKL1-blocked cells (right). Error bars denote standard deviation of triplicate samples (n = 3) in each one of three independent experiments (c, d, h)
