Project description:Safety issues associated with transcription factors or viruses may be avoided with the use of chemically induced pluripotent stem cells (CiPSCs), thus promoting their clinical application. Previously, we had successfully developed and standardized an induction method using small-molecule compound, with simple operation, uniform induction conditions, and clear constituents. In order to verify that the CiPSCs were indeed reprogrammed from mouse embryonic fibroblasts (MEFs), and further explore the underlying mechanisms, FSP-tdTomato mice were used to construct a fluorescent protein-tracking system of MEFs, for revealing the process of CiPSC reprogramming. CiPSCs were identified by morphological analysis, mRNA, and protein expression of pluripotency genes, as well as teratoma formation experiments. Results showed that after 40-day treatment of tdTomato-MEFs with small-molecule compounds, the cells were presented with prominent nucleoli, high core-to-cytoplasmic ratio, round shape, group and mass arrangement, and high expression of pluripotency gene. These cells could differentiate into three germ layer tissues in vivo. As indicated by the above results, tdTomato-MEFs could be reprogrammed into CiPSCs, a lineage that possesses pluripotency similar to mouse embryonic stem cells (mESCs), with the use of small-molecule compounds. The establishment of CiPSC lineage, tracked by fluorescent protein, would benefit further studies exploring its underlying mechanisms. With continuous expression of fluorescent proteins during cellular differentiation, this cell lineage could be used for tracking CiPSC transplantation and differentiation into functional cells.
Project description:β-actin is a crucial component of several chromatin remodeling complexes which control chromatin structure and accessibility. The mammalian Brahma-associated factor (BAF) is one such complex that plays essential roles in development and differentiation by regulating the chromatin state of critical genes and opposing the repressive activity of polycomb repressive complexes. While previous work has shown that β-actin loss can lead to extensive changes in gene expression and heterochromatin organization, it is not known if changes in β-actin levels can directly influence chromatin remodeling activities of BAF and polycomb proteins. Here we conduct a comprehensive genomic analysis of β-actin knockout mouse embryonic fibroblasts (MEFs) using ATAC-Seq, HiC-seq, RNA-Seq and ChIP-Seq of various epigenetic marks. We demonstrate that β-actin levels can affect the complex interplay between chromatin remodelers such as BAF/BRG1 and EZH2 in a dosage-dependent manner. Our results show that changes in β-actin levels and associated chromatin remodeling activities can not only impact local chromatin accessibility but also induce reversable changes in 3D genome architecture. Our findings support a novel role for β-actin levels in shaping the chromatin landscape during development and differentiation.
Project description:Signaling by the transforming growth factor-β (TGF-β) is an essential pathway regulating a variety of cellular events. TGF-β is produced as a latent protein complex and is required to be activated before activating the receptor. The mechanical force at the cell surface is believed to be a mechanism for latent TGF-β activation. Using β-actin null mouse embryonic fibroblasts as a model, in which actin cytoskeleton and cell-surface biophysical features are dramatically altered, we reveal increased TGF-β1 activation and the upregulation of TGF-β target genes. In β-actin null cells, we show evidence that the enhanced TGF-β signaling relies on the active utilization of latent TGF-β1 in the cell culture medium. TGF-β signaling activation contributes to the elevated reactive oxygen species production, which is likely mediated by the upregulation of Nox4. The previously observed myofibroblast phenotype of β-actin null cells is inhibited by TGF-β signaling inhibition, while the expression of actin cytoskeleton genes and angiogenic phenotype are not affected. Together, our study shows a scenario that the alteration of the actin cytoskeleton and the consequent changes in cellular biophysical features lead to changes in cell signaling process such as TGF-β activation, which in turn contributes to the enhanced myofibroblast phenotype.
Project description:ATAC-Seq analysis of beta actin knockout mouse embryonic fibroblasts expressing NLS-tagged beta-actin to study impact of actin levels on chromatin accessibility
Project description:The highly homologous β (βcyto) and γ (γcyto) cytoplasmic actins are hypothesized to carry out both redundant and unique essential functions, but studies using targeted gene knockout and siRNA-mediated transcript knockdown to examine βcyto- and γcyto-isoform--specific functions in various cell types have yielded conflicting data. Here we quantitatively characterized actin transcript and protein levels, as well as cellular phenotypes, in both gene- and transcript-targeted primary mouse embryonic fibroblasts. We found that the smooth muscle αsm-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary βcyto- or β/γcyto-actin double-knockout fibroblasts. Gene targeting of βcyto-actin, but not γcyto-actin, led to greatly decreased cell proliferation, decreased levels of cellular ATP, and increased serum response factor signaling in primary fibroblasts, whereas immortalization induced by SV40 large T antigen supported fibroblast proliferation in the absence of βcyto-actin. Consistent with in vivo gene-targeting studies in mice, both gene- and transcript-targeting approaches demonstrate that the loss of βcyto-actin protein is more disruptive to primary fibroblast function than is the loss of γcyto-actin.
