Reduction of Intracellular Tension and Cell Adhesion Promotes Open Chromatin Structure and Enhances Cell Reprogramming

Abstract The role of transcription factors and biomolecules in cell type conversion has been widely studied. Yet, it remains unclear whether and how intracellular mechanotransduction through focal adhesions (FAs) and the cytoskeleton regulates the epigenetic state and cell reprogramming. Here, it is shown that cytoskeletal structures and the mechanical properties of cells are modulated during the early phase of induced neuronal (iN) reprogramming, with an increase in actin cytoskeleton assembly induced by Ascl1 transgene. The reduction of actin cytoskeletal tension or cell adhesion at the early phase of reprogramming suppresses the expression of mesenchymal genes, promotes a more open chromatin structure, and significantly enhances the efficiency of iN conversion. Specifically, reduction of intracellular tension or cell adhesion not only modulates global epigenetic marks, but also decreases DNA methylation and heterochromatin marks and increases euchromatin marks at the promoter of neuronal genes, thus enhancing the accessibility for gene activation. Finally, micro‐ and nano‐topographic surfaces that reduce cell adhesions enhance iN reprogramming. These novel findings suggest that the actin cytoskeleton and FAs play an important role in epigenetic regulation for cell fate determination, which may lead to novel engineering approaches for cell reprogramming.


Figure S2. Cell mechanical phenotype was modulated during iN reprogramming as determined by quantitative deformability cytometry (q-DC).
A, Transit time of BAMtransduced fibroblasts at the indicated time points (day 0, n= 211; day 1 n= 257; day 3, n= 253) as derived by q-DC.B, Density scatter plots show the log of transit time as a function of cell diameter for BAM-transduced fibroblasts deforming through 9 x 10 μm constrictions at the indicated time points (day 0, n= 211; day 1= 257; day 3, n= 253).Dots represent singlecell data.Box plots show the ends at the quartiles, the median as a horizontal line in the box, the mean as a (+) symbol, and the whiskers extend from the minimum to maximum data point.Significance determined by one-way ANOVA using Tukey's correction for multiple comparisons (**p < 0.01, ****p < 0.0001).and BAM-transduced fibroblasts were treated with blebbistatin for 2 days followed by qRT-PCR analysis of neuronal gene expression at day 3 (n=3).Significance determined by oneway ANOVA and Tukey's multiple comparison test.Significance determined by two-tailed, student's t test, compared to DMSO condition for the same gene.Gene expression was normalized with 18S RNA levels.Bar graphs represent mean ± one standard deviation (*p < 0.05, **p < 0.01).

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Figure S41.Effect of biomaterials on cell stiffness.Box plots illustrate the variation in elastic modulus of non-transduced fibroblasts cultured on glass, flat PDMS membranes, 5 µm binary colloidal crystals (BCC) and 2 µm BCCs as acquired using AFM (n=31-32 per condition).Significance determined by two-way ANOVA using Tukey's correction for multiple comparisons.Box plots show the ends at the quartiles, the median as a horizontal line in the box, the mean as a (+) symbol, and the whiskers extend from the minimum to maximum data point (**p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure S3 .Figure S4 .Figure S5 .BFigure S6 .Figure S9 .Figure S11 .
Figure S3.Effect of transgenes on cell adhesion and cytoskeletal proteins.Fibroblasts were either non-transduced or transduced with individual or a combination of transgenes, followed by Western blot analysis of focal adhesion, nuclear membrane and cytoskeletal proteins at the indicated time points.GAPDH serves as a control.NT, non-transduced; A, Ascl1; B, Brn2; M, Myt1l.
Figure S15.Functional assessment of derived iN cells by electrophysiological analysis.A-D, Quantification of electrophysiological properties of iN cells derived with and without blebbistatin.Each circle represents an individual cell that was tested.The resting membrane potential, RMP (A), action potential amplitude (B) and half-width (C) and input resistance (D) were measured.E, Representative recordings of spontaneous excitatory postsynaptic currents (EPSCs) from iN cells derived in the absence and presence of blebbistatin.F-G, Quantification of EPSC frequency (F) and amplitude (G) of iN cells (n=6).H, Representative traces of spontaneous inhibitory postsynaptic currents (IPSCs) in iN cells derived in the absence and presence of blebbistatin.I-J.Quantification of IPSC frequency (I) and amplitude (J) of iN cells (n=4).Significance determined by two-tailed, student's t test, compared to DMSO condition.Bar graphs represent mean ± one standard error of mean (****p < 0.0001).
Figure S19.Blebbistatin upregulates neuronal genes in Ascl1-mediated iN reprogramming.qRT-PCR analysis of neuronal gene expression at day 7 in Ascl1transduced fibroblasts cultured in the absence and presence of blebbistatin (n=3).Significance determined by two-tailed, student's t test, compared to DMSO condition for the same gene.Gene expression was normalized with 18S RNA levels.Bar graphs represent mean ± one standard deviation (*p < 0.05, ***p < 0.001, ****p < 0.0001).

Figure S21 .Figure S25 .Figure S26 .Figure S28 .Figure S29 .Figure S31 .
Figure S21.UMAP clustering of single cell RNA sequencing samples.UMAP clustering of single cell RNA sequencing data from samples of BAM-transduced fibroblasts reprogrammed in the absence or presence of blebbistatin for 48 hours and collected on day 3 (n=2).
Figure S33.PF573228-derived iN cells display functionality.Quantification of electrophysiological properties of iN cells obtained in the absence and presence of 10 µM blebbistatin (Blebb) or 1 µM PF573228 (PF).The inhibitor was administered during the first 7 days of reprogramming.Each circle represents an individual cell that was tested.The resting membrane potential, RMP, input resistance and action potential amplitude and half-width were measured.Significance determined by one-way ANOVA using Tukey's correction for multiple comparisons (NS: not significant).

Figure S42 .
Figure S42.Summary on the role of the cytoskeleton and cell adhesion on the direct reprogramming of adult mouse fibroblasts into neurons.Disruption of cell adhesion and cytoskeletal tension modulates chromatin organization, the epigenetic state and neuronal gene expression to promote iN reprogramming.

Table S1 .
Antibody information for immunofluorescent staining and Western blotting analysis

Table S2 .
Primers used for qRT-PCR analysis

Table S3 .
Primers used for ChIP qRT-PCR analysis