Diseases and damage to the retina lead to losses in retinal neurons and eventual visual impairment. Although the mammalian retina has no inherent regenerative capabilities, fish have robust regeneration from Müller glia (MG). Recently, we have shown that driving expression of
Experimental studies have shown that chromatin modifiers have a critical effect on cellular reprogramming, i.e., the conversion of differentiated cells to pluripotent stem cells. Here, we develop a model of the OCT4 gene regulatory network that includes genes expressing chromatin modifiers TET1 and JMJD2, and the chromatin modification circuit on which these modifiers act. We employ this model to compare three reprogramming approaches that have been considered in the literature with respect to reprogramming efficiency and latency variability. These approaches are overexpression of OCT4 alone, overexpression of OCT4 with TET1, and overexpression of OCT4 with JMJD2. Our results show more efficient and less variable reprogramming when also JMJD2 and TET1 are overexpressed, consistent with previous experimental data. Nevertheless, TET1 overexpression can lead to more efficient reprogramming compared to JMJD2 overexpression. This is the case when the recruitment of DNA methylation by H3K9me3 is weak and the methyl-CpG-binding domain (MBD) proteins are sufficiently scarce such that they do not hamper TET1 binding to methylated DNA. The model that we developed provides a mechanistic understanding of existing experimental results and is also a tool for designing optimized reprogramming approaches that combine overexpression of cell-fate specific transcription factors (TFs) with targeted recruitment of epigenetic modifiers.
more » « less- NSF-PAR ID:
- 10484566
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- npj Systems Biology and Applications
- Volume:
- 10
- Issue:
- 1
- ISSN:
- 2056-7189
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Ascl1 in adult mouse MG stimulates neural regeneration. The regeneration observed in the mouse is limited in the variety of neurons that can be derived from MG;Ascl1 -expressing MG primarily generate bipolar cells. To better understand the limits of MG-based regeneration in mouse retinas, we used ATAC- and RNA-seq to compare newborn progenitors, immature MG (P8-P12), and mature MG. Our analysis demonstrated developmental differences in gene expression and accessible chromatin between progenitors and MG, primarily in neurogenic genes. Overexpression ofAscl1 is more effective in reprogramming immature MG, than mature MG, consistent with a more progenitor-like epigenetic landscape in the former. We also used ASCL1 ChIPseq to compare the differences in ASCL1 binding in progenitors and reprogrammed MG. We find that bipolar-specific accessible regions are more frequently linked to bHLH motifs and ASCL1 binding. Overall, our analysis indicates a loss of neurogenic gene expression and motif accessibility during glial maturation that may prevent efficient reprogramming. -
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Somatic cell transcription factors are critical to maintaining cellular identity and constitute a barrier to human somatic cell reprogramming; yet a comprehensive understanding of the mechanism of action is lacking. To gain insight, we examined epigenome remodeling at the onset of human nuclear reprogramming by profiling human fibroblasts after fusion with murine embryonic stem cells (ESCs). By assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing we identified enrichment for the activator protein 1 (AP-1) transcription factor c-Jun at regions of early transient accessibility at fibroblast-specific enhancers. Expression of a dominant negative AP-1 mutant (dnAP-1) reduced accessibility and expression of fibroblast genes, overcoming the barrier to reprogramming. Remarkably, efficient reprogramming of human fibroblasts to induced pluripotent stem cells was achieved by transduction with vectors expressing SOX2, KLF4, and inducible dnAP-1, demonstrating that dnAP-1 can substitute for exogenous human OCT4. Mechanistically, we show that the AP-1 component c-Jun has two unexpected temporally distinct functions in human reprogramming: 1) to potentiate fibroblast enhancer accessibility and fibroblast-specific gene expression, and 2) to bind to and repress
OCT4 as a complex with MBD3. Our findings highlight AP-1 as a previously unrecognized potent dual gatekeeper of the somatic cell state. -
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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.
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Abstract Probiotics, whether taken as capsules or consumed in foods, have been regarded as safe for human use by regulatory agencies. Being living cells, they serve as “tunable” factories for the synthesis of a vast array of beneficial molecules. The idea of reprogramming probiotics to act as controllable factories, producing potential therapeutic molecules under user‐specified conditions, represents a new and powerful concept in drug synthesis and delivery. Probiotics that serve as drug delivery vehicles pose several challenges, one being targeting (as seen with nanoparticle approaches). Here, we employ synthetic biology to control swimming directionality in a process referred to as “pseudotaxis.”
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Abstract Background In several eukaryotes, DNA methylation occurs within the coding regions of many genes, termed gene body methylation (GbM). Whereas the role of DNA methylation on the silencing of transposons and repetitive DNA is well understood, gene body methylation is not associated with transcriptional repression, and its biological importance remains unclear.
Results We report a newly discovered type of GbM in plants, which is under constitutive addition and removal by dynamic methylation modifiers in all cells, including the germline. Methylation at Dynamic GbM genes is removed by the DRDD demethylation pathway and added by an unknown source of de novo methylation, most likely the maintenance methyltransferase MET1. We show that the Dynamic GbM state is present at homologous genes across divergent lineages spanning over 100 million years, indicating evolutionary conservation. We demonstrate that Dynamic GbM is tightly associated with the presence of a promoter or regulatory chromatin state within the gene body, in contrast to other gene body methylated genes. We find Dynamic GbM is associated with enhanced gene expression plasticity across development and diverse physiological conditions, whereas stably methylated GbM genes exhibit reduced plasticity. Dynamic GbM genes exhibit reduced dynamic range in
drdd mutants, indicating a causal link between DNA demethylation and enhanced gene expression plasticity.Conclusions We propose a new model for GbM in regulating gene expression plasticity, including a novel type of GbM in which increased gene expression plasticity is associated with the activity of DNA methylation writers and erasers and the enrichment of a regulatory chromatin state.
