Project description:In direct lineage reprogramming, transcription factor (TF) overexpression reconfigures Gene Regulatory Networks (GRNs) to convert cell identities between fully differentiated cell types. We previously developed CellOracle, a computational pipeline that integrates single-cell transcriptome and epigenome profiles to infer GRNs. CellOracle leverages these inferred GRNs to simulate gene expression changes in response to TF perturbation, enabling network re-configuration during reprogramming to be interrogated in silico. Here, we integrate CellOracle analysis with lineage tracing of fibroblast to induced endoderm progenitor (iEP) conversion, a prototypical direct lineage reprogramming paradigm. By linking early network state to reprogramming success or failure, we reveal distinct network configurations underlying different reprogramming outcomes. Using these network analyses and in silico simulation of TF perturbation, we identify new factors to coax cells into successfully converting cell identity, uncovering a central role for the AP-1 subunit Fos with the Hippo signaling effector, Yap1. Together, these results demonstrate the efficacy of CellOracle to infer and interpret cell-type-specific GRN configurations at high resolution, providing new mechanistic insights into the regulation and reprogramming of cell identity.
Project description:In direct lineage conversion, transcription factor (TF) overexpression reconfigures gene regulatory networks (GRNs) to reprogram cell identity. We previously developed CellOracle, a computational method to infer GRNs from single-cell transcriptome and epigenome data. Using inferred GRNs, CellOracle simulates gene expression changes in response to TF perturbation, enabling in silico interrogation of network reconfiguration. Here, we combine CellOracle analysis with lineage tracing of fibroblast to induced endoderm progenitor (iEP) conversion, a prototypical direct reprogramming paradigm. By linking early network state to reprogramming outcome, we reveal distinct network configurations underlying successful and failed fate conversion. Via in silico simulation of TF perturbation, we identify new factors to coax cells into successfully converting their identity, uncovering a central role for the AP-1 subunit Fos with the Hippo signaling effector, Yap1. Together, these results demonstrate the efficacy of CellOracle to infer and interpret cell-type-specific GRN configurations, providing new mechanistic insights into lineage reprogramming.
Project description:Direct lineage reprogramming involves the conversion of cellular identity. Single-cell technologies are useful for deconstructing the considerable heterogeneity that emerges during lineage conversion. However, lineage relationships are typically lost during cell processing, complicating trajectory reconstruction. Here we present ‘CellTagging’, a combinatorial cell-indexing methodology that enables parallel capture of clonal history and cell identity, in which sequential rounds of cell labelling enable the construction of multi-level lineage trees. CellTagging and longitudinal tracking of fibroblast to induced endoderm progenitor reprogramming reveals two distinct trajectories: one leading to successfully reprogrammed cells, and one leading to a ‘dead-end’ state, paths determined in the earliest stages of lineage conversion. We find that expression of a putative methyltransferase, Mettl7a1, is associated with the successful reprogramming trajectory; adding Mettl7a1 to the reprogramming cocktail increases the yield of induced endoderm progenitors. Together, these results demonstrate the utility of our lineage-tracing method for revealing the dynamics of direct reprogramming.
Project description:How transcription factors (TFs) reprogram one cell lineage to another remains unclear. Here, we define chromatin accessibility changes induced by the proneural TF Ascl1 throughout conversion of fibroblasts into induced neuronal (iN) cells. Thousands of genomic loci are affected as early as 12 hours after Ascl1 induction. Surprisingly, over 80% of the accessibility changes occur between days 2 and 5 of the 3-week reprogramming process. This chromatin switch coincides with robust activation of endogenous neuronal TFs and nucleosome phasing of neuronal promoters and enhancers. Subsequent morphological and functional maturation of iN cells are accomplished with relatively little chromatin reconfiguration. Integrating chromatin accessibility and transcriptome changes, we built a network model of dynamic TF regulation during iN cell reprogramming, and identified Zfp238, Sox8 and Dlx3 as key TFs downstream of Ascl1. These results reveal a singular, coordinated epigenomic switch during direct reprogramming, in contrast to step-wise cell fate transitions in development.
Project description:Somatic cell reprogramming into pluripotent stem cells (iPSC) through the forced expression of defined factors induces changes in genome architecture reflective of the embryonic stem cell state. However, only a small minority of cells typically transition to pluripotency, which has limited our understanding of what defines cells that successfully reprogram. Here, we characterize the changes that occur across the DNA regulatory landscape during reprogramming by time-course profiling of isolated sub-populations of reprogramming intermediates poised to become iPSC. Widespread reconfiguration of chromatin states and transcription factor occupancy occurs early during reprogramming, and cells that fail to reprogram partially retain regulatory elements active in their somatic cell state. A second wave of reconfiguration occurs just prior to cells achieving pluripotency, where a majority of early changes revert to the somatic cell state and many of the changes that define the pluripotent state become established. Our comprehensive characterization of the molecular changes that occur during reprogramming broaden our understanding of the reprogramming process by providing crucial insights into iPSC generation, and shed light on how transcription factors in general access and change the chromatin during cell fate transitions.
Project description:Chickarmane2008 - Stem cell lineage determination
In this work, a dynamical model of lineage
determination based upon a minimal circuit, as discussed in PMID: 17215298
, which contains the Oct4/Sox2/Nanog core as well its interaction
with a few other key genes is discussed.
This model is described in the article:
A computational model for understanding stem cell, trophectoderm and endoderm lineage determination.
Chickarmane V, Peterson C
PloS one. 2008, 3(10):e3478
Abstract:
BACKGROUND: Recent studies have associated the transcription factors, Oct4, Sox2 and Nanog as parts of a self-regulating network which is responsible for maintaining embryonic stem cell properties: self renewal and pluripotency. In addition, mutual antagonism between two of these and other master regulators have been shown to regulate lineage determination. In particular, an excess of Cdx2 over Oct4 determines the trophectoderm lineage whereas an excess of Gata-6 over Nanog determines differentiation into the endoderm lineage. Also, under/over-expression studies of the master regulator Oct4 have revealed that some self-renewal/pluripotency as well as differentiation genes are expressed in a biphasic manner with respect to the concentration of Oct4. METHODOLOGY/
PRINCIPAL FINDINGS: We construct a dynamical model of a minimalistic network, extracted from ChIP-on-chip and microarray data as well as literature studies. The model is based upon differential equations and makes two plausible assumptions; activation of Gata-6 by Oct4 and repression of Nanog by an Oct4-Gata-6 heterodimer. With these assumptions, the results of simulations successfully describe the biphasic behavior as well as lineage commitment. The model also predicts that reprogramming the network from a differentiated state, in particular the endoderm state, into a stem cell state, is best achieved by over-expressing Nanog, rather than by suppression of differentiation genes such as Gata-6.
CONCLUSIONS: The computational model provides a mechanistic understanding of how different lineages arise from the dynamics of the underlying regulatory network. It provides a framework to explore strategies of reprogramming a cell from a differentiated state to a stem cell state through directed perturbations. Such an approach is highly relevant to regenerative medicine since it allows for a rapid search over the host of possibilities for reprogramming to a stem cell state.
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and identified
by: MODEL8390025091
.
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.
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domain worldwide. Please refer to CC0 Public Domain
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for more information.
Project description:Electromagnetic fields (EMF) are physical energy generated by electrically charged objects that can influence numerous biologic processes, including control of cell fate and plasticity. In this study, we show that magnetic gold nanoparticles in the presence of EMF can facilitate efficient direct lineage reprogramming to induced dopamine neurons both in vitro and in vivo. Remarkably, electromagnetic stimulation leads to the specific activation of the histone acetyl transferase Brd2, resulting in H3K27 acetylation and robust activation of neuronal-specific gene expression. In vivo reprogramming in conjunction with EMF stimulation efficiently alleviated symptoms in a mouse model of Parkinson’s disease (PD) in a noninvasive and controllable manner. These studies provide a proof of principle that EMF-based approaches may represent a viable and safe therapeutic strategy facilitating in vivo lineage conversion for neurodegenerative disorders.
Project description:Electromagnetic fields (EMF) are physical energy generated by electrically charged objects that can influence numerous biologic processes, including control of cell fate and plasticity. In this study, we show that magnetic gold nanoparticles in the presence of EMF can facilitate efficient direct lineage reprogramming to induced dopamine neurons both in vitro and in vivo. Remarkably, electromagnetic stimulation leads to the specific activation of the histone acetyl transferase Brd2, resulting in H3K27 acetylation and robust activation of neuronal-specific gene expression. In vivo reprogramming in conjunction with EMF stimulation efficiently alleviated symptoms in a mouse model of Parkinson’s disease (PD) in a noninvasive and controllable manner. These studies provide a proof of principle that EMF-based approaches may represent a viable and safe therapeutic strategy facilitating in vivo lineage conversion for neurodegenerative disorders.