43 research outputs found
Logical modelling of cellular decision processes with GINsim
International audienc
Constitutively active Smad2/3 are broad scope potentiators of transcription factor-mediated cellular reprogramming
Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering. Ruetz et al. show that constitutively active SMAD2/3 has a surprising ability to boost the efficiency of cell reprogramming both to iPSCs and across lineages and may therefore be a general factor that can enhance transcription-factor-mediated reprogramming in a variety of contexts
Investigation du réseau de régulation contrÎlant la spécification et la reprogrammation des cellules du sang
Immune cells arise from a common set of hematopoietic stem cells, which differentiate hierarchically into the myeloid and lymphoid lineages. This process is tightly regulated by an intertwined network of transcription and epigenetic factors, which control both the activation and repression of gene programs, to ensure cell commitment. However, recent work on cellular reprogramminghas shown that the ectopic expression of some specific factors can enforce the trans-differentiation of committed cells. The transcription factor C/EBPa can induce the reprogramming of B-cells into macrophages. Furthermore, a pulse of Cebpa expression in B cells followed by the expression of the four transcription factors Oct4-Sox2-Klf4-cMyc leads to an extremely fast and efficient reprogramming into induced pluripotent stem cells. Despite the many data we have on the molecular mechanisms by which specific genes are regulated, we are still lacking a global understanding of the interplay between these factors and how theycontrol cell fate. In order to decipher the molecular regulatory network controlling immune cell specification and their reprogramming, I have combined a variety of high-throughput methods to measure changes in gene expression and epigenetic regulation during B cells reprogramming. I have revealed the interplay between different transcription factors at enhancers regulating genes of the different programs (B cells, macrophages and pluripotent cells) and identified epigenetic regulators forming complexes and controlling enhancers activities (such as Lsd1, Hdac1, Brd4 and Tet2) and consequently regulating cell fate. Finally, I integrated these data together with published data, in a computational model of the regulatory network controlling the specification of B-cells and macrophages from multipotent progenitors. I used both analytic tools (stable states analysis) and simulations (logical asynchronous simulations, continuous time Markov chains) to study in silico differentiation and reprogramming.These analyses have revealed previouslyunknown transcriptional regulations, which weconfirmed experimentally, and allowed us to get abetter understanding of the regulatory circuitscontrolling cell fate commitment.Les cellules immunitaires proviennent d'un ensemble commun de cellules souches hĂ©matopoĂŻĂ©tiques qui se diffĂ©rencient hiĂ©rarchiquement en lignĂ©es myĂ©loĂŻdes et lymphoĂŻdes. Ce processus est Ă©troitement rĂ©gulĂ© par un rĂ©seau entrelacĂ© de facteurs de transcription et de rĂ©gulateurs Ă©pigĂ©nĂ©tiques, qui contrĂŽlent l'activation et la rĂ©pression des gĂšnes impliquĂ©s. Les travaux rĂ©cents sur la reprogrammation cellulaire ont montrĂ© que certaines protĂ©ines peuvent reprogrammer des cellules diffĂ©renciĂ©es, comme le facteur de transcription C/EBPa qui peut induire la trans-differenciation de cellules B en macrophages. De plus, une courte induction de Cebpa suivie de lâexpression des quatre facteurs de transcription Oct4-Sox2-Klf4-cMyc permet une reprogrammation extrĂȘmement rapide en cellules pluripotentes. Afin de dĂ©chiffrer le rĂ©seau de rĂ©gulation molĂ©culaire contrĂŽlant la spĂ©cification et la reprogrammation des cellules immunitaires, jâai combinĂ© diffĂ©rentes mĂ©thodes Ă haut dĂ©bit pour analyser lâexpression des gĂšnes et leur rĂ©gulation Ă©pigĂ©nĂ©tique, et ce au court de la reprogrammation des cellules B. Jâai dĂ©couvert des interactions entre diffĂ©rents facteurs de transcription, au niveau des rĂ©gions rĂ©gulatrices de gĂšnes des diffĂ©rents programmes gĂ©nĂ©tiques impliquĂ©s (lymphoide, myeloide et pluripotence), et jâai identifiĂ© des facteurs rĂ©gulant lâĂ©tat de la chromatine Ă©galement impliquĂ©s dans la reprogrammation (notamment Lsd1, Hdac1, Brd4 et Tet2). Enfin, Jâai intĂ©grĂ© ces donnĂ©es dans un modĂšle dynamique du rĂ©seau molĂ©culaire rĂ©gulant la spĂ©cification des cellules B et des macrophages Ă partir de progĂ©niteurs multipotents. Jâai utilisĂ© Ă la fois des mĂ©thodes analytiques (analyse des Ă©tats stables) et des simulations (simulations logiques asynchrones, chaĂźnes de Markov Ă temps continu) pour Ă©tudier in silico la diffĂ©renciation et la reprogrammation cellulaire. Ces analyses ont rĂ©vĂ©lĂ©s des rĂ©gulations transcriptionelles encore inconnues, que nous avons pu confirmer expĂ©rimentalement. Nous avons ainsi obtenu une meilleure comprĂ©hension des circuits de rĂ©gulation contrĂŽlant le destin cellulaire
Deciphering the regulatory network controlling blood cell specification and reprogramming
Les cellules immunitaires proviennent d'un ensemble commun de cellules souches hĂ©matopoĂŻĂ©tiques qui se diffĂ©rencient hiĂ©rarchiquement en lignĂ©es myĂ©loĂŻdes et lymphoĂŻdes. Ce processus est Ă©troitement rĂ©gulĂ© par un rĂ©seau entrelacĂ© de facteurs de transcription et de rĂ©gulateurs Ă©pigĂ©nĂ©tiques, qui contrĂŽlent l'activation et la rĂ©pression des gĂšnes impliquĂ©s. Les travaux rĂ©cents sur la reprogrammation cellulaire ont montrĂ© que certaines protĂ©ines peuvent reprogrammer des cellules diffĂ©renciĂ©es, comme le facteur de transcription C/EBPa qui peut induire la trans-differenciation de cellules B en macrophages. De plus, une courte induction de Cebpa suivie de lâexpression des quatre facteurs de transcription Oct4-Sox2-Klf4-cMyc permet une reprogrammation extrĂȘmement rapide en cellules pluripotentes. Afin de dĂ©chiffrer le rĂ©seau de rĂ©gulation molĂ©culaire contrĂŽlant la spĂ©cification et la reprogrammation des cellules immunitaires, jâai combinĂ© diffĂ©rentes mĂ©thodes Ă haut dĂ©bit pour analyser lâexpression des gĂšnes et leur rĂ©gulation Ă©pigĂ©nĂ©tique, et ce au court de la reprogrammation des cellules B. Jâai dĂ©couvert des interactions entre diffĂ©rents facteurs de transcription, au niveau des rĂ©gions rĂ©gulatrices de gĂšnes des diffĂ©rents programmes gĂ©nĂ©tiques impliquĂ©s (lymphoide, myeloide et pluripotence), et jâai identifiĂ© des facteurs rĂ©gulant lâĂ©tat de la chromatine Ă©galement impliquĂ©s dans la reprogrammation (notamment Lsd1, Hdac1, Brd4 et Tet2). Enfin, Jâai intĂ©grĂ© ces donnĂ©es dans un modĂšle dynamique du rĂ©seau molĂ©culaire rĂ©gulant la spĂ©cification des cellules B et des macrophages Ă partir de progĂ©niteurs multipotents. Jâai utilisĂ© Ă la fois des mĂ©thodes analytiques (analyse des Ă©tats stables) et des simulations (simulations logiques asynchrones, chaĂźnes de Markov Ă temps continu) pour Ă©tudier in silico la diffĂ©renciation et la reprogrammation cellulaire. Ces analyses ont rĂ©vĂ©lĂ©s des rĂ©gulations transcriptionelles encore inconnues, que nous avons pu confirmer expĂ©rimentalement. Nous avons ainsi obtenu une meilleure comprĂ©hension des circuits de rĂ©gulation contrĂŽlant le destin cellulaire.Immune cells arise from a common set of hematopoietic stem cells, which differentiate hierarchically into the myeloid and lymphoid lineages. This process is tightly regulated by an intertwined network of transcription and epigenetic factors, which control both the activation and repression of gene programs, to ensure cell commitment. However, recent work on cellular reprogramminghas shown that the ectopic expression of some specific factors can enforce the trans-differentiation of committed cells. The transcription factor C/EBPa can induce the reprogramming of B-cells into macrophages. Furthermore, a pulse of Cebpa expression in B cells followed by the expression of the four transcription factors Oct4-Sox2-Klf4-cMyc leads to an extremely fast and efficient reprogramming into induced pluripotent stem cells. Despite the many data we have on the molecular mechanisms by which specific genes are regulated, we are still lacking a global understanding of the interplay between these factors and how theycontrol cell fate. In order to decipher the molecular regulatory network controlling immune cell specification and their reprogramming, I have combined a variety of high-throughput methods to measure changes in gene expression and epigenetic regulation during B cells reprogramming. I have revealed the interplay between different transcription factors at enhancers regulating genes of the different programs (B cells, macrophages and pluripotent cells) and identified epigenetic regulators forming complexes and controlling enhancers activities (such as Lsd1, Hdac1, Brd4 and Tet2) and consequently regulating cell fate. Finally, I integrated these data together with published data, in a computational model of the regulatory network controlling the specification of B-cells and macrophages from multipotent progenitors. I used both analytic tools (stable states analysis) and simulations (logical asynchronous simulations, continuous time Markov chains) to study in silico differentiation and reprogramming.These analyses have revealed previouslyunknown transcriptional regulations, which weconfirmed experimentally, and allowed us to get abetter understanding of the regulatory circuitscontrolling cell fate commitment
AllProbes_AllReplicates.xls
<p>Gene expression profiling during iPS reprogramming of C/EBPa-pulsed B cells into iPS cells. The file contains normalized data for all the probes and replicates.</p
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RNA polymerase II depletion from the inactive X chromosome territory is not mediated by physical compartmentalization.
Subnuclear compartmentalization has been proposed to play an important role in gene regulation by segregating active and inactive parts of the genome in distinct physical and biochemical environments. During X chromosome inactivation (XCI), the noncoding Xist RNA coats the X chromosome, triggers gene silencing and forms a dense body of heterochromatin from which the transcription machinery appears to be excluded. Phase separation has been proposed to be involved in XCI, and might explain the exclusion of the transcription machinery by preventing its diffusion into the Xist-coated territory. Here, using quantitative fluorescence microscopy and single-particle tracking, we show that RNA polymerase II (RNAPII) freely accesses the Xist territory during the initiation of XCI. Instead, the apparent depletion of RNAPII is due to the loss of its chromatin stably bound fraction. These findings indicate that initial exclusion of RNAPII from the inactive X reflects the absence of actively transcribing RNAPII, rather than a consequence of putative physical compartmentalization of the inactive X heterochromatin domain
Krox20 hindbrain regulation incorporates multiple modes of cooperation between cis-acting elements
International audienceDevelopmental genes can harbour multiple transcriptional enhancers that act simultaneously or in succession to achieve robust and precise spatiotemporal expression. However, the mechanisms underlying cooperation between cis-acting elements are poorly documented, notably in vertebrates. The mouse gene Krox20 encodes a transcription factor required for the specification of two segments (rhombomeres) of the developing hindbrain. In rhombomere 3, Krox20 is subject to direct positive feedback governed by an autoregulatory enhancer, element A. In contrast, a second enhancer, element C, distant by 70 kb, is active from the initiation of transcription independent of the presence of the KROX20 protein. Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate the autoregulatory activity of element A, by increasing its chromatin accessibility. This work uncovers a novel, asymmetrical, long-range mode of cooperation between cis-acting elements that might be essential to avoid promiscuous activation of positive autoregulatory elements
Cooperation, cis-interactions, versatility and evolutionary plasticity of multiple cis-acting elements underlie krox20 hindbrain regulation.
Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes