Although acquisition of JAK2V617F mutation is an important pathogenetic event in many patients with polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF), the genetic events ...that contribute to most cases of JAK2V617F-negative MPD are not known. We used high throughput DNA sequence analysis of cytokine receptors critical for erythroid, megakaryocytic, and granulocytic proliferation and identified a somatic mutation in the thrombopoietin receptor (MPLW515L) in patients with JAK2V617F-negative MF. Expression of MPLW515L transforms hematopoietic cells to cytokine-independent growth, and activated components of the JAK-STAT signal transduction pathway including JAK2, STAT5, STAT3, ERK and AKT. We compared the disease phenotype induced by MPLW515L with that we have previously reported for the JAK2V617F allele in a murine bone marrow transplant model of myeloproliferative disease. In Balb/C mice, expression of MPLW515L caused a rapidly fatal, fully penetrant MPD (median latency of ~18 days) that was characterized by marked leukocytosis and thrombocytosis, with average platelet count of 3.4 million/ul as well as bone marrow reticulin fibrosis and thrombotic complications. A similar phenotype was observed in MPLW515L C57/Bl6 recipients, but with a longer median latency of ~50 days. In a subset of C57/Bl6 mice, the acute MPD was followed by a progressive reduction in platelet count and an increase in bone marrow fibrosis that occurred over 3 months, and was reminiscent of human MF. These findings stand in contrast to MPD induced by the JAK2V617F allele in C57/Bl6 mice, in which there is marked erythrocytosis, but not thrombocytosis or thrombotic complications or leukocytosis. Analysis of myeloid progenitor populations using multiparameter flow cytometry demonstrated an expansion of the common myeloid progenitor (CMP), the granulocyte-monocyte progenitor (GMP) population, and the megakaryocytic-erythroid progenitor (MEP) population in MPLW515L-expressing bone marrow and spleen, with up to a 10-fold increase in CMPs and an 8-fold increase in MEPs, whereas there were no differences observed in the relative proportion of CMP, GMP or MEP populations in JAK2V617F mice. These data demonstrate important phenotypic differences between disease induced by the MPLW515L or JAK2V617F alleles in C57/Bl6 mice. These include (i) marked differences in the degree of leukocytosis, with MPLW515L induced marked leukocytosis compared with JAK2V617F and (ii) the effect on the megakaryocyte lineage in which JAK2V617F induces megakaryopoiesis, but with reduced megakaryocyte ploidy with no thrombocytosis or thrombotic complications, whereas MPLW515L enhances megakarypoiesis and induces thrombocytosis with thrombotic complications. These findings suggest that JAK2V617F and MPLW515L - each of which is characterized by activation of JAK-STAT signaling in hematopoietic progenitors - have qualitative and quantitative differences in their ability to impact proliferation and/or survival of hematopoietic progenitors that presumably are the consequence of differences in signal transduction, and result in disparate phenotypes.
Cell replacement therapies for neurodegenerative disease have focused on transplantation of the cell types affected by the pathological process. Here we describe an alternative strategy for ...Parkinson's disease in which dopamine neurons are generated by direct conversion of astrocytes. Using three transcription factors, NEUROD1, ASCL1 and LMX1A, and the microRNA miR218, collectively designated NeAL218, we reprogram human astrocytes in vitro, and mouse astrocytes in vivo, into induced dopamine neurons (iDANs). Reprogramming efficiency in vitro is improved by small molecules that promote chromatin remodeling and activate the TGFβ, Shh and Wnt signaling pathways. The reprogramming efficiency of human astrocytes reaches up to 16%, resulting in iDANs with appropriate midbrain markers and excitability. In a mouse model of Parkinson's disease, NeAL218 alone reprograms adult striatal astrocytes into iDANs that are excitable and correct some aspects of motor behavior in vivo, including gait impairments. With further optimization, this approach may enable clinical therapies for Parkinson's disease by delivery of genes rather than cells.
MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES ...cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity.
Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct ...conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an “on-target” pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.
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•Ascl1 has pioneer activity, accessing closed chromatin to allow other factors to bind•Unlike other pioneer factors, Ascl1 binds its physiologic neural targets in fibroblasts•A trivalent chromatin domain predicts iN reprogramming ability in other cell types•Zfp238 is a direct Ascl1 target and critical mediator of iN cell reprogramming
Of three transcription factors that together convert different cell types into induced neuronal (iN) cells, Ascl1 leads the way as the “pioneer factor,” followed by Brn2 and Myt1l. A trivalent chromatin state at Ascl1 target genes predicts which types of cells will respond to reprogramming by these factors.
In post‐mitotic tissues, damaged cells are not replaced by new cells and hence effective local tissue repair mechanisms are required. In skeletal muscle, which is a syncytium, additional nuclei are ...obtained from muscle satellite (stem) cells that multiply and then fuse with the damaged fibres. Although insulin‐like growth factor‐I (IGF‐I) had been previously implicated, it is now clear that muscle expresses at least two splice variants of the IGF‐I gene: a mechanosensitive, autocrine, growth factor (MGF) and one that is similar to the liver type (IGF‐IEa). To investigate this activation mechanism, local damage was induced by stretch combined with electrical stimulation or injection of bupivacaine in the rat anterior tibialis muscle and the time course of regeneration followed morphologically. Satellite cell activation was studied by the distribution and levels of expression of M‐cadherin (M‐cad) and related to the expression of the two forms of IGF‐I. It was found that the following local damage MGF expression preceded that of M‐cad whereas IGF‐IEa peaked later than M‐cad. The evidence suggests therefore that an initial pulse of MGF expression following damage is what activates the satellite cells and that this is followed by the later expression of IGF‐IEa to maintain protein synthesis to complete the repair.
Cell lineage specification is accomplished by a concerted action of chromatin remodeling and tissue-specific transcription factors. However, the mechanisms that induce and maintain appropriate ...lineage-specific gene expression remain elusive. Here, we used an unbiased proteomics approach to characterize chromatin regulators that mediate the induction of neuronal cell fate. We found that Tip60 acetyltransferase is essential to establish neuronal cell identity partly via acetylation of the histone variant H2A.Z. Despite its tight correlation with gene expression and active chromatin, loss of H2A.Z acetylation had little effect on chromatin accessibility or transcription. Instead, loss of Tip60 and acetyl-H2A.Z interfered with H3K4me3 deposition and activation of a unique subset of silent, lineage-restricted genes characterized by a bivalent chromatin configuration at their promoters. Altogether, our results illuminate the mechanisms underlying bivalent chromatin activation and reveal that H2A.Z acetylation regulates neuronal fate specification by establishing epigenetic competence for bivalent gene activation and cell lineage transition.
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•Tip60 acetyltransferase is essential for neuronal specification•Primary substrate of Tip60 during neuronal induction is histone variant H2A.Z•Tip60/H2A.Zac promote H3K4me3 deposition and activation of bivalent promoters•Bivalent gene activation is necessary for neuronal induction
Janas et al. report that Tip60 acetyltransferase is essential for neuronal cell fate specification. The study reveals that one of the functions of Tip60 during neuronal induction is the acetylation of histone variant H2A.Z, which promotes H3K4me3 deposition and bivalent gene activation, thus establishing epigenetic competence for cell lineage transition.
The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins ...are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. Here we show that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, we found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed, and were preferentially activated on induction of differentiation. Our results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development.
Hallmarks of pluripotency De Los Angeles, Alejandro; Ferrari, Francesco; Xi, Ruibin ...
Nature (London),
09/2015, Letnik:
525, Številka:
7570
Journal Article
Recenzirano
Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem ...cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.
Mutations in β-amyloid (Aβ) precursor protein (
) cause familial Alzheimer's disease (AD) probably by enhancing Aβ peptides production from APP. An antibody targeting Aβ (aducanumab) was approved as ...an AD treatment; however, some Aβ antibodies have been reported to accelerate, instead of ameliorating, cognitive decline in individuals with AD. Using conditional APP mutations in human neurons for perfect isogenic controls and translational relevance, we found that the
-Swedish mutation in familial AD increased synapse numbers and synaptic transmission, whereas the
deletion decreased synapse numbers and synaptic transmission. Inhibition of BACE1, the protease that initiates Aβ production from APP, lowered synapse numbers, suppressed synaptic transmission in wild-type neurons, and occluded the phenotype of
-Swedish-mutant neurons. Modest elevations of Aβ, conversely, elevated synapse numbers and synaptic transmission. Thus, the familial AD-linked
-Swedish mutation under physiologically relevant conditions increased synaptic connectivity in human neurons via a modestly enhanced production of Aβ. These data are consistent with the relative inefficacy of BACE1 and anti-Aβ treatments in AD and the chronic nature of AD pathogenesis, suggesting that AD pathogenesis is not simply caused by overproduction of toxic Aβ but rather by a long-term effect of elevated Aβ concentrations.
Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four ...transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, was only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming.
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