ABSTRACT
The on-target pioneer factors Ascl1 and Myod1 are sequence-related but induce two developmentally unrelated lineages-that is, neuronal and muscle identities, respectively. It is unclear how these two basic helix-loop-helix (bHLH) factors mediate such fundamentally different outcomes. The chromatin binding of Ascl1 and Myod1 was surprisingly similar in fibroblasts, yet their transcriptional outputs were drastically different. We found that quantitative binding differences explained differential chromatin remodelling and gene activation. Although strong Ascl1 binding was exclusively associated with bHLH motifs, strong Myod1-binding sites were co-enriched with non-bHLH motifs, possibly explaining why Ascl1 is less context dependent. Finally, we observed that promiscuous binding of Myod1 to neuronal targets results in neuronal reprogramming when the muscle program is inhibited by Myt1l. Our findings suggest that chromatin access of on-target pioneer factors is primarily driven by the protein-DNA interaction, unlike ordinary context-dependent transcription factors, and that promiscuous transcription factor binding requires specific silencing mechanisms to ensure lineage fidelity.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , MyoD Protein/genetics , Nerve Tissue Proteins/genetics , Neurons/metabolism , Transcription Factors/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites , Cell Lineage/genetics , Cellular Reprogramming , Chromatin/chemistry , Chromatin/metabolism , Embryo, Mammalian , Fibroblasts/cytology , Mice , Mice, Inbred C57BL , Mice, Transgenic , MyoD Protein/metabolism , Nerve Tissue Proteins/metabolism , Neurons/cytology , Nucleotide Motifs , Protein Binding , Signal Transduction , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.
Subject(s)
Autism Spectrum Disorder/genetics , Cell Differentiation/genetics , Mutation , Neurodevelopmental Disorders/genetics , Neurons/metabolism , RNA, Long Noncoding/genetics , Autism Spectrum Disorder/pathology , Child , Chromosomes, Human, Pair 12/genetics , Chromosomes, Human, Pair 5/genetics , DNA Copy Number Variations , Female , Gene Expression Profiling/methods , Humans , Male , Neurodevelopmental Disorders/pathology , Neurogenesis/genetics , Neurons/cytology , Pedigree , Translocation, Genetic/geneticsABSTRACT
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 hr 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 is accomplished with relatively little chromatin reconfiguration. By 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 stepwise cell fate transitions in development.
Subject(s)
Chromatin/metabolism , Fibroblasts/metabolism , Neurons/metabolism , Cellular Reprogramming , HumansABSTRACT
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.
Subject(s)
Cellular Reprogramming , Embryo, Mammalian/cytology , Fibroblasts/cytology , Gene Regulatory Networks , Neurons/cytology , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation , Chromatin/metabolism , Fibroblasts/metabolism , Genome-Wide Association Study , Humans , Mice , Nerve Tissue Proteins/metabolism , Neurons/metabolism , POU Domain Factors/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolismABSTRACT
Long noncoding RNAs (lncRNAs) are thought to be prevalent regulators of gene expression, but the consequences of lncRNA inactivation in vivo are mostly unknown. Here, we show that targeted deletion of mouse Hotair lncRNA leads to derepression of hundreds of genes, resulting in homeotic transformation of the spine and malformation of metacarpal-carpal bones. RNA sequencing and conditional inactivation reveal an ongoing requirement of Hotair to repress HoxD genes and several imprinted loci such as Dlk1-Meg3 and Igf2-H19 without affecting imprinting choice. Hotair binds to both Polycomb repressive complex 2, which methylates histone H3 at lysine 27 (H3K27), and Lsd1 complex, which demethylates histone H3 at lysine 4 (H3K4) in vivo. Hotair inactivation causes H3K4me3 gain and, to a lesser extent, H3K27me3 loss at target genes. These results reveal the function and mechanisms of Hotair lncRNA in enforcing a silent chromatin state at Hox and additional genes.
Subject(s)
Bone and Bones/abnormalities , Gene Expression Regulation, Developmental , RNA, Long Noncoding/biosynthesis , RNA, Long Noncoding/genetics , Animals , Bone Development/genetics , Bone and Bones/embryology , Female , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Mice , Mice, Knockout , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
Long noncoding RNAs (lncRNAs) are increasingly appreciated as regulators of cell-specific gene expression. Here, an enhancer-like lncRNA termed NeST (nettoie Salmonella pas Theiler's [cleanup Salmonella not Theiler's]) is shown to be causal for all phenotypes conferred by murine viral susceptibility locus Tmevp3. This locus was defined by crosses between SJL/J and B10.S mice and contains several candidate genes, including NeST. The SJL/J-derived locus confers higher lncRNA expression, increased interferon-γ (IFN-γ) abundance in activated CD8(+) T cells, increased Theiler's virus persistence, and decreased Salmonella enterica pathogenesis. Transgenic expression of NeST lncRNA alone was sufficient to confer all phenotypes of the SJL/J locus. NeST RNA was found to bind WDR5, a component of the histone H3 lysine 4 methyltransferase complex, and to alter histone 3 methylation at the IFN-γ locus. Thus, this lncRNA regulates epigenetic marking of IFN-γ-encoding chromatin, expression of IFN-γ, and susceptibility to a viral and a bacterial pathogen.
Subject(s)
Disease Susceptibility , Epigenesis, Genetic , Interferon-gamma/genetics , RNA, Long Noncoding/genetics , Animals , CD8-Positive T-Lymphocytes/immunology , Cardiovirus Infections/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Salmonella Infections/immunology , Salmonella typhimurium/immunology , Theilovirus/immunologyABSTRACT
In adult stem cell lineages, progenitor cells commonly undergo mitotic transit amplifying (TA) divisions before terminal differentiation, allowing production of many differentiated progeny per stem cell division. Mechanisms that limit TA divisions and trigger the switch to differentiation may protect against cancer by preventing accumulation of oncogenic mutations in the proliferating population. Here we show that the switch from TA proliferation to differentiation in the Drosophila male germline stem cell lineage is mediated by translational control. The TRIM-NHL tumor suppressor homolog Mei-P26 facilitates accumulation of the differentiation regulator Bam in TA cells. In turn, Bam and its partner Bgcn bind the mei-P26 3' untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through distinct, sequential regulatory states, from Mei-P26 on/Bam off to Bam on/Mei-P26 off. TRIM-NHL homologs across species facilitate the switch from proliferation to differentiation, suggesting a conserved developmentally programmed tumor suppressor mechanism.
Subject(s)
Adult Stem Cells/cytology , Cell Differentiation , Cell Lineage , Drosophila/cytology , Gene Expression Regulation , Adult Stem Cells/metabolism , Animals , Cell Division , DNA Helicases/genetics , DNA Helicases/metabolism , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Germ Cells/cytology , Germ Cells/metabolism , MaleABSTRACT
The ubiquitin-proteasome system catalyzes the degradation of intracellular proteins. Although ubiquitination of proteins determines their stabilities, there is growing evidence that proteasome function is also regulated. We report the functional characterization of a conserved proteasomal regulatory complex. We identified DmPI31 as a binding partner of the F box protein Nutcracker, a component of an SCF ubiquitin ligase (E3) required for caspase activation during sperm differentiation in Drosophila. DmPI31 binds Nutcracker via a conserved mechanism that is also used by mammalian FBXO7 and PI31. Nutcracker promotes DmPI31 stability, which is necessary for caspase activation, proteasome function, and sperm differentiation. DmPI31 can activate 26S proteasomes in vitro, and increasing DmPI31 levels suppresses defects caused by diminished proteasome activity in vivo. Furthermore, loss of DmPI31 function causes lethality, cell-cycle abnormalities, and defects in protein degradation, demonstrating that DmPI31 is physiologically required for normal proteasome activity.
Subject(s)
Carrier Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/physiology , F-Box Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Caspases/metabolism , Cell Line , Drosophila Proteins/analysis , Drosophila Proteins/genetics , Humans , Male , Mice , Molecular Sequence Data , Proteome/analysis , Sequence Alignment , Spermatogenesis , Testis/metabolismABSTRACT
BACKGROUND: Hydrodynamic injection is an effective method for DNA delivery in mouse liver and is being translated to larger animals for possible clinical use. Similarly, phiC31 integrase has proven effective in mediating long-term gene therapy in mice when delivered by hydrodynamic injection and is being considered for clinical gene therapy applications. However, chromosomal aberrations have been associated with phiC31 integrase expression in tissue culture, leading to questions about safety. METHODOLOGY/PRINCIPAL FINDINGS: To study whether hydrodynamic delivery alone, or in conjunction with delivery of phiC31 integrase for long-term transgene expression, could facilitate tumor formation, we used a transgenic mouse model in which sustained induction of the human C-MYC oncogene in the liver was followed by hydrodynamic injection. Without injection, mice had a median tumor latency of 154 days. With hydrodynamic injection of saline alone, the median tumor latency was significantly reduced, to 105 days. The median tumor latency was similar, 106 days, when a luciferase donor plasmid and backbone plasmid without integrase were administered. In contrast, when active or inactive phiC31 integrase and donor plasmid were supplied to the mouse liver, the median tumor latency was 153 days, similar to mice receiving no injection. CONCLUSIONS/SIGNIFICANCE: Our data suggest that phiC31 integrase does not facilitate tumor formation in this C-MYC transgenic mouse model. However, in groups lacking phiC31 integrase, hydrodynamic injection appeared to contribute to C-MYC-induced hepatocellular carcinoma in adult mice. Although it remains to be seen to what extent these findings may be extrapolated to catheter-mediated hydrodynamic delivery in larger species, they suggest that caution should be used during translation of hydrodynamic injection to clinical applications.