ABSTRACT
Methylated cytosines are associated with gene silencing. The ten-eleven translocation (TET) hydroxylases, which oxidize methylated cytosines to 5-hydroxymethylcytosine (5hmC), are essential for cytosine demethylation. Gene silencing and activation are critical for intestinal stem cell (ISC) maintenance and differentiation, but the potential role of TET hydroxylases in these processes has not yet been examined. Here, we generated genome-wide maps of the 5hmC mark in ISCs and their differentiated progeny. Genes with high levels of hydroxymethylation in ISCs are strongly associated with Wnt signaling and developmental processes. We found Tet1 to be the most abundantly expressed Tet gene in ISCs; therefore, we analyzed intestinal development in Tet1-deficient mice and determined that these mice are growth-retarded, exhibit partial postnatal lethality, and have significantly reduced numbers of proliferative cells in the intestinal epithelium. In addition, the Tet1-deficient intestine displays reduced organoid-forming capacity. In the Tet1-deficient crypt, decreased expression of Wnt target genes such as Axin2 and Lgr5 correlates with lower 5hmC levels at their promoters. These data demonstrate that Tet1-mediated DNA hydroxymethylation plays a critical role in the epigenetic regulation of the Wnt pathway in intestinal stem and progenitor cells and consequently in the self-renewal of the intestinal epithelium.
Subject(s)
DNA Methylation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Developmental/genetics , Intestines/growth & development , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Stem Cells/physiology , Animals , Cell Differentiation/genetics , Cells, Cultured , Intestines/cytology , Mice , Mice, Inbred C57BL , Receptors, G-Protein-Coupled/genetics , Sequence Deletion , Stem Cells/cytology , Wnt Signaling Pathway/geneticsABSTRACT
The mammalian intestinal epithelium has a unique organization in which crypts harboring stem cells produce progenitors and finally clonal populations of differentiated cells. Remarkably, the epithelium is replaced every 3-5 d throughout adult life. Disrupted maintenance of the intricate balance of proliferation and differentiation leads to loss of epithelial integrity or barrier function or to cancer. There is a tight correlation between the epigenetic status of genes and expression changes during differentiation; however, the mechanism of how changes in DNA methylation direct gene expression and the progression from stem cells to their differentiated descendants is unclear. Using conditional gene ablation of the maintenance methyltransferase Dnmt1, we demonstrate that reducing DNA methylation causes intestinal crypt expansion in vivo. Determination of the base-resolution DNA methylome in intestinal stem cells and their differentiated descendants shows that DNA methylation is dynamic at enhancers, which are often associated with genes important for both stem cell maintenance and differentiation. We establish that the loss of DNA methylation at intestinal stem cell gene enhancers causes inappropriate gene expression and delayed differentiation.
Subject(s)
Cell Differentiation , DNA Methylation , Intestine, Small/cytology , Stem Cells/cytology , Animals , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation/genetics , Gene Deletion , Gene Expression Regulation, Developmental , MiceABSTRACT
BACKGROUND: Recent mapping of 5-hydroxymethylcytosine (5hmC) provides a genome-wide view of the distribution of this important chromatin mark. However, the role of 5hmC in specific regulatory regions is not clear, especially at enhancers. RESULTS: We found a group of distal transcription factor binding sites highly enriched for 5-hdroxymethylcytosine (5hmC), but lacking any known activating histone marks and being depleted for nascent transcripts, suggesting a repressive role for 5hmC in mouse embryonic stem cells (mESCs). 5-formylcytosine (5fC), which is known to mark poised enhancers where H3K4me1 is enriched, is also observed at these sites. Furthermore, the 5hmC levels were inversely correlated with RNA polymerase II (PolII) occupancy in mESCs as well as in fully differentiated adipocytes. Interestingly, activating H3K4me1/2 histone marks were enriched at these sites when the associated genes become activated following lineage specification. These putative enhancers were shown to be functional in embryonic stem cells when unmethylated. Together, these data suggest that 5hmC suppresses the activity of this group of enhancers, which we termed "silenced enhancers". CONCLUSIONS: Our findings indicate that 5hmC has a repressive role at specific proximal and distal regulatory regions in mESCs, and suggest that 5hmC is a new epigenetic mark for silenced enhancers.
Subject(s)
Cytosine/analogs & derivatives , Embryonic Stem Cells/metabolism , Enhancer Elements, Genetic/genetics , Epigenesis, Genetic , 5-Methylcytosine/analogs & derivatives , Animals , Binding Sites , Cell Lineage , Cytosine/metabolism , Embryonic Stem Cells/cytology , Histones/metabolism , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/metabolismABSTRACT
The differentiated features of postmitotic neurons are dictated by the expression of specific transcription factors. The mechanisms by which the precise spatiotemporal expression patterns of these factors are regulated are poorly understood. In C. elegans, the ceh-36 Otx homeobox gene is expressed in the AWC sensory neurons throughout postembryonic development, and regulates terminal differentiation of this neuronal subtype. Here, we show that the HMX/NKX homeodomain protein MLS-2 regulates ceh-36 expression specifically in the AWC neurons. Consequently, the AWC neurons fail to express neuron type-specific characteristics in mls-2 mutants. mls-2 is expressed transiently in postmitotic AWC neurons, and directly initiates ceh-36 expression. CEH-36 subsequently interacts with a distinct site in its cis-regulatory sequences to maintain its own expression, and also directly regulates the expression of AWC-specific terminal differentiation genes. We also show that MLS-2 acts in additional neuron types to regulate their development and differentiation. Our analysis describes a transcription factor cascade that defines the unique postmitotic characteristics of a sensory neuron subtype, and provides insights into the spatiotemporal regulatory mechanisms that generate functional diversity in the sensory nervous system.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/genetics , Cell Differentiation/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/physiology , Sensory Receptor Cells/physiology , Transcription Factors/genetics , Alleles , Amino Acid Sequence , Animals , Animals, Genetically Modified , Caenorhabditis elegans/embryology , Caenorhabditis elegans Proteins/metabolism , Embryo, Nonmammalian , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Molecular Sequence Data , Mutation/physiology , Otx Transcription Factors/genetics , Polymorphism, Single Nucleotide , Sensory Receptor Cells/classification , Sensory Receptor Cells/metabolismABSTRACT
Extravasation of monocytes into tissue and to the site of injury is a fundamental immunological process, which requires rapid responses via post translational modifications (PTM) of proteins. Protein arginine methyltransferase 7 (PRMT7) is an epigenetic factor that has the capacity to mono-methylate histones on arginine residues. Here we show that in chronic obstructive pulmonary disease (COPD) patients, PRMT7 expression is elevated in the lung tissue and localized to the macrophages. In mouse models of COPD, lung fibrosis and skin injury, reduced expression of PRMT7 associates with decreased recruitment of monocytes to the site of injury and hence less severe symptoms. Mechanistically, activation of NF-κB/RelA in monocytes induces PRMT7 transcription and consequential mono-methylation of histones at the regulatory elements of RAP1A, which leads to increased transcription of this gene that is responsible for adhesion and migration of monocytes. Persistent monocyte-derived macrophage accumulation leads to ALOX5 over-expression and accumulation of its metabolite LTB4, which triggers expression of ACSL4 a ferroptosis promoting gene in lung epithelial cells. Conclusively, inhibition of arginine mono-methylation might offer targeted intervention in monocyte-driven inflammatory conditions that lead to extensive tissue damage if left untreated.