The extent and biological impact of RNA cytosine methylation are poorly understood, in part owing to limitations of current techniques for determining the targets of RNA methyltransferases. Here we ...describe 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP), a technique that exploits the covalent bond formed between an RNA methyltransferase and the cytidine analog 5-azacytidine to recover RNA targets by immunoprecipitation. Targets are subsequently identified by high-throughput sequencing. When applied in a human cell line to the RNA methyltransferases DNMT2 and NSUN2, Aza-IP enabled >200-fold enrichment of tRNAs that are known targets of the enzymes. In addition, it revealed many tRNA and noncoding RNA targets not previously associated with NSUN2. Notably, we observed a high frequency of C→G transversions at the cytosine residues targeted by both enzymes, allowing identification of the specific methylated cytosine(s) in target RNAs. Given the mechanistic similarity of RNA cytosine methyltransferases, Aza-IP may be generally applicable for target identification.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The regulation of gene transcription involves a dynamic balance between packaging regulatory sequences into chromatin and allowing transcriptional regulators access to these sequences. Access is ...restricted by the nucleosomes, but these can be repositioned or ejected by enzymes known as nucleosome remodellers. In addition, the DNA sequence can impart stiffness or curvature to the DNA, thereby affecting the position of nucleosomes on the DNA, influencing particular promoter 'architectures'. Recent genome-wide studies in yeast suggest that constitutive and regulated genes have architectures that differ in terms of nucleosome position, turnover, remodelling requirements and transcriptional noise.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The packaging of chromosomal DNA by nucleosomes condenses and organizes the genome, but occludes many regulatory DNA elements. However, this constraint also allows nucleosomes and other chromatin ...components to actively participate in the regulation of transcription, chromosome segregation, DNA replication, and DNA repair. To enable dynamic access to packaged DNA and to tailor nucleosome composition in chromosomal regions, cells have evolved a set of specialized chromatin remodeling complexes (remodelers). Remodelers use the energy of ATP hydrolysis to move, destabilize, eject, or restructure nucleosomes. Here, we address many aspects of remodeler biology: their targeting, mechanism, regulation, shared and unique properties, and specialization for particular biological processes. We also address roles for remodelers in development, cancer, and human syndromes.
Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc.), trinucleotide ...expansion associated diseases (myotonic dystrophy, Huntington's, etc.) and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body). Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cells utilize diverse ATP-dependent nucleosome-remodelling complexes to carry out histone sliding, ejection or the incorporation of histone variants, suggesting that different mechanisms of action ...are used by the various chromatin-remodelling complex subfamilies. However, all chromatin-remodelling complex subfamilies contain an ATPase-translocase 'motor' that translocates DNA from a common location within the nucleosome. In this Review, we discuss (and illustrate with animations) an alternative, unifying mechanism of chromatin remodelling, which is based on the regulation of DNA translocation. We propose the 'hourglass' model of remodeller function, in which each remodeller subfamily utilizes diverse specialized proteins and protein domains to assist in nucleosome targeting or to differentially detect nucleosome epitopes. These modules converge to regulate a common DNA translocation mechanism, to inform the conserved ATPase 'motor' on whether and how to apply DNA translocation, which together achieve the various outcomes of chromatin remodelling: nucleosome assembly, chromatin access and nucleosome editing.
Adult germline stem cells (AGSCs) self-renew (Thy1+ enriched) or commit to gametogenesis (Kit+ enriched). To better understand how chromatin regulates AGSC biology and gametogenesis, we derived ...stage-specific high-resolution profiles of DNA methylation, 5hmC, histone modifications/variants, and RNA-seq in AGSCs and during spermatogenesis. First, we define striking signaling and transcriptional differences between AGSC types, involving key self-renewal and proliferation pathways. Second, key pluripotency factors (e.g., Nanog) are silent in AGSCs and bear particular chromatin/DNAme attributes that may “poise” them for reactivation after fertilization. Third, AGSCs display chromatin “poising/bivalency” of enhancers and promoters for embryonic transcription factors. Remarkably, gametogenesis occurs without significant changes in DNAme and instead involves transcription of DNA-methylated promoters bearing high RNAPol2, H3K9ac, H3K4me3, low CG content, and (often) 5hmC. Furthermore, key findings were confirmed in human sperm. Here, we reveal AGSC signaling asymmetries and chromatin/DNAme strategies in AGSCs to poise key transcription factors and to activate DNA-methylated promoters during gametogenesis.
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•Self-renewing (Thy1+) versus differentiating (Kit+) germline stem cells are profiled•Thy1+ to Kit+ comparisons reveal major differences in signaling and transcription•Promoters and enhancers for pluripotency genes are “poised” by chromatin•Gametogenesis involves transcription with DNA methylation at many promoters
By comparing high-resolution transcriptome and epigenome profiles of mouse adult spermatogonial stem cells and their progeny, Hammoud et al. provide a comprehensive resource useful for gaining insight into state transitions of self-renewing adult somatic stem cells.
Early vertebrate embryos must achieve totipotency and prepare for zygotic genome activation (ZGA). To understand this process, we determined the DNA methylation (DNAme) profiles of zebrafish gametes, ...embryos at different stages, and somatic muscle and compared them to gene activity and histone modifications. Sperm chromatin patterns are virtually identical to those at ZGA. Unexpectedly, the DNA of many oocyte genes important for germline functions (i.e., piwil1) or early development (i.e., hox genes) is methylated, but the loci are demethylated during zygotic cleavage stages to precisely the state observed in sperm, even in parthenogenetic embryos lacking a replicating paternal genome. Furthermore, this cohort constitutes the genes and loci that acquire DNAme during development (i.e., ZGA to muscle). Finally, DNA methyltransferase inhibition experiments suggest that DNAme silences particular gene and chromatin cohorts at ZGA, preventing their precocious expression. Thus, zebrafish achieve a totipotent chromatin state at ZGA through paternal genome competency and maternal genome DNAme reprogramming.
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•Sperm chromatin patterns mirror the blastomere at zygotic genome activation (ZGA)•Maternal chromatin patterns are reprogrammed to the paternal/sperm state by ZGA•Maternal reprogrammed/demethylated genes are all later methylated in development•Promoter DNA methylation prevents precocious expression of particular genes at ZGA
In the zebrafish zygote, the maternal genome undergoes considerable DNA methylation changes so that it conforms to the paternal pattern by the time zygotic genes are activated, a process that helps establish a totipotent zygotic genome.
To better understand transcriptional regulation during human oogenesis and preimplantation development, we defined stage-specific transcription, which highlighted the cleavage stage as being highly ...distinctive. Here, we present multiple lines of evidence that a eutherian-specific multicopy retrogene, DUX4, encodes a transcription factor that activates hundreds of endogenous genes (for example, ZSCAN4, KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably, mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells, measured here by the reactivation of '2C' genes and repeat elements, the loss of POU5F1 (also known as OCT4) protein and chromocenters, and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus, we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state.
Human testis development in prenatal life involves complex changes in germline and somatic cell identity. To better understand, we profiled and analyzed ∼32,500 single-cell transcriptomes of ...testicular cells from embryonic, fetal, and infant stages. Our data show that at 6–7 weeks postfertilization, as the testicular cords are established, the Sertoli and interstitial cells originate from a common heterogeneous progenitor pool, which then resolves into fetal Sertoli cells (expressing tube-forming genes) or interstitial cells (including Leydig-lineage cells expressing steroidogenesis genes). Almost 10 weeks later, beginning at 14–16 weeks postfertilization, the male primordial germ cells exit mitosis, downregulate pluripotent transcription factors, and transition into cells that strongly resemble the state 0 spermatogonia originally defined in the infant and adult testes. Therefore, we called these fetal spermatogonia “state f0.” Overall, we reveal multiple insights into the coordinated and temporal development of the embryonic, fetal, and postnatal male germline together with the somatic niche.
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•A transcriptional single-cell atlas of the fetal and postnatal human testes•Somatic niche cell types derive from a common progenitor ∼7 weeks after fertilization•PGCs transition directly into fetal state 0-like cells (state f0) starting at week 14•Fetal somatic niche cell specification precedes the PGC-to-state f0 transition
Guo et al. provide a transcriptional cell atlas of the fetal and postnatal human testes. Remarkably, starting from ∼14 weeks postfertilization, fetal primordial germ cells transition to a cell state highly similar to postnatal spermatogonial stem cells. Furthermore, somatic niche specification precedes this transition, which is consistent with guiding fetal germline development.
The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with ...other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously, transcriptome-wide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m⁵C, Ψ, and m¹A at single-base resolution transcriptome-wide. With RBS-Seq, m⁵C and m¹A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1–2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m⁵C, m¹A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m⁵C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting ∼10-fold fewer m⁵C sites in noncoding and coding RNAs and the absence of substantial m¹A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.
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