the-detection-5-hydroxymethylcytosine-neural-stem-cells-brains

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Zhejiang University

The pursuit of DNA demethylation has a colorful history, but it was not until 2009 that the stars of this story, the Ten-eleven-translocation (Tet) family of proteins, were really identified. Tet proteins convert 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which can be further oxidized to 5-formylcytosine and 5-cyboxycytosine by Tet proteins to achieve DNA demethylation. Recent studies have revealed that 5hmC-mediated DNA demethylation can play essential roles in diverse biological processes, including development and diseases. Here, we review recent discoveries in 5hmC-mediated DNA demethylation in the context of stem cells and development. 

5-hydroxymethylcytosine-mediated DNA demethylation in stem cells and development.

Epigenetic modulations play essential roles in diverse biological processes. During the past several years, DNA demethylation has been discovered in embryonic and postnatal development. Although some potential functions of DNA methylation have been demonstrated already, many questions remain in terms of unveiling the role of 5hmC; whether it serves either merely as an intermediate of DNA demethylation or as a stable epigenetic marker. 5-hydroxymethylcytosine (5hmC) is proved to be not merely serving as an intermediate of DNA demethylation, but also acts as a stable epigenetic marker. This review summarizes the current knowledge of the function of 5hmC in brain with the focus on the neuronal activity, neurodevelopment, aging, and neurological diseases. 

From development to diseases: the role of 5hmC in brain.

Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons. The precise control of the phosphorylation status of MeCP2 in neurons is critical for the normal development and function of the mammalian brain. However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts. Here we show that in neural progenitor cells isolated from the adult mouse hippocampus, cell cycle-linked phosphorylation at serine 421 on MeCP2 is directly regulated by aurora kinase B and modulates the balance between proliferation and neural differentiation through the Notch signalling pathway. Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types. 

Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signalling pathway.

Spinal cord injury (SCI) refers to the damage of spinal cord's structure and function due to a variety of causes. At present, many scholars have confirmed that apoptosis is the main method of secondary injury in spinal cord injury. In view of understanding the function of PI3K/Akt pathway on spinal cord injury, this study observed the temporal variation of key molecules (PI3K, Akt, p-Akt) in the PI3K/Akt pathway after spinal cord injury by immunohistochemistry and Western-blot. The results showed that the expression of PI3K, Akt and p-Akt display a sharp increase one day after the spinal cord injury, and then it decreased gradually with the time passing by, but the absolute expression was certainly higher than the normal group. These results indicate that the PI3K/Akt signaling pathway is involved in the spinal cord injury and the mechanism may be related to apoptosis. 

The change tendency of PI3K/Akt pathway after spinal cord injury.

Alzheimer's disease (AD) is the most common form of neurodegenerative disorder that leads to a decline in cognitive function. In AD, aggregates of amyloid β peptide precede the accumulation of neurofibrillary tangles, both of which are hallmarks of the disease. The great majority (>90 %) of the AD cases are not originated from genetic defects, therefore supporting the central roles of epigenetic modifications that are acquired progressively during the life span. Strong evidences have indicated the implication of epigenetic modifications, including histone modification and DNA methylation, in AD. Recent studies revealed that 5-hydroxymethylcytosine (5hmC) is dynamically regulated during neurodevelopment and aging.

Genome-wide alteration of 5-hydroxymenthylcytosine in a mouse model of Alzheimer's disease.

iPSCs: From Bench to Clinical Bed.

Fat mass and obesity-associated gene (FTO) is a member of the Fe (II)- and oxoglutarate-dependent AlkB dioxygenase family and is linked to both obesity and intellectual disability. The role of FTO in neurodevelopment and neurogenesis, however, remains largely unknown. Here we show that FTO is expressed in adult neural stem cells and neurons and displays dynamic expression during postnatal neurodevelopment. The loss of FTO leads to decreased brain size and body weight. We find that FTO deficiency could reduce the proliferation and neuronal differentiation of adult neural stem cells in vivo, which leads to impaired learning and memory. Given the role of FTO as a demethylase of N6-methyladenosine (m6A), we went on to perform genome-wide m6A profiling and observed dynamic m6A modification during postnatal neurodevelopment. The loss of FTO led to the altered expression of several key components of the brain derived neurotrophic factor pathway that were marked by m6A. These results together suggest FTO plays important roles in neurogenesis, as well as in learning and memory.

Fat mass and obesity-associated (FTO) protein regulates adult neurogenesis.

Emerging evidence suggests that active DNA demethylation machinery plays important epigenetic roles in mammalian adult neurogenesis; however, the precise molecular mechanisms and critical functional players of DNA demethylation in this process remain largely unexplored. Ten-eleven translocation (Tet) proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and its downstream derivatives. Here we show that 5hmC is elevated during the differentiation of adult neural stem cells (aNSCs), and Tet2 is primarily responsible for modulating 5hmC dynamics. Depletion of Tet2 leads to increased aNSC proliferation and reduced differentiation in vitro and in vivo. Genome-wide transcriptional analyses reveal important epigenetic roles of Tet2 in maintaining the transcriptome landscape related to neurogenesis. Mechanistically, transcription factor forkhead box O3 (Foxo3a) physically interacts with Tet2 and regulates the expression of genes related to aNSC proliferation. These data together establish an important role for the Tet2-Foxo3a axis in epigenetically regulating critical genes in aNSCs during adult neurogenesis.

Ten-eleven translocation 2 interacts with forkhead box O3 and regulates adult neurogenesis.

DNA demethylation, the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), plays important roles in diverse biological processes and multiple diseases by regulating gene expression.

The Dynamic DNA Demethylation during Postnatal Neuronal Development and Neural Stem Cell Differentiation.

Autism spectrum disorders (ASDs) include a group of syndromes characterized by impaired language, social and communication skills, in addition to restrictive behaviors or stereotypes. However, with a prevalence of 1.5% in developed countries and high comorbidity rates, no clear underlying mechanism that unifies the heterogeneous phenotypes of ASD exists. 5-hydroxymethylcytosine (5hmC) is highly enriched in the brain and recognized as an essential epigenetic mark in developmental and brain disorders. To explore the role of 5hmC in ASD, we used the genomic DNA isolated from the postmortem cerebellum of both ASD patients and age-matched controls to profile genome-wide distribution of 5hmC. We identified 797 age-dependent differentially hydroxymethylated regions (DhMRs) in the young group (age ≤ 18), while no significant DhMR was identified in the groups over 18 years of age. Pathway and disease association analyses demonstrated that the intragenic DhMRs were in the genes involved in cell-cell communication and neurological disorders. Also, we saw significant 5hmC changes in the larger group of psychiatric genes. Interestingly, we found that the predicted cis functions of non-coding intergenic DhMRs strikingly associate with ASD and intellectual disorders. A significant fraction of intergenic DhMRs overlapped with topologically associating domains. These results together suggest that 5hmC alteration is associated with ASD, particularly in the early development stage, and could contribute to the pathogenesis of ASD.

5-Hydroxymethylcytosine alterations in the human postmortem brains of autism spectrum disorder.

Ring1 and yin yang 1-binding protein (RYBP) are central components of noncanonical polycomb-repressive complex 1 (nc-PRC1), which represses target gene expression and is required for normal organismal development. However, the molecular function of RYBP in this complex is obscure. In this study, we showed that RYBP inhibits the polyubiquitination-mediated proteasomal degradation of Ring1B independently of its ubiquitin (Ub)-protein isopeptide ligase (E3) ligase activity, leading to its stabilization and increased catalytic activity toward monoubiquitination of histone H2A at lysine 119. Mechanistic dissection further disclosed that RYBP directly binds to ubiquitin protein ligase E3A (UBE3A) to promote its ubiquitination and proteasomal degradation in an autoubiquitination-independent manner. The resultant reduction of UBE3A protein level alleviates its effect on ubiquitination-mediated degradation of Ring1B, therefore resulting in increased stability and enhanced transcriptional repressor activity on its target genes. Thus, our current findings lay a foundation for understanding how RYBP functions in nc-PRC1 complexes, which is involved in development, stem cell maintenance, and carcinogenesis.-Li, M., Zhang, S., Zhao, W., Hou, C., Ma, X., Li, X., Huang, B., Chen, H., Chen, D. RYBP modulates stability and function of Ring1B through targeting UBE3A.

RYBP modulates stability and function of Ring1B through targeting UBE3A.

Noncoding RNAs and Base Modifications: Epigenomic Players Implicated in Neurological Disorders and Tumorigenesis.

Autism spectrum disorder (ASD), a pervasive developmental neurological disorder, is characterized by impairments in social interaction and communication, and stereotyped, repetitive patterns of interests or behaviors. The mechanism of ASDs is complex, and genetic components and epigenetic modifications play important roles. In this review, we summarized the recent progresses of ASDs focusing on the genetic and epigenetic mechanisms. We also briefly discussed current animal models of ASD and the application of high-throughput sequencing technologies in studying ASD.

Autism spectrum disorders: autistic phenotypes and complicated mechanisms.

Base Modifications: Regulation of Stem Cell Functions and Diseases.

Long non-coding RNAs (lncRNAs) are transcripts which are usually more than 200 nt in length, and which do not have the protein-coding capacity. LncRNAs can be categorized based on their generation from distinct DNA elements, or derived from specific RNA processing pathways. During the past several decades, dramatic progress has been made in understanding the regulatory functions of lncRNAs in diverse biological processes, including RNA processing and editing, cell fate determination, dosage compensation, genomic imprinting and development etc. Dysregulation of lncRNAs is involved in multiple human diseases, especially neurological disorders. In this review, we summarize the recent progress made with regards to the function of lncRNAs and associated molecular mechanisms, focusing on neuronal development and neurological disorders.

Long Non-coding RNA in Neuronal Development and Neurological Disorders.

N-methyladenosine (mA), catalyzed by the methyltransferase complex consisting of Mettl3 and Mettl14, is the most abundant RNA modification in mRNAs and participates in diverse biological processes. However, the roles and precise mechanisms of mA modification in regulating neuronal development and adult neurogenesis remain unclear. Here, we examined the function of Mettl3, the key component of the complex, in neuronal development and adult neurogenesis of mice. We found that the depletion of Mettl3 significantly reduced mA levels in adult neural stem cells (aNSCs) and inhibited the proliferation of aNSCs. Mettl3 depletion not only inhibited neuronal development and skewed the differentiation of aNSCs more toward glial lineage, but also affected the morphological maturation of newborn neurons in the adult brain. mA immunoprecipitation combined with deep sequencing (MeRIP-seq) revealed that mA was predominantly enriched in transcripts related to neurogenesis and neuronal development. Mechanistically, mA was present on the transcripts of histone methyltransferase Ezh2, and its reduction upon Mettl3 knockdown decreased both Ezh2 protein expression and consequent H3K27me3 levels. The defects of neurogenesis and neuronal development induced by Mettl3 depletion could be rescued by Ezh2 overexpression. Collectively, our results uncover a crosstalk between RNA and histone modifications and indicate that Mettl3-mediated mA modification plays an important role in regulating neurogenesis and neuronal development through modulating Ezh2.

mA Regulates Neurogenesis and Neuronal Development by Modulating Histone Methyltransferase Ezh2.

Human naïve pluripotency state cells can be derived from direct isolation of inner cell mass or primed-to-naïve resetting of human embryonic stem cells (hESCs) through different combinations of transcription factors, small molecular inhibitors, and growth factors. Long noncoding RNAs (lncRNAs) have been identified to be crucial in diverse biological processes, including pluripotency regulatory circuit of mouse pluripotent stem cells (PSCs), but few are involved in human PSCs' regulation of pluripotency and naïve pluripotency derivation. This study initially planned to discover more lncRNAs possibly playing significant roles in the regulation of human PSCs' pluripotency, but accidently identified a lncRNA whose knockdown in human PSCs induced naïve-like pluripotency conversion.

Article	Year
5-hydroxymethylcytosine-mediated DNA demethylation in stem cells and development. Stem cells and development\| PubMed ID: 24400731	2014
From development to diseases: the role of 5hmC in brain. Genomics\| PubMed ID: 25205306	2014
Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signalling pathway. Nature communications\| PubMed ID: 25420914	2014
The change tendency of PI3K/Akt pathway after spinal cord injury. American journal of translational research\| PubMed ID: 26807170	2015
Genome-wide alteration of 5-hydroxymenthylcytosine in a mouse model of Alzheimer's disease. BMC genomics\| PubMed ID: 27207465	2016
iPSCs: From Bench to Clinical Bed. Stem cells international\| PubMed ID: 27738437	2016
Fat mass and obesity-associated (FTO) protein regulates adult neurogenesis. Human molecular genetics\| PubMed ID: 28398475	2017
Ten-eleven translocation 2 interacts with forkhead box O3 and regulates adult neurogenesis. Nature communications\| PubMed ID: 28660881	2017
The Dynamic DNA Demethylation during Postnatal Neuronal Development and Neural Stem Cell Differentiation. Stem cells international\| PubMed ID: 29713349	2018
5-Hydroxymethylcytosine alterations in the human postmortem brains of autism spectrum disorder. Human molecular genetics\| PubMed ID: 29790956	2018
RYBP modulates stability and function of Ring1B through targeting UBE3A. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 30040487	2019
Noncoding RNAs and Base Modifications: Epigenomic Players Implicated in Neurological Disorders and Tumorigenesis. International journal of genomics\| PubMed ID: 30402455	2018
Autism spectrum disorders: autistic phenotypes and complicated mechanisms. World journal of pediatrics : WJP\| PubMed ID: 30607884	2019
Base Modifications: Regulation of Stem Cell Functions and Diseases. Stem cells international\| PubMed ID: 30627173	2018
Long Non-coding RNA in Neuronal Development and Neurological Disorders. Frontiers in genetics\| PubMed ID: 30728830	2018
mA Regulates Neurogenesis and Neuronal Development by Modulating Histone Methyltransferase Ezh2. Genomics, proteomics & bioinformatics\| PubMed ID: 31154015	2019
Long noncoding RNA CCDC144NL-AS1 knockdown induces naïve-like state conversion of human pluripotent stem cells. Stem cell research & therapy\| PubMed ID: 31358062	2019

Xuekun Li

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