Yi E. Sun

Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells

Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. The recent demonstration that members of the Ten-eleven translocation (Tet) family of proteins can convert 5-methylcytosine to 5-hydroxymethylcytosine raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, here we show in mouse ES cells that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite an increase in levels of DNA methylation at many Tet1-binding sites, Tet1 depletion does not lead to downregulation of all the Tet1 targets. Interestingly, although Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also involved in repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators.

Full-Length Human Mutant Huntingtin with a Stable Polyglutamine Repeat Can Elicit Progressive and Selective Neuropathogenesis in BACHD Mice

To elucidate the pathogenic mechanisms in Huntingtons disease (HD) elicited by expression of full-length human mutant huntingtin (fl-mhtt), a bacterial artificial chromosome (BAC)-mediated transgenic mouse model (BACHD) was developed expressing fl-mhtt with 97 glutamine repeats under the control of endogenous htt regulatory machinery on the BAC. BACHD mice exhibit progressive motor deficits, neuronal synaptic dysfunction, and late-onset selective neuropathology, which includes significant cortical and striatal atrophy and striatal dark neuron degeneration. Power analyses reveal the robustness of the behavioral and neuropathological phenotypes, suggesting BACHD as a suitable fl-mhtt mouse model for preclinical studies. Additional analyses of BACHD mice provide novel insights into how mhtt may elicit neuropathogenesis. First, unlike previous fl-mhtt mouse models, BACHD mice reveal that the slowly progressive and selective pathogenic process in HD mouse brains can occur without early and diffuse nuclear accumulation of aggregated mhtt (i.e., as detected by immunostaining with the EM48 antibody). Instead, a relatively steady-state level of predominantly full-length mhtt and a small amount of mhtt N-terminal fragments are sufficient to elicit the disease process. Second, the polyglutamine repeat within fl-mhtt in BACHD mice is encoded by a mixed CAA-CAG repeat, which is stable in both the germline and somatic tissues including the cortex and striatum at the onset of neuropathology. Therefore, our results suggest that somatic repeat instability does not play a necessary role in selective neuropathogenesis in BACHD mice. In summary, the BACHD model constitutes a novel and robust in vivo paradigm for the investigation of HD pathogenesis and treatment.

Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells

Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.

Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes.

Location, Location, Location The genome receives epigenetic marks throughout development that regulate the activity of multiple genes. One such mark is methylation, which usually represses gene transcription. Methylation has generally been studied in the promoters of genes, where many regulatory signals coordinate to control the expression of the gene. Studying neural stem cells from mice, Wu et al. (p. 444) now show that DNA methylation can be a double-edged sword. Although methylation of DNA sequences in promoters tends to be repressive, methylation of DNA sequences beyond the promoters can actually promote gene expression. Analysis of the methyltransferase Dnmt3a in mouse neural stem cells revealed that methylations around neurogenic genes—but outside their promoters—maintained the activity of these genes. DNA methylation is normally repressive but can activate genes when the methylated sites lie outside promoter regions. DNA methylation at proximal promoters facilitates lineage restriction by silencing cell type–specific genes. However, euchromatic DNA methylation frequently occurs in regions outside promoters. The functions of such nonproximal promoter DNA methylation are unclear. Here we show that the de novo DNA methyltransferase Dnmt3a is expressed in postnatal neural stem cells (NSCs) and is required for neurogenesis. Genome-wide analysis of postnatal NSCs indicates that Dnmt3a occupies and methylates intergenic regions and gene bodies flanking proximal promoters of a large cohort of transcriptionally permissive genes, many of which encode regulators of neurogenesis. Surprisingly, Dnmt3a-dependent nonproximal promoter methylation promotes expression of these neurogenic genes by functionally antagonizing Polycomb repression. Thus, nonpromoter DNA methylation by Dnmt3a may be used for maintaining active chromatin states of genes critical for development.

Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing

Mammalian pre-implantation development is a complex process involving dramatic changes in the transcriptional architecture. We report here a comprehensive analysis of transcriptome dynamics from oocyte to morula in both human and mouse embryos, using single-cell RNA sequencing. Based on single-nucleotide variants in human blastomere messenger RNAs and paternal-specific single-nucleotide polymorphisms, we identify novel stage-specific monoallelic expression patterns for a significant portion of polymorphic gene transcripts (25 to 53%). By weighted gene co-expression network analysis, we find that each developmental stage can be delineated concisely by a small number of functional modules of co-expressed genes. This result indicates a sequential order of transcriptional changes in pathways of cell cycle, gene regulation, translation and metabolism, acting in a step-wise fashion from cleavage to morula. Cross-species comparisons with mouse pre-implantation embryos reveal that the majority of human stage-specific modules (7 out of 9) are notably preserved, but developmental specificity and timing differ between human and mouse. Furthermore, we identify conserved key members (or hub genes) of the human and mouse networks. These genes represent novel candidates that are likely to be key in driving mammalian pre-implantation development. Together, the results provide a valuable resource to dissect gene regulatory mechanisms underlying progressive development of early mammalian embryos.

A selective TrkB agonist with potent neurotrophic activities by 7,8-dihydroxyflavone

Brain-derived neurotrophic factor (BDNF), a cognate ligand for the tyrosine kinase receptor B (TrkB) receptor, mediates neuronal survival, differentiation, synaptic plasticity, and neurogenesis. However, BDNF has a poor pharmacokinetic profile that limits its therapeutic potential. Here we report the identification of 7,8-dihydroxyflavone as a bioactive high-affinity TrkB agonist that provokes receptor dimerization and autophosphorylation and activation of downstream signaling. 7,8-Dihydroxyflavone protected wild-type, but not TrkB-deficient, neurons from apoptosis. Administration of 7,8-dihydroxyflavone to mice activated TrkB in the brain, inhibited kainic acid-induced toxicity, decreased infarct volumes in stroke in a TrkB-dependent manner, and was neuroprotective in an animal model of Parkinson disease. Thus, 7,8-dihydroxyflavone imitates BDNF and acts as a robust TrkB agonist, providing a powerful therapeutic tool for the treatment of various neurological diseases.

DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling

DNA methylation is a major epigenetic factor that has been postulated to regulate cell lineage differentiation. We report here that conditional gene deletion of the maintenance DNA methyltransferase I (Dnmt1) in neural progenitor cells (NPCs) results in DNA hypomethylation and precocious astroglial differentiation. The developmentally regulated demethylation of astrocyte marker genes as well as genes encoding the crucial components of the gliogenic JAK-STAT pathway is accelerated in Dnmt1–/– NPCs. Through a chromatin remodeling process, demethylation of genes in the JAK-STAT pathway leads to an enhanced activation of STATs, which in turn triggers astrocyte differentiation. Our study suggests that during the neurogenic period, DNA methylation inhibits not only astroglial marker genes but also genes that are essential for JAK-STAT signaling. Thus, demethylation of these two groups of genes and subsequent elevation of STAT activity are key mechanisms that control the timing and magnitude of astroglial differentiation.

A positive autoregulatory loop of Jak-STAT signaling controls the onset of astrogliogenesis

During development of the CNS, neurons and glia are generated in a sequential manner. The mechanism underlying the later onset of gliogenesis is poorly understood, although the cytokine-induced Jak-STAT pathway has been postulated to regulate astrogliogenesis. Here, we report that the overall activity of Jak-STAT signaling is dynamically regulated in mouse cortical germinal zone during development. As such, activated STAT1/3 and STAT-mediated transcription are negligible at early, neurogenic stages, when neurogenic factors are highly expressed. At later, gliogenic periods, decreased expression of neurogenic factors causes robust elevation of STAT activity. Our data demonstrate a positive autoregulatory loop whereby STAT1/3 directly induces the expression of various components of the Jak-STAT pathway to strengthen STAT signaling and trigger astrogliogenesis. Forced activation of Jak-STAT signaling leads to precocious astrogliogenesis, and inhibition of this pathway blocks astrocyte differentiation. These observations suggest that autoregulation of the Jak-STAT pathway controls the onset of astrogliogenesis.

CD133+ neural stem cells in the ependyma of mammalian postnatal forebrain

The postnatal forebrain subventricular zone (SVZ) harbors stem cells that give rise to olfactory bulb interneurons throughout life. The identity of stem cells in the adult SVZ has been extensively debated. Although, ependymal cells were once suggested to have stem cell characteristics, subsequent studies have challenged the initial report and postulated that subependymal GFAP+ cells were the stem cells. Here, we report that, in the adult mouse forebrain, immunoreactivity for a neural stem cell marker, prominin-1/CD133, is exclusively localized to the ependyma, although not all ependymal cells are CD133+. Using transplantation and genetic lineage tracing approaches, we demonstrate that CD133+ ependymal cells continuously produce new neurons destined to olfactory bulb. Collectively, our data indicate that, compared with GFAP expressing adult neural stem cells, CD133+ ependymal cells represent an additional—perhaps more quiescent—stem cell population in the mammalian forebrain.

Leukemia inhibitory factor mediates an injury response but not a target-directed developmental transmitter switch in sympathetic neurons

Leukemia inhibitory factor (LIF; also known as cholinergic differentiation factor) is a multifunctional cytokine that affects neurons, as well as many other cell types. To examine its neuronal functions in vivo, we have used LIF-deficient mice. In culture, LIF alters the transmitter phenotype of sympathetic neurons, inducing cholinergic function, reducing noradrenergic function, and altering neuropeptide expression. In vivo, a noradrenergic to cholinergic switch occurs in the developing sweat gland innervation, and changes in neuropeptide phenotype occur in axotomized adult ganglia. We find that the gland innervation of LIF-deficient mice is indistinguishable from normal. In contrast, neuropeptide induction in ganglia cultured as explants or axotomized in situ is significantly suppressed in LIF-deficient mice. Thus, LIF plays a role in transmitter changes induced by axotomy but not by developmental interactions with sweat glands.